Characterisation of protein dual targeting to energy ... · Characterisation of protein dual targeting to energy organelles in Arabidopsis thaliana Chris Carrie This thesis was submitted

Characterisation of protein dual targeting to energy

organelles in Arabidopsis thaliana

Chris Carrie

This thesis was submitted as part of the requirement for the degree of Doctor of

Philosophy at the University of Western Australia

December 2010

ARC Centre of Excellence in Plant Energy Biology

School of Biomedical, Biomolecular and Chemical Sciences

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“The price of success is hard work, dedication to the job at hand,

and the determination that whether we win or lose, we have

applied the best of ourselves to the task at hand.”

Vince Lombardi

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Declaration The research presented in this thesis is my own work unless otherwise stated. This work

was carried out in the Australian Research Council Centre of Excellence in Plant

Energy Biology at the University of Western Australia. The material presented in this

thesis has not been submitted for any other degree

Chris Carrie

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Acknowledgements First and foremost I would like to thank my supervisor Jim for all his expertise,

guidance, and support over the past few years, and also for allowing me to follow some

abstract ideas, like seeing if ND proteins can target peroxisomes. I would also like to

thank all of the members, past and present, from the Whelan lab for making a great

working environment and for putting up with me even when I was a little grumpy.

Special mentions must go to Monika, Reena, and Estelle for not only providing your

expertise and knowledge for various parts of this thesis, but also for your friendship

throughout the course of my PhD. I want to thank everyone else from the Centre of

Plant Energy Biology, as it really has been a great place to come to work everyday.

Special mentions must go to Harvey, Etienne, and Holger for kindly giving me some of

your hard earned peroxisomes for use in some of my experiments.

I would like to thank my family who have supported me throughout this journey, even

when I missed family events to be in the lab. Part of this thesis really belongs to all of

you as well because without you I probably never would have been able to do it. I wont

mention everyone by name there really are to many of you but special must go to my

Mum. Lastly I would like to thank Amy for the unconditional love and support I get

from you everyday.

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Publications Primary studies published during my PhD: Study I: Carrie C, Murcha MW, Millar AH, Smith SM, Whelan J (2007) Nine 3-

ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria. Plant Mol Biol 63: 97-108.

Study II: Carrie C, Kuhn K, Murcha MW, Duncan O, Small ID, O'Toole N,

Whelan J (2009) Approaches to defining dual-targeted proteins in Arabidopsis. Plant J 57: 1128-1139.

Study III: Carrie C, Murcha MW, Kuehn K, Duncan O, Barthet M, Smith PM,

Eubel H, Meyer E, Day DA, Millar AH, Whelan J (2008) Type II NAD(P)H dehydrogenases are targeted to mitochondria and chloroplasts or peroxisomes in Arabidopsis thaliana. FEBS Lett 582: 3073-3079.

Study IV: Lister R, Carrie C, Duncan O, Ho LH, Howell KA, Murcha MW,

Whelan J (2007) Functional definition of outer membrane proteins involved in preprotein import into mitochondria. Plant Cell 19: 3739-3759.

Study V: Carrie C, Giraud E, Duncan O, Xu L, Wang Y, Huang S, Clifton R,

Murcha M, Filipovska A, Rackham O, Vrielink A, Whelan J (2010) Conserved and Novel Functions for Arabidopsis thaliana MIA40 in Assembly of Proteins in Mitochondria and Peroxisomes. J Biol Chem 285: 36138-36148

Study VI: Carrie C, Murcha MW, Whelan J (2010) An in Silico Analysis of the

Mitochondrial Protein Import Apparatus of Plants. BMC Plant Biol 10: 249

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Additional publications Albrecht V, Simkova K, Carrie C, Delannoy E, Giraud E, Whelan J, Small ID, Apel K,

Badger MR, Pogson BJ (2010) The Cytoskeleton and the Peroxisomal-Targeted SNOWY COTYLEDON3 Protein Are Required for Chloroplast Development in Arabidopsis. Plant Cell, in press

Carrie C, Giraud E, Whelan J (2009) Protein transport in organelles: Dual targeting of

proteins to mitochondria and chloroplasts. FEBS J 276: 1187-1195 Giraud E, Ng S, Carrie C, Duncan O, Low O, Lee CP, Van Aken O, Millar AH,

Murcha MW, Whelan J (2010) TCP transcription factors link the regulation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis thaliana. Plant Cell, in press

Ho LH, Giraud E, Lister R, Thirkettle-Watts D, Low J, Clifton R, Howell KA, Carrie C, Donald T, Whelan J (2007) Characterization of the regulatory and expression context of an alternative oxidase gene provides insights into cyanide-insensitive respiration during growth and development. Plant Physiol 143: 1519-1533

Jia L, Wu Z, Hao X, Carrie C, Zheng L, Whelan J, Wu Y, Wang S, Wu P, Mao C

(2010) Identification of a novel mitochondrial protein, short postembryonic roots 1 (SPR1), involved in root development and iron homeostasis in Oryza sativa. New Phytol, in press

Millar AH, Carrie C, Pogson B, Whelan J (2009) Exploring the function-location

nexus: using multiple lines of evidence in defining the subcellular location of plant proteins. Plant Cell 21: 1625-1631

Murcha MW, Elhafez D, Lister R, Tonti-Filippini J, Baumgartner M, Philippar K,

Carrie C, Mokranjac D, Soll J, Whelan J (2007) Characterization of the preprotein and amino acid transporter gene family in Arabidopsis. Plant Physiol 143: 199-212

Thatcher LF, Carrie C, Andersson CR, Sivasithamparam K, Whelan J, Singh KB

(2007) Differential gene expression and subcellular targeting of Arabidopsis glutathione S-transferase F8 is achieved through alternative transcription start sites. J Biol Chem 282: 28915-28928

Van Aken O, Zhang B, Carrie C, Uggalla V, Paynter E, Giraud E, Whelan J (2009)

Defining the Mitochondrial Stress Response in Arabidopsis thaliana. Mol Plant 2:1310-1324

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Abbreviations ACAT Acetoacetyl-CoA thiolase

AGAT Alanine/glyoxylate aminotransferase

AGL Agamous like protein

AOX Alternative oxidase

APL Altered phloem development

CaS Calcium-sensing receptor

Ccs1 Copper/zinc chaperone for superoxide dismutase

CP Carrier protein

CSD Copper/zinc superoxide dismutase

Erv1 Essential for respiration and vegetative growth

ER Endoplasmic reticulum

FAD Flavin adenine dinucleotide

GDP Guanosine diphosphate

GeBP GL1 enhancer binding protein

GFP Green flouresence protein

GST Glutathione S-transferase

Hot13 Helper of Tim protein of 13 kDa

Icp55 Intermediate cleavage peptidase of 55 kDa

KAT 3-Ketoacyl-CoA thiolase

MCD Malonyl CoA decarboxylase

Mdm10 Mitochondria distribution and morphology protein 10

MIA Mitochondrial import and assembly

Mim1 Mitochondrial import 1

MPP Mitochondrial processing peptidase

ND Type II alternative NAD(P)H dehydrogenase

NDC1 Type II alternative NAD(P)H dehydrogenase C1

OM64 mitochondrial outer membrane protein of 64 kDa

Omp85 Outer membrane protein of 85 kDa

PRAT Preprotein and amino acid transporter

PQ Plastoquinone

PTS1 Peroxisomal targeting signal type 1

RFP Red flouresence protein

SPP Stromal processing peptidase

SSU Small subunit of Ribulose 1,5-bisphosphate carboxylase/oxygenase

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TLP Tubby like protein

TIC Translocase of the inner envelope of chloroplasts

TIM Translocase of the inner mitochondrial membrane

TOC Translocase of the outer envelope of chloroplasts

TOM Translocase of the outer mitochondrial membrane

TPR Tetratricopeptide repeat

SAM Sorting and assembly machinery

UTR Untranslated region

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Abstract Eukaryotic cells are defined by their containment of membrane bound

compartments, termed organelles. The majority of proteins found within a particular

organelle are encoded by genes in the nucleus, synthesised in the cytosol, and

subsequently targeted to specific organelles. The traditional view of biology is that one

gene gives rise to one protein, targeted to one location. However, in the past 15 years an

increasing number of proteins have been found to be localised in more than one

organelle, a phenomenon called dual targeting.

In the model plant Arabidopsis thaliana, only a limited number of proteins have

been identified to date as being dual targeted. The work carried out in studies I, II and

III aimed to identify new dual targeted proteins in Arabidopsis. A list of candidate dual

targeted proteins was defined by cross-referencing a number of publically available

subcellular localisation datasets, generated by large scale proteomic studies. In addition,

candidate genes were also identified by computational predictions, based on the protein

amino acid sequences. A selection of proteins were then selected for targeting analysis

by in vivo green fluorescent protein (GFP) tagging, results were then confirmed by

either in vitro import assays or Western blot analysis.

In this way, studies I, II, and III led to the identification of 12 new dual targeted

proteins in Arabidopsis. Five proteins were found to target both mitochondria and

plastids, one was found to target mitochondria and the nucleus, and five were found to

target both mitochondria and peroxisomes. The latter is particularly significant as this

was the first time that dual targeting between mitochondria and peroxisomes had been

demonstrated in plants. Of these, three of the alternative NAD(P)H dehydrogenases of

the inner mitochondrial membrane were subsequently found to also be targeted to the

peroxisome. This is mediated by an N-terminal mitochondrial targeting signal and a C-

terminal peroxisomal targeting signal. The peroxisomal targeting was missed in

previous studies due to the detection of localisation using only C-terminal GFP fusions.

By analysing the subcellular localisation of Arabidsis thiolase proteins, study I revealed

that the β-oxidation of fatty acids does not occur in plant mitochondria, given that no

thiolases were found to be targeted to mitochondria in Arabidopsis. This was in contrast

to previous proteomic and activity assays which determined that some plant thiolases

were mitochondrial. These results confirmed the requirement for multiple lines of

complimentary evidence when performing subcellular localisation studies.

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While analysing the potential dual targeted proteins, it become evidenced that a

number of experimental parameters are critical for designing GFP based studies,

especially with regards to dual targeted proteins. It was shown that the location of the

passenger protein, whether it be C- or N-terminally based, the type of tissue used and

the analyses of all possible gene models are critical for accurate determination of

localisation (Study II). Furthermore, all results must also be verified by a second

technique, so that multiple lines of complementary data are used before subcellular

localisation is accurately determined. These guidelines were proposed after it was

discovered that a number of proteins previously assigned to only one location, were

subsequently found to be dual targeted after using these techniques. Also, some proteins

previously thought to be dual targeted were found to be only targeted to one organelle.

In order to better understand the mechanisms of dual targeting in Arabidopsis,

study IV aimed to determine the mitochondrial receptors involved in the import of dual

targeted proteins. It was demonstrated that dual targeted proteins appear to use a

different import pathway than mitochondrial specific proteins. This was proposed to be

mediated by the mitochondrial import receptor Metaxin, which was demonstrated to

interact with dual targeted proteins (Study IV). In addition, a new plant specific

mitochondrial import receptor was identified, OM64, (Study IV). Taking this further,

study VI analysed the mitochondrial import apparatus in a number of different plant

species and other organisms, revealing that the evolution of the mitochondrial import

apparatus of plants is still an on going process. The identified differences between plant

import components and their yeast and human counterparts is thought to be due to the

selective pressure to sort proteins between mitochondria and chloroplasts, suggesting a

novel mode of evolution in plants.

Whilst analysing the mitochondrial import components of Arabidopsis, it

became apparent that the protein orthologues to the yeast Mia40 in Arabidopsis

contained a peroxisomal targeting signal. Upon closer, in vivo analysis of Mia40

localisation, it was evidenced that in Arabidopsis, Mia40 is dual targeted to

mitochondria and peroxisomes, unlike the yeast Mia40, which is localised only in the

mitochondria (Study V). Upon functional analysis of Arabidopsis Mia40, it was also

found to be different to yeast, in that it was not required for the inner membrane space

disulfide relay cycle. It was shown that Arabidopsis Mia40 is involved in the formation

of complex I of the mitochondrial respiratory chain, and also in the oxidative folding of

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the copper/zinc chaperone for SOD (Ccs1) and copper/zinc superoxide dismutase

(SOD), in both the mitochondria and peroxisomes. Thus, plants have gained an extra

function for Mia40, in addition to having it dual targeted to the peroxisomes and

mitochondria. These findings support the theory that proteins are dual targeted in order

to increase the functions of some genes.

Overall, a number of novel dual targeted proteins were identified in Arabidopsis,

not only between mitochondria and chloroplasts, but also between mitochondria and

peroxisomes, and mitochondria and the nucleus, giving novel insights into functions of

these proteins. A number of factors that can influence dual targeting were also shown,

including the type of tissues and techniques used. While the extent of dual targeting and

the exact mechanisms by which it occurs are only just beginning to be understood, the

reasons why some proteins are dual targeted are still largely unknown. However, by

utilising some of the techniques used in this thesis it may eventually possible to identify

all dual targeted proteins in plants, which upon closer inspection could give insights into

the purpose for dual targeting in plants

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Contents

DECLARATION .......................................................................................................... III

ACKNOWLEDGEMENTS ......................................................................................... IV

PUBLICATIONS ........................................................................................................... V

ADDITIONAL PUBLICATIONS .............................................................................. VI

ABBREVIATIONS ..................................................................................................... VII

ABSTRACT .................................................................................................................. IX

CONTENTS ................................................................................................................. XII

CHAPTER 1 .................................................................................................................... 1

GENERAL INTRODUCTION ...................................................................................... 1

1.0 EUKARYOTIC CELL EVOLUTION – ENDOSYMBIOSIS .................................................. 2

1.1 GENE TRANSFER ....................................................................................................... 2

1.2 PEROXISOME – ENDOSYMBIOSIS VS NON-ENDOSYMBIOTIC ORIGIN ........................... 3

1.3 PROTEIN TARGETING AND IMPORT ............................................................................ 4

1.3.1 Plastid protein targeting and import ................................................................ 4

1.3.2 Plastid targeting signals .................................................................................. 4

1.3.3 Outer envelope membrane import machinery: TOC complex ......................... 5

1.3.4 Inner envelope import machinery: TIC complex ............................................. 7

1.3.5 Thylakoid protein import .................................................................................. 8

1.4 MITOCHONDRIAL PROTEIN TARGETING AND IMPORT ................................................ 8

1.4.1 Mitochondrial targeting signals ....................................................................... 8

1.4.2 Mitochondrial protein import .......................................................................... 9

1.5 PROTEIN IMPORT INTO PLANT MITOCHONDRIA ....................................................... 12

1.6 PEROXISOMAL PROTEIN IMPORT ............................................................................. 14

1.6.1 Receptor cargo interaction and membrane docking ...................................... 14

1.6.2 Receptor cargo translocation cargo release and receptor recycling ............ 15

1.7 DUAL TARGETING ................................................................................................... 17

1.7.1 Ambiguous targeting signals .......................................................................... 18

1.7.2 Alternative mechanisms of dual targeting ...................................................... 19

1.7.3 Systematic studies of dual targeted proteins .................................................. 20

1.8 RESEARCH PROPOSAL ............................................................................................. 21

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CHAPTER 2 .................................................................................................................. 26

FOREWORD TO STUDY I ......................................................................................... 27

CHAPTER 3 .................................................................................................................. 40

FOREWORD TO STUDY II ....................................................................................... 41

CHAPTER 4 .................................................................................................................. 54

FOREWORD TO STUDY III ...................................................................................... 55

CHAPTER 5 .................................................................................................................. 62

FOREWORD TO STUDY IV ...................................................................................... 63

CHAPTER 6 .................................................................................................................. 86

FOREWORD TO STUDY V ........................................................................................ 87

CHAPTER 7 .................................................................................................................. 99

FOREWORD TO STUDY VI .................................................................................... 100

CHAPTER 8 ................................................................................................................ 116

GENERAL DISCUSSION .......................................................................................... 117

7.1 DEFINING DUAL TARGETED PROTEINS: TARGETING VS ACCUMULATION STUDIES . 117

7.2 MECHANISMS OF DUAL TARGETING – SIGNALS AND IMPORT RECEPTORS .............. 122

7.3 REASONS FOR DUAL TARGETING ........................................................................... 124

7.4 FUTURE PERSPECTIVES ......................................................................................... 126

REFERENCES ............................................................................................................ 127

Chapter 1 General Introduction

1

Chapter 1

General Introduction


2

1.0 Eukaryotic cell evolution – Endosymbiosis

The defining feature of eukaryotic cells is that they contain membrane bound

compartments termed organelles. Plant cells contain three organelles involved in energy

metabolism; plastids, mitochondria, and peroxisomes. Chloroplasts, (specialised

plastids) produce energy through the conversion of light energy into chemical energy.

Chloroplasts also synthesise amino acids, lipids, and many other specialised

compounds. Mitochondria are involved in energy metabolism through the oxidation of

organic acids into reduced nucleotides via the tricarboxcyclic acid cycle (TCA cycle),

which are finally oxidised into chemical energy by the electron transport chain.

Peroxisomes play an important role in plant energy metabolism in a number of ways,

such as lipid metabolism, photorespiration, nitrogen metabolism, detoxification, and the

synthesis of some plant hormones. Integral to the function of plant cells is the

integrated nature of metabolism, as all three organelles work together to produce the

energy required for cellular growth and maintenance (Siedow and Day, 2000).

Except for rare exceptions in prokaryotes, the metabolic compartmentalisation

of organelles is specific to eukaryotic cells (Martin, 2010). The endosymbiotic origin of

organelles is explained by the hypothesis that mitochondria and plastids were once free-

living bacteria that underwent evolutionary transformation into complex metabolic

compartments (Tielens et al., 2002; van der Giezen, 2009). Both mitochondria and

plastids have retained their own DNA and as such, the sequence and structure provides

compelling evidence that they were once free living prokaryotes (Gray, 1999).

However, these organellor genomes are highly reduced in comparison to their free-

living ancestors (Gray, 1999). Plastid genomes have been shown to encode between 20

– 200 proteins, ywhilst mitochondrial genomes encode for as little as 3 proteins in

Plasmodium falciparum, ranging up to 67 proteins in Reclinomonas americana (Timmis

et al., 2004). In the particular case of highly specialised mitochondria called mitosomes

and hydrogenosomes, organelles which have lost their entire genomes (Tovar et al.,

2003; Boxma et al., 2005; van der Giezen and Tovar, 2005; van der Giezen, 2009),

energy metabolism still occurs though they have lost their ability to carry out oxidative

phosphorylation.

1.1 Gene transfer

Despite the reduction in genome size, plastids and mitochondria still contain

more than one thousand proteins (Millar et al., 2006). During millions of years of


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evolution these organelles lost or transferred most of their genes to the nucleus (Martin

et al., 1993). The transferred genes acquired expression and targeting signals such that

the encoded protein could be translated on cytosolic ribosomes and imported into the

organelle to achieve function (Martin, 2010). Once the host copy of a particular gene

acquired all the mechanisms for correct expression, translation, import, and function,

the organelle copy was no longer required and was lost, thus completing endosymbiotic

gene transfer (Allen, 2003). It could readily be imagined that at the onset of

endosymbiosis, which led to the formation of mitochondria and plastids, the

endosymbiont may have underwent lysis, providing a pool of total ‘genome’ DNA, that

may have been subsequently incorporated into the host genomic DNA. This is referred

to as genome transfer. This type of genome transfer, or partial genome transfer, can be

observed even today between organelles and the nucleus (Timmis et al., 2004; Kleine et

al., 2009). However, it has yet to be shown that this results in functional gene relocation

between organelles and the nucleus. In contrast, both for mitochondria and plastids, the

transfer of individual genes likely occurs via a reverse transcribed cDNA from organelle

mRNA, a process shown to be still ongoing (Adams and Palmer, 2003).

1.2 Peroxisome – Endosymbiosis vs non-endosymbiotic origin

There are currently two theories to explain the origin of peroxisomes; the first is

that peroxisomes originate from an ancient endosymbiont, and the second suggests that

peroxisomes are derived from the endoplasmic reticulum (ER) (Gabaldon, 2010). The

fact that core peroxisomal mechanisms for division, biogenesis, and maintenance are

conserved across a diverse range of organisms (Gabaldon et al., 2006) suggests that

peroxisomes have likely evolved from a common ancestor arising from a single

evolutionary event. The endosymbiotic origin for peroxisomes was proposed after it

was realised that peroxisomes are formed by division and have the ability to import

proteins in a post-translational manner. These features are common with the

endosymbiotically derived mitochondria and plastids (Lazarow and Fujiki, 1985). An

endosymbiotic origin for peroxisomes provides an appealing metabolic scenario, which

takes into account the role of enzymes involved in the detoxification of highly reactive

oxygen species (de Duve, 1982). According to this theory, the proto-peroxisome was

acquired at a time when the level of atmospheric oxygen was increasing and represented

a toxic compound for the majority of organisms at the time (de Duve, 1982), proposing

that peroxisomes originated from an ancient actinobacterium (Duhita et al.).


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However there is considerable experimental evidence demonstrating that the

biogenesis of peroxisomes is tightly linked to the ER. Evidence supporting this proposal

include the demonstration that some peroxisomal membrane proteins are first targeted

to the ER prior to reaching peroxisomes. Additionally, peroxisome-less yeast mutants

can form peroxisomes de novo from the ER upon introduction of the wildtype gene

(Erdmann and Kunau, 1992; Tabak et al., 2003). Independent phylogenetic evidence

also links peroxisome evolution to the ER, showing that components of the peroxisomal

import machinery are related to the components of the ER associated decay pathway

(Gabaldon et al., 2006; Schluter et al., 2006). It should also be noted that some plastid

proteins are initially targeted via the ER, suggesting that the endosymbiotic evidence for

the origin of peroxisomes may have been lost over time (Villarejo et al., 2005).

1.3 Protein targeting and import

Irrespective of the evolutionary origin, the majority of organellor proteins are

encoded by the nucleus. These cytosolically synthesized proteins must be targeted to the

correct organelle (Figure 1.1). Organellor proteins contain within their amino acid

sequences all the information required for targeting to the correct organelle. A brief

description of plastid, mitochondrial, and peroxisomal targeting signals and import

apparatus is outlined below.

1.3.1 Plastid protein targeting and import

Most prior research on plastid protein import has been carried out using

chloroplasts, which as the site of photosynthesis, are the most abundant type of plastids.

It is proposed that other types of plastids contain the same import apparatus on the

envelope membranes (Strzalka et al., 1987; Wan et al., 1996; Davila-Aponte et al.,

2003). A general import pathway of the chloroplast envelopes has been described which

constitutes the translocons at the outer and inner envelope of chloroplasts (TOC and

TIC respectively) (Figure 1)(Balsera et al., 2009).

1.3.2 Plastid targeting signals

The majority of nuclear encoded chloroplast proteins contain an N-terminal

targeting signal, which is cleaved upon import by the stromal processing peptidase

(SPP) (Bruce, 2000, 2001). Early research into chloroplast targeting signals proposed

that they would contain definite motifs and structural features specifically designed to

avoid mis-targeting to other organelles. However, plastid targeting signals are in fact


5

heterogeneous in sequence and are mostly unstructured (Bruce, 2000, 2001). The

general features of chloroplast targeting signals are that they lack acidic residues giving

an overall positive charge, they are enriched in hydroxylated amino acids (mainly

serine) and vary in length between 20 – 150 amino acids. These features also resemble

the targeting signals of mitochondrial proteins. In contrast to mitochondrial targeting

signals, chloroplast targeting signals show no secondary structure in aqueous solution

(Krimm et al., 1999). It has been proposed that chloroplast targeting signals can form a

perfect random coil (von Heijne and Nishikawa, 1991). The lack of a secondary

structure is thought to be due to the recruitment of cytosolic factors following

translation (May and Soll, 2000; Zhang and Glaser, 2002). It has also been observed

that some targeting signals acquire a typical structure upon contact with the lipid

environment of the outer envelope membrane, which has a unique composition that

distinguishes it from the outer membrane of mitochondria (Wienk et al., 2000; Bruce,

2001). The interaction between the targeting signals and the galactolipids specific to the

outer envelope membrane has been speculated to stimulate import (Chen and Li, 1998).

A recent study using hierarchical clustering of targeting signals defined motifs

contained within the targeting signals into seven distinct groups (Lee et al., 2008).

1.3.3 Outer envelope membrane import machinery: TOC complex

The TOC complex is composed of a channel protein (Toc75), two GTPase

receptors (Toc159 and Toc34), and two dynamically associated components (Toc64 and

Toc12) (Oreb et al., 2008). The import channel forming protein, Toc75, is a highly

conserved β-barrel protein which is evolutionary related to Omp85, a protein involved

in the integration of proteins into the bacterial outer membrane in gram negative

bacteria (Bolter et al., 1998; Gentle et al., 2005). The Toc75 protein contains two

domains; an N-terminal cytosolic domain termed the recognition complex assembly

unit, and a C-terminal membrane embedded domain, forming the β-barrel type protein

channel (Hinnah et al., 2002; Ertel et al., 2005). The main preprotein receptor of the

TOC complex has been identified as Toc34 (Balsera et al., 2009). Toc34 is anchored to

the membrane by a single transmembrane helix in the C-terminal end of the protein and

contains a large GTPase domain located in the cytosol (May and Soll, 1998; Sun et al.,

2002; Koenig et al., 2008). Based on specific energy requirements, the import of

proteins into plastids can be initially divided into three steps (Perry and Keegstra, 1994;

Kouranov and Schnell, 1997): (1) reversible binding on the plastid surface independent

of nucleotides; (2) stable binding and insertion at < 100 µM ATP in the presence of


6

GTP (Kessler et al., 1994); and (3) translocation across the outer and inner envelope

requiring > 100 µM ATP (Pain and Blobel, 1987; Theg et al., 1989).

The recognition of plastid precursor proteins by the GTPase receptors on the

outer envelope surface has been intensively studied (Balsera et al., 2009). There are

currently two models for precursor recognition. The first favours Toc159 as the initial

receptor (Hiltbrunner et al., 2001; Bauer et al., 2002; Smith et al., 2004). The process of

GTP hydrolysis and oligomerisation of both GTPases results in the transfer of precursor

proteins to the Toc75 import channel (Hirsch et al., 1994; Schnell et al., 1994; Ma et al.,

1996). Transport through the Toc75 channel and across the membrane is then furthered

by the inter-envelope space located Hsp70 (Perry and Keegstra, 1994). This hypothesis

has been challenged by a second model, which proposes that Toc34 is the initial

receptor for plastid precursor proteins (Sveshnikova et al., 2000; Schleiff et al., 2003;

Becker et al., 2004). Electron microscopy of purified TOC complex suggests that the

TOC complex contains a toroidal structure composed of four protein channels enclosing

a central protruding domain (Schleiff et al., 2003). Stoichiometric analysis of the TOC

complex further identified that it contained four Toc75 proteins, four Toc34 proteins,

and a single Toc159 protein (Schleiff et al., 2003). It has been speculated that the

central Toc159 represents the GTP-driven import motor, which moves preproteins

through the import channel after receiving them from Toc34 (Balsera et al., 2009). A

single Toc159 molecule could alternatively interact with the four Toc34 receptors in a

rotational ‘on and off mode’ (Sveshnikova et al., 2000; Schleiff et al., 2002; Schleiff et

al., 2003). It has also been observed that a TOC complex consisting of Toc159 and

Toc75 can import precursor proteins into liposomes demonstrating a minimal TOC

complex (Balsera et al., 2009). A different study led to the conclusion that the G-

domain of Toc159 was not necessary for protein import (Chen et al., 2000; Lee et al.,

2003, precursor proteins could completely bypass the receptor subunits and bind

directly to Toc75 {Chen, 2000 #161)(Chen et al., 2000). Consequently, import was

achieved without the G-domain of Toc159. Although the two models agree that the

GTP mediated regulation of the Toc34/Toc159 receptors and the central channel

function of Toc75 are both required for protein import, there are still conflicting views

about the exact specifics of recognition and translocation.


7

1.3.4 Inner envelope import machinery: TIC complex

The composition and stoichiometry of the membrane complex for import into

chloroplasts at the inner envelope membrane is not as well defined as the TOC

machinery. The TIC complex is composed of between 7 and 8 proteins in higher plants,

including Tic110 and Tic20, which form the putative constituents of the translocon

channel, the cochaperone Tic40 and the translocon associated proteins Tic55, Tic32 and

Tic62 (Stengel et al., 2007). The individual and cooperative functions of these proteins

are still unknown. The identity of the most important part of the complex, the translocon

protein conducting channel, still remains elusive. In the case of the TOC translocon,

Toc75 has been unequivocally identified as an essential channel forming protein. In the

case of the TIC complex there is still much discussion as to the identity of the

translocation channel. Several candidates have been put forward to fulfil this essential

role, including Tic110, Tic20, and Tic21 (Chen et al., 2002; Heins et al., 2002; Teng et

al., 2006). Tic21 and Tic20 both show structural similarities to the translocation channel

proteins from the mitochondrial inner membrane Tim17 and Tim23. However,

biochemical evidence for this function is lacking (Rassow et al., 1999; Teng et al.,

2006). Tic20 has been shown to be an essential protein for plant development (Chen et

al., 2002). It has been proposed that Tic20 plays a regulatory role and may also be

involved in TIC complex assembly (van Dooren et al., 2008).In contrast, Tic110

displays features that make it a good candidate for the translocation channel: it is

conserved throughout plastid types across multiple species (Davila-Aponte et al., 2003);

is expressed in cells in comparative amounts to Toc75 (Vojta et al., 2004); shows

channel activity in vitro (Heins et al., 2002); has been found to be associated with

precursor proteins and chaperones (Lubeck et al., 1996; Nielsen et al., 1997); can form

super complexes with the TOC complex (Schnell and Blobel, 1993); and is essential for

plant viability (Inaba et al., 2005). In any case, Tic110 has an essential role in preprotein

recognition on the trans side of the outer membrane and associates with Tic40 to link

the central translocation channel and import motor (Kovacheva et al., 2005).

Apart from the main channel forming components of the motor module, the TIC

complex also contains a number of regulatory members (Balsera et al., 2009). They all

contain features for sensing the redox state of the chloroplasts and thereby regulate

import at the inner envelope, according to the specific requirements of the plastid

(Balsera et al., 2009). It has been known for sometime that at least two precursors; non-


8

photosynthetic ferredoxin (FdIII) and FNR isoform II (FNRII), are differentially

imported in the light and dark (Hirohashi et al., 2001).

1.3.5 Thylakoid protein import

The thylakoid membrane is a highly specialised membrane that contains the

photosystems and the ATPase complex, which are involved in photosynthetic electron

transfer, coupled to the chemiosomotic process. The abundant photosynthetic machinery

of the thylakoid membrane is composed of subunits encoded by both nuclear and

plastidic genomes, in contrast to all known plastid luminal proteins, which are nuclear

encoded (Balsera et al., 2009). Proteomics and other analyses have identified that

systems homologous to the Sec, Tat, and YidC machineries are found in the thylakoids,

termed the cpSec, cpTat and Alb3 machinery respectively (Schunemann, 2007). These

machineries are responsible for the import and assembly of thylakoidal proteins

(Schunemann, 2007). Some subunits of the thylakoids are targeted by a bipartite

targeting signal at their N-terminus. The first part directs the proteins through the outer

and inner envelopes to the stroma, where the protein is cleaved by the SPP into an

intermediate form of the protein. The second part of the targeting signal guides the

intermediate form of the protein into the thylakoids, where it is processed by the

thylakoidal processing peptidase, thereby generating the mature form of the protein

(Sakamoto, 2006).

1.4 Mitochondrial protein targeting and import

Most research concerning the targeting and import of proteins into mitochondria

originates from studies on the model organism Saccharomyces cerevisiae (yeast)

(Figure 1.2). Import into mitochondria requires several membrane bound complexes:

the translocase of both the outer and inner membranes (TOM and TIM respectively); the

sorting and assembly machinery (SAM) which inserts β-barrel proteins into the outer

membrane; and the presequence translocase associated motor (PAM) which is required

for translocation into the matrix (Figure 1)(Chacinska et al., 2009). There is also another

pathway termed the mitochondria import and assembly (MIA) pathway for the import

of intermembrane space proteins (Chacinska et al., 2004).

1.4.1 Mitochondrial targeting signals

Mitochondrial targeting signals are generally located at the N-terminus and are

between 20 and 60 amino acids in length, are enriched in positively charged and


9

hydroxylated residues, and have the ability to form an α-amphipathic helix.

Mitochondrial targeting signals are also generally cleaved upon import. Although

sequence conservation at the cleavage site is low, most contain an arginine residue at

position -2 or -3 (Schneider et al., 1998). The mitochondrial targeting signal is crucial

for interaction with the TOM complex on the outer membrane of mitochondria. A large

number of mitochondrial proteins have also been shown to contain internal targeting

signals; the characteristics of which are much less defined than N-terminal targeting

signals (Neupert and Herrmann, 2007). This class of targeting signals is found on all

outer membrane proteins and also a number of proteins destined for the matrix and

inner membrane (Neupert and Herrmann, 2007). Hydrophobic proteins destined for the

inner membrane, including mitochondrial carrier proteins, mainly contain internal

targeting information within their transmembrane domains (Neupert and Herrmann,

2007). All mitochondrial targeting signals are recognized by specific receptors of the

TOM complex and guided to their specific translocons of the inner membrane.

1.4.2 Mitochondrial protein import

TOM complex

With a few exceptions, proteins that are imported into mitochondria must first

interact with the TOM complex. The TOM complex is a 400 kDa protein complex

consisting mainly of the integral membrane protein Tom40, which forms the protein

channel and a number of smaller single α-helical transmembrane subunits. Also

associated with the TOM complex are the primary receptors for mitochondrial targeting

signals, anchored to the membrane by a single transmembrane helix. Tom40 contains a

predicted amphipathic β-barrel structure embedded into the membrane (Hill et al.,

1998). The receptor domains of the TOM complex contain a hydrophobic groove,

which interacts with the hydrophobic signals or the hydrophobic surface of

mitochondrial targeting signals (Abe et al., 2000; Chan et al., 2006). Upon receptor

binding of mitochondrial targeting signals, the protein is transferred to the translocation

pore of Tom40. According to the binding chain hypothesis, the translocation of proteins

through the TOM complex does not rely on ATP or the membrane potential but rather

on an interaction with several binding sites with increasing affinity to cross the outer

membrane (Meisinger et al., 2001).

SAM complex

β-barrel proteins of the mitochondrial outer membrane are inserted by a

specialised integration pathway. After transport through the TOM complex, β-barrel


10

proteins are recognised by small TIMs in the intermembrane space (IMS) and guided to

the SAM complex. The SAM complex is made up of a number of integral membrane

subunits, the most notable Sam50. Sam50 is a member of the Omp55 family of proteins,

involved in the insertion of outer membrane proteins in bacteria (Paschen et al., 2003).

Sam50, which is conserved in most organisms, is thought to be the integrase subunit of

the SAM complex. The integration of β-barrel proteins into the outer membrane is

independent of ATP and is thought to be an energetically favourable process. However,

the actual mechanistic action of the SAM complex is yet to be determined. Recently the

SAM complex has also been implicated in the insertion of α-helical proteins into the

outer membrane (Stojanovski et al., 2007).

IMS import

All mitochondrial proteins destined for the IMS are nuclear encoded and

generally do not contain classical mitochondrial targeting signals. There are two main

pathways for proteins to be imported into the IMS (Herrmann and Hell, 2005). The first

describes the import of IMS proteins that contain a mitochondrial targeting signal with a

bipartite presequence, which is characterised by a hydrophobic sorting signal

downstream of the targeting signal. The first most N-terminal part of the targeting

signal directs the protein to the TIM23 complex, where the hydrophobic region anchors

the protein into the inner membrane. A processing step in the IMS proteolytically

removes the presequence behind the transmembrane region, leaving behind a soluble

mature IMS protein [In yeast both cytochrome C1 and cytochrome b2 are processed in

this manner (Gasser et al., 1982)]. However, most IMS proteins do not contain a

cleavable N-terminal mitochondrial targeting signal. Following TOM complex

translocation IMS proteins interact with specific factors within the IMS. These factors

promote oxidative folding events which lead to the trapping of the proteins in the IMS.

The IMS of mitochondria contains a complete set of machinery to catalyse the oxidative

folding, and reflects the evolution of these components from the periplasmic space of

bacteria (Mesecke et al., 2005). Substrate proteins, which are specific for the

mitochondrial IMS import and assembly (MIA) pathway contain conserved cysteine

residues. After passage through the TOM complex, IMS destined proteins interact with

the essential protein Mia40, which forms intermolecular disulfide bridges within the

IMS destined protein (Chacinska et al., 2004). Upon release from Mia40, the substrate

proteins are oxidised into functionally folded proteins. For reoxidation, Mia40 must

interact with the sulfhydryl oxidase Erv1 which itself is regenerated by transferring


11

electrons to cytochrome c and the respiratory chain (Mesecke et al., 2005). The final

release of electrons to molecular oxygen completes the electron transfer chain of the

intermembrane space assembly pathway.

TIM23 and TM22 complexes

There are two distinct pathways for import into the mitochondrial matrix and

insertion into the inner membrane. The general import pathway directs proteins with

mitochondrial targeting signals to the TIM23 complex, whilst the carrier import

pathway inserts proteins into the membrane via the TIM22 complex. Once the precursor

proteins reach the matrix, the presequence is removed by the mitochondrial processing

peptidase and molecular chaperones assist the folding and assembly of precursor

proteins into functional complexes.

TIM23 complex

The TIM23 complex is responsible for the translocation of proteins into the

matrix and for the import of a limited number of inner membrane and IMS proteins. In

all cases, TIM23 substrates contain an N-terminal targeting signal. Inner membrane

destined proteins containing a single transmembrane helix use a stop transfer

mechanism, where the transmembrane helix acts as the stop transfer signal. If an inner

membrane protein contains more than one transmembrane region, it may be fully

imported into the matrix first and then subsequently relocated to the inner membrane by

Oxa1p, which is a YisC/Alb3 homolog (Hell et al., 1998). Oxa1p is also involved in the

insertion of mitochondrially encoded proteins into the inner membrane from the matrix

(Hell et al., 1998; Luirink et al., 2001). In general, proteins are passed from the TOM

complex to the TIM23 complex by interacting with receptor like domains of the TIM23

complex in the IMS (Mokranjac et al., 2003). The membrane potential across the inner

membrane provides the energy source for translocation. Similar to translocation through

the TOM complex, precursors moving through the TIM23 complex interact with a

number of different subunits until fully translocated into the matrix, upon which the

precursor proteins are cleaved and folded into fully functional proteins. During the

import of matrix targeted proteins, the TIM23 complex becomes associated with the

PAM complex. The PAM complex is comprised of a number of small subunits, the

major constituents being mtHsp70 and the nucleotide exchange factor Mge1. The exact

role of the PAM complex is not yet completely understood, although it is thought to

provide two important roles during the import of matrix targeted proteins: the first is the


12

active pulling of preproteins through the TIM23 channel and the second is the passive

trapping of preproteins within the matrix by binding mtHsp70. The cooperation between

the TIM23 and PAM complex results in the import and correct folding of matrix

targeted proteins.

TIM22

Most inner membrane proteins that are synthesised in the cytosol do not contain

a cleavable targeting signal and characteristically contain an even number of

transmembrane helices with both the N or C-termini oriented to the IMS. These internal

targeting signals direct the protein to the TIM22 complex of the inner membrane for

insertion into the inner membrane. This import pathway is termed the carrier import

pathway due to the initial characterisation using the highly abundant carrier proteins.

This pathway not only includes the membrane bound TIM22 subunits but also requires

the small TIMs from the IMS. The voltage dependent channel forming protein TIM22 is

predicted to contain four transmembrane helices (Kovermann et al., 2002). Import of

TIM22-mediated proteins comprises several steps, which start with initial binding at the

TOM complex on the mitochondrial surface (Rehling et al., 2004). After emerging from

the TOM complex, carrier proteins bind to the small TIMs in the IMS, shielding the

hydrophobic domains from unproductive interactions in the IMS and guiding the

protein to TIM22. The insertion of proteins by TIM22 into the inner membrane is

strictly dependent on the membrane potential. Once carrier proteins reach the

membrane, they are assembled into functional dimers (Neupert and Herrmann, 2007).

1.5 Protein import into plant mitochondria

Although the plant mitochondrial import apparatus displays many similarities

with yeast, significant differences have been observed (Figure 1.3). With reference to

the main translocases, the pore or channel forming subunits; Tom40, Tim22, Tim17,

and Tim23, are highly conserved across all known organisms and the same is true for

plants, with one major exception (Lithgow and Schneider, 2010). The plant Tim17

protein contains an extra C-terminal extension when compared to yeast, which has been

demonstrated to be inserted into the outer membrane (Murcha et al., 2003; Murcha et

al., 2005). The exact role of this extension is still unclear. While the main channel

forming subunits of the translocases are highly conserved, this cannot be said for a

number of other components. When the TOM complex from plants was first purified

and components identified, a number of differences compared to the model organism


13

yeast was observed (Jansch et al., 1998; Werhahn et al., 2001). Only two proteins from

plants were shown to be related to yeast (Tom40 and Tom7), the rest do not display

significant sequence similarity to the known components of the yeast TOM complex.

One of the major differences between the yeast TOM complex and the plant TOM

complex is in the receptor subunits. Firstly, the proteins identified as the plant Tom20s

showed no sequence similarity to the yeast Tom20 protein (Werhahn et al., 2001). In

fact the plant Tom20 proteins are an elegant case of convergent evolution (Lister and

Whelan, 2006; Perry et al., 2006). Although the yeast and plant proteins are not

evolutionary related to each other they have been shown to have similar tertiary

structures, and in fact contain very similar domains (Perry et al., 2006). The yeast

Tom20 contains a N-terminal transmembrane domain with the receptor domain at the

C-terminus, the plant proteins show the opposite orientation with the transmembrane

domain at the C-terminus and receptor domain at the N-terminus.

In analysing the TOM complex it was discovered that plants do not contain a

Tom22 receptor but contain a protein related to Tom22 of 9 kDa in size, called Tom9

(Werhahn et al., 2001). Further analysis of the plant Tom9 protein showed that contains

a single transmembrane segment similar to Tom22 and a C-terminal trans domain

located in the IMS (Macasev et al., 2004). This trans domain from plants was shown to

have the same function as yeast by complementing a yeast strain deficient in Tom22

(Macasev et al., 2004). Thus the plant Tom9 is the equivalent of yeast Tom22 lacking

the cytosolic receptor domain. One of the most surprising observations about the plant

TOM complex is the absence of a Tom70 like protein (Jansch et al., 1998; Werhahn et

al., 2001). Extensive database and sequence searches failed to identify a Tom70 like

protein in plant genomes. Thus it was of great interest when a Toc64 like protein was

identified on the outer mitochondrial membrane of Arabidopsis (Chew et al., 2004).

Toc64 is a TPR protein found on the outer envelope of chloroplasts and is involved in

chloroplast protein import (Qbadou et al., 2006). It has been hypothesised that this

mitochondrial Toc64 like protein may if fact be a plant Tom70 protein (Chew et al.,

2004). This is due to the similar features of Toc64 and Tom70. Tom70 contains an N-

terminus transmembrane domain followed by 11 TPR segments, which are responsible

for precursor binding and interacting with chaperones during mitochondrial protein

import (Li et al., 2009; Mills et al., 2009). Toc64 also contains an N-terminus

transmembrane domain and contains 3 TPR segments at the C-terminus, which are

thought to be required for precursor binding and also interacting with cytosolic

chaperones (Qbadou et al., 2006). Finally, a unique feature of the plant import apparatus


14

is the location of the mitochondrial processing peptidases (MPP). Despite the high

sequence similarity of plant and yeast MPP, plant MPP is an integral component of the

cytochrome bc1 complex of the inner membrane, whereas yeast and mammalian MPPs

are located within the matrix (Glaser and Dessi, 1999).

1.6 Peroxisomal protein import

The targeting and subsequent import of peroxisomal proteins can be divided into

four steps: receptor cargo interaction; docking at the peroxisomal membrane;

translocation and cargo release; and finally, receptor recycling back to the cyctosol

(Brown and Baker, 2008).

1.6.1 Receptor cargo interaction and membrane docking

Peroxisomal proteins are synthesised on free polyribosomes in the cytosol

(Lazarow and Fujiki, 1985). Peroxisomal proteins are imported via two conserved

pathways requiring conserved peroxisomal targeting signals (PTS). The major

difference between peroxisomal and mitochondrial or chloroplast targeting is that PTSs

are recognised by soluble receptors in the cytosol, as opposed to membrane bound

receptors. The most common PTS is the PTS1 signal, which is a carboxy terminal

tripeptide motif with the consensus sequence (S/A/C)(K/R/H)(L/M) (Lametschwandtner

et al., 1998). The predominantly cytosolic receptor for PTS1 containing proteins is

Pex5p, which is structurally divided into two domains. The carboxy domain of the

receptor has a high affinity for PTS1 signals and contains a seven TPR motif helix

bundle which forms a ring structure for ligand binding (Gatto et al., 2000; Stanley et

al., 2006). The deduced crystal structure of Pex5p when cocrystalized to a PTS1 peptide

contained two clusters of three TPRs (1-3 and 5-7) enclosing the peptide. The TPR4

hinge region was shown not to be directly involved in PTS1 binding (Klein et al., 2001).

The amino terminal region of Pex5p has some strictly conserved residues and it is

thought that the peroxisomal targeting information is contained within this region of the

protein (Saidowsky et al., 2001; Otera et al., 2002). Recently it has been demonstrated

that a N526K mutation in the carboxy terminus of Pex5p results in conformational

alterations in the amino terminus, which mimic those induced by PTS1 binding

(Carvalho et al., 2007). As the mutation still allows import of Pex5p into peroxisomes

without a bound cargo, it is thought that the triggering mechanism for docking and

translocation into peroxisomes originates from Pex5p.


15

The second pathway for peroxisomal import involves the peroxisomal targeting

type 2 signal (PTS2) which is located near the N-terminus and consists of the sequence

RLXXXXX(H/Q)L (Lazarow, 2006). While only a small number of proteins utilise this

pathway in yeast, there appear to be many more in plants. This pathway appears to have

been lost in Caenorhabditis elegans (Motley et al., 2000; Reumann et al., 2004). The

cytosolic receptor for PTS2 containing proteins is Pex7p, predicted to contain a seven

bladed β-propellor domain with each blade consisting of a WD40 repeat (Marzioch et

al., 1994; Zhang and Lazarow, 1995). Similar to Pex5p, Pex7p also shuttles between the

cytosol and the peroxisome during cargo translocation (Nair et al., 2004). However,

Pex7p does not work independently, as there are several accessory proteins required for

delivery of PTS2 containing proteins to peroxisomes (Stein et al., 2002). In yeast, there

are two structurally related peroxins, Pex18p and Pex21p, which are crucial for the

import of PTS2 containing proteins (Purdue et al., 1998). In plants, however, the

situation is slightly different as Pex5p and Pex7p form a PTS1/PTS2 receptor complex,

with the amino terminal domain of Pex5p interacting with the carboxy terminal of

Pex7p (Nito et al., 2002). This interaction has been shown experimentally, as a down

regulation of Pex5p results in a PTS2 import defect, suggesting that PTS1 and PTS2

protein import is coupled in plants in a similar manner to that seen in mammals

(Hayashi et al., 2005).

Once the respective PTS receptors Pex5p and Pex7p have bound their correct

cargo, they are then targeted to the peroxisomal membrane surface. At the peroxisomal

membrane surface the receptor cargo complex interacts with a number of membrane

proteins, before being translocated into the peroxisomal matrix (Brown and Baker,

2008).

1.6.2 Receptor cargo translocation, cargo release and receptor recycling

The translocation of the receptor cargo complex into peroxisomes has been

operationally defined as a peroxisome associated protease resistant state (Brown and

Baker, 2008). This can be explained by the two current hypotheses for the translocation

of proteins into the peroxisome: the extended shuttle hypothesis, where the receptor

cargo complex completely enters the peroxisomal matrix; or the simple shuttle

hypothesis, where the receptor cargo complex is embedded into the membrane, with the

cargo released into the matrix and the receptor remaining protease protected in the

membrane (Brown and Baker, 2008).


16

There has been much debate in the literature over the extended versus simple

shuttle hypotheses, with evidence readily found for both (Rachubinski and Subramani,

1995; Kunau, 2001; Smith and Schnell, 2001). It has been demonstrated that when GFP

is fused to the carboxy terminus of Pex7p, the intracellular distribution shifted from

mainly cytosolic to peroxisomal. When the GFP was subsequently cleaved, the GFP

remained in the peroxisome whereas Pex7p was observed to exit the peroxisome back to

the cytosol (Nair et al., 2004). While it has now been clearly demonstrated that both

Pex5p and Pex7p receptors do enter the peroxisome, it is still unclear whether they

simply remain embedded in the membrane, with their cargo binding site exposed to the

matrix or whether they are fully translocated into the matrix along with their cargo

(Dammai and Subramani, 2001).

In both mammals and plants the PTS2 containing sequence of a peroxisomal

protein is proteolytically removed after import. The removal of the PTS2 sequences is

not tightly linked with import, as both cleaved and uncleaved forms of thiolase have

been observed with in vitro imports into rat liver peroxisomes (Miura et al., 1994). The

enzyme responsible for this cleavage in mammals is termed trypsin domain-containing

domain 1 (TYSND1). A related enzyme in plants, Deg15, has been identified to carry

out the same processing step in Arabidopsis and watermelon (Helm et al., 2007;

Kurochkin et al., 2007).

The exact mechanistic details underlying the translocation and components of

the translocon are still lacking. It has been proposed that the components that make up

the docking complex on the peroxisomal membrane form part of the translocon (Brown

and Baker, 2008). The possible multiple binding sites for the Pex5p receptor on the

peroxisomal membrane have suggested the existence of an import cascade of a cargo

loaded receptor, as it interacts with different components of the import machinery

(Baker and Sparkes, 2005). It has been observed that Pex5p changes its characteristics

when it is associated with the peroxisomal membrane, as it behaves as an integral

membrane protein when it interacts with the docking complex (Gouveia et al., 2000).

Taken together with the observation that Pex5p can spontaneously insert into lipid

membranes, this suggests that a population of Pex5p receptors actually form the import

pore via protein lipid interactions, leading to an opening of the membrane allowing the

entry of a second cargo loaded Pex5p (Erdmann and Schliebs, 2005; Kerssen et al.,


17

2006). This hypothesis is referred to as the transient pore model (Erdmann and Schliebs,

2005).

Once the loaded cargo receptor complex enters the peroxisome the cargo must

be released into the matrix by the receptor, although little is known about the exact

mechanism of the process. In vitro experiments have indicated a displacement model for

receptor release, where a protein actively displaces the loaded cargo from the receptor

(Agne et al., 2003). Once the cargo is unloaded, both Pex5p and Pex7p can return to the

cytosol and take part in further rounds of import (Baker and Sparkes, 2005). The

dislocation and recycling of receptors from the peroxisome requires the action of the

receptor recycling complex, the mechanism for which is not yet fully understood (Agne

et al., 2003).

1.7 Dual targeting

The traditional dogma of molecular biology is that one gene gives rise to one

protein, which subsequently has one location. However, this no longer appears valid in

post-genomic biology. It has become clear with the sequencing of a number of

genomes, that the complexity of the proteome exceeds that of the genome in terms of

functional units, (i.e., there are more proteins than genes). This observed complexity

could be achieved in a number of different ways. Alternative splicing and protein

modifications are the best characterised processes to date (Kazan, 2003; Siuti and

Kelleher, 2007; Witze et al., 2007). Another mechanism that can increase the

complexity of proteomes is transcript editing of both nuclear and organellor genomes

(Nishikura, 2006; Takenaka et al., 2008). The dual targeting of proteins does not

increase the number of proteins, but it can expand the function(s) of a protein located in

two or more locations, because presumably, it functions in a distinct biochemical

process at each different location. A dual targeted protein is defined as the product(s) of

one gene targeted to two or more locations and was first characterised in 1995 for the

Pea glutathione reductase (GR), which was reported to be targeted to both chloroplasts

and mitochondria (Creissen et al., 1995). The number of identified dual targeted

proteins represents only a small proportion of the organeller proteomes. However, the

small number of characterised dual targeted proteins may only represent the tip of the

iceberg. While the majority of dual targeted proteins in plants are targeted to

mitochondria and chloroplasts, there are many other examples of dual targeting,

including mitochondria, plastids, and cytosol (Small et al., 1998), mitochondria and ER


18

(Bhagwat et al., 1999), mitochondria and the nucleus (Krause and Krupinska, 2009),

peroxisomes and mitochondria (Petrova et al., 2004), plastids and the cytosol (Kiessling

et al., 2004) and mitochondria and the cytosol (Regev-Rudzki et al., 2005). With the

amount of knowledge being gained from complete genome sequencing, combined with

the emerging information from organelle proteomic studies, GFP studies and prediction

programs, the number of dual targeted proteins has been increasing steadily over the

past 15 years since their discovery (Cho et al., 1999; Koroleva et al., 2005; Heazlewood

et al., 2007). Much work has been carried out to understand the mechanisms involved in

dual targeting. In particular alternative transcriptional initiation or splicing and

ambiguous targeting signals have been investigated (Peeters and Small, 2001; Karniely

and Pines, 2005). Alternative transcriptional initiation or splicing represent

transcriptional or post transcriptional events, which produce proteins translated with two

different targeting signals (Dinkins et al., 2008). Ambiguous targeting signals target a

protein to two locations, with the signals being indistinguishable from each other.

1.7.1 Ambiguous targeting signals

As discussed previously, mitochondria and chloroplasts have separate and

distinct targeting signals that can mediate their targeting and import into each organelle.

However, a small subset of these proteins contain ambiguous targeting signals that

target proteins to both organelles. This definition applies to the product of a single gene,

which gives rise to one protein, which is then targeted and imported into both

mitochondria and chloroplasts (Peeters and Small, 2001). Since the discovery of the

first ambiguously dual targeted protein, GR, a range of different proteins from various

biosynthetic pathways (transcription, translation and protein degradation) have been

demonstrated to be dual targeted by ambiguous targeting signals (Peeters and Small,

2001; Elo et al., 2003; Silva-Filho, 2003).

Analysis of ambiguous targeting signals has shown that they are similar to both

chloroplast and mitochondrial targeting signals, in that they are enriched in positively

charged residues and deficient in acidic residues such as glycine (Pujol et al., 2007).

However, there are no known distinguishing features that can separate ambiguous dual

targeting signals from mitochondrial and/or chloroplastidic specific targeting signals.

They appear to fall somewhere in between mitochondrial and chloroplast targeting

signals in their content of serine and arginine residues and are possibly slightly enriched

in hydrophobic residues. It has been shown that, in yeast, for a protein targeted to the

mitochondria and another location, its mitochondrial targeting signal is weaker


19

compared to mitochondrial proteins determined using the MITOPROT prediction

program. However no such evidence has been found in plants (Claros and Vincens,

1996; Dinur-Mills et al., 2008).

To date the most characterised ambiguous dual targeted signal has been that of

the Pea GR (Rudhe et al., 2002; Chew et al., 2003; Rudhe et al., 2004). Studies

involving deletion and site directed mutagenesis have revealed that some regions in the

targeting signal are more important for targeting to one organelle, but overall the dual

targeting signal overlaps (Chew et al., 2003). This study is consistent with other studies

carried out on tandem arrangements of mitochondrial and chloroplast targeting signals,

which demonstrated that passenger proteins are targeted by the most N-terminal signal

(de Castro Silva Filho et al., 1996). In the case of GR, it was found that positive

residues throughout the signal and hydrophobic residues at the N-terminus affected the

import into mitochondria whilst the hydrophobic residues had the greatest affect on

chloroplast import (Chew et al., 2003). In addition, it has also been observed that

arginine plays an important role in the mitochondrial import of three dual targeted

tRNA synthetases (Pujol et al., 2007). A recent study into dual targeting signals

concluded that while there is no general rule for the determinants of dual targeting, the

N-terminal portion is essential for the import into both mitochondria and chloroplast

(Berglund et al., 2009).

1.7.2 Alternative mechanisms of dual targeting

Post-translational mechanisms that result in the dual targeting of a protein are

usually found in non-plant organisms. In yeast, two enzymes of the TCA cycle,

fumarase and aconitase, have both been shown to be distributed between the cytosol and

mitochondria (Karniely and Pines, 2005). The mechanism for this dual distribution

involves the reverse translocation of a subset of molecules back into the cytosol

(Karniely and Pines, 2005). While the cytosolic presence of fumarase is quite obvious

(50% of the total fumarase is located in the cytosol), the amount of aconitase in the

cytosol is very small (less than 5%) (Sass et al., 2003; Regev-Rudzki et al., 2005).

Recently the abundance of fumarase in the cytosol compared to the mitochondria was

shown to be controlled by intracellular metabolite clues (Regev-Rudzki et al., 2009).

More specifically, it was suggested that metabolites from the glyoxylate shunt can act as

nanosensors for fumarase distribution (Regev-Rudzki et al., 2009), showing a complex

mechanism of control. Whilst no such pathway has been identified in plants, external


20

factors such as light and stress have been suggested to influence dual targeting (Silva-

Filho, 2003).

A single gene may also be alternatively transcribed from two different exons or

alternatively spliced to produce two separate messages encoding proteins targeted to

different locations (Peeters and Small, 2001). Some genes use multiple translation start

sites to determine dual targeting, for example, the longer protein is targeted to one

organelle and the shorter protein targeted to a second organelle (Chabregas et al., 2001;

Kobayashi et al., 2001; Watanabe et al., 2001; Hedtke et al., 2002). This example has

been reported in Arabidopsis with DNA polymerase γ2, which is dual targeted via the

use of a non AUG start codon (CUG), which adds an additional seven amino acids to

the N-terminus (Christensen et al., 2005). When translation starts at the standard AUG,

the protein is targeted to plastids, but when translation starts at the alternative CUG site,

the protein is targeted to both mitochondria and plastids (Christensen et al., 2005).

1.7.3 Systematic studies of dual targeted proteins

Studies that have investigated the biochemical processes common to both

mitochondria and chloroplasts have identified a number of dual targeted proteins. The

ascorbate glutathione cycle of Arabidopsis was originally thought to be housed solely in

chloroplasts, to remove the large amounts of H2O2 generated by photosynthetic

reactions. However biochemical studies have also measured the activity of ascorbate

glutathione cycle enzymes in the mitochondria of various plants. A study demonstrated

that the enzymes involved in the ascorbate glutathione cycle, (ascorbate peroxidase

(APX), monodehydroascorbate reductase (MDHAR) and GR) were in fact dual targeted

to both mitochondria and chloroplasts in Arabidopsis (Chew et al., 2003). This was the

first evidence for proteins of an entire biochemical pathway to be targeted to both

mitochondria and chloroplasts.

A study involving organellor tRNA synthetases has also demonstrated that most

organellor tRNA synthetases are dual targeted to mitochondria and chloroplasts

(Duchene et al., 2005). This is not surprising, as both mitochondria and chloroplasts

contain their own genome, which must be replicated, transcribed and translated. It has

also been suggested that most of the proteins involved in organelle DNA and RNA

metabolism are in fact dual targeted (Elo et al., 2003).


21

These previous studies have identified dual targeted proteins by focusing on the

targeting ability and/or location of single gene product(s) or small gene families. So far

there has been no genome-wide search for dual targeted proteins in Arabidopsis.

Therefore, it is possible that there are many more dual targeted proteins in Arabidopsis

that have yet to be identified. It was hypothesised that a global approach to the analysis

of protein locations may assist in the identification of additional dual targeted proteins.

The overall aim of this research was therefore to employ a systematic and global

approach to the identification of dual targeted proteins. This was attempted in order to

determine the extent of dual targeting of proteins in plants, with particular emphasis on

the dual targeting of proteins to the mitochondria and another location. A further aim of

this research was to investigate the mechanisms (i.e., receptors and signals) involved in

the dual targeting of proteins to mitochondria.

1.8 Research proposal

The specific aims of this PhD study are:

1. To identify proteins present in multiple organelles in plants (plastids,

mitochondria and/or peroxisomes).

2. To investigate the mechanism of dual targeting by identifying the import

receptors and machinery responsible for import of dual targeted proteins to the

mitochondria.

To achieve these aims, it was necessary to identify proteins present in multiple

organelles. This list of proteins was generated using bioinformatic resources available

from previous studies. First, a list of proteins experimentally defined as located in

mitochondria was compiled using the SUBA database (Heazlewood et al., 2007), and a

list of proteins predicted to target to peroxisomes was compiled using the Araperox

database (Reumann et al., 2004). These lists were cross-referenced to form a list of

candidate dual targeted proteins for mitochondria and peroxisomes. Second, the protein

prediction program, Predotar was used in two different modes (animal only and plant

only) to generate a ranked list of proteins targeted to mitochondria and plastids (Small

et al., 2004). Third, a list of proteins defined experimentally by proteomic approaches as

being present in two or more locations was compiled using the SUBA database

(Heazlewood et al., 2007). Finally a list of candidate dual targeted proteins likely to be

located in both mitochondria and chloroplasts was also compiled (i.e., proteins involved

in DNA transcription and replication).


22

The compiled lists were then merged into a single list of candidate dual targeted

proteins. Dual targeting of proteins was initially tested using GFP tagging. Results for

selected dual targeted proteins were subsequently confirmed using Western blotting,

mass spectrometry, or in vitro protein import assays.

To gain further insights into the mechanisms of dual targeting, a putative

receptor protein likely to be involved in dual targeting was tested. OM64, a protein

previously identified as located on the outer mitochondrial membrane, shares 70%

amino acids sequence identity with Toc64, a protein that has been proposed to act as a

receptor for plastid protein import (Chew et al., 2004). The functional role of OM64 in

the import of dual targeted proteins into plant mitochondria was analysed in variety of

assays.

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Chapter 2 Subcellular localisation of Arabidopsis thiolases

26

Chapter 2

Subcellular localisation of Arabidopsis thiolases

Chapter 2 Subcellular localisation of Arabidopsis thiolases

27

Foreword to Study I Knowledge of a proteins subcellular localisation is critical in understanding its

function within plant metabolism and the biochemical pathways its involved in. Nearly

a decade ago, it was proposed that the β-oxidation of fatty acids may take place within

the mitochondria of plants (Masterson and Wood, 2001). However, it has also been

proposed that β-oxidation occurs within the peroxisomes of plant cells (Graham and

Eastmond, 2002). A mitochondrial and peroxisomal location for the β-oxidation

pathway in plants is supported by the finding of 3-ketoacyl-CoA thiolase 2 (KAT2)

activity within both organelles (Footitt et al., 2002). KAT2 is the terminal enzyme in the

β-oxidation pathway in plants. In Arabidopsis KAT2, has been identified as a

mitochondrial protein in proteomic studies using purified mitochondria (Kruft et al.,

2001),(Heazlewood et al., 2004). Continuing from these, another study proposed a role

for KAT2 in isoleucine catabolism in plant mitochondria (Taylor et al., 2004). KAT2 is

predicted by three different subcellular prediction programs to be mitochondrial

(Heazlewood et al., 2005), however its protein sequence has also been shown to contain

a PTS2 sequence within the N-terminal part of the protein (Baker and Sparkes, 2005).

Based on prediction, proteomic, and activity analysis, KAT2 seemed a likely

candidate for a dual targeted protein between mitochondria and peroxisomes. Study I

aimed to clarify the subcellular localisation of KAT2 and define the localisation of any

other known thiolase like proteins in Arabidopsis. This was achieved by identifying all

thiolase like genes from Arabidopsis, and performing in vitro and in vivo organelle

targeting assays to define their subcellular localisations. It was found that the

Arabidopsis genome contains three genes encoding 3-ketoacyl-CoA thiolases (KAT),

one of which encodes two proteins with different N-terminal sequences. The

Arabidopsis genome also contains two genes encoding acetoacetyl-CoA thiolases

(ACAT), producing a total of five different proteins. In vitro and in vivo organelle

targeting studies identified that 3 KATs and 1 ACAT were peroxisomal, 1 KAT and 4

ACATs were cytosolic, and none were found to be mitochondrial. Its was concluded

that β-oxidation of fatty acids in Arabidopsis does not take place within mitochondria

and the final steps of isoleucine catabolism are most likely carried out in the cytosol.

Abstract The sub-cellular location of enzymes of

fatty acid b-oxidation in plants is controversial. In the

current debate the role and location of particular

thiolases in fatty acid degradation, fatty acid synthesis

and isoleucine degradation are important. The aim of

this research was to determine the sub-cellular location

and hence provide information about possible func-

tions of all the putative 3-ketoacyl-CoA thiolases

(KAT) and acetoacetyl-CoA thiolases (ACAT) in

Arabidopsis. Arabidopsis has three genes predicted to

encode KATs, one of which encodes two polypeptides

that differ at the N-terminal end. Expression in

Arabidopsis cells of cDNAs encoding each of these

KATs fused to green fluorescent protein (GFP) at their

C-termini showed that three are targeted to peroxi-

somes while the fourth is apparently cytosolic. The four

KATs are also predicted to have mitochondrial tar-

geting sequences, but purified mitochondria were un-

able to import any of the proteins in vitro. Arabidopsis

also has two genes encoding a total of five different

putative ACATs. One isoform is targeted to peroxi-

somes as a fusion with GFP, while the others display

no targeting in vivo as GFP fusions, or import into

isolated mitochondria. Analysis of gene co-expression

clusters in Arabidopsis suggests a role for peroxisomal

KAT2 in b-oxidation, while KAT5 co-expresses with

genes of the flavonoid biosynthesis pathway and

cytosolic ACAT2 clearly co-expresses with genes of

the cytosolic mevalonate biosynthesis pathway. We

conclude that KATs and ACATs are present in the

cytosol and peroxisome, but are not found in

mitochondria. The implications for fatty acid

b-oxidation and for isoleucine degradation in

mitochondria are discussed.

Keywords Thiolase Æ Mitochondria Æ Peroxisomes Æb-oxidation Æ Sub-cellular localization

Abbreviations

AOX Alternative oxidase

ACAT Acetoacetyl-CoA thiolase

KAT 3-Ketoacyl-CoA thiolase

Rubisco SSU Small subunit of Ribulose 1,5-

bisphosphate carboxylase/oxygenase

Introduction

It is widely accepted that the complete b-oxidation of

medium- and long-chain fatty acids in plants takes

place in the peroxisomes (Hooks 2002), as it does in

yeast (van Roermund et al. 2003). However, some

biochemical evidence suggests that plant mitochondria

can also carry out such b-oxidation of fatty acids

(Masterson and Wood 2001). It has also become clear

recently that plant mitochondria catalyse at least the

initial steps in the degradation of branched-chain

a-keto acids, derived from leucine, isoleucine and

Electronic supplementary material Supplementary materialis available in the online version of this article at http://dx.doi.org/10.1007/s11103-006-9075-1 and is accessible forauthorized users.

C. Carrie Æ M. W. Murcha Æ A. H. Millar Æ S. M. Smith ÆJ. Whelan (&)ARC Centre of Excellence in Plant Energy Biology,University of Western Australia, MCS building M316,35 Stirling Highway, Crawley 6009 WA, Australiae-mail: [email protected]

Plant Mol Biol (2007) 63:97–108

DOI 10.1007/s11103-006-9075-1

123

Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoAthiolases (ACATs) encoded by five genes in Arabidopsisthaliana are targeted either to peroxisomes or cytosolbut not to mitochondria

Chris Carrie Æ Monika W. Murcha Æ A. Harvey Millar ÆSteven M. Smith Æ James Whelan

Received: 25 June 2006 / Accepted: 10 August 2006 / Published online: 21 November 2006� Springer Science+Business Media B.V. 2006

valine, through a branched chain a-keto acid dehy-

drogenase complex similar to the pyruvate and 2-oxo-

glutarate dehydrogenase complexes of the TCA cycle

(Fujiki et al. 2000; Graham and Eastmond 2002; Taylor

et al. 2004). In the case of the leucine carbon skeleton,

the later steps of degradation are carried out entirely

within the mitochondrion (Graham and Eastmond

2002; Taylor et al. 2004). In contrast, final degradation

of valine derivatives requires both mitochondrial and

peroxisomal steps (Lange et al. 2004). Meanwhile the

complete oxidation of the products from the isoleucine

carbon skeleton includes a b-oxidation step that

requires a 3-ketoacyl-CoA thiolase (KAT) for the

removal of an acetyl-CoA from 2-methylaceto-acetyl

CoA to form propionyl-CoA. However, it is unclear if

this thiolase catalysed b-oxidation of 2-methylaceto-

acetyl CoA occurs in the mitochondrion in plants, as it

does in mammals (Fukao et al. 2001), or whether it

occurs in the peroxisome in plants akin to the final

steps of valine metabolism (Lange et al. 2004).

Comparisons to Saccharomyces cerevisiae are not

informative as yeast degrades branched chain amino

acids not via the branched chain dehydrogenase com-

plex route in mitochondria, but via the Erhlich path-

way involving pyruvate decarboxylase to form the

corresponding aldehydes and an aldehyde dehydroge-

nase to form the corresponding alcohol in the cytosol

(Derrick and Large 1993). Thus yeast does not need a

thiolase for isoleucine degradation.

Two distinct forms of 3-ketoacyl-CoA thiolase are

known. Type 1 3-ketaoacyl-CoA thiolase (KAT; EC

2.3.1.16) is typically involved in the degradative pro-

cess of fatty acid b-oxidation. The Type II enzyme is an

acetoacetyl-CoA thiolase (ACAT; EC 2.3.1.9), typi-

cally involved in the mevalonate pathway where it

functions in the biosynthetic direction. However,

ACATs are not exclusively involved in mevalonate

synthesis. In mammals that undertake both fatty acid

b-oxidation and isoleucine catabolism in mitochondria,

the former is performed by a KAT while the later is

performed by an ACAT (Pereto et al. 2005).

In Arabidopsis, thiolase has been reported to be

associated with both peroxisomes and mitochondria in

sucrose density gradients (Footitt et al. 2002). Kruft

et al (2001) and Heazelwood et al (2004) have both

claimed the thiolase KAT2 encoded by At2g33150 to

be present in purified mitochondria in large-scale

proteome analyses. This thiolase has previously been

proposed to be a component of isoleucine catabolism

in mitochondria (Taylor et al. 2004). However, the

thiolase is question is a type I enzyme, while in mam-

mals it is the mitochondrial type II ACATs that have

been implicated in isoleucine catabolism (Pereto et al.

2005). The KAT2 thiolase encoded by At2g33150 has a

predicted type 2 peroxisomal targeting sequence

(PTS2) conforming to the consensus R-(X)6-H/Q-A/L/F

with a downstream cysteine residue required for pro-

teolytic cleavage (Baker and Sparkes 2005), and is

imported into peroxisomes in vitro (Johnson and

Olsen 2003). At2g33150 is well known to be essential

for peroxisomal b-oxidation (Germain et al. 2001).

However, the protein encoded by At2g33150 is also

predicted to be targeted to mitochondria by three dif-

ferent targeting prediction programs (Heazlewood

et al. 2004). Furthermore, changing a single amino acid

in the peroxisomal targeting signal of the KAT

precursor in mammals, a glutamic acid residue to any

non-acidic residue, resulted in targeting to both mito-

chondria and peroxisomes (Tsukamoto et al. 1994).

This raises the possibility that the thiolase encoded by

At2g33150 is targeted to peroxisomes and mitochon-

dria, representing a dual targeted protein.

This study was carried out to define the subcellular

localization of the products from the putative KATs

and ACATs in Arabidopsis. This was achieved by

identifying all possible thiolase genes in Arabidopsis

and comparing their sequences to known-location type

I and type II thiolases in yeast, mammals and fungi. We

then used transcript sequence data to produce all the

possible cDNAs for these gene products. These

cDNAs were used with in vivo and in vitro organelle

targeting assays to define subcellular localization of

type I and II thiolases in Arabidopsis and gene

expression profiling data was compared to define likely

functional links.

Materials and methods

Identification of genes and cDNAs encoding

thiolase

The predicted protein sequence encoded by At2g33150

previously shown to be a thiolase was used to define

other thiolase encoding genes in Arabidopsis (Germain

et al. 2001), and the sequences from other species as

previously published (Pereto et al. 2005). A similarity

tree was made using ClustalW multiple sequence

alignment and neighbour joining (Thompson et al.

1994, 1997). The gene structures were obtained from

The Arabidopsis Information Resource annotation

version 6 (TAIR6) and three individual cDNAs were

amplified for all possible cDNAs. The cDNAs pro-

duced from the genes were defined using the Arabid-

opsis genome tiling array (Mockler et al. 2005;

Yamada et al. 2003). Targeting predictions of the

98 Plant Mol Biol (2007) 63:97–108

123

encoded proteins were carried out using a variety of

prediction programs: TargetP (Emanuelsson et al.

2000), Mitoprot (Claros and Vincens 1996), Subloc

(Hua and Sun 2001), Ipsort (Bannai et al. 2002),

Predotar (Small et al. 2004), Mitpred (Kumar et al.

2006) and PeroxP (Emanuelsson et al. 2003). Percent-

age identity and similarity was calculated using Mat-

GAP v2.02 (Campanella et al. 2003).

Subcellular targeting of predicted thiolase proteins

The coding sequences of the predicted thiolase pro-

teins were cloned in frame with the coding region of

GFP in pGEM 3Zf(+) containing the 35S CaMV pro-

moter (Chew et al. 2003). The alternative oxidase

(AOX) coding region fused to GFP (Lee and Whelan

2004), and the red fluorescent protein (RFP) fused to a

type 1 peroxisomal SRL targeting signal from pumpkin

(Pracharoenwattana et al. 2005), were used as mito-

chondrial and peroxisomal controls respectively. The

constructs were used to transform Arabidopsis

suspension culture cells by biolistic transformation as

previously outlined (Thirkettle-Watts et al. 2003).

Fluorescence patterns were obtained 48 h after trans-

formation by visualization under an Olympus BX61

fluorescence microscope and imaged using the CellR

imaging software. In vitro protein import assays into

mitochondria isolated from Arabidopsis suspension

cell cultures were carried out as described in Lister

et al. (2004). In vitro mitochondrial uptake assays were

performed by adding precursor protein to 100 lg of

isolated mitochondria in 200 ll in the presence of

respiratory substrate (succinate 5 mM) and ATP

(1 mM) and ADP (200 lM) in import buffer (0.3 M

sucrose, 50 mM KCl, 10 mM MOPS pH 7.2, 5 mM

KH2PO4, 1% (w/v) BSA, 1 mM MgCl2, 1 mM methi-

onine and 5 mM DTT). Reactions were incubated at

24�C for 20 min then divided into two equal aliquots

and placed on ice. To one aliquot Proteinase K was

added to a final concentration of 40 lg/ml and incu-

bated for 15 min on ice, followed by the addition of

PMSF to 2 mM to terminate protease digestion. The

mitochondria were pelleted, washed twice in ice-cold

import buffer. The final pellet was resuspended in

SDS-PAGE sample buffer and proteins separated in

12% (w/v) polyacrylamide gels, then dried. Radiola-

belled proteins were visualized by exposing to a BAS

TR2040 imaging plate for 24 h and reading on a BAS

2500 Bio imaging analyser (Fuji, Tokyo). Outer

membrane ruptured mitochondria (Mit-OM) were

prepared after the import assay to test for the intra-

organelle location of imported protein. Rupture of the

outer membrane allowed access of added protease to

intermembrane space components or inner membrane

proteins exposed to the intermembrane space. Mit-OM

were prepared by resuspending 100 lg of mitochon-

drial protein in 10 ml SEH buffer (250 mM sucrose,

1 mM EDTA, 10 mM Hepes pH 7.4) and then adding

155 ll of 20 mM Hepes pH 7.4 and incubating on ice

for 20 min. To restore osmolarity 25 ll of 2 M sucrose

and 10 ll of 3 M KCl was added and mixed, re-pelleted

and washed in import buffer. Valinomycin was added

to a final concentration of 1 lM where indicated prior

to the addition of the precursor protein to mitochon-

dria and commencement of the import assay. AOX was

used as a positive control and the small subunit of

Ribulose 1, 5-bisphosphate carboxylase/oxygenase

(Rubisco SSU) as a negative control. As some pre-

cursor proteins displayed protease insensitivity even in

the presence of valinomycin the sensitivity of the pre-

cursor proteins alone to added protease was tested.

Sensitivity of the in vitro synthesized radiolabelled

proteins was tested by adding proteinase K to the

synthesized protein alone in the absence of mitochon-

dria to ensure that the added protease could digest the

protein.

In silico expression analysis

Expression correlation for genes encoding KATs and

ACATs was performed using the Expression Angler

program on the Botany Array Resource (Toufighi

et al. 2005). The Genevestigator Arabidopsis micro-

array database was used to analyse the response of the

genes of interest in this study in a variety of tissue types

(Zimmermann et al. 2004). The meta-analyser tool was

the function utilized, ATH1 22k array wild type only

arrays were chosen and the genes of interest were

selected. The data was visualized using a linear scale

from a total of 1860 array experiments. TMeV (TIGR

Multiple Experiment Viewer) programme was used to

cluster the genes and stresses, and Euclidean distance

and complete linkage were chosen for the hierarchal

clustering (Eisen et al. 1998; Saeed et al. 2003).

Results

The Arabidopsis thiolase gene families

Searches of the Arabidopsis genome identify five loci

with sequence similarity to genes encoding known

thiolase proteins (Germain et al. 2001). Comparison of

predicted amino acid sequences shows that they fall

into two classes. Three loci encode the Type I class of

enzyme, KAT 1, 2 and 5, typically involved in

Plant Mol Biol (2007) 63:97–108 99

123

acetyl-CoA formation in fatty acid b-oxidation by

removal of a 2-carbon chain from a 3-ketoacyl-CoA.

The other two genes encode the type II class of

enzyme, ACAT 1 and 2, typically involved in aceto-

acetyl-CoA formation from two molecules of acetyl-

CoA (Fig. 1A, Supplementary Fig. 1A). The type I

genes have previously been annotated as KAT1, KAT2

and KAT5 (3-ketoacyl-CoA thiolase) (Germain et al.

2001), based on the chromosome on which they are

found (At1g04710, At2g33150 and At5g48880, respec-

tively). All three are closely related to known peroxi-

somal located type I thiolases in human, mouse and

yeast and this cluster also contains representatives from

the fungus Neurospora crassa and the monocot rice

(Oryza sativa). The matrix and membrane-bound type I

mitochondrial thiolases involved in fatty acid degrada-

tion in human, mouse and Drosophila cluster separately

and do not contain members from the completed

genome sequences of fungi, Arabidopsis or rice.

The two Arabidopsis type II thiolases (here referred

to as ACAT1 and ACAT2, At5g47720 and At5g48230,

respectively) cluster with the known cytosolic type II

thiolases from yeast and the cytosolic and mitochon-

drial type II thiolases from human, mouse and Dro-

sophila. The monocot rice also has two type II thiolases

that cluster in this set and N. crassa contains a single

type II gene that clusters with the yeast cytosolic type

II protein.

The sequence divergence of mitochondrial type I

thiolases in mammals from the peroxisomal type I

KATs in plants, fungi and animals makes the presence

of KAT2 in Arabidopsis mitochondria appear unlikely

based on phylogenetic evidence if thiolase location is

conserved. However, the sequence analysis does not

define the location of the type II ACAT proteins in

Arabidopsis and leaves open the possibility of a mito-

chondrial location of at least one of these proteins,

especially given the presence of multiple type II thio-

lases in both plants and mammals.

Definition of the number of products from each

Arabidopsis thiolase gene

Analysis of EST sequences and tiling array data shows

that KAT1 and KAT2 loci each encode single poly-

peptide sequences (Fig. 1B) (Mockler et al. 2005;

Yamada et al. 2003). In contrast, KAT5 encodes two

proteins that differ at the N-terminus due to alternative

RNA splicing that generates either 13 (KAT5.1) or 14

(KAT5.2) exons (Fig. 1B). The N-terminal sequences

of proteins encoded by KAT1, KAT2 and KAT5.2

include putative PTS2-type sequences, while the

protein encoded by KAT5.1 does not (Fig. 1B).

Interestingly, proteins encoded by KAT1, KAT2,

KAT5.1 and KAT5.2 proteins are predicted to be tar-

geted to mitochondria by at least two of three different

programs (Table 1). Analysis of EST sequences and

tiling array data shows that ACAT1 and ACAT2 loci

also encode three and two proteins respectively

(Fig. 1B). Differential RNA splicing results in the

protein encoded by ACAT1.1 lacking ten amino acids

at the C-terminus relative to the protein encoded by

ACAT1.2. The protein ACAT1.3 has 20 different

amino acid residues at the N-terminus relative to

ACAT1.1. None of the proteins has predicted orga-

nelle-targeting information (Table 1). Differential

RNA splicing also accounts for the N-terminal 6 amino

acid residues of the protein encoded by ACAT2.1

being replaced by 11 different amino acid residues in

the case of the protein encoded by ACAT2.2 (Fig. 1B).

Neither protein has predicted organelle-targeting

information (Table 1).

Targeting of type I and II thiolases in vivo

To localize thiolases in vivo, GFP and RFP fusions

were employed. GFP and RFP have been used exten-

sively to study protein targeting to mitochondria,

peroxisomes and chloroplasts (Heazlewood et al.

2005). To demonstrate specific mitochondrial and

peroxisomal targeting in vivo and our ability to

distinguish the two, an AOX–GFP construct (Lee and

Whelan 2004) and an RFP–PTS1 construct (Pracha-

roenwattana et al. 2005), were employed. The two

gene constructs were co-delivered into Arabidopsis

suspension culture cells using a biolistic gene gun

(Thirkettle-Watts et al. 2003). After 48 h individual

cells expressing both GFP and RFP fluorescence were

imaged. The results show that GFP and RFP were

targeted to discrete organelles consistent with specific

targeting to mitochondria and peroxisomes respec-

tively (Fig. 2).

To examine thiolase targeting we made translational

fusions with GFP at the C-terminus since peroxisomal

(PTS2) and mitochondrial targeting sequences are both

N-terminal. Thiolase cDNAs encoding all nine pro-

teins were linked to the GFP coding region and cloned

downstream of the CaMV 35S promoter. They were

Fig. 1 Classification of thiolase (KAT and ACAT) genes andgene structure in Arabidopsis. (A) A phylogenetic tree wasgenerated using the neighbour joining method, using ClustalW ofthiolase proteins from a variety of organisms. KAT = 3-ketoacyl-CoA thiolases, ACAT = acetoacetly-CoA thiolases. (B) Genestructure and predicted proteins encoded by thiolase genes. Thedifferences in the proteins encoded by each locus are indicated inbold where evidence for more than one cDNA exists. The openwhite boxes indicate exons

c

100 Plant Mol Biol (2007) 63:97–108

123

MEKATERQRI LLRHLQPSSS SDASLSASAC LSKDSAAYQY

MEKAIERQRV LLEHLRPSSS SSHNYEASLS ASACLAGDSA

MAPPVSDDSL QPRDVCVVGV ARTPIGDFLG SLSSLTATRL

MNVDESDVCI VGVARTPMGG FLGSLSSLPA TKLGSLAIAA

MAHTSESVNP RDVCIVGVAR TPMGGFLGSL SSLPATKLGS

KKGKYGVASI CNGGGGASAL VLEFMSEKTI GYSAL

MERAMERQKI LLRHLNPVSS SNSSLKHEPS LLSPVNCVSE

MAAFGDDIVI VAAYRTAICK ARRGGFKDTL PDDLLASVLK

KAT1At1g04710

KAT2At2g33150

At5g47720.1

At5g47720.2

At5g48230.1

At5g48230.2

KAT5.1 At5g48880.1

KAT5.2At5g48880.2

ACAT1.1

ACAT 1.2

ACAT 2.2

ACAT 2.1

ACAT 1.3

At5g47720.3

MYLSFDPAVM ATYSSVPVCA DVCVVGVART PIGDFLGSLS

A

B

Plant Mol Biol (2007) 63:97–108 101

123

each delivered into Arabidopsis suspension culture

cells together with the RFP–PTS1 construct. After 48 h

individual cells expressing both GFP and RFP fluo-

rescence were imaged, and the images merged. The

results show that KAT1, KAT2 and KAT5.2 and

ACAT 1.3 were targeted to peroxisomes, as indicated

by coincidence of GFP and RFP images (Fig. 2). In

contrast, ACAT1.1, ACAT1.2, ACAT2.1, ACAT2.2

and KAT5.1 show diffuse fluorescence throughout the

cell indicating that no specific targeting to any orga-

nelle has occurred, suggesting a cytosolic localization.

With peroxisomal targeting of KAT1, KAT2, KAT5.2

and ACAT 1.3, although it was apparent that the

patterns of GFP and RFP were essentially identical,

the higher intensity of the former means that when

merged the green fluorescence was dominant in some

cells.

Protein import into isolated mitochondria

None of the thiolases were apparently targeted to

mitochondria in vivo. However, it is possible that up-

take was prevented by the GFP fusion, or that mito-

chondria normally take up less thiolase than

peroxisomes, such that GFP fluorescence from mito-

chondria did not reach an intensity to be detected. To

test these possibilities we examined the ability of iso-

lated mitochondria to take up all nine thiolases. Each

protein was synthesized in a rabbit reticulocyte lysate

translation system in the presence of radiolabelled

methionine, and then tested for import into mito-

chondria isolated from Arabidopsis cell cultures. As a

control the import and processing of AOX and Rubisco

SSU were examined, the former as a positive control

for import and the latter as a control to demonstrate

the specificity of import into isolated mitochondria

(Chew et al. 2003; Chew and Whelan 2004). In this

case the AOX precursor protein (36 kDa) was

imported and cleaved to a mature protein (32 kDa) as

previously demonstrated (Fig. 3, lanes 1 and 2)

(Whelan et al. 1995). Addition of protease resulted in

the 32-kDa mature form being resistant to protease

digestion indicating uptake by mitochondria. This

resistance was abolished by the addition of valinomy-

cin that dissipates the inner membrane potential

(Fig. 3, lanes 4 and 5) (Tanudji et al. 2001). The

phosphate translocator from maize was used as an

additional control, after uptake into mitochondria and

rupture of the outer membrane protease digestion

results in a small cleaved protected fragment of

33 kDa, indicating that the added protease has access

to inside the outer membrane (Bathgate et al. 1989;

Murcha et al. 2004, 2005; Winning et al. 1992). In

contrast to the mitochondrial controls, Rubisco SSU

was not protected from protease digestion indicating it

was not imported into mitochondria (Fig. 3).

When the nine thiolase proteins were tested for

mitochondrial uptake two distinct patterns were ob-

served, KAT1, ACAT 1.1, ACAT 1.2, ACAT 1.3,

KAT5.1 and KAT5.2 did not yield any protease pro-

tected products upon incubation with mitochondria

and thus were deemed not to be imported (Fig. 3).

KAT2, ACAT2.1 and ACAT2.2 yielded resistant

products upon incubation with mitochondria. Although

KAT2 was not proteolytically cleaved by mitochon-

dria, a protease resistant product with a lower mol

mass was obtained when mitochondria were treated

with Proteinase K (Fig. 3, lanes 1–3). Notably this was

also generated in the presence of valinomycin (Fig. 3,

lanes 4–5). However, upon rupture of the outer

Table 1 Summary of the subcellular location of thiolase proteins

Protein Locus Target prediction Peroxisomaltargeting

Proteomic In vivo In vitro Location Function

KAT 1 At1g04710 M PTS2 P NM PeroxisomeKAT 2 At2g33150 M PTS2 Ma,b, Cc, Nd P NM Peroxisome b-oxidationKAT 5.1 At5g48880.1 M – NT NM Cytosol Flavonoid biosynthesisKAT 5.2 At5g48880.2 M PTS2 P NM Peroxisome Flavonoid biosynthesisACAT 1.1 At5g47720.1 None – NT NM CytosolACAT 1.2 At5g47720.2 None – NT NM CytosolACAT 1.3 At5g47720.3 None – P NM PeroxisomeACAT 2.1 At5g48320.1 None – NT NM Cytosol Mevalonate pathwayACAT 2.2 At5g48230.2 None – NT NM Cytosol Mevalonate pathway

Targeting prediction = M (mitochondria) if two or more predictions indicate a mitochondrial location. Peroxisomal Targeting = thepresence of a Type 1 or 2 peroxisomal targeting signal. Proteomic = evidence for location from independent proteomic studies,M = Mitochondria, C = chloroplast and N = nuclear.a,b Kruft et al (2001) and Heazlewood et al (2004), c Kleffmann et al (2004), d Pendle et al (2005). In vivo = targeting ability as byGFP tagging, P = peroxisomal and NT = no targeting. In vitro tested ability to target to mitochondria, NM = not taken up intoisolated mitochondria. Final column indicates the location concluded and suggested role in metabolism

102 Plant Mol Biol (2007) 63:97–108

123

membrane this product was absent (Fig. 3, lanes 6–9).

In the case of ACAT2.1 and ACAT2.2 a similar pat-

tern was observed except that the protected protein

had the same molecular mass as the protein added to

the import assay (Fig. 3, lanes 1–5). Again with rupture

of the outer membrane no protease protection was

observed (Fig. 3, lanes 6–9).

Although the protease protected fragments pro-

duced upon incubation of KAT2, ACAT2.1 and

ACAT2.2 may suggest uptake by mitochondria, their

presence when valinomycin was added to the import

assay and their sensitivity when the outer mitochon-

drial membrane is ruptured suggests that they may

represent protease resistant products in the presence of

intact mitochondria. The protease susceptibility of

KAT2, ACAT2.1 and ACAT2.2 was tested by the

ability of added protease to digest the radiolabelled

precursor protein. Incubation of KAT2.1, ACAT2.1

and ACAT2.2 with proteinase K alone indicated that

they were resistant to protease digestion; in contrast

AOX was completely digested (Fig. 4). Thus it was

concluded that there was no uptake of any radiola-

belled thiolase proteins into isolated mitochondria, in

agreement with the GFP targeting (Fig. 2).

Co-expression analysis

The probable functions of these type I and type II thio-

lases in their determined subcellular locations was

examined by analysis of co-expression of these genes in

microarray data from Arabidopsis (Fig. 5). We used the

Expression Angler co-expression correlation tool from

the Botany Array Resource (Toufighi et al. 2005) to find

the most co-expressed genes based on microarray

hybridization data on Arabidopsis 22K genechips; the

top 25 co-expressed genes are shown in each case

(Fig. 5A, Supplementary Table 1). This analysis shows

that KAT2 co-expresses (Correlation >0.65–0.79) more

highly with a range of peroxisomal fatty acid degrada-

tion components in the peroxisome than with any other

nuclear genes in Arabidopsis. These included the fatty

acid multifunction protein MFP2 (At3g06860), citrate

synthase (At2g42790), acyl-CoA oxidases (At5g65110,

At3g51840) and enoyl-CoA hydratase (At4g16210)

Fig. 2 In vivo targeting ability of thiolases in Arabidopsis. ThecDNA coding sequences of thiolase from Arabidopsis weretagged with GFP to assess targeting ability. Each cell shown wastransformed with both a GFP construct and with RFP with a typeI PTS. Each panel shows the localization of GFP targeted eitherby the mitochondrial protein alternative oxidase (AOX) or bythiolases (GFP panel). A peroxisomal pattern obtained in thesame cell with the RFP with a type I PTS is shown (RFP-SRLpanel) together with the merged images (Merged panel)

bGFP RFP-SRL Merged

AOX

KAT1At1g04710

KAT2At2g33150

ACAT 1.1At5g47720.1

ACAT 1.2At5g47720.2

ACAT 2.1At5g48230.1

ACAT 2.2At5g48230.2

KAT 5.1At5g48880.1

KAT 5.2At5g48880.2

ACAT 1.3At5g47720.3

20 µm

Plant Mol Biol (2007) 63:97–108 103

123

(Fig. 5A). Curiously, Expression Angler showed KAT5

is not co-expressed with b-oxidation enzymes, but

instead with a series of flavonoid biosynthesis enzymes

(Correlation >0.6–0.71), including flavanone 3-hydrox-

ylase (At3g51240) 4-courmarate CoA ligase

(At1g65060), chalcone synthase (At5g13930), chalcone

isomerase (At5g05270). Notably this pathway requires

short acyl-CoAs for biosynthesis.

The gene encoding a type II enzyme ACAT2

(At5g48230) was found by Expression Angler to be co-

expressed with a range of genes, but notably, hydrox-

ymethylglutaryl-CoA synthase (At4g11820) and

mevalonate diphosphate decarboxylase (At2g38700,

At3g54250) were highly co-expressed (Correlation

>0.80) (Fig. 5, Supplementary Table 1). This is con-

sistent with the role of type II genes in the cytosolic

mevalonate pathway leading to isoprene-containing

compounds such as sterols and terpenoids.

The isoleucine catabolism pathway involves the

branched chain amino acid dehydrogenase complex

(At5g09300, At3g13450, At3g06850), isovaleryl-CoA

dehydrogenase (At3g45300), enoyl-CoA hydratase

(At4g31810) in mitochondria, and then 3-hydroxy-2-

methylbutyryl-CoA dehydrogenases and the fatty acid

multifunction proteins (At4g29010, At3g06860,

At3g15290), in addition to a thiolase, but these fore-

mentioned genes do not appear to be co-expressed

with any of the thiolase genes (data not shown).

pAOX

KAT 2At2g33150

ACAT 2.1At5g48230.1

ACAT 2.2At5g48230.2

36 kDa

48 kDa

40 kDa

41 kDa

PKPrecursor

Lane 1 2+ +

+-

Fig. 4 Protease susceptibility of KAT 2, ACAT 2.1 and ACAT2.2. The ability of proteinase K to digest thiolases was tested byincubating the protease with radiolabelled protein. Alternativeoxidase was used as a control and apparent mol mass areindicated in kDa

ValPK

Mit-OMMit

Lane-

+-

1 2 3 4 5 6 7 8 9

pAOX

pPic

KAT 1 At1g04710

pTIM23

pRubisco SSU

mAOX

mPic

KAT 2At2g33150

ACAT 1.1At5g47720.1

ACAT 1.2At5g47720.2

ACAT 2.1At5g48230.1

ACAT 2.2At5g48230.2

KAT 5.1At5g48880.1

KAT 5.2At5g48880.2

46 kDa

48 kDa

42 kDa

43 kDa

40 kDa

41 kDa

43 kDa

48 kDa

36 kDa

32 kDa

20 kDa

14 kDa

38 kDa34 kDa33 kDa

20 kDa

---

-- -

-- - - - -- -- - -

-

+ + + ++ + +

+ + + ++ + + +

ACAT 1.3At5g47720.3 43 kDa

Fig. 3 In vitro import of radiolabelled thiolase proteins intomitochondria isolated from Arabidopsis. Lane 1, precursorprotein alone. Lane 2, precursor protein incubated withmitochondria under conditions that support import into mito-chondria. Lane 3, as lane 2 with proteinase K added afterincubation of precursor with mitochondria. Lane 4 and 5, as lane2 and 3 with valinomycin added to the import assay prior to theaddition of precursor protein. Lanes 6–9 as 2–5 except that themitochondrial outer membrane was ruptured after the incuba-tion period with precursor protein but prior to addition ofproteinase K. Apparent mol mass are indicated in kDa.Abbreviations: Mit = mitochondria, Mit-OM = mitochondriawith outer membrane ruptured, PK = proteinase K, Val = vali-nomycin, AOX = alternative oxidase, Pic = phosphate carrier,Rubisco SSU = small subunit of ribulose-1, 5 bisphosphatecarboxylase/oxygenase, p = precursor protein band, m = matureprotein band

104 Plant Mol Biol (2007) 63:97–108

123

To confirm the co-expression groups in Fig. 5A, we

used Genevestigator (Zimmermann et al. 2004) to

cluster KAT2, KAT5 and ACAT2 and their cohort of

highlighted co-expressed genes across a series of

microarray data based on tissue specific expression

(Fig. 5B). This bootstrapped cluster tree showed three

separate groupings of genes, confirming the Expression

Angler analysis of distinct expression patterns of these

three thiolases, correlating with distinct roles in

metabolism. Note this type of expression analysis

cannot distinguish differential roles for isoforms of

proteins resulting from alternative splicing as the probe

sets used to determine expression do not distinguish

between splice forms.

Discussion

Table 1 summarizes the results of our knowledge on

the subcellular localization of thiolase protein in

Arabidopsis. Although some thiolase proteins contain

a predicted mitochondrial targeting signal both in vitro

and in vivo protein localization assays indicate that

they are not imported into mitochondria. This conflicts

with proteome analysis of isolated mitochondria sug-

gesting a mitochondrial localization for KAT2

(Heazlewood et al. 2004; Kruft et al. 2001). We pro-

pose that this is due to contamination by peroxisomal

proteins and that KAT2 is not an authentic mito-

chondrial protein. Heazlewood et al (2004) reported a

low level of contamination of their mitochondrial

samples with peroxisomes, consistent with the poten-

tial for some false positive identifications in this shot-

gun proteomic study. The apparent requirement of a

thiolase for isoleucine catabolism in mitochondria

(Taylor et al. 2004) is not a strong argument for the

role of KAT2 in mitochondria, as mitochondrial type I

enzymes in animals are structurally distinct from

Arabidopsis KATs (Fig. 1). The terminal step and

isoleucine degradation might be best served by the

type II rather than a type I enzyme, and we have now

shown convincingly that type II thiolases are in the

cytosol and/or peroxisome in Arabidopsis (Fig. 2).

Our data suggests that at least in Arabidopsis,

thiolases involved in b-oxidation are not present in

mitochondria, despite the fact that some biochemical

evidence has suggested this may take place in mito-

chondria of pea (Masterson and Wood 2001). The

results presented here however cannot be definitive for

all plant species as it is possible that genes encoding

thiolases in other plant species may have mitochondrial

targeting ability due to the fact that at least some

thiolase genes in Arabidopsis encode proteins that

have predicted mitochondrial targeting ability. Thus

relatively small changes are likely required to achieve

mitochondrial targeting of plant thiolases, as has been

observed with some peroxisomal thiolases from other

organisms (Danpure et al. 2003; Tsukamoto et al.

1994).

0.5

0.6

0.7

0.8

0.9

1

At3g0

6860

At2g4

2790

At5g6

5110

At4g1

6210

At3g5

1840

0.5

0.6

0.7

0.8

0.9

1

At3g5

1240

At5g0

5270

At5g1

3930

KAT5 (At5g48880)

0.5

0.6

0.7

0.8

0.9

1

At4g1

1820

At2g3

8700

At3g5

4250

Acyl-CoA oxidase (At3g51840)Acyl-CoA oxidase (At5g65110)KAT2 (At2g33150)Citrate synthase (At2g42790)Enoyl-CoA hydratase (At4g16210)Fatty acid multifunction protein MFP2 (At3g06860)Flavanone 3-hydroxylase (At3g51240)Chalcone synthase (At5g13930)Chalcone isomerase (At5g05270)4-courmarate CoA ligase (At1g65060)KAT5 (At5g48880)Mevalonate diphosphate decarboxylase (At2g38700)ACAT2 (At5g48230)Hydroxymethylglutaryl-CoA synthase (At4g11820)Mevalonate diphosphate decarboxylase (At3g54250)

100%

100%

KAT2 (At2g33150)

At1g6

5060

ACAT2 (At5gt48230)

A

B

70%

100%

Fig. 5 In silico expression analysis. (A) The 25 genes co-expressed to the greatest extent with each thiolase gene weredetermined using the Expression Angler tool from the BotanyArray Resource. The annotated genes indicated for KAT2,KAT5 and ACAT2 are those for which the proteins encoded bythese genes could function in peroxisomal fatty acid degradation,flavonoid biosynthesis and the mevalonate pathway, respectively.(B) Clustering analysis of co-expressed genes with KAT2, KAT5and ACAT2 to determine which (KAT or ACAT) branch withco-expressed genes as determined by Expression Angler. EachKAT or ACAT is located in a distinct group in agreement withanalysis by Expression Angler, with good bootstrap valuessupporting the branch points

Plant Mol Biol (2007) 63:97–108 105

123

The data on enzymes required for isoleucine deg-

radation from 2-methyl-3-hydroxybutyryl-CoA

through to propionyl-CoA increasingly suggests that

this part of the biochemical pathway is a non-mito-

chondrial activity. Of the three 3-hydroxy-2-methyl-

butyryl-CoA dehydrogenases in Arabidopsis

(At4g29010, At3g06860, At3g15290), At3g06860 has

been located to peroxisomes by three separate reports

using GFP tagging (Cutler et al. 2000; Koh et al. 2005;

Tian et al. 2004) and At3g15290 has been located to

chloroplasts by mass spectrometry (Kleffmann et al.

2004). The type II ACAT thiolases are all non-mito-

chondrial (Fig. 2), being present in either the cytosol

or peroxisome from our own data. Transport of

2-methyl-3-hydroxybutyryl-CoA out of mitochondria

has not been investigated, but the substrate specificity

of an array of known mitochondrial carriers from the

Mitochondrial Carrier Protein (MCP) family, the

Preprotein and Amino acid Transporter (PRAT)

family and ATP Binding Cassette (ABC) transporters

remain to be studied in Arabidopsis (Pohlmeyer et al.

1997; Rassow et al. 1999; Brugiere et al. 2004; Picault

et al. 2004). The distribution of pathways of amino

acid biosynthesis and metabolism between organelles

and the cytosol is relatively common in plants, but in

the case of isoleucine metabolism, although the met-

abolic enzymes involved are now relatively clear, the

transport activities that facilitate this pathway be-

tween mitochondria, the cytosol and the peroxisome

remain to be elucidated.

Co-expression analysis of transcript data can be a

powerful tool to confirm other data or provide leads

for further analysis. In this case, the co-expression

results for KAT2 are consistent with all our

experimental data. This gene encodes a peroxisomal

thiolase and is co-expressed with other peroxisomal

proteins involved in the same process, namely

b-oxidation of fatty acids. For ACAT2 the subcellu-

lar location, enzyme class and co-expression also

coincide to suggest a role in mevalonate biosynthesis

leading to isoprenes. The KAT5 co-expression result

was a surprise as this protein was suspected to be

involved in b-oxidation based on its enzyme class.

However, the different location of KAT5.1 and

KAT5.2 (Table 1), the fact that KAT5 does not

maintain b-oxidation in seedlings of the KAT

knockout but can partially complement for the lack

of KAT2 when driven by 35S expression (Germain

et al 2001), and the co-expression link with flavonoid

biosynthesis rather than b-oxidation genes (Fig. 5),

suggests that while KAT5 encodes a thiolase, it has a

distinct role to KAT2 in acyl-CoA metabolism in

plants.

Acknowledgements This work was funded through grants fromthe Australian Research Council (ARC) Centre of ExcellenceProgramme to JW, SS and AHM. AHM is funded as an ARCQueen Elizabeth II Fellow and SS as an ARC Federation Fellow.

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123

Chapter 3 Approaches to defining dual targeted proteins

40

Chapter 3

Approaches to defining dual targeted proteins

Chapter 3 Approaches to defining dual targeted proteins

41

Foreword to Study II Study I revealed that dual targeted proteins can be difficult to determine even

with substantial experimental evidence. To date, most dual targeted proteins identified

have been identified in single or small gene family studies. With the exception of

aminoacyl-tRNA synthetases, there has been no genome wide, systematic study to

identify dual targeted proteins in Arabidopsis thaliana (Duchene et al., 2005). Study II

aimed to take a broader view of dual targeting in Arabidopsis by seeking to identify a

large number of dual targeted proteins. A list of candidates for dual targeted proteins

was generated using computational prediction of subcellular location of proteins (Small

et al., 2004), cross-over sets in subcellular proteomes of mitochondria, chloroplasts and

peroxisomes (Heazlewood et al., 2007) and a list of proteins predicted to be located in

the mitochondria and the nucleus (Schwacke et al., 2007). The subcellular localisation

of a number of these candidate proteins were then tested experimentally.

Using this approach a total of 12 new dual targeted proteins were identified in

Arabidopsis. Five proteins were dual targeted to mitochondria and plastids, six were

dual targeted to mitochondria and peroxisomes, and one that was dual targeted to

mitochondria and the nucleus. In the course of this analysis, it became evident that a

number of technical parameters need to be taken into account when performing

subcellular localisation experiments using GFP fusions. First, the position of GFP with

respect to the tagged polypeptide is important, (e.g. N or C-terminal tagging is

important, as only analysing one over the other, can mask organelle targeting signals).

Second, the segment of the candidate protein chosen to be fused with GFP can also

greatly affect the results obtained. Third, the use of different tissue types or cells was

found to affect the level of dual targeting observed. It should also be noted that testing

of all available gene models is also required if more than one model exists for a gene

locus.

TECHNICAL ADVANCE

Approaches to defining dual-targeted proteins in Arabidopsis

Chris Carrie, Kristina Kuhn, Monika W. Murcha, Owen Duncan, Ian D. Small, Nicholas O’Toole and James Whelan*

ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, MCS Building M316, 35 Stirling

Highway, Crawley 6009, WA, Australia

Received 31 July 2008; revised 25 October 2008; accepted 30 October 2008; published online 12 December 2008.*For correspondence (fax +61 8 64884401; e-mail [email protected]).

Summary

A variety of approaches were used to predict dual-targeted proteins in Arabidopsis thaliana. These predictions

were experimentally tested using GFP fusions. Twelve new dual-targeted proteins were identified: five that

were dual-targeted to mitochondria and plastids, six that were dual-targeted to mitochondria and

peroxisomes, and one that was dual-targeted to mitochondria and the nucleus. Two methods to predict

dual-targeted proteins had a high success rate: (1) combining the AraPerox database with a variety of

subcellular prediction programs to identify mitochondrial- and peroxisomal-targeted proteins, and (2) using a

variety of prediction programs on a biochemical pathway or process known to contain at least one dual-

targeted protein. Several technical parameters need to be taken into account before assigning subcellular

localization using GFP fusion proteins. The position of GFP with respect to the tagged polypeptide, the tissue

or cells used to detect subcellular localization, and the portion of a candidate protein fused to GFP are all

relevant to the expression and targeting of a fusion protein. Testing all gene models for a chromosomal locus is

required if more than one model exists.

Keywords: Arabidopsis, dual targeting, mitochondria, chloroplasts, peroxisomes, nucleus.

Introduction

An essential step in defining the function of any protein is

clarifying its subcellular location. The completely sequenced

genomes of plants such as Arabidopsis thaliana (Arabidop-

sis Genome Initiative, 2000) (2005), Physcomitrella patens

(Rensing et al., 2008) and Chlamydomonas reinhardtii

(Merchant et al., 2007) and extensive ESTs from various

plant species encode many proteins for which the function

is either unknown or is annotated based on sequence

similarity alone. However, the function of any protein in

eukaryotic cells is not solely a definition of its catalytic

activity; rather it is the product of the catalytic activity and

the subcellular location of the protein. Within a plant cell,

mitochondria and chloroplasts have many enzymatic steps

in common. Both contain ATP synthase complexes with

similar subunits, a partly shared genetic apparatus, and a

variety of related enzymes involved in metabolism and

REDOX biology (Buchanan et al., 2002). Even though many

proteins in these two organelles may have identical catalytic

activity, their cellular function depends on their location.

Various approaches have been used to define the location

of proteins, from computational prediction (Chou and Shen,

2007), GFP tagging (Heazlewood et al., 2007; Koroleva et al.,

2005) and subcellular proteomics (Heazlewood et al., 2007;

Lilley and Dupree, 2007) to direct studies on individual

gene products. One disadvantage evident with large-scale

approaches is that they usually are ‘winner takes all’

approaches, and often define a single location for any given

protein. Each approach has limitations, e.g. computational

prediction usually only works reliably for soluble proteins

with clear organelle-targeting signals, GFP tagging may

result in artefacts, such as accumulation in the nucleus, or

alterations in the targeting ability of the protein due to a

foreign passenger protein (Chew et al., 2003a), while sub-

cellular proteomics often focuses on one organelle, and

proteins ‘claimed’ by two organelles are left unresolved

(Heazlewood et al., 2007).

Defining location is even more complicated for pro-

teins that are located in two compartments, so-called

1128 ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd

The Plant Journal (2009) 57, 1128–1139 doi: 10.1111/j.1365-313X.2008.03745.x

dual-targeted proteins. The first dual-targeted protein

reported in plants was glutathione reductase from Pisum

sativum (pea) (Creissen et al., 1995). Since then, the list of

dual-targeted proteins has grown to approximately 40 from

a variety of species (Duchene et al., 2005; Mackenzie, 2005;

Peeters and Small, 2001; Silva-Filho, 2003). Dual targeting of

a protein can be achieved in a number of ways (Peeters and

Small, 2001; Regev-Rudzki and Pines, 2007; Silva-Filho,

2003): (1) via alternative transcriptional initiation to provide

a protein with different targeting signals, e.g. glutathione

S-transferase F8 (Thatcher et al., 2007), (2) via a targeting

signal directing a protein to two locations, referred to as an

ambiguous targeting signal, e.g. glutathione reductase

(Chew et al., 2003a; Creissen et al., 1995), (3) by two different

targeting signals within one polypeptide, e.g. catalase A in

yeast (Petrova et al., 2004), (4) through utilization of alter-

native translation start sites, currently suggested for a DNA

polymerase targeted to mitochondria and chloroplasts

(Christensen et al., 2005), and also for holocarboxylase

synthetase 1 located in mitochondria and the cytosol

(Puyaubert et al., 2007), and (5) via retrograde translocation,

best known in yeast in which a single fumarase gene

encodes both the mitochondrial and cytosolic forms of

fumarase (Regev-Rudzki and Pines, 2007).

Defining the subcellular localization of a protein with

accuracy is important, as large-scale phenotyping screens,

microarray experiments and protein–protein interaction

assays all rely on such information to build hypotheses or

define models. However, in the case of dual-targeted

proteins, there was no systematic analysis or approach to

define such proteins. With the exception of aminoacyl-tRNA

synthetases (Duchene et al., 2005), most dual-targeted pro-

teins identified to date were identified in studies on individ-

ual proteins. One potential drawback of such approaches is

that dual-targeted proteins may be missed or ascribed to a

single organelle.

Here we describe an approach to define dual-targeted

proteins in Arabidopsis thaliana. Lists of proteins that are

candidates for dual targeting have been generated using

computational prediction of the subcellular location of

proteins (Small et al., 2004), from cross-over sets in subcel-

lular proteomes of mitochondria, chloroplast and peroxi-

somes (Heazlewood et al., 2007), and from a set of proteins

predicted to be located in mitochondria and the nucleus

(Schwacke et al., 2007). We experimentally tested the sub-

cellular localization of subsets of these proteins, thereby

defining dual-targeted proteins found in mitochondria and

peroxisomes, mitochondria and plastids, and mitochondria

and the nucleus. We observed that the tissue type used to

test for dual targeting can greatly affect results, and that both

N- and C-terminal GFP tagging of the investigated polypep-

tide as well as co-transformation of GFP constructs with

appropriate organelle-targeted RFP control constructs are

essential to accurately define subcellular location.

Results

Various approaches were used to computationally predict

dual-targeted proteins in Arabidopsis. To predict dual tar-

geting to mitochondria and plastids, we used the neural-

network-based program Predotar (Small et al., 2004). The

Predotar networks were not trained using any dual-targeted

proteins, and thus the mitochondrial and plastid scores from

the networks tend to be antagonistic. Indeed, the majority of

known dual-targeted plastid/mitochondrial plant proteins

are predicted as uniquely plastidial by Predotar. Therefore,

the complete set of annotated Arabidopsis protein

sequences were run through Predotar twice, once using the

plant prediction networks and once using animal/fungal

prediction networks that were not trained with plastid

sequences. A total of 803 protein sequences were predicted

to contain a plastid-targeting sequence by the plant networks

and to contain a mitochondrial-targeting sequence by the

animal/fungal networks (Table S1a). This list contains 15

proteins that were previously reported to be dual-targeted in

Arabidopsis, which represents a significant enrichment

compared to the number of known dual-targeted proteins

expected to occur in a random group of this size of organelle-

targeted proteins (Table 1), suggesting that this approach is

useful in narrowing down candidates. The dual-targeting

candidates were then ranked by their mitochondrial score

using the animal/fungal networks minus the mitochondrial

score using the plant networks. This ranking places most of

the known dual-targeted proteins towards the top of the list

(Table S1a). As a second strategy, we used available

proteome datasets to predict dual targeting. The reported

proteomes of mitochondria and chloroplasts have an over-

lap of 97 proteins (Arabidopsis subcellular proteomic (SUBA)

database; Heazlewood et al., 2007 and references therein),

three of which have been previously reported to be dual-

targeted (Table S1b), again significantly more than expected

in any random group of 100 proteins (Table 1). In a third

computational approach, all known and putative peroxi-

somal proteins in the Araperox database (Reumann et al.,

2004) were cross-referenced against proteins that had been

experimentally identified in mitochondria or chloroplasts or

that were predicted to be mitochondrial or plastidial using 10

prediction programs. This produced a list of nine proteins

(Table S1c). As there are no known proteins targeted to

mitochondria and peroxisomes, it was not possible in

advance to determine the significance of these predictions.

To further identify potentially dual-targeted proteins, we

focused on processes that occur in different organelles,

specifically DNA replication and transcription. Several

known dual-targeted proteins, such as two DNA polymer-

ases (Christensen et al., 2005; Elo et al., 2003), a RecA

homologue (Shedge et al., 2007) and an RNA polymerase

(Hedtke et al., 2000), which are targeted to both mitochon-

dria and chloroplasts, fit into this functional group. From

Dual targeting of proteins in Arabidopsis thaliana 1129

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 1128–1139

computational screens of the Arabidopsis genome using

publicly available information resources and databases (see

Experimental procedures), we identified seven additional

uncharacterized genes encoding proteins potentially

involved in DNA replication, maintenance or structural

organization that were predicted to be mitochondrial or

plastidial according to the Predotar and TARGETP programs

(Emanuelsson et al., 2000; Small et al., 2004). This group of

proteins was selected for experimental studies. Eleven

transcription factors that have been predicted, but as yet not

experimentally confirmed, to be located in mitochondria as

well as in the nucleus were also tested (Schwacke et al.,

2007) (Table S1d,f).

Taken together, all computational approaches produced a

non-redundant list of 905 proteins, 19 of which have

previously been reported to be dual-targeted. In order to

experimentally test the predicted dual targeting, we gener-

ated GFP reporter constructs for 41 of these proteins. The

Predator list of 803 was too long to test systematically, so we

chose five proteins from the top 20 and 10 more throughout

the list, allowing assessment of the rank order (Supplemen-

tary Table 1A). For the ‘Mass spec’ list of 97 proteins that

have been determined to be located in mitochondria and

plastids (Table 1, Supplementary Table 1B), we tested 10 of

these proteins, and for the other lists we tested more than

50% of the proteins. In the case of the Predator and ‘Mass

spec’ lists, the proteins were chosen at random within the

parameters outlined above. Mitochondrial and plastid

targeting were tested using C-terminal tagging with GFP,

and peroxisomal targeting was studied using N-terminal

tagging. GFP was fused to the full or partial sequence of

candidate proteins, and fluorescence was monitored follow-

ing biolistic transformation of Arabidopsis cell cultures with

these constructs. This approach allowed a relatively large

number of samples to be tested, as each test only requires

2 ml of culture cells on a filter support on an MS agar plate.

When investigating dual targeting to mitochondria and

plastids, Arabidopsis seedlings and onion epidermal cells

were transformed in addition to cell cultures. To define the

subcellular localization of GFP fluorescence with certainty,

we generated RFP control constructs driving expression of

mitochondrial-, plastid- or peroxisomal-targeted RFP (Carrie

et al., 2007; Murcha et al., 2007). For each protein to be

tested, GFP constructs were co-transformed with the appro-

priate RFP control constructs. This avoided using stains such

as Mitotracker to define organelles and provided a positive

control for the success of transformation and protein uptake

into the appropriate organelle. The subcellular localizations

of all proteins tested are shown in Figures 1, 2, 3 and 5 and

Figures S1 and S2.

To experimentally define proteins that were targeted

to mitochondria and peroxisomes, N- and C-terminal GFP

fusions were constructed and targeting was studied for

three proteins (Table S1c). When GFP was placed at the

C-terminus of a carrier protein (At3g55640), malonyl CoA

decarboxylase (At4g04320) or alanine/glyoxylate amino-

transferase (At4g39660), a fluorescence pattern that was

identical to that obtained with mitochondrial RFP was

observed (Figure 1, images 1a–1c, 3a–3c and 5a–5c). How-

ever, when GFP was placed at the N-terminus, a pattern that

overlapped with peroxisomal RFP was obtained (data not

shown). To confirm that the peroxisomal targeting was due

to the predicted peroxisomal-targeting signal type 1 (PTS1)

at the C-terminus of the proteins, the last 10 amino acids of

each of the three proteins were fused to the C-terminus of

GFP (Figure 1, images 2d–2f, 4d–4f and 6d–f). Peroxisomal

GFP fluorescence was again observed. Thus dual targeting

was detected for the three proteins tested from the group of

Table 1 Summary of predicted and exper-imentally determined dual-targeted pro-teins in Arabidopsis

Method ofprediction

Total no.proteins

No. knowndual-targetedproteins inthis list P-values

No.proteinstested

No. knowndual-targetedproteins afterthis study

Predotar 803 15 0.0003 14 17Mass spec 97 3 0.08 11 4AraPerox 9 0 N/A 6a 6a

Transcription factors 11 0 N/A 8 1DNA maintenance 9 2 N/A 9 5Totals 905 19 N/A 41a 31a

A number of approaches were used to predict dual-targeted proteins in Arabidopsis. The totalnumber of proteins predicted by each approach to be dual-targeted and the number ofexperimentally verified dual-targeted proteins is indicated for each approach. The enrichment ofdual-targeting proteins in each list was determined compared to groups of randomly selectedmitochondrial and chloroplasts proteins as outlined in Experimental procedures. The targetingability of the number of proteins tested from each group and the number of dual-targetedproteins from each group after this study are also shown. The totals represent the non-redundantnumbers tested due to overlap between groups.aThis group includes three alternative NAD(P)H dehydrogenases that we have shown to bedual-targeted to mitochondria and peroxisomes (Carrie et al., 2008).

1130 Chris Carrie et al.


putative peroxisomal and mitochondrial proteins, but this

depended on the nature of the GFP construct.

When testing the targeting properties of the seven

proteins that may function in DNA maintenance, replication

or transcription, we detected dual targeting to mitochondria

and chloroplasts for three proteins, a topoisomerase I TopI-

like protein (At4g31210, AtTopI), a protein with similarity to

the human mitochondrial DNA helicase Twinkle (At1g30680,

AtTwinkle) and a protein related to human RCC1 and

Arabidopsis Uvr8 (At5g08710; RFP tagging was used for

this gene for cloning reasons) (Figure 2, image series 3, 4

and 5). For AtTopI, as for the dual-targeted DNA polymerase

Polc1 (Elo et al., 2003), which we used as a control (Fig-

ure S2, image series 1), the intensity of fluorescence from

mitochondria and plastids was equal in most transformed

suspension cells. For AtTwinkle, the plastid fluorescence

was much stronger than mitochondrial fluorescence in

suspension cells and onion cells, but the intensity of

fluorescence from mitochondria was equal to that from

plastids in Arabidopsis seedlings (Figure 2, images series 3).

The different intensities of GFP fluorescence in the two

organelles were not restricted to this group of proteins.

When we tested the targeting of calcium-sensing receptor

(CaS, At5g23060), a phosphoprotein that has been shown to

be located in thylakoid membranes (Vainonen et al., 2008)

but is potentially dual-targeted according to Predotar and

organelle proteome overlap (Table S1), dual targeting was

observed with onion epidermal cells and with Arabidopsis

seedlings, but not with suspension cell cultures (Figure 2,

image series 6). We previously observed a similar tissue-

specific variation of GFP fluorescence for dual-targeted

type II NAD(P)H dehydrogenases (Figure S2, image series

2) (Carrie et al., 2008). Thus the ability to detect dual

targeting depended on the nature of the tissue.

The above findings prompted us to analyse the targeting

ability of DNA polymerase c2 (Polc2), which has been

MergedRFPSRLMergedAOXRFP

10 µm

GFP AA 1-518

GFP AA 322-332

GFPAA 1-332

GFP AA 466-476

GFP AA 1-476

GFP AA 508-518

Alanine/Glyoxylate aminotransferase (AGAT)

(At4g39660)

Malonyl CoA decarboxylase (MDC)(At4g04320)

Carrier protein (CP)(At3g55640)

Test for mitochondrial targeting ability Test for peroxisomal targeting ability

3a 3b 3c 3d 3e 3f

4a 4b 4c 4d 4e 4f

5a 5b 5c 5d 5e 5f

6a 6b 6c 6d 6e 6f

1a 1b 1c 1d 1e 1f

2a 2b 2c 2d 2e 2f

Figure 1. Proteins found to be dual-targeted to mitochondria and peroxisomes.

N- and C-terminal GFP fusion proteins were constructed for proteins predicted to be located in mitochondria and peroxisomes. The amino acids (AA) fused to GFP

are indicated in a schematic representation of each construct (left). Targeting ability was tested in Arabidopsis suspension cells using AOX–RFP as a control for

mitochondrial targeting and RFP–SRL as a peroxisome-specific control.



previously reported as a dual-targeted protein (Christensen

et al., 2005). Christensen et al. (2005) detected targeting

exclusively to plastids if translation was initiated at the first

in-frame AUG codon. When using a construct that included

the annotated 5¢ UTR, dual targeting was observed, which

was proposed to be due to translation starting at a CUG

codon seven amino acids upstream of the AUG (Christensen

et al., 2005). The authors proposed that the targeting of

Polc2 is regulated by alternative translation initiation. In

contrast, we observed that, when translation started at the

first in-frame AUG, GFP was targeted to both mitochondria

and plastids, even though mitochondrial fluorescence was

much weaker than plastid fluorescence in suspension cells

(Figure 3a, image series 1 and 2). Mitochondrial GFP fluo-

rescence was detected in onion cells and Arabidopsis

seedlings (Figure 3b, image series 4), and exclusive mito-

chondrial localization was occasionally observed in Arabid-

opsis seedlings (Figure 3b, image 4c). Addition of seven

amino acids was performed as described by Christensen

et al. (2005), resulting in targeting to both organelles with

equal intensity of fluorescence in Arabidopsis cell suspen-

sions and onion cells (Figure 3a,b), but only to mitochondria

in Arabidopsis seedlings (Figure 3b, image 5b).

To further study the import of Polc2 into mitochondria

and plastids, we performed in vitro import assays on the

DNA polymerase protein starting with the standard AUG

codon. Although such large proteins are usually difficult

to study with in vitro import assays, we readily detected

the import of this protein into mitochondria isolated from

Arabidopsis seedlings (Figure 4a, left panel). Import

was abolished by addition of valinomycin to the import

assay, indicating that it was dependent on the membrane

potential, as expected for a protein translocated into the

mitochondrial matrix. No import of the precursor of the

small subunit of 1,5-ribulose bisphosphate carboxylase/

oxygenase (Rubisco SSU) into isolated mitochondria was

observed (Figure 4b, right panel), indicating that the

import of Polc2 observed was not due to plastid contam-

ination or non-specific import of chloroplast proteins into

isolated mitochondria, as previously reported for isolated

pea mitochondria (Cleary et al., 2002), but not for isolated

Arabidopsis mitochondria as used in this study (Chew

et al., 2003b).

We next analysed the kinetics of import into mitochon-

dria isolated from suspension cell cultures and Arabidop-

sis seedlings used in the above import assays (Figure 3b).

Arabidopsis cell culture

MergedSSURFPMergedAOXRFP

ArabidopsisseedlingsOnion cells

RFPAA 1-117

RCC1/Urv8 like protein (434 AA)At5g08710

GFPAA 1-387

CaS like protein (387 AA)At5g23060

GFPAA 1-192

Topoisomerase I (AtTopI) (1280 AA)At4g31210

GFPAA 1-42

Alternative oxidase (AOX) (321 AA)

GFPAA 1-180

Small subunit of 1,5 ribulose bisphosphatecarboylase/oxygenase (SSU Rubisco) (180 AA)

GFPAA 1-184

DNA helicase twinkle (Atwinkle) (709 AA)At1g30680

Test for mitochondrial targeting ability Test for chloroplastidic targeting ability

10 µm

1a 1b 1c 1d 1e 1f 1g 1h

2a 2b 2c 2d 2e 2f 2g 2h

3a 3b 3c 3d 3e 3f 3g 3h

4a 4b 4c 4d 4e 4f 4g 4h

f5e5d5c5b5a5

h6f6e6d6c6b6a6 6g

5g 5h

Figure 2. Proteins found to be dual-targeted to mitochondria and chloroplasts.

Subcellular localization was predicted for a list of proteins involved in DNA maintenance replication and transcription (Table S1d). One other protein (CaS) predicted

to be targeted to mitochondria and plastids was also tested. For the RCC1/Uvr8-like protein, RFP was used as the reporter and GFP as the control; all other candidate

proteins were fused to GFP. The numbers refer to the amino acids used in each construct. The total number of amino acids in each protein is shown after the name if

the full-length protein was not used to make the fusion protein. Targeting ability was tested in Arabidopsis suspension cell cultures, onion epidermal cells and

2-week-old Arabidopsis seedlings.



After 10 min, when the import rate was still relatively

linear, the import of alternative oxidase (AOX) was almost

twofold higher into mitochondria isolated from seedlings

compared to cultured cells, and a large difference per-

sisted even after 20 min. For import of Polc2, a difference

of 40% at 20 min was also observed between mitochondria

isolated from seedlings compared to cultured cells. As

equal amounts of mitochondrial protein were used in the

above import assays, we analysed the abundance of

several components of the mitochondrial import appa-

ratus; a representative immunoblot from several mito-

chondrial preparations is shown (Figure 4c). Differences

were observed for some components, e.g. TOM20-2 was

approximately twice as abundant in mitochondria isolated

from cell cultures compared to mitochondria isolated from

plant material. The opposite was seen with TOM20-4. Thus

isolated mitochondria from different tissues import pro-

teins at different rates, which might be related to differ-

ences in the abundance of particular components of the

mitochondrial import apparatus.

A number of transcription factors have been predicted to

be targeted to mitochondria and nuclei in Arabidopsis and

rice (Schwacke et al., 2007). We tested the targeting of

eight of these transcription factors to mitochondria. For

three of these, we initially fused full-length cDNAs to the

GFP coding sequence. With one exception, this resulted

in no GFP fluorescence. A mitochondrial pattern was

obtained for the transcription factor APL (altered phloem

development, At1g79430), indicating that the protein had

mitochondrial-targeting ability (Figure 5a). As GFP fused to

the N-terminus of this protein had been shown previously

to target to nuclei (Bonke et al., 2003), we concluded that

this transcription factor is dual-targeted. For the seven

other transcription factors predicted to possess N-terminal

mitochondrial transit peptides, we fused GFP to the first 60

amino acids of these proteins. GFP fluorescence was seen

Arabidopsis cell culture

GFPAA -7-120

GFPAA 1-120

DNA polymerase γ2 (Polγ2) (1050 AA)At1g50840

DualMitochondrialPlastidDualArabidopsis seedlingsOnion cells

MergedSSURFPMergedAOXRFP

Test for mitochondrial targeting ability Test for chloroplastidic targeting ability

AUG

CUGAUG

1 120

1 120–710 µm

1a 1b 1c 1d 1e 1f

2a 2b 2c 2d 2e 2f

3a 3b 3c 3d 3e 3f

GFPAA -7-120

GFPAA 1-120

DNA polymerase γ2 (Polγ2) (1050 AA)At1g50840AUG

AUG1 120

1 120–7

4a

4b

4c 4d 4e

5a 5b

4b

CUG

(b)

(a)

Figure 3. Targeting of DNA polymerase c2 to mitochondria and chloroplasts.

The targeting ability of the 120 N-terminal amino acids (with the AUG start codon) or the 127 N-terminal amino acids (with the upstream CUG start changed to AUG

to ensure fidelity of translation) of DNA polymerase c2 was tested in three tissue types:

(a) Arabidopsis suspension cell culture and (b) 2-week-old Arabidopsis seedlings and onion epidermal cells. Dual targeting of the native AUG constructs was easier

to visualize in onion cells (image 4a) and Arabidopsis seedlings (images 4d and 4e). In some cases, an exclusive mitochondrial localization was observed in the latter

(image 4c). Dual targeting of the CUG constructs (changed to AUG to ensure fidelity of translation) was observed in Arabidopsis cell suspension and onion cells, but

mitochondrial targeting only was detected in Arabidopsis seedlings (image 5b). The gene models show the sequences used to generate the proteins: the upstream

CUG was converted to AUG, and the native AUG was changed to CUG so that a single protein would be produced in each case. The constructs with the native AUG

did not contain any upstream nucleotides to ensure that translation could only commence at this AUG.



mainly in the nucleus and also in the cytosol but never in

mitochondria for these constructs (Figure S1). For two of

the proteins, AtTLP9 (At3g06380) and AtTLP7 (At1g53320),

we cloned full-length cDNAs in order to perform in vitro

import assays into isolated Arabidopsis mitochondria.

No import was detected (data not shown), indicating that

TOM20-2

TOM20-3

TOM20-4

OM64

TOM40

TIM17-2

Plants

Cell cu

lture

Plants Cell culture

Time (min) 2 5 10 20 2 5 10 20

pAOX 36 kDa

mAOX 32 kDa

Lane

MitPKVal

1 2 3 4 5

+ + + ++ +

+ +

–– – –– – –

pAOX 36 kDa

pPolγ2 120 kDa

mAOX 32 kDa

mPolγ2 116 kDa

Lane 1 2 3 4 5MitChlProt + +– – –

+ ++ +–

–– –

– –

pSSU 20 kDa

mSSU14 kDa

pPolγ2 120 kDa

mPolγ2 116 kDa

0.00

0.20

0.40

0.60

0.80

1.00

0 5 10 15 20Time (min)

Plants

AOX

n = 3

0.00

0.20

0.40

0.60

0.80

1.00

0 5 10 15 20

Plants

n = 3

Time (min)

Cell culture Cell culture

Polγ2

(a) (c)

(b)

Figure 4. In vitro import of DNA polymerase c2 into mitochondria, and analysis of the kinetics of protein import into mitochondria from Arabidopsis suspension

cells and seedlings.

(a) In vitro import of DNA polymerase c2 (translated from AUG) into mitochondria. Incubation of the full-length precursor protein with an apparent molecular mass of

120 kDa (lane 1) with mitochondria (lane 2) resulted in a protease-protected band with an apparent molecular mass of 116 kDa (lane 3). The protease protection was

abolished when valinomycin was added to mitochondria prior to the import assay (lanes 4 and 5). A similar pattern was observed with the mitochondrial protein

alternative oxidase. The third panel shows the specificity of import into mitochondria: the precursor of the Rubisco SSU (lane 1) is not imported into a protease-

protected location when incubated with mitochondria (lanes 2 and 3), but is readily imported and protease-protected when incubated with chloroplasts (lanes 4 and 5).

(b) Analysis of the kinetics of protein import into mitochondria isolated from Arabidopsis suspension cell cultures and 2-week-old Arabidopsis seedlings. The

alternative oxidase and Polc2 precursors were incubated with 250 lg of isolated mitochondria from both tissues, and the amount of imported protein was assessed at

various times. The maximum amount of import was set to 1, and other values are expressed as relative amounts. Error bars show standard errors from three

independent uptake experiments.

(c) Western blot analysis of various components involved in the import of proteins into mitochondria: 30 lg of isolated mitochondria were loaded onto each lane.



none of the seven transcription factors is targeted to

mitochondria.

Discussion

A number of approaches were used to search for dual-

targeted proteins, leading to identification of 12 new

examples, including proteins targeted to mitochondria and

peroxisomes or mitochondria and nuclei (Table 2). From

these studies, it has emerged that caution must be exercised

when assigning a location to a protein from GFP assays, as a

single targeting location may be deduced when in fact the

protein is dual-targeted or has dual-targeting ability.

The location of the GFP passenger is critical in determin-

ing dual targeting. This was evident for proteins targeted to

mitochondria and peroxisomes via two different signals, an

N-terminal mitochondrial transit peptide and a C-terminal

PTS-1 signal. In mammalian cells, serine:pyruvate/ala-

nine:glyoxylate aminotransferase and 2-methylacyl CoA

racemase have also been shown to target to both mitochon-

dria and peroxisomes via two separate targeting signals, an

N-terminal mitochondrial-targeting signal and a C-terminal

peroxisomal-targeting signal (Amery et al., 2000; Oda et al.,

2000). Alternative transcript initiation has been shown to

produce two mRNAs yielding two different proteins for

serine:pyruvate/alanine:glyoxylate aminotransferase, one of

which lacks the N-terminal mitochondrial-targeting signal

(Oda et al., 2000). A similar mechanism for producing

mitochondrial and peroxisomal proteins from a single gene

may operate in plants. However, 5¢ RACE did not reveal

alternative transcripts for two alternative NAD(P)H dehydro-

genases recently shown to be targeted to mitochondria

and peroxisomes via an N-terminal mitochondrial-targeting

signal and a C-terminal PTS-1 (Carrie et al., 2008).

In the case of the APL transcription factor, two gene

models exist; the longer mRNA has an additional N-terminal

sequence and is not predicted to be targeted to mitochon-

dria, whereas the shorter transcript yields a smaller protein

that is predicted to possess a mitochondrial transit peptide

(Figure 5b,c). Previously, it has been demonstrated that GFP

GFPAA 1-358

APL like transcription factor (358 AA)At1g79430

At1g79430.1

At1g79430.2

Predicted mitochondrialtargeting signal

Nuclear localisationsignal

Locus TargetP MitoProt2 Subloc Ipsort Predotar Mitopred Peroxp Wolfpsort Multiloc LoctreeAt1g79430.1 mito mito nuclear mito mito nuclear mito nuclearAt1g79430.2 nuclear nuclear nuclear nuclear

MergedAOXRFP

10 µm

(a)

(b)

(c)

Figure 5. Analysis of the targeting ability of the transcription factor APL, predicted to be targeted to mitochondria and the nucleus.

(a) When GFP is fused to the C-terminus of the shorter of two proteins encoded by APL (b), only mitochondrial targeting is evident.

(b) Models of two mRNAs that have been proposed for this gene are shown; the positions of a predicted mitochondrial transit peptide and a nuclear localization

signal are shown.

(c) Subcellular targeting for the two forms of APL as predicted by 10 programs.

Table 2 Locations of the dual-tragted proteins in Arabidopsis

Mitochondria/chloroplast Mitochondria/peroxisome Mitochondria/nucleus Chloroplast/nucleus

Dual-targeted proteinsin Arabidopsis

42 (37) 6a (0) 1 (0) 0 (0) 49

The table shows the total number of known dual-targeted proteins in Arabidopsis after this study, and with the number known before this study inparentheses.aThis group includes three alternative NAD(P)H dehydrogenases that we have shown to be dual-targeted to mitochondria and peroxisomes (Carrieet al., 2008).



fused to the N-terminus of the larger protein is targeted to

the nucleus (Bonke et al., 2003). We detected mitochondrial

targeting but no nuclear signal when GFP was fused to the

C-terminus of the smaller protein. APL has a predicted

nuclear localization signal (KKRP) starting at amino acid 190

that appears to direct nuclear import only when the

mitochondrial-targeting signal is blocked with additional

amino acids at the N-terminus. Several studies have dem-

onstrated that either removing some N-terminal amino acids

from mitochondrial-targeting signals or placing other amino

acids in front can affect mitochondrial targeting (de Castro

Silva Filho et al., 1996; Chaumont et al., 1994; Rudhe et al.,

2002). For the smaller form of APL in which the mitochon-

drial transit peptide is unmasked, mitochondrial import

appears to be dominant over nuclear targeting. Alternative

splicing may represent a more widely used mechanism to

achieve dual targeting. It has been shown for glutathione

S-transferase F8 that alternative transcript initiation leads to

different subcellular localizations (Thatcher et al., 2007).

Furthermore, improved annotation of genomes often results

in several gene models for a single chromosomal locus.

Thus, although some studies may have determined a single

localization for a protein, newer gene models produced after

these studies must be checked with respect to localization,

as observed here with the APL transcription factor.

We observed that, in the case of dual targeting to

mitochondria and chloroplasts, the tissue used to test

targeting can play a major role in determining whether a

protein is dual-targeted. While some proteins such as

AtTopI and Polc1 were clearly dual-targeted in all three

tissue types tested, dual targeting was difficult to detect for

AtTwinkle, RCC1/Uvr8-like and CaS in Arabidopsis suspen-

sion cells as the fluorescence from mitochondria was

much weaker, but still detectable, than fluorescence from

plastids. However, dual targeting of these proteins was

more readily detected in onion cells and Arabidopsis

seedlings. This finding prompted us to test the targeting

of Arabidopsis Polc2. This protein has previously been

proposed to be dual-targeted by the use of two different

translation start sites, a standard AUG codon and an

upstream CUG (Christensen et al., 2005). We detected dual

targeting with translation starting from the standard AUG

codon in Arabidopsis seedlings, cell culture and onion

cells, and moreover confirmed by in vitro uptake assays

using the full-length Polc2 polypeptide that the protein

translated from the AUG is imported into mitochondria.

These data are consistent with data from tobacco, in which

two DNA polymerases homologous to Polc1 and Polc2

have been reported to be dual-targeted to mitochondria

and plastids when translated from the standard AUG

codon (Ono et al., 2007).

Because we observed differences between different

tissues used to detect dual targeting, we tested the kinetics

of protein import into mitochondria isolated from two

tissue types (Arabidopsis suspension cell cultures and

2-week-old seedlings) in order to determine whether the

import capacity of mitochondria from some tissues affects

the ability to detect dual targeting. We observed that

mitochondria from Arabidopsis seedlings had a faster rate

of import. This may affect the ability to detect dual targeting

as the threshold for GFP fluorescence detection may not be

reached if the rate of mitochondrial protein import is low.

Additionally, an immunoblot analysis of selected compo-

nents of the mitochondrial protein import apparatus

showed that some of the receptor components differed in

abundance between tissues, i.e. the level of TOM20-2 was

lower but that of TOM20-4 was higher in seedlings than in

mitochondria from cell culture. In a previous study analy-

sing the functionality of all three isoforms of TOM20, it was

observed that, in a double knock-out of tom20-2 and

tom20-3, in which TOM20-4 alone was present, import of

dual-targeted glutathione reductase was greater compared

to wild-type or any double knock-out that inactivated

TOM20-4 (i.e. tom20-3/tom20-4 or tom20-2/tom20-4) (Lister

et al., 2007). The relative abundance of a protein receptor

isoform may affect the import of a specific set of proteins,

in this instance Polc2, while others such as AtTopI may be

unaffected. Thus the relative abundance of various iso-

forms of the protein import receptors appears to differ

between tissues, and this may contribute to the ability to

detect dual targeting.

Overall, this study identified 12 new dual-targeted pro-

teins, and increased the number of dual-targeted proteins

detected in Arabidopsis to 51 (Table 2). It has identified more

proteins targeted to both mitochondria and chloroplasts,

and extended the concept of dual targeting in plant cells to

mitochondria and peroxisomes as well as mitochondria and

the nucleus.

Experimental procedures

Construction of GFP fusion proteins to analyse targeting

A Gateway� cloning cassette was constructed (Figure S3) to allowrecombination cloning of cDNA clones using Gateway� cloningtechniques according to the manufacturer’s instructions (Invitro-gen, http://www.invitrogen.com/). This strategy was chosen for allbut nine genes (see Table S2). For those nine genes, reporter geneconstructs were produced by replacing the AOX sequence in thetargeting control vectors described below with the cDNA sequenceof the gene to be tested. The Arabidopsis gene locus and theprimers used for each constructs are listed in Table S2.

Five GFP or RFP fusions were produced as controls for subcellularlocalization. The alternative oxidase (AOX) targeting signal of 42amino acids and the full-length cDNA of the small subunit of 1,5-ribulose bisphosphate carboylase/oxygenase (Rubisco SSU) werefused to GFP and RFP as mitochondrial and chloroplast controls,respectively (Carrie et al., 2007). The peroxisomal-targeting controlcontained the PTS-1 targeting signal of pumpkin (Cucurbita sp.)malate synthase fused to the C-terminus of RFP (Carrie et al., 2007;Pracharoenwattana et al., 2005).



Determination of the targeting ability of GFP/RFP

fusion proteins

Biolistic transformations of GFP and RFP constructs wereperformed on Arabidopsis cell culture, seedlings and onionepidermal cells as previously reported (Carrie et al., 2007; Thir-kettle-Watts et al., 2003). The GFP and RFP plasmids (5 lg each)were co-precipitated onto gold particles and transformed usingthe biolistic PDS-1000/He system (Bio-Rad, http://www.bio-rad.-com/). Particles were bombarded onto either 2 ml of Arabidopsiscell suspension resting on filter paper on osmoticum plates, 7- to14-day-old Arabidopsis seedlings placed on filter paper on stan-dard MS plates, or onion cell epidermal peels placed on filterpaper on standard MS plates. After bombardment, all Arabidopsiscell suspension, seedlings and onion cells were placed in the darkat 22�C for 24–48 h before visualization of GFP/RFP. Localization oftransient GFP and RFP expression was performed using anOlympus BX61 fluorescence microscope (http://www.olympusmicro.com) with excitation wavelengths of 460/480 nm (GFP) and 535/555 nm (RFP), and emission wavelengths of 495–540 nm (GFP)and 570–625 nm (RFP). Subsequent images were captured usingCell� imaging software as previously described (Carrie et al.,2007; Murcha et al., 2007).

Prediction of dual-targeted proteins

Dual-targeted proteins were predicted in a number of differentways. The Predator prediction program can be used in ‘animal’ or‘plant’ mode (Small et al., 2004). The entire predicted Arabidopsisproteome was analysed twice to produce a mitochondrial-targetingprediction in the animal mode, and a plastid-targeting prediction inthe plant mode. The 803 proteins that passed both screens wereranked using the score for targeting to mitochondria using theanimal mode minus the score for mitochondrial targeting in theplant mode (Table S1a).

To predict proteins that are dual-targeted between mitochon-dria and peroxisomes, the list of predicted peroxisomal proteinsfrom Araperox (Reumann et al., 2004) was compared to knownmitochondrial proteins using the SUBA database (Heazlewoodet al., 2007) (Table S1b,c). The subcellular localizations of theoverlapping list of proteins were predicted using 10 predictors ofsubcellular localization: Target P (Emanuelsson et al., 2000, 2007),Predotar (Small et al., 2004), MitoprotII (Claros and Vincens, 1996),iPSORT (Bannai et al., 2002), Subloc (Hua and Sun, 2001),Mitopred (Guda et al., 2004), Wolfpsort (Horton et al., 2007),Multiloc (Hoglund et al., 2006), Loctree (Nair and Rost, 2005) andPeroxP (Emanuelsson et al., 2003). Proteins were selected ascandidates if either a mitochondrial location had already beenpublished or a protein showed strong mitochondrial prediction byat least five predictors.

Nuclear loci encoding proteins potentially involved inmitochondrial DNA maintenance replication and transcription(Table S1e) were identified from computational screens of theArabidopsis genome using publicly available databases at NCBI(National Centre for Biotechnology Information), TAIR (TheArabidopsis Information Resource) and AMPDB (ArabidopsisMitochondrial Protein Database) (Heazlewood et al., 2007). Thesescreens were particularly directed at identifying homologues ofestablished fungal and animal mitochondrial DNA-associatedproteins (BLAST screens at NCBI), mitochondrial homologues ofproteins reported to associate with the plastid genome (BLAST),and genes co-expressed with Arabidopsis c-type DNA polymeras-es and predicted to be involved in DNA-related processes [ATTED-II (Obayashi et al., 2007) and Expression Angler (Toufighi et al.,

2005)]. The identified loci were screened for previously uncharac-terized putative mitochondrial and plastidial gene products usingSUBA (Heazlewood et al., 2007).

Additionally, 11 transcription factors previously predicted tobe targeted to mitochondria and the nucleus (Schwackeet al., 2007) were tested for mitochondrial-targeting ability(Table S1d).

Significance of predictions

We suspected that the number of dual-targeted proteins found inthe list produced by the Predotar method (15 from 803 proteins)was greater than expected by chance. The very low proportion ofknown dual-targeted proteins among proteins predicted to beeither mitochondrial or plastidial (31 in 4320) prevents standardstatistical techniques being used to demonstrate this, so empiricalP-values were calculated by taking 10 000 random sets of 803proteins from the set of 4320 proteins predicted to be mito-chondrial or plastidial and identifying the number of known dual-targeted proteins in each of these. The P-values were taken as thenumber of random sets with at least 15 dual-targeted proteinsdivided by 10 000. Three sets of proteins contained 15 or moredual-targeted proteins, so there is an enrichment of dual-targetedproteins in the set produced by the Predotar method with aP-value = 0.0003. Similarly, empirical P-values were calculated todemonstrate the enrichment of dual-targeted proteins in the‘Mass spec’ set. In this case, 10 000 random sets of 97 proteinswere selected from the 2913 proteins with experimental evidencefor localization in the mitochondria or plastids according to theSUBA database (http://www.plantenergy.uwa.edu.au/applications/suba2/). The P-value was taken as the number of randomsets with at least three dual-targeted proteins divided by 10 000(Table 1).

In vitro import assays and Western blot analysis

In vitro import of proteins into isolated mitochondria and Westernblot analysis ere performed as previously described (Lister et al.,2007). To generate the TOM40 antibody, a recombinant proteincontaining the first 200 amino acids from the N-terminus of TOM40-1 (At3g20000) fused to an N-terminal 6· His affinity purificationtag was expressed in Escherichia coli strain BL21(DE3). Therecombinant protein was purified by denaturing Immobilized metalaffinity chromatography (IMAC), using the Bio-Rad ProfiniaTM pro-tein purification system. The resultant eluate was separated by 12%v/v SDS–PAGE, and the recombinant protein was extracted using aBio-Rad Model 422 electro-eluter. Buffer exchange was performedusing an Amicon Ultracel-5 k centrifugal filter device (http://www.millipore.com) such that the antigen was re-suspended in PBSsolution, recovering a total of 3 mg of protein for inoculation. Fourseparate doses were administered to a rabbit at regular intervalsover a 3-month period using standard protocols and Freud’scomplete adjuvant (Cooper and Paterson, 2008).

Supporting Information

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Targeting ability of all proteins analysed in this study,except those shown in Figures 1, 2, 3 and 5.Figure S2. Proteins found to be dual-targeted to mitochondria andchloroplasts.



Figure S3. Plasmid maps of the two GFP gateway destinationvectors used in this study.Table S1. List of proteins predicted to be dual-targeted usingvarious approaches as outlined in the text.Table S2. List of chromosomal loci investigated and primers used inthis study.Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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Chapter 4 Arabidopsis NAD(P)H dehydrogenases are dual targeted

54

Chapter 4

Arabidopsis NAD(P)H dehydrogenases are dual targeted

Chapter 4 Arabidopsis NAD(P)H dehydrogenases are dual targeted

55

Foreword to Study III Upon closer examination of the list of candidate dual targeted proteins defined

in study II, it was observed that a number of type II NAD(P)H dehydrogenases (ND)

were predicted to target both mitochondria and peroxisomes. Typically, ND proteins

have been located on the mitochondrial inner membrane where they can oxidise

NAD(P)H (Michalecka et al., 2003; Rasmusson et al., 2004; Elhafez et al., 2006). In

Arabidopsis there are seven genes, that encode for ND proteins, three have been defined

as external (NDB1, 2 and 4) and three have been defined as internal (NDA1 and 2 and

NDC1) (Michalecka et al., 2003; Elhafez et al., 2006) whilist the remaining gene,

NDB3, is thought to be a pseudogene (Elhafez et al., 2006). Over the past decade a

number of different studies have defined ND proteins as mitochondrial using GFP

tagging (Michalecka et al., 2003), in vitro uptake experiments (Elhafez et al., 2006),

Western blotting (Rasmusson and Agius, 2001), and enzymatic activity assays

(Svensson and Rasmusson, 2001; Geisler et al., 2004). However, when looking for

proteins targeted to mitochondria and peroxisomes, a number of ND proteins were

predicted to contain not only mitochondrial targeting signals but also PTS1 like signals

at their C-terminus (NDA1 and 2 and NDB1). During this analysis it was also predicted

that NDC1 was a plastid protein, which is not surprising considering its cyanobacterial

ancestry (Michalecka et al., 2003).

Using the established techniques from study II, GFP fusions were made of all

Arabidopsis ND proteins at both the N and C-terminus. It was found that NDA1 and 2

and NDB1 were dual targeted to mitochondria and peroxisomes by two separate

targeting signals: an N-terminal targeting signal for mitochondria, and a C-terminal

PTS1 for peroxisomal targeting. This second signal had previously been missed, due to

studies only using the N-terminal part of the protein for targeting studies. It was also

discovered that NDC1 was targeted to both mitochondria and plastids, using GFP

tagging and in vitro import experiments. It is proposed that the reason why the plastid

targeting was missed in previous studies was because only the N-terminal part of the

protein was used (Michalecka et al., 2003), not the full length protein as was used in

this study. The dual targeting of ND proteins raises interesting questions as to their roles

within plant metablolism.

FEBS Letters 582 (2008) 3073–3079

Type II NAD(P)H dehydrogenases are targeted to mitochondriaand chloroplasts or peroxisomes in Arabidopsis thaliana

Chris Carriea, Monika W. Murchaa, Kristina Kuehna, Owen Duncana,Michelle Barthetb, Penelope M. Smithb, Holger Eubela, Etienne Meyera,

David A. Dayb, A. Harvey Millara, James Whelana,*

a ARC Centre of Excellence in Plant Energy Biology, MCS Building M316 University of Western Australia,35 Stirling Highway, Crawley 6009, Western Australia, Australia

b ARC Centre of Excellence in Plant Energy Biology, School of Biological Sciences, University of Sydney, NSW, Australia

Received 21 July 2008; revised 30 July 2008; accepted 31 July 2008

Available online 12 August 2008

Edited by Ulf-Ingo Flugge

Abstract We found that four type II NAD(P)H dehydrogen-ases (ND) in Arabidopsis are targeted to two locations in thecell; NDC1 was targeted to mitochondria and chloroplasts, whileNDA1, NDA2 and NDB1 were targeted to mitochondria andperoxisomes. Targeting of NDC1 to chloroplasts as well as mito-chondria was shown using in vitro and in vivo uptake assays anddual targeting of NDC1 to plastids relies on regions in the ma-ture part of the protein. Accumulation of NDA type dehydrogen-ases to peroxisomes and mitochondria was confirmed usingWestern blot analysis on highly purified organelle fractions. Tar-geting of ND proteins to mitochondria and peroxisomes isachieved by two separate signals, a C-terminal signal for peroxi-somes and an N-terminal signal for mitochondria.� 2008 Federation of European Biochemical Societies. Pub-lished by Elsevier B.V. All rights reserved.

Keywords: Chloroplast; Mitochondria; Peroxisome; Dualtargeting; Green fluorescent protein; Alternative NAD(P)Hdehydrogenase

1. Introduction

A hallmark of eukaryotic cells is the partitioning of various

biochemical pathways out of the cytosolic milieu and into dis-

crete organelles. Although the compartmentalisation of vari-

ous biochemical functions allows specialisation, it requires

that many functions are duplicated and thus many enzymatic

activities take place in more than one organelle. In the majority

of cases these common functions are performed by different

proteins, encoded by distinct genes, that are each targeted to

a single location in the cell [1]. However, in other cases it ap-

pears that the same function in different organelles is carried

out by the same protein that is targeted to two locations, a

Abbreviations: AOX, alternative oxidase; GFP, green fluorescentprotein; KAT2, 3-ketoacyl-CoA thiolase; ND, type II alternativeNAD(P)H dehydrogenase; RFP, red fluorescent protein; SSU, Rubi-sco small subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase;TIM17-2, translocase of the inner mitochondrial membrane

*Corresponding author. Fax: +61 8 93801148.E-mail address: [email protected] (James Whelan).

0014-5793/$34.00 � 2008 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2008.07.061

process called dual targeting. This was first reported for gluta-

thione reductase from pea, which is targeted to both mitochon-

dria and chloroplasts [2]. To date, studies in several plants

suggest that more than 30 proteins are dual targeted to mito-

chondria and chloroplasts [3].

The targeting of proteins is routinely assessed by attaching a

reporter, most often green fluorescent protein (GFP), to the

protein being studied and the intra-cellular distribution of fluo-

rescence measured [4]. This approach is convenient and sensi-

tive and has been used widely to define dual targeting to

mitochondria and chloroplasts [5–8]. However, this approach

has some limitations that depend on the nature of the con-

structs. Firstly, for proteins that may be targeted to two loca-

tions using two signals in different parts of the protein

sequence, GFP fusion to one part of the protein can mask

an adjacent signal – resulting in localisation to only one of

its in vivo destinations. Secondly, targeting ability can be af-

fected by the nature of the passenger protein. This occurs even

for proteins targeted to a single location [9,10], but it seems to

be even more pronounced for dual targeted proteins. In two

independent studies examining the role of the mature protein

for dual targeted proteins to mitochondria and chloroplasts,

both concluded that the passenger or mature protein influ-

enced dual targeting ability [11,12].

Type II NAD(P)H dehydrogenases are typically located on

the mitochondrial inner membrane where they can oxidise

NAD(P)H and are insensitive to the complex I inhibitor rote-

none [13–15]. Seven genes encode putative type II NAD(P)H

dehydrogenases in Arabidopsis, three have been defined as

external (NDB 1, 2 and 4) and three defined as internal

NAD(P)H dehydrogenases (NDA 1 and 2 and NDC1)

[13,14]. The remaining gene encoding a putative external

NAD(P)H dehydrogenase, NDB3, could not be cloned by a

number of groups and thus is either a pseudogene or its expres-

sion is very restricted [13,16]. Previous studies using GFP tag-

ging have shown NDA1, NDA2, NDB1, NDB2 and NDC1 to

be targeted to mitochondria [14], in vitro mitochondrial uptake

assays have shown NDA1, NDA2, NDB1, NDB2, NDB4 and

NDC1 to be imported into mitochondria [13], and a number of

studies using Western blot analysis of mitochondrial proteins

and/or cellular fraction with antibodies raised against peptides

from potato NDA1 and NDB1 have all concluded a mitochon-

drial localisation for these proteins [17–19]. Additionally over

two decades of biochemical analysis have shown that the

blished by Elsevier B.V. All rights reserved.

3074 C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079

activities associated with these proteins are located in mito-

chondria [15,20]. Thus it can be concluded that these proteins

are located in mitochondria.

However the set of proteins predicted to be located in per-

oxisomes by the AraPerox database, with medium to high con-

fidence, identifies three of these mitochondrial type II

NAD(P)H dehydrogenases [21]. Thus we re-assessed the tar-

geting ability of all six NAD(P)H dehydrogenases with the

view that they may also be located in other cellular organelles

in addition to mitochondria.

2. Materials and methods

2.1. Sequence analysis and cloningThe full length coding sequences of NDA1, NDA2, NDB1, NDB2,

NDB4 and NDC1 were cloned as both N- and C-terminal GFP fusionsby Gateway cloning under the control of the 35S CaMV promoter.Additionally the last 10 amino acids of NDA1, NDA2, NDB1 andNDB2 were cloned to the C-terminus of GFP. The alternative oxidase(AOX) targeting signal, the full length targeting sequence of small sub-unit of 1,5 ribulose bisphosphate carboxylase/oxygenase (SSU Rubi-sco) and the peroxisomal targeting signal SRL of pumpkin malatesynthase, were fused to red fluorescent protein (RFP) and used asmitochondrial, chloroplast and peroxisomal controls, respectively[22–24].

The constructs were used to transform Arabidopsis suspension cul-ture cells, Arabidopsis seedlings (1–2 weeks old) and onion epidermalcells by biolistic transformation as previously outlined [25]. Fluores-

Physcomitrella NDB1 RPhyscomitrella NDB2 SPhyscomitrella NDB3 S

Arabidopsis NDB1 SRI Potato NDB1 SRI

Grape NDB1 SRIRice NDB1 SRIArabidopsis NDB2 SSI

Grape NDB2 SRIGrape NDB3 SRI

Arabidopsis NDB4 SSIRice NDB2 SSL

Rice NDB3 LCS

Fig. 1. ClustalW alignment of type II NAD(P)H dehydrogenases from a vaNAD(P)H dehydrogenases from a variety of plants revealed that several coterminal end of the protein. The predicted strength of the PTSI signal wamitochondria and/or chloroplasts is shown. Arabidopsis thaliana NDAt2g2990;NP_180560, NDB1 At4g28220;NP_567801, NDB2 At4g05020;NCAB52796, NDB1 CAB52797, Populus trichocarpa NDA1 ABK95883, VitNDB1 CAO41235, NDB2 CAO16606, NDB3 CAO41237, Saccharomyces cersativa NDC1 Os06g11140:BAD35311, NDA1 Os01g61410:NP_915326.1, NDOs05g26660:AAV43826, NDB3 Os08g04630:XP_480031.1, ChlamydomonXP_001702271, NDB1 XP_001703643, Physcomitrella patens NDC1 manNDA1 manually annotated from scaffold 28 of Physcomitrella genome [39],NDB3 XP_001764062. Targeting prediction for all proteins are shown in Su

cence patterns were obtained 24 h after transformation by visualizationunder an Olympus BX61 fluorescence microscope, with excitationwavelengths of 460–480(GFP) and 535–555(RFP). Emissions were col-lected for GFP between 495 and 540 and RFP between 570 and 625,and imaged using the CellR imaging software. To ensure no cross overin detection of signals AOX-RFP and SSU-GFP were co-transformedto ensure that the filters were detecting the appropriate signal.

2.2. Determination of subcellular targeting abilityN- and C-terminal GFP-tagged proteins were used to transform

Arabidopsis cell suspension culture, 1–2 week old Arabidopsis seed-lings and onion epidermal cells by biolistic transformation as previ-ously outlined [25]. For each construct to be tested threetransformations were carried out, the test construct with a mitochon-drial, plastidic and peroxisomal control. In vitro import assays intoisolated Arabidopsis mitochondria and pea chloroplasts were carriedout as previously outlined [23,25].

2.3. Antibody production and Western blottingAntibodies were raised in rabbit against NDA1, amino acids 57–236

and the NDB2 specific peptide at amino acids 438–452(ETDDVSKNNIELKIE). The specificity of the antibodies was testedagainst recombinant proteins synthesised in a wheat germ translationlysate according to manufacturers instructions (Roche, Sydney), pro-grammed to synthesise NDA1, NDA2 and NDB2 by making lineartemplates by PCR as per manufactures instructions (Roche, Sydney).

Mitochondria and peroxisomes were purified from 7 day old cell sus-pension culture using free flow electrophoresis as described by Eubel etal. [26]. Western blot analysis was carried out against 20 lg of mito-chondrial and peroxisomal proteins separated by SDS–PAGE aspreviously outlined [27].

Grape NDC1Rice NDC1

Physcomitrella NDC1Chlamydomonas NDC1

Arabidopsis NDA1 SRIArabidopsis NDA2 SRI

Poplar NDA1 SRIGrape NDA1 SRIPotato NDA1 SRIRice NDA1 SRI

Grape NDA2 RIGRice NDA2 RIG

Physcomitrella NDA1 SRFPhyscomitrella NDA2 SRF

Chlamydomonas NDA1 SRWChlamydomonas NDA2 SLF

Yeast NDI KGLYeast NDE1 SSIYeast NDE2 SSV

Chlamydomonas NDB1 SRVVERMRM

Arabidopsis NDC1

Strong PTS-1Moderate PTS-1Weak PTS-1MitochondrialPlastid

riety of plants and yeast. Alignment of the sequences encoding type IIntained putative peroxisomal type I targeting signals (PTSI) at the C-s taken from AraPerox [21]. The predicted ability to be targeted toC1 At5g08740;NP_568205, NDA1 At1g07180;NP_563783, NDA2P_180560, NDB4 At2g20800;NP_179673, Solanum tuberosum NDA1is vinifera NDA1 CAO21440, NDC1 CAO71655, NDA2 CAO67571,evisiae NDI NP_013586, NDE1 NP_013865, NDE2 NP_010198, OryzaA2 Os07g377730:NP911221.1, NDB1 Os06g47000:BAD45556, NDB2as reinhardtii NDC1 ABR53723, NDA1 XP_001698901, NDA2ually annotated from scaffold 101 of Physcomitrella genome [39],NDA2 XP_001769969, NDB1 XP_001766162, NDB2 XP_001759207,pplementary Table 1.

C. Carrie et al. / FEBS Letters 582 (2008) 3073–3079 3075

3. Results

A ClustalW alignment of all ND sequences available from

various plant species and yeast revealed the amino acid se-

quence SRI at the C-terminal end of Arabidopsis NDA1,

NDA2 and NDB1 (and NDB3), and in a variety of ND pro-

teins from other plants. Other PTS I type targeting signals,

most notably SRM or SSI, were also found in ND sequences

[28] (Fig. 1). The fact that these amino acids are not present

in all ND sequences suggests that this tripeptide is not required

for function, opening the possibility that it may play a role in

defining subcellular localisation via its peroxisomal targeting

activity [15]. Analysis of NDC1 sequences from Arabidopsis,

rice and Chlamydomonas reinhardtii predicted plastid-targeting

in all three species based on the N-terminal region (Supple-

mentary Table 1), even though these proteins display very

low levels of sequence identity in this region (data not shown).

Examination of the gene constructs used in a previous study

that indicated an exclusive mitochondrial localisation for these

proteins, revealed that only the N-terminal region was used in

the GFP fusions [14], amino acids 1–55 for NDA1, 1–60 for

NDA2, 1–59 for NDB1 and NDB2 and amino acids 1–83

for NDC1.

3.1. NDC1 is targeted to mitochondria and chloroplasts

The full-length cDNA for NDC1 was placed in front of GFP

and its subcellular localisation examined by particle bombard-

ment. As controls, the cells transformed with the NDC1-GFP

construct were co-transformed either with plastid targeted

RFP using the targeting signal of the small subunit of 1,5

Fig. 2. Subcellular targeting of NDC1 using GFP tagging (A) In vitro uptakframe with GFP and co-transformed into Arabidopsis cells with mitochonpanel). (B) In vitro uptake of NDC1 into isolated mitochondria and chmitochondria (lane 2) and chloroplasts (lane 3) under conditions that suppassessed by insensitivity to added protease. Both organelles processed themitochondria, uptake was sensitive to the addition of valinomycin (lanes 5 an7 and 8). (C) The specificity of import of protein into isolated organellesoxygenase (SSU) that was only imported into chloroplasts and alternative o

ribulose bisphosphate carboxylase/oxygenase (SSU Rubisco-

RFP) or the mitochondrial alternative oxidase targeting signal

(AOX-RFP). Targeting of NDC1-GFP to chloroplasts was

clearly observed in Arabidopsis suspension cells ( Fig. 2A),

the pattern was clearly not identical to AOX-RFP but resem-

bled that of SSU-RFP quite closely. This is in contrast to what

has been previously reported where a mitochondrial localisa-

tion was concluded when the first 83 amino acids of NDC1

was used [14]. However we routinely observed a weaker signal,

similar to the pattern obtained with AOX-RFP. Thus we

tested the targeting ability in a variety of tissues, namely Ara-

bidopsis seedlings and onion epidermal cells. Transformation

of these tissues resulted in the detection of two distinct signals,

a plastid signal evidenced by relatively large organelles, 2–

4 lM in diameter and few in number and smaller organelles,

1 or less lM in diameter typical of a mitochondrial pattern.

The mitochondrial targeting ability of NDC1 that we observed

in this study is consistent with previous results using GFP and

in vitro uptake assays [13,14].

To confirm that NDC1 could target to both chloroplasts and

mitochondria, in vitro uptake assays with isolated Arabidopsis

mitochondria and pea chloroplasts were carried out. Upon

incubation with isolated chloroplasts and mitochondria the

NDC1 precursor protein with a mol mass of 70 kDa was im-

ported into a protease resistant location and processed to a

mature size with a mol mass of 60 kDa (Fig. 2B, lanes 1–3).

Both organelles appeared to process the precursor protein to

the same mature protein, to confirm this import reactions into

mitochondria and chloroplasts were loaded into the same lane

to determine any small difference in mobility, none was

e assays (B and C). A) The full-length cDNA for NDC1 was fused indrial targeted RFP (top panel) or chloroplast targeted RFP (bottomloroplasts. Precursor proteins (lane 1) were incubated with isolatedort the uptake of proteins into the respective organelles. Uptake wasprecursor to a mature protein with the same mobility (lane 4). Ford 6). For chloroplasts uptake was inhibited by addition of CuCl2 (laneswas confirmed using the precursor of 1,5 bisphosphate carboxylase

xidase (AOX) that was only imported into mitochondria.


detected (Fig. 2B, lane 4). As the translation of the precursor

alone also produces a protein with a mol mass of 60 kDa,

likely due to translation initiation at an internal methionine,

such as amino acid 47 in NDC1. Translation initiation at

Fig. 3. Subcellular targeting of NDA1, NDA2, NDB1 and NDB2. GFP wastargeting assessed by particle bombardment of Arabidopsis suspension cellsmitochondrial targeted RFP or peroxisomal targeted RFP as controls. (A) SNDB2-GFP. (B) Subcelluar targeting pattern obtained with NDA1, NDA2amino acids used is shown for each construct.

internal methionine residues is frequently observed with in vi-

tro translation lysates [27]. Thus we confirmed that the prote-

ase resistance was due to import into the respective organelle.

Import into mitochondria was inhibited by the addition of

fused to the different proteins at the N- or C-terminal and subcellular, 1–2 weeks old Arabidopsis seedlings and onion epidermal cells withubcelluar targeting pattern obtained with AOX-GFP, KAT2-GFP andand NDB1 fused to GFP. The position of the GFP and the number of


valinomycin (Fig. 2B, lanes 5 and 6) [27], and import into chlo-

roplast inhibited by the addition of CuCl2 (Fig. 2B, lanes 7 and

8) [29]. The specificity of import into the respective organelles

was confirmed as the small subunit of 1,5 bisphosphate (SSU)

was only imported into chloroplasts and the alternative oxi-

dase precursor only imported into mitochondria (Fig. 2C, left

panel).

3.2. NDA1, NDA2 and NDB1 are targeted to mitochondria and

peroxisomes

In order to determine the localisation of the other ND pro-

teins, N- and C-terminal GFP fusions were made followed by

particle bombardment. To determine a mitochondrial and per-

oxisomal pattern chimeric constructs with the AOX and KAT2

linked to GFP were used (Fig. 3A, image series 1 and 2). In the

case of NDB2 attaching GFP to the C-terminal resulted in tar-

geting to mitochondria as evidenced by co-localisation with

AOX-RFP (Fig. 3A, images 3a–3c). Attaching the last 10 ami-

no acids of NDB2 to the C-terminal end of GFP resulted in a

cytosolic localisation for GFP, as evidenced by fluorescence

throughout the cell, in all tissues tested (Fig. 3A, image series

4). In contrast when NDA1, NDA2 and NDB1 were tested in a

similar manner both mitochondrial and peroxisomal targeting

ability was detected. C-terminal fusions gave an exclusively

Anti NDA1

Anti NDB2

Anti TIM17-2

Anti Kat2

Mit

A1 B2---A

Anti 6-His

Anti A1

Anti B2

60 kDa

62 kDa

C

Fig. 4. Western blot analysis of mitochondrial and peroxisomal fractions prantibodies. Wheat germ lysate (20 lg) programmed to synthesise each of themembrane and probed with antibodies raised against NDA1 and NDB2 totranslation lysate programmed to synthesis b-glucuronidase (GUS), NDA1 aflow electrophoresis were separated by SDS–PAGE, blotted to a nitrocelluloused is indicated to the right of the panel and the apparent mol mass of the cand NDA2 the precursor size of the protein is detected when probing in vitrowhen probing organelle fractions.

mitochondrial localisation, based on co-localisation with

AOX-RFP (Fig. 3B, images 1a–1c, 3a–3c and 5a–5c). This is

consistent with the mitochondrial targeting ability previously

observed with these proteins [14]. However when the last 10

amino acids of NDA1, NDA2 and NDB1 were placed at the

C-terminal region of GFP peroxisomal targeting was observed

(Fig. 3B, images 2d–2f, 4d–4f and 6d–6f). The peroxisomal tar-

geting ability of these constructs was also detected in Arabid-

opsis seedlings and onion cells (Fig. 3B, images 2g and 2h, 4g

and 4h and 6g and 6h). Thus we concluded that these proteins

were targeted to peroxisomes in addition to mitochondria.

NDB4 targeted GFP to mitochondria as previously reported

(Supplementary Fig. 1) [14].

To confirm the dual location of NDA1 in mitochondria and

peroxisomes we raised antibodies against NDA1, expected to

be located in both locations from results above, and NDB2,

expected to be located only in mitochondria from results

above. We confirmed that the NDA1 and NDB2 antibodies

did not cross react with the other antigen by over-expression

of the respective proteins in an in vitro translation lysate prob-

ing with Anti 6-His antibodies that detected both proteins,

Anti A1 antibodies that detected only NDA1 and Anti B2 anti-

bodies that detected only B2 (Fig. 4A). As the NDA1 antibody

was raised against a fragment of the NDA1 protein on 180

55 kDa

62 kDa

32 kDa

44 kDa

Per

Anti A1

B

Anti 6-His

GUS A1 A2

60 kDa

68 kDa

59 kDa

60 kDa

obed with various antibodies. (A) Confirmation of NDA1 and NDB2ND proteins was separated by SDS–PAGE, blotted to a nitrocelluloseconfirm that they detected their target antigens. (B) As A except thatnd NDA2. (C) 20 lg of mitochondria or peroxisomes purified by freese membrane and probed with antibodies as indicated. The antibodyross reacting protein indicated in the left in kDa. Note that for NDA1

synthesised protein whereas the mature size of the protein is detected


amino acids that displayed 83% sequence identity with the cor-

responding region of NDA2 we tested if the NDA1 antibody

cross reacted with NDA2. No cross reactivity was detected

with full length in vitro synthesised NDA2 (Fig. 4B).

Highly purified mitochondria and peroxisome fractions were

isolated from Arabidopsis cells [26] and proteins separated by

SDS–PAGE and subjected to Western blotting. In control

experiments, we used antibodies against proven markers of

mitochondria (TIM17-2 (Translocase of the Inner Mitochon-

drial membrane; [27]), and peroxisomes (KAT2 (3-ketoacyl-

CoA thiolase; [22,30]). These antibodies reacted strongly with

mitochondrial and peroxisomal fractions, respectively, and

much more weakly with the other fraction, indicating a small

degree of cross-contamination between the fractions (Fig.4B).

Densitometric analysis revealed the KAT2 signal in mitochon-

dria was �5% of that detected in peroxisomes, whilst the

TIM17-2 signal in peroxisomes was �1–2% of the signal that

could be detected in mitochondria (when the blot was overex-

posed).

Probing with antibodies raised against NDA1 resulted in the

detection of a single protein band with an apparent molecular

mass of 55 kDa, in both mitochondrial and peroxisome frac-

tions ( Fig. 4B). The blots indicated that there was more

NDA1 protein in the peroxisomal fraction than in the mito-

chondrial one, confirming that these proteins are found in both

compartments. Probing mitochondrial and peroxisomal frac-

tions with antibodies raised against the NDB2 specific peptide

produced a band only in the mitochondrial fraction (Fig. 4B),

confirming that it can target to mitochondria but not to per-

oxisomes. Importantly, this latter result also shows that the

very small amount of cross-contamination between the two

isolated fractions cannot explain the dual localisation of the

NDA1 signal. Thus the Western blot results confirm the

GFP data.

4. Discussion

In this study we have shown that four ND proteins, NDA1,

NDA2, NDB1 and NDC1 are dual targeted. The dual target-

ing ability of ND proteins was overlooked in previous GFP

studies due to a number of technical parameters, namely the

nature of the GFP-protein constructs used in each study. In

the case of NDC1, it appears that the mature protein sequence

is required for its dual localisation by GFP (Fig. 2), as ob-

served for other dual targeted proteins [12,31]. The dual target-

ing of NDA1, NDA2 and NDB1 to mitochondria and

peroxisomes is dictated by two distinct signals. In the case of

the NDAs, the apparent Mr of the mature protein observed

in peroxisomes and mitochondria was identical (Fig. 4C). As

NDA proteins are processed upon import into mitochondria

[13], this strongly suggests that they are also processed upon

import into peroxisomes. It has been shown previously that

peroxisomes recognise N-terminal PTS2 type targeting signals

that are removed upon import and the processing of the NDA

proteins could be carried out by the same peptidase as both

NDA1 and NDA2 have a cysteine residue at amino acids 35

and 38, respectively, which defines the processing site by this

peptidase [32]. Alternatively, the NDA proteins may be pro-

cessed by pitrilysin-like metallopeptidase present in peroxi-

somes [32]. These enzymes belong to the same family of

proteases as the mitochondrial processing peptidase [33].

The mitochondrial pattern obtained with GFP with NDA1,

NDA2 and NDB1 differed slightly to that obtained from

AOX-RFP. Close examination of the merged images revealed

that the GFP fluorescence appeared at the periphery of the

mitochondrion, thus the GFP and RFP fluorescence co-local-

ise, but are not identical. A similar pattern of GFP fluores-

cence is routinely obtained when using outer membrane

mitochondrial proteins in humans and Arabidopsis [34,35].

This pattern may be due to the fact that GFP attached to

the C-terminal of an inner membrane protein will not be

�pulled� into mitochondria. The C-terminal of the ND proteins

may be located in the intermembrane space and thus never en-

ter the mitochondrial matrix. Thus the GFP attached to the C-

terminal end of these proteins remains outside the mitochon-

drion. Using only the N-terminal predicted targeting region re-

sults in a typical mitochondrial pattern as previously observed

[14], as the default targeting information for mitochondria dic-

tated a matrix location [36]. Secondary signals dictate the in-

tra-organelle location and topology of proteins, such as

transmembrane regions and the location of positive residues

relative to transmembrane regions [36].

The cellular role of various ND proteins now needs to be

re-evaluated in light of their dual localisation. For instance

NDC1 gene expression is enhanced by light treatments [16]

but the protein is also known to be halved in abundance in

plastoglobules during high light treatment [37]. So what im-

pact does this transcriptional light response have on the mito-

chondrial pool of NDC1 protein? Likewise, Western blot

analysis with potato mitochondria revealed changes in

NDA protein in a diurnal manner [18], and it is now unclear

how much of this may be attributed to a mitochondrial func-

tion as opposed to a peroxisomal function, or differential

contamination of mitochondria with peroxisomes. Further,

loss of or over-expression of potato NDB1 has been shown

to alter NADPH/NADH ratio in cells [38], but this may be

related to its activity in peroxisomes rather than mitochon-

dria.

Seven genes encode alternative ND proteins in Arabidopsis,

two NDA like proteins, four NDB type proteins and a single

NDC type protein, the latter proposed to be derived from

the cyanobacterial ancestor that gave rise to the plastid endo-

symbiosis [14]. It is tempting to speculate from the prediction

of targeting ability of these proteins from a variety of plants

( Fig. 1, Supplementary Table 1) that genes encoding single

NDA and NDB type proteins underwent duplication followed

by acquisition of additional targeting signals by some proteins.

In the case of NDC it may have acquired dual targeting ability

upon transfer of the gene from the organelle to the nucleus, or

alternatively a location specific signal subsequently acquired

dual targeting ability over time.

Acknowledgements: This work was supported by an AustralianResearch Council Grant DP0664692, ARC Australian PostdoctoralFellowships to M.W.M. and H.E., and an ARC AustralianProfessorial Fellowship to A.H.M.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.febslet.2008.

07.061.

http://dx.doi.org/10.1016/j.febslet.2008.07.061

http://dx.doi.org/10.1016/j.febslet.2008.07.061


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Chapter 5 Plant mitochondrial protein import receptors

62

Chapter 5

Plant mitochondrial protein import receptors

Chapter 5 Plant mitochondrial protein import receptors

63

Foreword to Study IV Studies I, II and III identified new dual targeted proteins in Arabidopsis. Study

IV, therefore aimed to identify how these dual targeted proteins are imported into

Arabidopsis mitochondria, by investigating the mitochondrial outer membrane protein

import receptors. While this study contains work on all the putative import receptors

(Tom20, Metaxin and OM64), only the work regarding OM64 is being presented for

this thesis, the work carried out on Tom20 and Metaxin was not part of this thesis (see

declaration form).

Using genome searches and biochemical purification of the plant TOM complex

there has been no evidence found for a Tom70 orthologue in plants (Jansch et al., 1998;

Chan et al., 2006). However, it was discovered that Arabidopsis mitochondria contain a

protein anchored to the outer membrane with a large cytosolic domain termed OM64

(Chew et al., 2004). Interestingly, it was seen that OM64 contains three TPR motifs and

is related to the Toc64 protein from plastids (Chew et al., 2004). Toc64 is an outer

envelope protein of plastids and is thought to be involved in protein import into plastids

(Qbadou et al., 2006). To date, no functional role for OM64 has been demonstrated,

however it has been proposed that OM64 may play a similar role to Tom70 in yeast

(Chew et al., 2004). Thus OM64, was of interest as it is related to an import component

of plastids and is found on the outer mitochondrial membrane. Study IV aimed to test

the hypothesis that OM64 is involved in the import of dual targeted proteins into

mitochondria. However, no specific role for OM64 in the import of dual targeted

proteins was found. Insertional inactivation, in vitro competition experiments with

overexpressed OM64, antibody inhibition assays, and direct interaction assays all

suggested a role for OM64 in the import of proteins into mitochondria.

Functional Definition of Outer Membrane Proteins Involved inPreprotein Import into Mitochondria W

Ryan Lister,1 Chris Carrie,2 Owen Duncan,2 Lois H.M. Ho, Katharine A. Howell,Monika W. Murcha, and James Whelan3

Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009, Western

Australia, Australia

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of

the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three

outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochon-

drial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis

TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous

inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited

isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some

but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of

mitochondrial precursor proteins and likely plays a role in the assembly of b-barrel proteins into the outer membrane. An

outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor

was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and

interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays.

Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy.

Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of

mitochondrial protein import.

INTRODUCTION

The mitochondrial protein import machinery has been most com-

prehensively characterized in yeast (Saccharomyces cerevisiae),

Neurospora crassa, and to a lesser extent in mammalian sys-

tems. Hetero-oligomeric translocation complexes in the outer

and inner membranes mediate the recognition, import, and sub-

organellar sorting of mitochondrial precursor proteins (Neupert,

1997; Pfanner and Geissler, 2001; Hoogenraad et al., 2002;

Truscott et al., 2003; Wiedemann et al., 2004). The translocase of

the outer mitochondrial membrane (TOM) complex facilitates the

recognition of precursor proteins and their translocation through

the outer membrane (Taylor and Pfanner, 2004). In yeast, the

outer membrane receptors TOM20, TOM22, and TOM70 asso-

ciate with the general import pore that consists of the pore-

forming TOM40, and TOM7, TOM6, and TOM5. TOM20 and

TOM70 are N-terminal anchored primary receptor proteins that

recognize precursor proteins with N-terminal and internal tar-

geting information, respectively (Wiedemann et al., 2004). Pre-

cursor proteins subsequently interact with the TOM22 receptor,

which delivers them to the general import pore (Taylor and

Pfanner, 2004). Operating in concert with the TOM complex, the

sorting and assembly machinery (SAM) complex (also called

topogenesis of mitochondria outer membrane b-barrel proteins

[TOB]) in the outer mitochondrial membrane inserts proteins into

the outer membrane (Pfanner et al., 2004; Taylor and Pfanner,

2004; Habib et al., 2005; Paschen et al., 2005). Although initially

thought to be a protein import receptor of the TOM complex

(Gratzer et al., 1995), SAM37 (also called MAS37/TOM37) was

subsequently reported to be located in a distinct outer mem-

brane complex, SAM, and was demonstrated to function in the

sorting and assembly of b-barrel proteins (Wiedemann et al.,

2003). The SAM complex, consisting of SAM50 (TOB55), SAM35

(TOB38), SAM37, and MDM10 (TOM13), in yeast and N. crassa,

is required for the correct assembly of complex b-barrel proteins

into the outer membrane after import through the TOM complex,

but the precise molecular functions of SAM35 and SAM37 have

not yet been elucidated (Wiedemann et al., 2003; Paschen et al.,

2005; Neupert and Herrmann 2007). Proteins destined for the

inner membrane and matrix interact with two discrete TIM

(translocase of the inner mitochondrial membrane) complexes

(Wiedemann et al., 2004).

Experimental isolation of mitochondrial proteins coupled with

genome sequence analysis in animals and plants has revealed that

much of the import apparatus is conserved throughout diverse

eukaryotic lineages (Herrmann, 2003; Dyall et al., 2004; Lister

et al., 2005; Dolezal et al., 2006). However, considerable dif-

ferences exist in the TOM complex subunit composition between

1 Current address: Plant Biology Laboratory, Salk Institute for BiologicalStudies, La Jolla, CA 92037.2 These authors contributed equally to this work.3 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: James Whelan([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.107.050534

The Plant Cell, Vol. 19: 3739–3759, November 2007, www.plantcell.org ª 2007 American Society of Plant Biologists

species; only the core translocase module of TOM40, TOM22, and

TOM7 is conserved (Dolezal et al., 2006), and the plant TOM22 has

lost the N-terminal receptor domain present in yeast and animals

(Macasev et al., 2004). Isolation of the plant TOM complex also

identified small proteins analogous to yeast TOM5 and TOM6 and

a 23-kD protein (plant TOM20) analogous to yeast/animal TOM20,

but none displayed significant protein sequence similarity to the

yeast or mammalian counterparts (Heins and Schmitz, 1996;

Jansch et al., 1998; Werhahn et al., 2003). Subsequent elucidation

of the solution structure of the plant TOM20 showed it to have a

similar tertiary structure but reverse domain arrangement to yeast/

animal TOM20 (Perry et al., 2006). Given that there is no signif-

icant sequence similarity between plant TOM20 and that of yeast

or mammals, it is proposed that convergent evolution has lead to

a receptor with the same function but reversed orientation (Lister

and Whelan, 2006; Perry et al., 2006). As plant TOM20 is not

orthologous to yeast or mammalian TOM20, it is of interest to

determine if it plays the same role in the TOM complex.

No clear plant homolog of the yeast and animal TOM70 re-

ceptor can be identified in the genomes of Arabidopsis thaliana

or rice (Oryza sativa; Chan et al., 2006), in agreement with the lack

of any biochemical evidence for this component in the purified

plant TOM complex (Jansch et al., 1998). Interestingly, anchored

in the outer membrane of Arabidopsis mitochondria is mtOM64, a

paralog of the chloroplast outer envelope protein import receptor

At TOC64-III (translocase of the outer chloroplast envelope)

(Chew et al., 2004; Qbadou et al., 2006). Notably, inactivation of

both plastid-localized TOC64 orthologs in Physcomitrella patens

and inactivation of TOC64 in Arabidopsis had no effect on protein

import into plastids (Hofmann and Theg, 2005; Aronsson et al.,

2007). To date, no functional role has been demonstrated for

mtOM64 in Arabidopsis. Plants also display differences in the

SAM complex in comparison to yeast; in plants, only SAM50 can

be clearly identified by sequence similarity (Lister et al., 2005).

Arabidopsis METAXIN was identified by sequence similarity to the

human METAXIN 1 protein; the latter displays limited sequence

similarity to yeast SAM37 and has been implicated in mitochon-

drial protein import (Armstrong et al., 1997; Abdul et al., 2000).

METAXIN1 and METAXIN2 in mammals have been reported to

be involved in the import of b-barrel proteins but in a different

complex compared with SAM50 (Kozjak-Pavlovic et al., 2007).

METAXIN protein has been shown to be present in Arabidopsis

mitochondria (Lister et al., 2004), but no role in mitochondrial

protein import or sorting has been demonstrated.

Another notable difference between yeast and higher eukary-

otes, such as plants and mammals, is that the yeast subunits of

the TOM, TIM, and SAM complexes are encoded by single

nuclear genes, while in higher organisms, components of TOM,

TIM, and SAM are often encoded by small multigene families. It is

not clear what the function of these multigene families are,

whether they represent functionally distinct isoforms or provide

greater ability for regulation of these components at a transcrip-

tional level (i.e., subfunctionalization verses neofunctionaliza-

tion). In Drosophila melanogaster, it has been documented that

TOM20 and TOM40 are each encoded by two differentially

expressed genes (Hwa et al., 2004). Detailed expression analysis

of almost all genes encoding mitochondrial protein import com-

ponents in Arabidopsis indicates differential expression patterns

of genes within each family (Lister et al., 2004). Furthermore,

functional analysis of the Arabidopsis TIM17 gene family sug-

gests some differences in function based on the ability to

complement a tim17 mutant in yeast (Murcha et al., 2003).

Although yeast is an excellent model to provide the basic

mechanism of how proteins are imported into mitochondria, it

cannot give insights into the functions of nonhomologous com-

ponents in other organisms, such as plants, in which the putative

receptor components are not orthologous to yeast receptor

subunits. Furthermore, the yeast model system cannot be used

to explore any functional diversification of these multiple genes

encoding import components. Here, we investigate the function

of three outer mitochondrial membrane proteins in Arabidopsis

with respect to protein import into mitochondria. We demon-

strate that the three highly expressed TOM20 isoforms in

Arabidopsis are predominantly functionally equivalent but dis-

play limited specialization and together are important, but not

essential, for the import of a wide range of mitochondrial pro-

teins. Furthermore, we demonstrate that mtOM64 is a mitochon-

drial protein that can interact with a variety of precursor proteins,

increases in abundance when two or more TOM20 isoforms are

inactivated, and plays a role in the import for at least some

mitochondrial proteins. Finally, we identify that METAXIN inter-

acts with a wide variety of precursor proteins and is involved in

their import, and results suggest that it plays more than one role

in the import and assembly of proteins into mitochondria.

RESULTS

The TOM20 Gene Family Encodes Functionally Redundant

Proteins Involved in Mitochondrial Protein Import

TOM20 is encoded by four paralogous genes in the Arabidopsis

genome, TOM20-1 to TOM20-4 (Werhahn et al., 2001). TOM20-1

and TOM20-3 are tandemly duplicated genes, the predicted

proteins of which display 60% amino acid identity. TOM20-2,

TOM20-3, and TOM20-4 are highly expressed in diverse plant

organs (Lister et al., 2004), whereas by contrast, TOM20-1 tran-

script is rarely detectable (see Supplemental Figure 1A online)

(Lister et al., 2004; Murcha et al., 2007), and TOM20-1 is the only

TOM20 protein that has not been directly identified in isolated

plant mitochondria (Werhahn et al., 2001; Heazlewood et al.,

2004; Lister et al., 2004). Antibodies raised against TOM20-3

cross-react with in vitro–translated TOM20-1 (see Supplemental

Figure 1B online), but TOM20-1 could not be detected with this

antibody in mitochondria isolated from whole seedlings of any of

the Arabidopsis genotypes used in this study. Mitochondria were

isolated from wild-type Arabidopsis (ecotype Columbia-0) and

two independent T-DNA insertion lines of each highly expressed

TOM20 gene (Sessions et al., 2002; Alonso et al., 2003; Rosso

et al., 2003), and the absence of transcript derived from the

specific TOM20 isoforms was verified by RT-PCR (data not

shown) and immunodetection of total mitochondrial protein sam-

ples with specific antibodies raised to each of these TOM20

isoforms (Figure 1A). No severe phenotypic abnormalities were

observed in any of the single insertion mutants; however, tom20-2

had a slightly delayed flowering time, 4 to 7 d later than wild-type

plants (Figure 2A).

3740 The Plant Cell

In vitro import of radiolabeled plant mitochondrial precursor

proteins into mitochondria isolated from wild-type and tom20

plants was performed. While the selection of precursor proteins

represented a wide range of mitochondrial protein import path-

ways (general import pathway, alternative oxidase [AOX] and the

10-kD protein of the small mitochondrial ribosomal subunit

[RPS10]; carrier import pathway, mitochondrial phosphate car-

rier [PiC]; dual-targeted proteins, glutathione reductase [GR];

plant specific protein, FAd-subunit of mitochondrial ATP syn-

thase) (Murcha et al., 1999; Heazlewood et al., 2003), no signif-

icant differences in the rate of protein import were observed

between wild-type and tom20 mitochondria for any precursor

protein (see Supplemental Figure 2 online). Therefore, none of

the Tom20 proteins alone appeared to have an essential function

in mitochondrial protein import.

Different TOM20 Isoforms Display Some Precursor

Recognition Specificity

The single TOM20 isoform mutant plants were crossed to gen-

erate double TOM20 knockout plants deficient in each combi-

nation of the three highly expressed TOM20 isoforms. Notably,

the resulting double knockout plants were viable and did not

display severe phenotypic abnormalities, although again slight

delays in flowering time were noticeable (Figure 2A). Mitochon-

dria were isolated from wild-type and double tom20 knockout

plants and the protein complexes solubilized by digitonin treat-

ment and separated by one-dimensional blue native PAGE

(BN-PAGE), followed by immunodetection of the remaining

TOM20 protein (Figure 1B). TOM20-3 in both wild-type mitochon-

dria and the tom20-2 tom20-4 mitochondria was detected in a

protein complex of a similar apparent molecular mass, indicating

that the loss of two TOM20 isoforms did not disrupt the integrity or

structure of the TOM complex. This was also observed for

TOM20-4 in wild-type and tom20-2 tom20-3 mitochondria. Re-

sidual Coomassie blue staining of the mitochondrial inner mem-

brane respiratory complexes I, III, IV, and V enabled estimation of

the TOM complex molecular mass at ;230 kD, based on a

comparison with a previous study of potato (Solanum tuberosum)

mitochondrial membrane protein complexes (Jansch et al., 1998).

Interestingly, a second complex containing TOM20-2 was always

detected in wild-type mitochondria (Figure 1B, open arrowhead),

indicating that some TOM20-2 is located in a higher molecular

weight complex. Furthermore, in mitochondria isolated from

tom20-3 tom20-4 plants, a larger proportion of TOM20-2 was

present at a higher molecular weight, abolishing the two discrete

complexes and forming a continuous distribution of larger com-

plexes. Thus, when mitochondria only contain TOM20-2 it ap-

pears to lead to an ectopic size distribution of TOM complexes.

The rate of protein import into mitochondria with only one

functional TOM20 protein was measured in vitro for a range of

plant mitochondrial precursor proteins (Figure 2B). The import of

TOM40 and PiC was unaffected; however, for the other precur-

sor proteins, significant differences in the rate of protein import in

various mutants were observed (P < 0.05). AOX import into

tom20-2 tom20-3 and tom20-2 tom20-4 mitochondria was re-

duced to 71 and 64%, respectively, compared with AOX import

into wild-type mitochondria. FAd was imported into tom20-2

tom20-4 mitochondria at 85% of the wild-type rate of import.

Finally, the import of the dual-targeted protein GR into tom20-2

tom20-3 mitochondria was 31% higher after 10 min and 71%

higher after 20 min, at which the rate was still linear. Therefore,

generation of mutant plants with only one functional TOM20

protein revealed that a functional TOM complex can be formed

with a single TOM20 and that different TOM20 isoforms display

some precursor recognition specificity that is evident when

import of a variety of precursor proteins is assessed.

Tom20 Is a Nonessential Protein

Plants were generated that lacked all the three highly expressed

TOM20 isoforms, tom20-2 tom20-3 tom20-4 (tom20 triple knockout).

Figure 1. The TOM Complex Can Form with Only One TOM20 Isoform.

(A) Immunodetection of Tom20 isoforms in mitochondrial protein sam-

ples isolated from wild-type and tom20 T-DNA insertional mutant plants

and separated by SDS-PAGE.

(B) Immunodetection of Tom20 proteins in mitochondrial protein sam-

ples isolated from wild-type and tom20 double knockout plants after

digitonin solubilization and separation of protein complexes by first

dimension BN-PAGE. The open arrowhead indicates the higher molec-

ular weight complex containing TOM20-2. The position and approximate

molecular mass of the inner membrane respiratory complexes I, III, IV,

and V are indicated, as detected by the residual Coomassie blue staining

and reported in a previous study of potato mitochondrial protein

complexes (Jansch et al., 1998).

Mitochondrial Preprotein Import 3741

Figure 2. Arabidopsis TOM20 Is Encoded by a Multiple Gene Family of Predominantly Functionally Equivalent Isoforms That Plays a Role in the Import

of Preproteins into Mitochondria.

3742 The Plant Cell

These plants were viable but displayed a slightly slower growth

rate (Figure 2A). In vitro import of radiolabeled precursor proteins

into mitochondria isolated from tom20 triple knockout plants

indicated that TOM20 is involved in the import of a diverse range

of mitochondrial precursor proteins (Figure 2C). Import of AOX,

TOM40, and PiC was reduced to only 20 to 30% of the amount of

import into wild-type mitochondria (P < 0.01), indicating that

TOM20 is involved in the general, carrier, and outer membrane

b-barrel protein import pathways. However, the import of GR

was unaffected, and import of FAd did not decrease significantly

(P > 0.05), indicating that TOM20 has little or no involvement in

the import of these precursor proteins or that its absence can be

compensated for completely by another import component.

mtOM64 Is Involved in Mitochondrial Protein Import

mtOM64 is a protein anchored in the mitochondrial outer mem-

brane that has 67% protein sequence identity to At TOC64-III, a

chloroplast outer envelope protein import receptor (Chew et al.,

2004; Qbadou et al., 2006). Sequence analysis indicated that

mtOM64 has three C-terminal tetratricopeptide repeat (TPR)

motifs, resembling the C-terminal TPR domains of At TOC64-III

and TOM70 that function to recognize chloroplast and mito-

chondrial proteins bound to HSP90 (Young et al., 2003; Qbadou

et al., 2006). To investigate the role of mtOM64, two Arabidopsis

lines with independent T-DNA insertions in the coding region of

mtOM64 were obtained and a specific antibody was raised to

verify the absence of mtOM64 protein in the knockout plants

(Figure 3A). BN-PAGE analysis revealed that mtOM64 did not

migrate with a complex, but rather it was consistently detected at

the bottom of the gel, indicating that it did not form part of a larger

complex under the conditions tested (data not shown). In vitro

protein import reactions were conducted with a wide range of

mitochondrial precursor proteins into mitochondria isolated from

the two mtom64 plant lines (Figure 3B). In comparison with wild-

type mitochondria, no difference in the rate of protein import was

observed for AOX, PiC, GR, and TOM40. However, the import of

FAd was consistently 30 to 40% lower in plants lacking mtOM64

(P < 0.05), indicating that it is involved in the import of this plant-

specific protein. This is in agreement with a previous study that

suggested that FAd does not solely rely on TOM20 for import

(Dessi et al., 1996; Murcha et al., 1999).

To investigate the interaction of mtOM64 with mitochondrial

precursor proteins, import competition experiments were per-

formed with mtOM64 competitor protein that was synthesized in

an in vitro wheat germ lysate transcription/translation system.

First, radiolabeled precursor proteins were preincubated with

expressed competitor proteins in a wheat germ lysate mix (wheat

germ lysate resuspended in wheat germ reconstitution buffer),

either mtOM64 or an equivalent volume, and a quantity of

b-glucuronidase (GUS). Following preincubation, the precursor/

competitor protein mix was added to wild-type mitochondria

under conditions that support import, and the amount of pre-

cursor protein imported after 10 min was quantitated (Figure 3C).

Preincubation with the wheat germ reconstitution buffer alone

decreased protein import of AOX and FAd by 69 and 53%,

respectively (see Supplemental Figure 3 online). Preincubation

with wheat germ lysate mix inhibited AOX import by 65%,

indicating that the wheat germ reconstitution buffer, and not the

wheat germ lysate, was responsible for the decrease in AOX im-

port. By contrast, preincubation with the wheat germ lysate mix

did not inhibit FAd import, indicating that a factor present in the

wheat germ lysate stimulates FAd import, thus abrogating the in-

hibitory effect of the wheat germ reconstitution buffer. To control

for the effects of wheat germ lysate, wheat germ reconstitution

buffer, and protein overexpression, an identical volume of wheat

germ lysate mix in which GUS was overexpressed was used as a

control competitor protein; thus, the specific effect of mtOM64

on the import of a variety of proteins could be assessed. Prein-

cubation of AOX or TIM23 with the mtOM64 competitor protein

did not result in a significant change in the amount of precursor

protein import relative to the GUS control. However, preincuba-

tion of FAd with mtOM64 resulted in a significant reduction (P <

5.0 3 10�4) of FAd import into mitochondria of 32% compared

with GUS (Figure 3C), in close agreement with the decrease in the

amount of import into mtom64 mitochondria (Figure 3B). There-

fore, exogenous mtOM64 protein was able to specifically com-

pete with the FAd precursor protein but not other precursors

tested. Preincubation of FAd with the chloroplast import receptor

At TOC64-III did not affect FAd import, in contrast with the 32%

reduction after preincubation with mtOM64 (Figure 3D), again

indicating a specific effect of mtOM64. The ability of mtOM64 to

compete with the FAd precursor could result either from a direct

interaction with this precursor or interaction with a common compo-

nent on the outer membrane that enables import into mitochondria.

METAXIN Is an Outer Mitochondrial Membrane Protein

Essential for Normal Cellular Development, Starch

Metabolism, and Plant Growth

Although the tom20 triple and mtom64 knockout plants ap-

peared to interact with a range of precursor proteins, the lack of a

Figure 2. (continued).

(A) Five-week-old Arabidopsis plants with different genotypes indicated. Plants lacking Tom20-2 display a delayed-growth phenotype.

(B) [35S]-labeled precursor proteins AOX, FAd, TOM40, GR, and PiC were incubated with mitochondria isolated from wild-type and mutant plants

deficient in two of the TOM20 isoforms under conditions that support import. Aliquots were removed at 2, 5, 10, and 20 min and treated with PK. PK-

protected mature radiolabeled protein was quantitated at each time point and normalized to the highest time point for replicate experiments (n $ 3 6

SE). Where indicated, mutant mitochondria had a significantly lower amount of protein import than wild-type mitochondria, with a P value < 0.05 (*) and

0.01 (#) using Student’s t test. m, mature protein; p, precursor protein.

(C) [35S]-labeled precursor proteins AOX, FAd, TOM40, GR,x and PiC were incubated with mitochondria isolated from wild-type and tom20-2 tom20-3

tom20-4 plants (triple KO). The import time course and analysis were performed as above. Where indicated, mutant mitochondria had a significantly

lower amount of protein import than wild-type mitochondria, with a P value < 0.01 (#) and 0.005 (circles) using Student’s t test.


striking phenotype, as observed previously when plastid import

receptors were inactivated (Soll and Schleiff, 2004; Bedard and

Jarvis, 2005; Kessler and Schnell, 2006), suggested that addi-

tional components may also be present. Experiments were

conducted to identify additional mitochondrial outer membrane

proteins involved in protein import. As human METAXIN1 has

previously been implicated in mitochondrial protein import

(Armstrong et al., 1997), the role of Arabidopsis METAXIN was

investigated. Arabidopsis lines with independent T-DNA inser-

tions in the METAXIN coding sequence were obtained and the

Figure 3. mtOM64 Is a Mitochondrial Preprotein Import Component That Is Involved in the Import of FAd into Mitochondria.

(A) Immunodetection of mtOM64 in mitochondrial protein samples isolated from wild-type and mtom64 T-DNA insertional mutant plants and separated

by SDS-PAGE.

(B) [35S]-labeled precursor proteins AOX, FAd, Tom40, GR, and PiC were incubated with mitochondria isolated from wild-type and mtom64 plants under

conditions that support protein import. Aliquots were removed at 2, 5, 10, and 20 min and treated with PK. PK-protected mature radiolabeled protein was

quantitated at each time point and normalized to the highest time point for replicate experiments (n $ 3 6 SE). Where indicated, mutant mitochondria had a

significantly lower amount of protein import than wild-type mitochondria, with a P value < 0.05 (*), 0.02 (#), and 0.01 (circles) using Student’s t test.

(C) [35S]-labeled precursor proteins AOX, FAd, and TIM23 were preincubated with overexpressed GUS or mtOM64 competitor protein prior to

incubation with mitochondria from wild-type plants. PK-protected mature radiolabeled protein was quantitated and normalized against the amount of

protein imported after preincubation with GUS (n ¼ 3 6 SE). Where indicated, the mtOM64 competitor protein decreased protein import significantly

compared with GUS, with a P value < 5.0 3 10�4 (*) using Student’s t test.

(D) [35S]-labeled FAd precursor protein was preincubated with overexpressed GUS, At TOC64-III, or mtOM64 competitor proteins prior to incubation

with mitochondria from wild-type plants. PK-protected mature radiolabeled FAd was quantitated and normalized against the amount of protein imported

after preincubation with GUS. Lane 1, AOX precursor only; lane 2, AOX precursor incubated with GUS; lane 3, AOX precursor incubated with At

TOC64-III; lane 4, AOX precursor incubated with mtOM64. Where indicated, the competitor protein decreased protein import significantly compared

with GUS, with a P value < 0.01 (*).

3744 The Plant Cell

absence of METAXIN transcript confirmed (see Supplemental

Figure 4 online). metaxin plants displayed severe phenotypic

abnormalities following leaf emergence, including diminished

growth, abnormal leaf morphology and ectopic floral develop-

ment and sterility (Figures 4A and 4B). Iodine staining of plants at

the end of both the light and dark photoperiods revealed that

metaxin plants accumulated higher levels of starch (Figure 4C).

Light microscopy of leaf cross sections indicated that the me-

sophyll cells of the metaxin plants contained more chloroplasts

compared with the wild type (Figures 4D and 4E). Transmission

electron microscopy of leaf mesophyll cells revealed large starch

deposits within the chloroplasts of metaxin cells, which was not

observed in wild-type cells (Figures 4F and 4G). Genetic trans-

formation of the METAXIN cDNA sequence under the 35S

promoter of Cauliflower mosaic virus into metaxin plants rescued

the mutant phenotype (data not shown). Sequence analysis

indicated that METAXIN has two highly hydrophobic segments

near the C terminus, possibly acting as transmembrane regions

that anchor the protein in a membrane (Figure 4H). Arabidopsis

suspension cell culture was biolistically transformed with a

chimeric construct that fused green fluorescent protein (GFP)

to the N terminus of METAXIN. The full-length METAXIN protein

was able to direct N-terminal GFP to mitochondria, as indicated

by the colocalization of GFP with the mitochondrial-targeted

AOX-RFP (red fluorescent protein) control construct (Figure 4H).

Interestingly, the GFP signal formed hollow circular structures,

suggesting that the GFP was targeted only to the outer mito-

chondrial membrane. These structures closely resemble those

observed by Setoguchi et al. (2006) upon immunofluorescence

microscopy–based detection of the mammalian mitochondrial

outer membrane proteins TOM22 and Bak.

To independently confirm the outer membrane localization of

METAXIN, intact wild-type mitochondria were isolated and incu-

bated in increasing concentrations of proteinase K (PK). Immu-

nodetection of TOM20-2 and TOM20-4 using specific polyclonal

antibodies raised to their cytosolic domains revealed that

1.7 mg/mL PK resulted in the complete digestion of the cyto-

solic portion of these proteins (Figure 4I). Immunodetection of

METAXIN by a specific polyclonal antibody raised to the pre-

dicted METAXIN cytosolic domain indicated that the cytosolic

portion of METAXIN was completely degraded in the presence of

53.3 mg/mL of PK (Figure 4I). At up to 106.7 mg/mL PK, there was

no noticeable degradation of VDAC (voltage-dependent anion

channel) or AOX, which are located in the outer membrane and on

the matrix side of the inner mitochondrial membrane, respec-

tively, indicating that the PK did not compromise the integrity of

either of the mitochondrial membranes at the concentrations

used. Therefore, METAXIN is accessible to externally added PK,

indicating that the predicted cytosolic domain to which the

antibody was raised is exposed to the cytosol. Membrane pro-

tein complexes from both wild-type and metaxin mitochondria

were solubilized by digitonin and separated by one-dimensional

BN-PAGE, followed by immunodetection of METAXIN, TOM20,

and the COXII subunit (cytochrome c oxidase) of mitochondrial

respiratory Complex IV (Figure 4J). COXII colocalized with Com-

plex IV, as determined by Coomassie blue staining of the res-

piratory complexes (data not shown), whereas METAXIN was

detected in a high molecular weight complex that is distinct from

the smaller TOM complex (Figure 1B). Furthermore, this high mo-

lecular weight complex could not be detected in metaxin mito-

chondria, indicating that it is an authentic mitochondrial protein

complex containing METAXIN. Therefore, while METAXIN is

exposed to the cytosol on the outer mitochondrial membrane,

it is located in a distinct complex that has not previously been

characterized in plant mitochondria.

Mitochondria from Metaxin Plants Have Reduced Rates

of Protein Import

A range of radiolabeled mitochondrial proteins were incubated

with mitochondria isolated from wild-type and metaxin plants

(Figure 5). As the yeast SAM37 forms part of the SAM complex,

the import of two b-barrel proteins, TOM40 and VDAC, was also

studied. The rate of protein import into metaxin mitochondria was

dramatically reduced for all precursors tested, especially TOM40

and VDAC. This indicates that METAXIN is involved in the import

of these proteins into mitochondria, either because it is directly

required for import by all of these mitochondrial proteins or

because it is required for correct TOM40 import and assembly

and thus needed for generation of a functional general import

pore, as is observed for SAM37 in yeast (Paschen et al., 2003;

Wiedemann et al., 2003). Although the decrease in protein import

of all the precursor proteins may be due to insufficient import or

assembly of TOM40, two-dimenstional PAGE analysis indicated

that TOM40 was present in the same amount in metaxin plants as

in the wild type (see Supplemental Figure 6 online). This indicates

that the inactivation of METAXIN does not completely block the

import of TOM40.

As it cannot be ruled out that the decrease in the rate of protein

import into metaxin mitochondria was a downstream conse-

quence of the disruption of TOM complex structure/organization

or a process in mitochondria not directly related to protein import,

an alternative approach was required to determine if it played any

direct role in the import of a variety of precursor proteins destined

to various intramitochondrial locations. METAXIN was expressed

in a wheat germ lysate and preincubated with radiolabeled mito-

chondrial precursor proteins before incubation with mitochondria

to determine if exogenous METAXIN could compete with mito-

chondria for interaction with the precursor proteins. The addition

of METAXIN resulted in a large decrease in the import of diverse

precursor proteins into mitochondria, relative to the amount of

protein import when precursors were incubated with GUS (Figure

6A). METAXIN preincubation inhibited the import of the general

pathway proteins AOX and RPS10, the carrier pathway protein

PiC, and the dual-targeted GR by 50 to 70%, while the b-barrel

proteins TOM40 and VDAC were imported 30 to 40% less.

Notably, very little reduction (;10%) was observed for FAd, and

TIM23 import was not significantly reduced (P > 0.1), indicating

that the addition of METAXIN to the in vitro protein import reac-

tion was not causing general disruption of the protein import

machinery or general import pore but was likely specifically inter-

acting with certain precursor proteins.

Comparative sequence alignment of plant METAXIN, animal

METAXIN1, and fungal SAM37 protein sequences from nine

diverse species revealed that the sequence of SAM37 from the


Figure 4. METAXIN Is an Outer Mitochondrial Membrane Protein Required for Normal Plant Metabolism, Growth, and Development.

(A) METAXIN-deficient Arabidopsis (metaxin) displays retardation of growth.

(B) metaxin is sterile and displays floral abnormalities, including incomplete anther and style maturation.

(C) Iodine staining of starch at 0 and 12 h after initiation of the light photoperiod.

(D) Light microscopy of wild-type leaf cross section (arrow indicates chloroplast).

(E) Light microscopy of metaxin leaf cross section (arrows indicate chloroplasts).

(F) Transmission electron microscopy of wild-type leaf mesophyll cell.

(G) Transmission electron microscopy of metaxin leaf mesophyll cell (arrows indicate starch granules within chloroplast).

(H) Subcellular localization of METAXIN was tested by fluorescence microscopy visualization of Arabidopsis suspension cells biolistically

cotransformed with plasmids encoding NAOX-RFPC and NGFP-METAXINC. TM, transmembrane domain.

3746 The Plant Cell

fungi Schizosaccharomyces pombe and Aspergillus clavulata

were more similar to the animal METAXIN1 proteins than to yeast

SAM37 (Figure 6B; see Supplemental Figure 5 online). Two puta-

tive conserved domains were identified at similar positions in

each protein, in plant METAXIN the N-terminal GST-N-METAXIN,

and C-terminal GST-C-METAXIN domains, and in animals/fungi

the N-terminal GST-N-METAXIN1-like and C-terminal GST-C-

METAXIN1-3 domains (Figure 6B) (Marchler-Bauer and Bryant,

2004). Alignment of the METAXIN/SAM37 protein sequences

revealed regions of significant sequence similarity in these

domains. Furthermore, a region of 40 to 52 amino acids from

the C-terminal end of the GST-N-METAXIN domain displayed

significant similarity between all species, suggesting it may be

important for protein function (see Supplemental Figure 5 online).

Progressive truncations of METAXIN from the C terminus were

made to disrupt the three putative conserved domains, and the

proteins were translated in a wheat germ lysate (Figure 6C).

These METAXIN deletions were used in competition import

assays with wild-type mitochondria to ascertain which regions

of the METAXIN protein were required for it to compete with

mitochondrial-located METAXIN for the import of radiolabeled

AOX (Figure 6C). The first 112 amino acids of METAXIN inhibited

AOX import; however, further deletion to only the first 72 or

37 residues of METAXIN abolished the competitive inhibition of

AOX import, suggesting that the conserved region (or competitor

domain) identified between amino acids 72 and 112 may be

important for interaction of the METAXIN protein with AOX

(Figure 6C). Preincubation with METAXIN that lacked only

the competitor domain resulted in a significant decrease in

import compared with GUS, but the amount of import inhibition

was less than when full-length METAXIN was used as a com-

petitor protein, further supporting a role for this region in the

import of preproteins across the outer membrane. Finally, in-

creasing the concentration of the METAXIN1-112 competitor

protein resulted in increased levels of AOX import inhibition

(Figure 6D).


(I) Mitochondria were purified from wild-type plants and incubated with increasing concentrations of PK. The mitochondrial proteins were separated by

SDS-PAGE and probed with specific antibodies as indicated.

(J) Immunodetection of CoxII and METAXIN in mitochondrial protein samples isolated from wild-type and metaxin plants after digitonin solubilization

and separation of protein complexes by first dimension BN-PAGE. The position and approximate molecular mass of the inner membrane respiratory

complexes I, III, IV, and V are indicated.

Figure 5. METAXIN-Deficient Mutant Plants Have Reduced Rates of Mitochondrial Protein Import.

[35S]-labeled precursor proteins TOM40, VDAC, AOX, FAd, PiC, and GR were incubated with mitochondria isolated from wild-type and METAXIN-

deficient mutants under conditions that support protein import. Aliquots were removed at 2, 5, 10, and 20 min and treated with PK. PK-protected mature

radiolabeled protein was quantitated at each time point and normalized to the highest time point for replicate experiments (n $ 3 6 SE). Where indicated,

mutant mitochondria had a significantly lower amount of protein import than wild-type mitochondria, with a P value < 0.05 (*), 0.01 (#), and 0.001 (circles)

using Student’s t test.


Figure 6. The Cytosolic Portion of METAXIN Can Interact with Mitochondrial Precursor Proteins.

(A) [35S]-labeled precursor proteins AOX, RPS10, TOM40, VDAC, PiC, TIM23, FAd, and GR were preincubated with overexpressed GUS or metaxin

competitor protein prior to incubation with mitochondria from wild-type plants under conditions that support protein import. PK-protected mature

radiolabeled protein was quantitated and normalized against the amount of protein imported after preincubation with GUS (n ¼ 3 6 SE). Where

indicated, the METAXIN competitor protein decreased protein import significantly compared with GUS, with a P value < 0.05 (*), 0.02 (#), 0.001 (circles)

using Student’s t test.

(B) Phylogenetic tree of protein sequences of plant METAXIN, animal METAXIN1, and fungal SAM37/METAXIN from Arabidopsis, M. truncatula,

O. sativa, C. elegans, A. mellifera, M. musculus, A. clavulata, S. pombe, and S. cerevisiae. METAXIN/SAM37 proteins from plants, animals, and fungi

display overlapping GST-N-METAXIN/GST-N-METAXIN1-like and GST-C-METAXIN/GST-C-METAXIN1-3 conserved domains.

(C) [35S]-labeled AOX precursor protein was preincubated with overexpressed GUS or truncated/deleted METAXIN proteins prior to incubation with

mitochondria from wild-type plants under conditions that support protein import. METAXIN (DComp) refers to a recombinant protein comprised of

the full-length METAXIN protein with the putative competitor domain between amino acids 72 and 112 removed. PK-protected mature radiolabeled

3748 The Plant Cell

TOM20, mtOM64, and METAXIN Interact with a Variety of

Precursor Proteins

To determine if these proteins could interact directly with precur-

sor proteins, pull-down and yeast two-hybrid interaction assays

were performed. For the pull-down assays, full-length TOM20-4,

METAXIN, and mtOM64 proteins were synthesized in a wheat

germ lysate and added to a variety of radiolabeled precursor

proteins. The interactions of each of these proteins with various

precursors was tested by the ability to pull down radiolabeled

precursor protein with antibodies raised against TOM20-4,

METAXIN, and mtOM64, respectively (Figure 7A). All three pro-

teins interacted with various precursor proteins, AOX, PiC, FAd,

TIM23, and TOM40. However, the precursor protein GR could

not be pulled down in this assay with any of the antibodies tested

(Figure 7A). Although it is not possible to compare how efficiently

each antibody pulls down each precursor due to variable anti-

body affinities, it was apparent that although the precursor pro-

tein FAd could be pulled down by all three antibodies, it was weak

in comparison to the other precursor proteins. For both AOX and

FAd, removal of the mitochondrial targeting signal resulted in

no interaction (Figure 7A). In contrast with the positive interaction

detected with the various precursor proteins and TOM20,

METAXIN, and mtOM64, using the same lysate programmed to

synthesize GUS, or using the lysate alone (Figure 7A) resulted in

no detectable interaction, indicating that the antibodies were not

directly binding to any of the precursor proteins (Figure 7A). The

binding of the Protein A Sepharose to the antibody was con-

firmed with protein gel blotting (Figure 7B). To further determine

if these proteins interacted with mitochondrial precursor pro-

teins, a yeast two-hybrid interaction screen was performed. The

cDNAs encoding the three TOM20 proteins, METAXIN, mtOM64,

and At TOC64-III, were each cloned to produce a recombinant

fusion protein with the GAL4 binding domain (bait), while cDNAs

encoding the precursor proteins were cloned to produce a fusion

protein with the GAL4 activation domain (prey). Interactions were

determined by the ability to grow without the addition of His to

the media and the ability to grow in the absence of adenine to

give a red/orange color. All mitochondrial precursor proteins

supported growth in the absence of His when TOM20, METAXIN,

or mtOM64 was used as bait, and colony numbers were typically

5- to 10-fold higher than that obtained when At TOC64-III was

used as bait (data no shown). To visualize the difference, colonies

were grown in the absence of adenine (Figure 7C). Whereas a

red/orange color is clearly evident for all mitochondrial precursor

proteins with the mitochondrial proteins as bait, At TOC64-III

colonies are clearly white. Notably, nonrecombinant bait and/or

prey constructs did not produce any interaction (see Supple-

mental Figure 7 online). Removing the mitochondrial targeting

signal from AOX or FAd resulted in no interaction. One exception

was when GR was used as the prey fusion protein; a weak orange

color was evident with At TOC64-III as bait. Arabidopsis GR is a

dual-targeted protein (Chew et al., 2003a), although the plastid

receptor for this protein has not yet been determined. It contains

the consensus phosphorylation motif in the targeting signal that

has been reported to increase targeting ability to chloroplasts

(Chew et al., 2003a, 2003b; Martin et al., 2006). Thus, it is possi-

ble that it can interact with At TOC64-III, although the color was

weak compared with the color development with mitochondrial

precursor proteins and mitochondrial outer membrane compo-

nents. In summary, the two assays show the ability of the three

mitochondrial proteins, TOM20, METAXIN, and mtOM64, to in-

teract with a variety of mitochondrial precursor proteins.

A Flexible Regulatory Response to Mitochondrial Protein

Import Dysfunction

Adaptive responses of the plant cell to ablation of the different

protein import components were monitored using antibodies

raised to each TOM20 isoform, mtOM64, METAXIN, and several

other mitochondrial proteins (Figure 8A). Overall, these results

indicated a complicated series of responses. The absence of any

one of the TOM20 isoforms did not result in an increase in the

abundance of either of the remaining two TOM20 proteins (Figure

8A). However, in plants lacking two TOM20 isoforms, the amount

of the remaining TOM20 protein increased at least twofold,

supporting the observation from in vitro protein import experi-

ments (Figure 2B) that the TOM20 isoforms are largely function-

ally redundant. Furthermore, the amount of METAXIN was

increased in each TOM20 double mutant. The level of the inner

mitochondrial membrane translocase component TIM17 in-

creased in tom20-2 tom20-4, tom20-3 tom20-4, and the tom20

triple knockout, indicating that the retrograde regulatory mech-

anism that increases TOM20 abundance may also control other

mitochondrial protein import components. Interestingly, mtOM64

abundance increased in all tom20 plants except tom20-2 (Figure

8A). Together, the alteration in abundance of each component is

consistent with the proposal of overlapping roles for these pro-

teins. Notably, no signal was evident in the tom20 triple knockout

when probed with the TOM20-3 antibody. As this antibody

clearly interacts with TOM20-1 (see Supplemental Figure 1B

online), and as the abundance of TOM20-2, TOM20-3, and

TOM20-4 increased in the respective TOM20 double knockout

lines, it appears that TOM20-1 abundance is not being increased


AOX was quantitated and normalized against the amount of protein imported after preincubation with GUS. Where indicated, the METAXIN competitor

protein decreased protein import significantly compared with GUS, with a P value < 0.01 (#) using Student’s t test. The circle indicates that the full-length

METAXIN competitor protein inhibited AOX import significantly more than METAXIN (DComp), with a P value < 0.02 using Student’s t test.

(D) [35S]-labeled AOX precursor protein was preincubated with overexpressed GUS or METAXIN1-112 (the N-terminal 112 amino acids of METAXIN) prior

to incubation with mitochondria from wild-type plants under conditions that support protein import. PK-protected mature radiolabeled AOX was

quantitated and normalized against the amount of protein imported after preincubation with GUS. Where indicated, the METAXIN1-112 competitor protein

decreased protein import significantly compared with GUS, with a P value < 0.01 (#) using Student’s t test. Circles indicate that addition of METAXIN1-112

competitor protein inhibited AOX import significantly more than the next lower concentration of competitor, with a P value < 0.01 using Student’s t test.


Figure 7. Interaction of Precursor Proteins with TOM20, mtOM64, or METAXIN.

(A) The ability of TOM20, mtOM64, or METAXIN to pull down precursor protein in solution was tested for various precursor proteins. The lanes indicate

the antibody used to pull down the precursor proteins from a solution containing the corresponding protein synthesized in the RTS lysate. The left-hand

panel represents the reaction where the RTS lysate was programmed to synthesize the target protein. The right-hand panel represents the negative

control, where RTS lysate was not programmed to synthesize any protein, and thus the respective pull-down reactions contained no TOM20, METAXIN,

mtOM64, or GUS, respectively. AOXDp and FAdDp represent altered precursor proteins where the mitochondrial targeting presequence is not present.

(B) Protein gel blot analysis of the pull-down reactions to verify that the target protein was being specifically pulled down. Pull-down reactions were

performed and 10% of this reaction was analyzed by protein gel blotting. For each protein synthesized in the RTS reaction, the pull-down reaction was

performed and separated by SDS-PAGE, blotted, and probed with antibodies to TOM20, METAXIN, mtOM64, and anti-rabbit IgG. Lanes 1 to 4 in each

panel represent lysate programmed to synthesize TOM20, METAXIN, mtOM64, and GUS, respectively. A positive product was detected with the

TOM20 and METAXIN antibody (indicated by cirlces). As the mtOM64 protein has a similar molecular mass to that of the heavy IgG chains, a specific

product for mtOM64 could not be resolved; the rabbit IgG molecules are present as these were used to pull down the target proteins.

(C) Yeast two-hybrid interaction assays. The mitochondrial outer membrane proteins TOM20-2, TOM20-3, TOM20-4, mtOM64, and METAXIN and the

chloroplast outer envelope protein At TOC64-III were cloned into the bait vector and tested for interaction with the five precursor proteins cloned into the

prey vector. A red/orange color indicates an interaction. AOXDp and FAdDp represent these precursor proteins where the mitochondrial targeting

presequence had been removed.

3750 The Plant Cell

perceptibly to compensate for the loss of the three highly expressed

isoforms. As TOM20-1 transcript abundance is extremely low (see

Supplemental Figure 1C online) and detection of the TOM20-1

protein has not been reported, it appears that this isoform does not

play an important role in mitochondrial protein import.

AOX protein abundance, a marker for retrograde signaling in

plant mitochondria (Rhoads et al., 2006), increased in all mutants

lacking TOM20-2, most noticeably in tom20-2/tom20-4 and

the tom20 triple knockout. The in vitro protein import experi-

ments revealed that TOM20-2 plays a specialized role in AOX

Figure 8. Protein Levels in Mutant Plants Indicate Retrograde-Regulated Compensation by TOM20 Isoforms, Disruption of the TOM Complex in

metaxin, VDAC Accumulation in the Cytosol of metaxin Cells, and Upregulation of mtOM64 and METAXIN When Multiple TOM20 Isoforms Are Depleted.

(A) Mitochondria were purified from wild-type and mutant plants, and the mitochondrial proteins separated by SDS-PAGE and probed with specific

antibodies to import components and other mitochondrial proteins.

(B) Detection of marker proteins in mitochondrial and cytosolic protein fractions from wild-type and METAXIN-deficient plants. Lane 1, the wild type;

lane 2, metaxin-1; lane 3, metaxin-2.

(C) Abundance of TOM40, VDAC, UBC (ubiquitin conjugating enzyme), and ACT2 (Actin2) transcripts in metaxin-1 and metaxin-2 rosette leaves relative

to wild-type levels. Where indicated, the transcript abundance in metaxin was significantly higher than in the wild type, with a P value < 0.05 (*) using

Student’s t test. cFBPase, cytosolic fructose-1,6-bisphosphatase; E1a, E1 a-subunit of pyruvate dehydrogenase.


recognition and import (Figure 2B). AOX is a marker of stress-

induced mitochondrial retrograde pathways in plants, and its

upregulation in tom20-2 indicates that this isoform alone appears

to trigger this response (Rhoads et al., 2006). Similarly, the E1a

subunit of pyruvate dehydrogenase (E1a) was less abundant in

mitochondria isolated from tom20-2/tom20-3 and the tom20

triple knockout, indicating that TOM20-2 and TOM20-3 may both

have a higher affinity for this mitochondrial protein. The levels of

the inner membrane uncoupling protein (UCP), VDAC, cyto-

chrome c oxidase subunit II (COXII), and heat shock protein 60

(HSP60) were unchanged in the tom20 mutant plants, including

the tom20 triple knockout, demonstrating that despite lacking all

TOM20 receptors, the mitochondria are able to import a wide

range of proteins to a final abundance very similar to wild-type

levels. This again demonstrates that TOM20 is not an essential

protein import component and that the plant cell lacking TOM20

is able to generate a mitochondrial proteome quite similar to

wild-type mitochondria. This may be achieved by the operation

of the other protein import components mtOM64 and METAXIN.

Furthermore, it suggests that the upregulation of TOM20,

mtOM64, and TIM17 in the tom20 double and triple knockout

plants is not the consequence of an extensive alteration of a wide

range of mitochondrial proteins, but rather the outcome of a

precise retrograde regulatory pathway that specifically targets

the mitochondrial protein import apparatus.

The mtom64 mitochondria did not display significant changes

in the abundance of the proteins tested, except that the abun-

dance of METAXIN was consistently higher (Figure 8A). The

metaxin mitochondria showed dramatic decreases in the abun-

dance of several mitochondrial proteins, including an almost

total absence of all TOM20 isoforms and mtOM64 and a large

reduction in TIM17 and UCP abundance (Figure 8A). Interest-

ingly, AOX protein levels increased significantly in the metaxin

mitochondria, as did the alternative respiratory pathway activity

catalyzed by AOX (Ho et al., 2007), suggesting that this protein is

required at a greater abundance in the mitochondria in response

to aberrant mitochondrial behavior. This is in agreement with

the suggestion that elevated AOX abundance in mitochondria

lacking TOM20-2 is an adaptation to increased stress. By con-

trast, the level of VDAC, E1a, COXII, and HSP60 were unaltered

compared with wild-type mitochondria, suggesting that despite

a partially dysfunctional protein import apparatus in metaxin, the

plant cell can achieve normal levels of many mitochondrial pro-

teins. To determine if the abundance of VDAC throughout the

plant cell was unaltered by ablation of METAXIN, cytosolic pro-

teins were isolated from wild-type and metaxin leaves and the

amount of VDAC measured by immunodetection (Figure 8B).

metaxin plants had a much higher abundance of VDAC in the

cytosol, in contrast with the very low levels of VDAC in the wild-

type cytosolic fraction. Equivalent abundance of the cytosolic

fructose-1,6-bisphosphatase between wild-type and metaxin

cytosolic fractions demonstrated that not all cytosolic proteins

in the mutant had increased abundance. Cytosolic accumulation

of mitochondrial E1a was not observed, indicating that the cyto-

solic accumulation does not occur for all mitochondrial proteins

and that METAXIN may be particularly important for the recog-

nition and import of VDAC. Together with the increased VDAC

and TOM40 transcript abundance in metaxin plants (Figure 8C),

this suggests that the metaxin cells upregulate the expression

of this critical protein to attain normal levels in mitochondria,

potentially by saturating the residual import capability of the

metaxin mitochondria. To determine if TOM40 was present in the

metaxin and tom20 triple knockout lines, two-dimensional iso-

electric focusing (IEF)/SDS-PAGE analysis was performed with

purified mitochondrial proteins, and the amount of TOM40 was

determined. Overall, there appeared to be no significant changes

in TOM40 abundance in any of the mutant plant lines (see

Supplemental Figure 6 online). Likewise, for the FAd subunit, it

was evident that it was present at normal levels in the mtom64

plants (see Supplemental Figure 6 online). Finally, it was evident

that AOX was more abundant in tom20-2, tom20-2 tom20-4,

tom20-2 tom20-3 tom20-4, and metaxin lines (Figure 8A), even

though import was reduced from ;30 to 80%, respectively. Thus,

the steady state levels of proteins present in mitochondria isolated

from 4-week-old mutant plants was not affected by the absence

of the respective import components, even though a reduced rate

of protein import for these proteins was measured in vitro.

DISCUSSION

Investigation of the function of TOM20, mtOM64, and METAXIN

using a variety of approaches indicated that they all play a role in

the import of proteins into mitochondria. Insertional inactivation

is a powerful tool to determine function but cannot distinguish

between primary (direct) and secondary (indirect) effects. Here,

we observed that (1) altering any one of the three components

resulted in changes in abundance of at least one of the other two

that could possibly compensate for the function (Figure 8); and

(2) in the metaxin mutant, a reduced amount of other compo-

nents was observed (Figure 8). To overcome these limitations, a

variety of alternative approaches was undertaken. The potential

for all three components to directly interact with precursor

proteins was demonstrated (Figure 7), which combined with

the import and competition assays, strongly suggests a model of

overlapping ability to interact with precursor proteins on the outer

surface of mitochondria. Thus, although the insertional inactiva-

tion of mtOM64 only affected the import of the FAd precursor

(Figure 3), the interaction assays indicate that it can bind and

affect the import of a variety of precursor proteins (Figure 7).

However, as METAXIN abundance increased in the mtom64

mutants (Figure 8) and TOM20 is present, an in vitro assay where

excess precursor protein is added the absence of a nonlimiting

component may result in normal rates of import being observed

even if that component can interact with precursor proteins when

present. It was notable that in the mtom64 mutants, only the

import of the FAd precursor was affected (Figure 3), and the

competition assays indicated that METAXIN competitor protein

could only compete weakly for the import of this precursor

protein (Figure 6). Thus, the upregulated abundance of METAXIN

and the fact that the import of the FAd precursor is still high in the

presence of the triple tom20 knockout (Figure 2C) are consistent

with a role for mtOM64 in the import of this precursor protein.

However, TOM20 and METAXIN can also play a role, as they can

interact directly with this precursor protein (Figure 7). mtOM64

may play a role in the import of other precursor proteins, but due

to the apparent functional redundancy of the import machinery,

3752 The Plant Cell

this is not detected in knockout mutants, and other approaches

are required to elucidate its interaction with these precursor

proteins, such as direct interaction assays. In the case of GR,

only the yeast two-hybrid assay indicated an interaction. The in

vitro import assays in the triple tom20 and mtom64 mutants

revealed no effect on import. Addition of full-length METAXIN

could compete for import, but this may be due to competition for

a common import component. Alternatively, as GR is located in

the intermembrane space and the matrix (Chew et al., 2003a,

2003b), it may interact with METAXIN via the import route to the

intermembrane space or directly with GR via the intermembrane

space exposed domains of METAXIN. A recent study of METAXIN

from human cell lines using coimmunoprecipitation revealed that

it interacted with an inner membrane protein present in a larger

complex with several other proteins (Xie et al., 2007), while

another study in humans using BN-PAGE concluded that it was

not in a complex with SAM50 (Kozjak-Pavlovic et al., 2007). Thus,

the role of METAXIN may differ considerably between species

and/or it may play a variety of roles in various organisms.

The Tom20 Multiple Gene Family

Duplication of the TOM20 gene family has apparently resulted in

limited functional specialization, as evidenced by the formation

of a second and larger complex containing TOM20-2 and differ-

ences in the rate of import of some precursor proteins. As the rice

genome encodes only a single TOM20 isoform, this gene family

duplication and subfunctionalization has most likely occurred

subsequent to the divergence of monocotyledonous and dicot-

yledonous plants. Studies on duplicated genes suggest that

subfunctionalization is an important transition state to neofunc-

tionalization and acts to increase the preservation of duplicated

genes (Rastogi and Liberles, 2005). TOM20 was identified in

plants from direct protein analysis of the isolated TOM complex

that contained TOM40 (Jansch et al., 1998). Subsequent analysis

of TOM20 indicated that it is not orthologous to yeast or mam-

malian TOM20 (Perry et al., 2006), thus necessitating reevalua-

tion of its role in mitochondrial protein import. Based on the fact

that the majority of the protein is exposed on the cytosolic side of

the outer membrane (Figure 4), that the nuclear magnetic reso-

nance structure of TOM20-3 indicates a very similar prese-

quence binding fold to mammalian TOM20 (Likic et al., 2005;

Perry et al., 2006), that inactivation of all three TOM20 isoforms

leads to a substantial decrease in the rate of protein import

(Figure 2), that TOM20 directly interacts with mitochondrial

precursor proteins (Figure 7), and that overexpressed Arabidop-

sis TOM20-3 can compete for protein import into yeast mito-

chondria (Perry et al., 2006), we conclude that TOM20 likely

functions as a mitochondrial protein import receptor.

Although we could not detect TOM20-1 protein and transcript

was not detected in a variety of materials we have previously

examined (see Supplemental Figure 1 online) (Lister et al., 2004;

Murcha et al., 2007), it is possible that its expression at a protein

level is below the limits of detection or limited to specific cell

types. An examination of 2509 arrays in Genevestigator indicates

that it is called present in 347 (14%) (Zimmermann et al., 2004),

although this does not incorporate false discovery rate correction

that should be used (Nettleton, 2006). Analysis of these arrays

indicated that TOM20-1 transcript abundance was higher in

roots compared with other organs. Thus, we performed quanti-

tative RT-PCR and could detect some expression (see Supple-

mental Figure 1C online); however, the corresponding protein

could not be detected in mitochondria isolated from the same

triple tom20 mutant plants (water culture) used to carry out the

in vitro import assays (Figure 2) or from a variety of other mito-

chondrial preparations (cell culture, water culture or plant or root

material; data not shown). Thus, given the reduction we observe

in import with the tom20 triple knockout for several precursor

proteins, combined with the fact that we cannot detect the

protein, it is unlikely that TOM20-1 is highly expressed and could

compensate to function as the predominant import receptor.

Is mtOM64 a Receptor?

The absence of a plant homolog of TOM70 has frequently been

noted as one of the fundamental differences in the plant mito-

chondrial import apparatus (Lister et al., 2005; Chan et al., 2006).

In this study, mtOM64 could not be identified in the TOM

complex by immunodetection (data not shown), suggesting

that any attachment is peripheral and that it is dynamically

associated with the TOM complex, as observed for the interac-

tion of At TOC64-III with the TOC complex (Schleiff et al., 2003).

Insertional inactivation, in vitro competition experiments with

overexpressed mtOM64, antibody inhibition assays, and direct

interaction assays all suggest a role for mtOM64 in the import of

proteins into mitochondria. For the competition experiments,

mtOM64 (or METAXIN) was synthesized in a wheat germ tran-

scription/translation system where waste products are continu-

ously removed to achieve high levels of protein expression. It has

been reported that a different wheat germ lysate system than the

one used in this study can have an inhibitory effect on protein

import into mitochondria and plastids (Schleiff et al., 2002; Dessi

et al., 2003). However, the following should be noted: (1) the FAd

precursor is as efficiently imported into mitochondria from a

wheat germ lysate compared with a rabbit reticulocyte lysate

(Dessi et al., 1996; Tanudji et al., 2001), and (2) a study analyzing

the inhibitory effect of wheat germ on import indicated that it is

due to the folding status of the mature part of the protein (Dessi

et al., 2003). Other precursor proteins can also be imported into

mitochondria from a wheat germ lysate (Biswas and Getz, 2004).

Thus, it appears that some formulations of the wheat germ

translation lysate contain factors that inhibit import, and, notably,

it has been reported that other factors, such as mitochondrial

import stimulating factor, can relieve this inhibition (Hachiya

et al., 1993). The type of wheat germ translation system used

here is optimized to produce enzymatically active protein. Over-

all, although addition of this lysate mix alone reduces the rate of

protein import, it can be readily used, as it is a plant-based lysate,

and the inhibition observed in these studies was dependent on

the mRNA used to program the lysate and thus was not a general

effect of the lysate itself on import as was the topic of investi-

gation in previous studies. Furthermore, as evidenced by the

immunodetection (Figure 8A) and two-dimensional IEF/SDS-

PAGE (see Supplemental Figure 6 online) analyses, the steady

state level of mitochondrial proteins is not directly related to the

in vitro rate of import.


Several independent approaches used here all indicate a role

for mtOM64 in the import of at least some mitochondrial proteins;

however, its exact role cannot yet be concluded. Although it

can clearly interact directly with a variety of precursor proteins,

the binding chain hypothesis for mitochondrial import proposes

that many components contain such binding properties. For in-

stance, it has been recently shown that the pore-forming SAM50

(TOB55) from yeast contains such a domain (Rehling et al., 2001;

Habib et al., 2007). The localization of mtOM64 on the cytosolic

face of the outer membrane is evidenced by digestion with

externally added protease (Chew et al., 2004) and supports

an interaction with precursor proteins on the outer face of

mitochondria. At TOC64-III, TOM70, and mtOM64 have three

C-terminal TPR motifs that are predicted to form a superhelical

structure (Scheufler et al., 2000). Recently, the C-terminal TPR

motifs of Arabidopsis At TOC64-III were demonstrated to medi-

ate its recognition of chloroplast precursor proteins via interac-

tion with the chaperone HSP90 (Qbadou et al., 2006). In this

study, the competitive inhibition of FAd import by mtOM64 was

not abolished by addition of geldanamycin, a chemical that binds

to HSP90 and inhibits its chaperone activity (data not shown)

(Young et al., 2003). Thus, it can be concluded that mtOM64

does specifically interact with mitochondrial precursor proteins

and therefore may function as a receptor in the early stages of

precursor recognition and import. mtOM64 is a rate-limiting com-

ponent for the import of the plant-specific protein FAd (Figure 3),

although this may be an indirect affect due to the inactivation of

mtOM64 altering the level of as yet unknown component in-

volved or rate-limiting for the import of this precursor protein.

A Multifunctional METAXIN?

As the yeast SAM complex contains two of the three outer mem-

brane proteins known to be essential for cell viability, SAM50 and

SAM35 (Milenkovic et al., 2004), it was surprising to find that only

SAM50 is highly conserved in diverse eukaryotic organisms.

Animals possess METAXIN1 and METAXIN2, which display just

36 and 28% sequence similarity to yeast SAM37 and SAM35,

respectively (Mus musculus sequences). METAXIN2 was dem-

onstrated to interact with METAXIN1 and to be peripherally

associated with the mitochondrial outer membrane (Armstrong

et al., 1999), akin to yeast SAM35 (Waizenegger et al., 2004).

Taken together, the sequence similarity and protein–protein

interactions suggest that the animal METAXIN proteins are

orthologs of SAM35 and SAM37. By contrast, Arabidopsis

possesses only one METAXIN protein, which displays 21%

sequence similarity to mouse METAXIN1 and only 11% similarity

to yeast SAM35. Thus, it appears that the plant METAXIN is a

highly diverged form of yeast SAM37. The location of Metaxin on

the outer mitochondrial membrane and sensitivity to externally

added protease (Figure 4), the ability of various regions of the

METAXIN protein to inhibit import of some precursor proteins

(Figure 6), and the ability to interact with precursors directly

(Figure 7) together strongly suggest that METAXIN can interact

with precursor proteins on the outside of mitochondria. However,

given that METAXIN is not located in the TOM complex with

TOM20 (Figure 4), it would be premature to conclude that it plays

a primary role as a preprotein receptor.

The accumulation of VDAC in the cytosol of metaxin plants

(Figure 8) and the negligible rate of VDAC and TOM40 import into

metaxin mitochondria in vitro (Figure 5) suggest that METAXIN

also performs a role in b-barrel protein import. The competition

and interaction experiments indicated that plant METAXIN can

bind both VDAC and TOM40 (Figures 6 and 7), and the abun-

dance of transcripts encoding these proteins increased signifi-

cantly in metaxin plants (Figure 8). Therefore, plant METAXIN likely

functions in a plant SAM complex, which remains to be charac-

terized. The significant sequence similarity of plant and animal

METAXIN proteins and the conservation of the plant GST-N-

METAXIN and GST-C-METAXIN motifs and the functional

Competitor domain in animal METAXIN and yeast SAM37 sug-

gests that animal METAXIN and yeast SAM37 may have some

functional similarities to the plant METAXIN. Mutant yeast cells

lacking SAM37 displayed reduced import of several mitochon-

drial proteins, and anti-SAM37 antibodies were able to inhibit

protein import, leading to the initial characterization of SAM37 as

an import receptor that cooperated with TOM70 to recognize a

range of mitochondrial proteins (Gratzer et al., 1995). Subse-

quently it was reported that SAM37 was not involved in the initial

binding to mitochondria of the inner membrane metabolite

carrier protein AAC, and its depletion did not affect the general

or carrier import pathways (Ryan et al., 1999). However, if TOM20

and TOM70 were still present, their receptor capabilities may

have compensated for the absence of SAM37. Indeed, inactiva-

tion of SAM37 was synthetically lethal with the deletion of either

TOM20 or TOM70 (Gratzer et al., 1995). Thus, the role of the

cytosolic domain of METAXIN remains unclear in other orga-

nisms; however, its ability to interact with a variety of precursor

proteins suggests it may play another role in addition to the

assembly of b-barrel proteins and thus interact with proteins on

both the outside and inside of the outer membrane. Notably, At

TOC64-III in chloroplasts has been proposed to play a role in

precursor binding on the cytosolic and inter envelope space

(Qbadou et al., 2007).

A Flexible Import Apparatus with Overlapping Specificity

Overall, TOM20, mtOM64, and METAXIN possess cytosolic

domains that are located on the outer face of the mitochondria

and are required for normal rates or protein import. Overex-

pressed protein competes for import, and they can interact with a

variety of precursor proteins, all indicating that it is likely that

TOM20, mtOM64, and METAXIN directly interact with mitochon-

drial precursor proteins to function in the initial stages of the

import process. Given the NMR structure of TOM20 combined

with the results presented here, it is likely that it is a receptor for at

least some precursor proteins, while mtOM64 and METAXIN

may play similar roles.

These findings provide a unique insight into the complexity of

mitochondrialpreprotein importmachinery inamulticellulareukary-

ote. Analysis of genome sequences from a variety of organisms

suggests that plants do not have orthologs to TOM20 and TOM70

and that the receptor domain of TOM22 is lacking, compared

with yeast and mammalian systems (Macasev et al., 2004). Here,

we have defined three plant outer mitochondrial membrane pro-

teins that may fulfill the roles of preprotein receptors to produce a

3754 The Plant Cell

flexible and redundant set of receptor subunits in plants. SAM37/

35 and METAXIN appear to play a role in b-barrel assembly in all

lineages but may have acquired lineage-specific functions as a

receptor subunit, especially evident in plants. Finally, the use of

very similar protein import components in both plant mitochon-

dria and plastids suggests coevolution of the import machineries

of both organelles, which may have been a significant impetus in

the development of this unique import apparatus.

METHODS

Plant Growth

All plants were grown at 228C under long-day conditions (16 h of 100 mE

m�2 s�1 light, 8 h dark), except for metaxin plants that were iodine stained

for starch content analysis, which were grown with a 12-h-light/12-h-dark

photoperiod. For T-DNA insertion line genotyping, Arabidopsis thaliana

seeds were grown on soil after stratification for 2 d. For mitochondrial

isolation, Arabidopsis seeds were sterilized in 70% (v/v) ethanol and 5%

(v/v) bleach/0.1% (v/v) Tween 20 and grown for 14 d on an orbital shaker

at 80 rpm in 80 mL of sterile liquid growth media (0.53 Murashige and

Skoog media, 0.53 Gamborgs B5 vitamins, 2% [w/v] sucrose, 50 mg/mL

cefotaxime, and 2 mM MES KOH, pH 5.7). As metaxin homozygotes are

infertile, seeds from plants heterozygous for the metaxin-1 or metaxin-2

null allele were sterilized as above and grown for 14 d on agar growth media

(13 Gamborgs B5 salts, 3% [w/v] sucrose, 50 mg/mL cefotaxime, 2 mM

MES KOH, pH 5.7, and 0.75% [w/v] agar). metaxin homozygote plants were

then transplanted to soil and grown until flowering. Columbia-0 control

plants were grown under the same conditions as metaxin.

Iodine Staining

For iodine staining of starch, plant tissue was boiled for 5 min in 80% (w/v)

ethanol, washed in water, and then incubated for 5 min in 50% (v/v)

Lugols’s solution (Sigma-Aldrich). Plant tissue was then destained for

90 min in water.

T-DNA Insertion Lines

The following T-DNA insertion lines were obtained from SALK (Alonso

et al., 2003), SAIL (Sessions et al., 2002), and GABI-KAT (Rosso et al.,

2003) collections and genotyped to confirm homozygosity for the T-DNA

insert: tom20-2 (At1g27390): SALK_067986, SALK_134973; tom20-3

(At3g27080): GABI_554C03, SAIL_88_A03; tom20-4: SALK_147093,

SALK_004057; mtOM64 (At5g09420): SALK_068772, SALK_089921;

and metaxin (At2g19080): SALK_107629, SALK_039892.

Mitochondrial Isolation

Approximately 10 g (fresh weight) of aerial tissue from soil-grown plants or

20 g (fresh weight) of 14-d-old seedlings from liquid-grown (water culture)

plants were used to isolate mitochondria as described previously (Day

et al., 1985), with 10 mM L-Cys added to the grinding medium. Typically,

10 g of soil-grown and 20 g of liquid-grown plant tissue yielded ;2 mg of

mitochondrial protein. For isolation of mitochondria for protein gel blot

analysis, BSA was omitted from the final washes. All mitochondria were

isolated from liquid-grown seedlings except for the metaxin genotypes,

for which aerial tissue was used due to sterility of the metaxin null mutant.

Cytosol Isolation

Rosette tissue was homogenized in grinding buffer without BSA (Day

et al., 1985), centrifuged at 2500g for 5 min, and then the supernatant

centrifuged at 40,000g for 40 min. Crude cytosolic supernatant was

centrifuged at 100,000g for 1 h at 48C. The supernatant was concentrated

in a >5 kD centrifugal filter unit (Millipore).

Immunodetection of Proteins, Pull-Down Assays, and Antibody

Inhibition of Import

Mitochondrial or cytosolic proteins (50 mg) were resolved by SDS-PAGE,

transferred to Hybond-C extra nitrocellulose membrane, and immuno-

detection performed as previously outlined (Murcha et al., 2005). Poly-

clonal antibodies were raised in rabbits against recombinant protein

encoded by the predicted cytosolic regions of METAXIN, mtOM64,

TOM20-2, and TOM20-4. TOM20-3 polyclonal antibody was obtained

from Trevor Lithgow (University of Melbourne, Vic, Australia) (Taylor et al.,

2003). Antibodies to HSP60 were obtained from Stress-Gen, and anti-

bodies to COXII and cFBPase were obtained from Agrisera. Monoclonal

antibodies against VDAC (PM035) and E1a (PM030) were obtained from

Tom Elthon (University of Nebraska, Lincoln, NE). Antibodies to AOX

(Elthon et al., 1989), TIM17 (Murcha et al., 2005), and UCP (Considine

et al., 2001) have been described previously. For the immunodetection

experiments, mitochondrial proteins were isolated from the same water

culture-grown seedling tissue that was used to prepare mitochondria for

the in vitro import experiments, except for metaxin plants, for which tissue

was always obtained from aerial rosettes.

IgG was purified for pull-down and inhibition studies using the Pierce

Melon IgG purification kit according to the manufacturer’s instructions

(Pierce). For the pull-down assays, TOM20, mtOM64, and METAXIN were

expressed in the wheat germ RTS (Roche), and on completion of the 24-h

synthesis reaction, 1 mL of freshly synthesized radiolabeled precursor

was incubated with 15 mL of freshly synthesized TOM20, mtOM64, or

METAXIN and incubated at 248C for 1 h. RTS lysate programmed to

synthesize GUS was used as a control, antibody obtained from Sigma-

Aldrich. At the end of the incubation period, the volume was adjusted to

300 mL in PBS with 1% (w/v) BSA, and 50 mL of Protein A Sepharose 4B

conjugate (Sigma-Aldrich) was added to preclear the lysate for any

nonspecific binding to the Protein A Sepharose (Sambrook et al., 1989).

After incubation with gentle mixing for 1 h, the Protein A Sepharose was

removed by centrifugation. Five microliters of the appropriate purified

antibody was added and incubated for 1 h, followed by addition of 50 mL

of Protein A Sepharose and incubation for a further 1 h. The beads were

pelleted by centrifugation at 5000 rpm, washed in 200 mL of PBS with 1%

(w/v) BSA, and repelleted. Products were analyzed by SDS-PAGE

followed by exposure to a BAS-TR2040 plate for 48 h and imaged in a

BAS2500 (Fuji). Four different sets of negative controls were performed to

ensure that the antibody was only pulling down the target proteins and

that interactions of the precursor protein with the test proteins was

mediated by the mitochondrial targeting signal. First, AOX and FAd

proteins were engineered that lacked the mitochondrial targeting signal,

called AOXDp and FAdDp, respectively (see below for details), to test if

interaction was with this region of the protein. Second, the pull downs

were performed in the absence of TOM20, METAXIN, mtOM64, or GUS to

ensure that any radiolabeled precursor was not being pulled down

directly with the antibody. Third, the nonmitochondrial protein GUS was

used as a comparison. Finally, the specificity of the pull down was tested

by protein gel blotting.

Yeast Two-Hybrid Interaction Assay

The Clontech Matchmaker two-hybrid system was used to determine

interactions between the precursor proteins AOX, AOXDp, FAd, FAdDp,

PiC, GR, and TOM40 with TOM20-2, TOM20-3, TOM20-4, mtOM64,

METAXIN, and At TOC64-III. The latter were cloned into the bait vector

pGADT7-Rec[2] (Leu selection) and the precursor proteins cloned into the

prey vector pGBKT7 (Trp selection) by recombination cloning according


to the manufacturer’s instructions. Positive interactions were screened

via two rounds of selection, first by growth on -His (with -Leu and -Trp)

media and secondly by growth on -Ade media (with -Leu, -Trp, and -His);

this higher stringency protocol reduces the rate of false positives (James

et al., 1996). In the case of the AOX and FAd precursor proteins, the first 42

and 31 amino acids were removed as these regions had been previously

shown to contain the mitochondrial targeting activity (Dessi et al., 2003;

Lee and Whelan, 2004).

BN-PAGE

Mitochondrial membrane complexes were solubilized in 5% (v/v) digito-

nin and separated by first dimension BN-PAGE as described previously

(Eubel et al., 2005). Proteins from the first dimension gel were transferred

onto nitrocellulose membrane and immunodetection performed as de-

scribed above.

Clones/Constructs

The cDNAs encoding the following proteins have been described previ-

ously: AOX (GenBank accession number X68702) (Whelan et al., 1995),

FAd (GenBank accession number X79057) (Dessi et al., 1996), GR

(At3g54660) (Chew et al., 2003a), PiC (GenBank accession number

ABO16064) (Bathgate et al., 1989; Murcha et al., 2004), and RPS10

(At3g22300) (Adams et al., 2002). METAXIN (At2g19080), mtOM64

(At5g09420), At TOC64-III (At3g17970), TOM40 (At3g20000), and VDAC

(At3g01280) were amplified from Arabidopsis cDNA as described previ-

ously (Murcha et al., 2003).

GFP Subcellular Localization

GFP subcellular localization was performed by cloning GFP-5 in frame

with the N or C terminus of the cDNA clone and subsequent transformation

of Arabidopsis suspension cells by biolistic transformation (Thirkettle-

Watts et al., 2003; Lee and Whelan, 2004). RFP was fused to the targeting

signal of soybean (Glycine max) alternative oxidase (AOX-RFP) as a

mitochondrial control (Murcha et al., 2007). Fluorescence patterns were

visualized after 48 h under an Olympus BX61 fluorescence microscope

and imaged using CellR imaging software.

In Vitro Mitochondrial Protein Import

[35S]-Met–labeled precursor proteins were synthesized using rabbit re-

ticulocyte TNT in vitro transcription/translation lysate (Promega) as de-

scribed previously (Whelan et al., 1995). The use of equivalent quantities

of mitochondria from different genotypes in import reactions was ensured

by triplicate measurement of protein concentration with the Coomassie

protein assay reagent (Pierce). Time-course analysis of precursor protein

import into intact mitochondria isolated from wild-type or mutant plants

was performed as described previously (Whelan et al., 1995), but with the

addition of 1 mM GTP and 1 mM NADH to the import master mix. Briefly,

250 mg of mitochondria were added to 450 mL of ice-cold import master

mix (0.3 M sucrose, 50 mM KCl, 10 mM MOPS, 5 mM KH2PO4, 0.1% [w/v]

BSA, 1 mM MgCl2, 1 mM Met, 0.2 mM ADP, 0.75 mM ATP, 5 mM suc-

cinate, 5 mM DTT, 1 mM GTP, and 1 mM NADH, pH 7.5) and incubated on

ice for 3 min. Twenty-five microliters of radiolabeled precursor protein

was added and the import reaction initiated by incubation at 268C with

gentle rocking. One hundred microliters containing 50 mg mitochondrial

protein was removed at 2, 5, 10, and 20 min. Upon removal, each aliquot

was mixed with 3.2 mg PK and incubated on ice for 30 min. Proteolysis

was inhibited by the addition of 1 mL of 100 mM PMSF. Mitochondria were

reisolated by centrifugation at 20,000g for 3 min at 48C, the mitochondrial

pellet resuspended in sample buffer, and the protein sample separated by

SDS-PAGE gel and imported radiolabeled proteins detected as outlined

previously (Murcha et al., 1999). PK protected mature radiolabeled pro-

tein was quantitated at each time point and normalized to the highest time

point measurement for replicate experiments (n ¼ 3 6 SE).

For import competition assays, competitor proteins were synthesized

using the RTS wheat germ lysate in vitro transcription/translation system

(Roche), according to the manufacturer’s instructions. Competitor pro-

tein was used in the wheat germ lysate mix (consisting of wheat germ

lysate resuspended in wheat germ reconstitution buffer) in which it was

synthesized, without further purification from the wheat germ lysate mix.

Five microliters of radiolabeled precursor protein was mixed with 15 mL

of competitor protein (2 mg) in wheat germ lysate mix and preincubated

at room temperature for 15 min. A control competition reaction was

performed by preincubation of precursor protein with 15 mL (2 mg) of GUS

in wheat germ lysate mix. Therefore, the same quantity of wheat germ

lysate mix was added to both competitor and control competition import

reactions. The expressed competitor protein was quantitated by sepa-

ration of the proteins by SDS-PAGE and protein gel blotting with Anti-6-

His antibodies, as all proteins expressed in this system contained a 6-His

tag. The radiolabeled precursor/competitor solution was added to an

import master mix containing mitochondria and incubated at 268C for 10

min, after which the import reaction was stopped by incubation on ice and

PK treatment. PK-protected mature radiolabeled protein was quantitated

at each time point and normalized to the amount of protein imported after

preincubation with GUS. To test for the effect of the wheat germ lysate

mix upon mitochondrial protein import, AOX and FAd precursor proteins

were preincubated for 15 min at room temperature with either 15 mL of

wheat germ lysate mix or 15 mL of wheat germ reconstitution buffer alone,

after which protein import was performed as described above. Mature

imported radiolabeled protein was quantitated after import and normal-

ized to the amount of protein imported in a control import reaction in

which precursors had not been preincubated in wheat germ lysate mix or

wheat germ reconstitution buffer.

PK Digestion of Mitochondrial Outer Membrane Proteins

Mitochondria were isolated from liquid grown plants as described above

and 500 mg of mitochondria incubated with 0 to 32 mg PK in a final volume

of 300 mL of 13 wash buffer (Day et al., 1985) on ice for 15 min. Proteolysis

was inhibited by the addition of 2 mL of 100 mM PMSF. Mitochondria were

reisolated by centrifugation at 20,000g for 5 min and resuspended in

SDS-PAGE sample buffer. Fifty micrograms was separated in each lane

by SDS-PAGE and immunodetection with specific antibodies performed.

Two-Dimensional IEF/SDS-PAGE

Mitochondria for two-dimensional IEF/SDS PAGE separation were further

purified by centrifugation on a 45% Percoll gradient in 13 wash buffer

minus BSA (Day et al., 1985). The mitochondrial fraction was washed

twice in 13 wash buffer minus BSA, then pelleted by centrifugation at

31,000g for 15 min. Mitochondrial proteins were separated by two-

dimensional IEF/SDS-PAGE as described previously (Ito et al., 2006).

Proteomic Analysis of Mitochondrial Proteins by

Mass Spectrometry

Trypsin digestion and tandem mass spectrometry identification of pro-

teins from two-dimensional PAGE gels was performed as described

previously (Chew et al., 2003a).

Transcript Analysis

Quantitative RT-PCR was performed as per the manufacturer’s instructions

using the Roche LC480 and LightCycler480 SYBR Green I Master (Roche)

3756 The Plant Cell

in a total volume of 10 mL. Gene-specific primers were used with cDNA

pools synthesized from wild-type and metaxin rosette leaf total RNA as

described previously (Murcha et al., 2003). Primers for measurement of

transcript abundance are listed in Supplemental Table 1 online. Transcript

abundance for each amplicon was normalized to the wild-type sample.

Phylogenetic Analysis

The phylogenetic relationship was inferred using the neighbor-joining

method. Sequence alignment was performed using ClustalW (see Sup-

plemental Figure 5 online). The bootstrap consensus tree inferred from

5000 replicates was taken to represent the relatedness of the sequences

analyzed. Branches corresponding to partitions reproduced in <50%

bootstrap replicates are collapsed. The percentage of replicate trees in

which the associated sequences clustered together in the bootstrap test

(5000 replicates) are shown next to the branches. The tree is drawn to

scale, with branch lengths in the same units as those of the evolutionary

distances used to infer the phylogenetic tree. All positions containing

gaps and missing data were eliminated from the data set (complete

deletion option). There were a total of 263 positions in the final data set.

Phylogenetic analyses were conducted in MEGA4.

Accession Numbers

Sequence data from this article can be found in the National Center for

Biotechnology Information and Arabidopsis Genome Initiative databases

under the following accession numbers: TOM20-1, NP_189343 and

At3g27070; TOM20-2, NP_174059 and At1g27390; TOM20-3,

NP_189344 and At3g27080; TOM20-4, NP_198909 and At5g40930;

MEXAXIN, NP_565446 and At2g19080; mtOM64, NP_196504 and

At5g09420; Pic, X57566; AOX, X68702; FAd, X79057; GR, X27456;

TOM40, NP_188634 and At3g2000; VDAC, Q9SMX3 and At5g15090;

ACT2, NM_112764 and At3g18780; UBC, ABF59034 and At5g25760.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Analysis of the Expression of TOM20-1.

Supplemental Figure 2. Null Mutations in Any of the TOM20 Genes

Do Not Lead to Deficiencies in Mitochondrial Protein Import.

Supplemental Figure 3. Effect upon Protein Import by Components

of the Wheat Germ Lysate Expression System.

Supplemental Figure 4. metaxin Mutants Are Depleted in METAXIN

Transcript.

Supplemental Figure 5. ClustalW Protein Sequence Alignment of Plant

METAXIN, Animal METAXIN1, and Fungal SAM37 Protein Sequences.

Supplemental Figure 6. The Mitochondrial Proteome Is Not Signif-

icantly Altered by Lesions in METAXIN, mtOM64, or Any TOM20

Isoform.

Supplemental Figure 7. Negative and Positive Controls for Yeast

Two-Hybrid Assay.

ACKNOWLEDGMENTS

This work was supported by the Australian Research Council Centre of

Excellence Program CEO561495. We thank Harvey Millar and Ian Small

for useful suggestions.

Received January 17, 2007; revised October 8, 2007; accepted October

16, 2007; published November 2, 2007.

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Chapter 6 Arabidopsis Mia40

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Chapter 6

Arabidopsis Mia40

Chapter 6 Arabidopsis Mia40

87

Foreword to Study V Whilst searching for proteins that were potentially dual targeted to mitochondria

and peroxisomes, it was uncovered that the Arabidopsis homolog of the yeast Mia40

contains a PTS1 signal, indicating a peroxisomal location for this protein in plants.

Mia40 is part of an import pathway in the intermembrane space of yeast and humans

(Chacinska et al., 2004). This pathway, termed the mitochondrial import and assembly

pathway (MIA), contains two essential proteins in yeast: Erv1 and Mia40 (Chacinska et

al., 2004; Rissler et al., 2005). The MIA pathway is a disulfide relay system, involved

in forming disulfide bonds into newly imported intermembrane space proteins such as

the small Tims (Sideris and Tokatlidis, 2010). As there was no functional data on the

Arabidopsis Mia40, a study was undertaken to confirm its subcellular location and

function within plant cells.

In contrast to findings in yeast, this study showed that Arabidopsis Mia40 is a

dual targeted protein in plants, targeted to both peroxisomes and the mitochondrial

intermembrane space. The Arabidopsis Mia40 was also found to be non-essential, with

knockout plants displaying no abnormal phenotype. However, at a molecular level, the

absence of Mia40 had a number of interesting phenotypes. The absence of Mia40 in

Arabidopsis led to a decrease in protein levels in both peroxisomes and mitochondria of

the copper/zinc chaperone for superoxide dismutase 1 (Ccs1) and the mitochondrial and

peroxisomal copper/zinc superoxide dismutases (CSD1 and CSD3 respectively). The

loss of Mia40 also led to a decrease in the capacity of complex I of the respiratory chain

and ΔMia40 plants also demonstrated compromised assembly of complex I. While

Mia40 in yeast has been implicated in the import of small Tim proteins (Chacinska et

al., 2004), the absence of Arabidopsis Mia40 led to no change in the import of small

Tim proteins in plants. Thus Arabidopsis Mia40 has taken on new roles in peroxisomes

and mitochondria in plants and the disulfide relay system operates in a mechanistically

different manner in plant mitochondria compared to yeast.

Conserved and Novel Functions for Arabidopsis thalianaMIA40 in Assembly of Proteins in Mitochondria andPeroxisomes□S

Received for publication, March 7, 2010, and in revised form, September 4, 2010 Published, JBC Papers in Press, September 9, 2010, DOI 10.1074/jbc.M110.121202

Chris Carrie‡, Estelle Giraud‡, Owen Duncan‡, Lin Xu‡, Yan Wang‡, Shaobai Huang‡, Rachel Clifton‡,Monika Murcha‡, Aleksandra Filipovska§, Oliver Rackham§, Alice Vrielink¶, and James Whelan‡1

From the ‡Australian Research Council Centre of Excellence in Plant Energy Biology, the §Western Australian Institute for MedicalResearch, Centre for Medical Research, and the ¶School of Biomedical, Biomolecular, and Chemical Sciences, University of WesternAustralia, Crawley, Western Australia 6009, Australia

The disulfide relay system of the mitochondrial intermem-brane space has been extensively characterized in Saccharomy-ces cerevisiae. It contains two essential components, Mia40 andErv1. The genome of Arabidopsis thaliana contains a singlegene for each of these components. Although insertional inacti-vation of Erv1 leads to a lethal phenotype, inactivation ofMia40results in no detectable deleterious phenotype. A. thalianaMia40 is targeted to and accumulates in mitochondria and per-oxisomes. Inactivation of Mia40 results in an alteration of sev-eral proteins in mitochondria, an absence of copper/zinc su-peroxide dismutase (CSD1), the chaperone for superoxidedismutase (Ccs1) that inserts copper into CSD1, and a decreasein capacity and amount of complex I. In peroxisomes theabsence of Mia40 leads to an absence of CSD3 and a decrease inabnormal inflorescencemeristem 1 (Aim1), a�-oxidation path-way enzyme. Inactivation of Mia40 leads to an alteration of thetranscriptome of A. thaliana, with genes encoding peroxisomalproteins, redox functions, and biotic stress significantly chang-ing in abundance. Thus, the mechanistic operation of the mito-chondrial disulfide relay system is different inA. thaliana com-pared with other systems, and Mia40 has taken on new roles inperoxisomes and mitochondria.

Characterization of the proteomes of mitochondria fromSaccharomyces cerevisiae (yeast), mammals, and plants indi-cates that they contain from 1000 proteins in yeast to �2000 inhigher organisms (1). As the coding capacity of mitochondria islimited to between 8 and approximately 50 proteins in yeast andplants, respectively (2), the majority of mitochondrial proteinsare encoded by nuclear located genes, translated in the cytosol,and imported into mitochondria. The import of hundreds ofdifferent proteins is achieved by the combined action of a num-ber of multisubunit, integral membrane protein complexes,known as translocases. These translocases work in conjunctionwith a variety of soluble components located in the cytosol,intermembrane space, andmitochondrialmatrix, such as chap-erones, peptidases, and assembly factors (3, 4).

The outer mitochondrial membrane contains the TOMcomplex (translocase of the outer membrane) and the sortingand assembly machinery; the latter is also known as the TOBcomplex (topogenesis of�-barrel proteins) (4). Almost allmito-chondrial proteins are initially recognized by the outer mem-brane complex and are passed to other components dependingon their final location.�-Barrel proteins of the outermembranesuch as Tom40 and voltage-dependent anion channel protein(VDAC) are passed to the sorting and assembly machinerycomplex (5). Proteins imported via the general and carrierimport pathways are passed to the translocases of the innermembrane 23 (TIM23) and 22 (TIM22), respectively (3, 4). Pro-teins are passed to the TIM23 complex directly from the outermembrane complex with the aid of Tim50 (6, 7) and also pos-sibly Tim23, where the N-terminal region has been shown totransiently associate with the outer membrane (8). Proteins aretransferred to the TIM22 complex via the aid of the small inter-membrane space proteins Tim9 and -10 (3, 4). A number of vari-ations of thesemain pathways can occur. These include the cross-ing over between pathways (9) and the utilization of differentsorting processes. Proteins can be either stop-transfer-sorted orconservative-sorted, with the latter pathway using the Oxa1ptranslocase located on the innermitochondrialmembrane (10). Avariety of other protein import pathways also exist, such as thoseutilized for the import of cytochrome c (11) and the small Timproteins of the intermembrane space (12, 13).The import pathway of the small intermembrane space pro-

teins, such as Tim8, -9, -10, and -13, is the most recentlydescribed protein import pathway. Their import is achieved viathe mitochondrial intermembrane space assembly machinery(MIA)2 that consists of Mia40 and Erv1 (3, 12, 14), both ofwhich are essential proteins in yeast (15, 16). Characterizationof the import pathway of Tim9 and -10 in yeast revealed that aslittle as nine amino acids are required to achieve transfer fromthe outer membrane complex on the outer membrane toMia40, which acts as a receptor in the intermembrane space(17, 18). These proteins then undergo oxidative folding in theintermembrane space. Conserved cysteine residues in these

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Tables 1 and 2 and Figs. 1– 4.

1 To whom correspondence should be addressed: ARC COE PEB, University ofWestern Australia, 35 Stirling Highway, Crawley WA 6009, Australia. Tel.:61-8-64881749; Fax: 61-8-64884401; E-mail: [email protected].

2 The abbreviations used are: MIA, mitochondrial intermembrane assembly;CA2, carbonic anhydrase 2; Kat2, 3-ketoacyl-CoA thiolase; Aim1, alteredinflorescence meristem 1; Sod1, superoxide dismutase 1; Ccs1, copperchaperone for CSD1; RFP, red fluorescent protein; BN, blue native; TES,2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 46, pp. 36138 –36148, November 12, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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proteins are oxidized byMia40, which is subsequently oxidizedby Erv1. Erv1 is oxidized by cytochrome c, which is in turnoxidized by cytochrome c oxidase, with molecular oxygen act-ing as the final electron acceptor (19).As mitochondrial endosymbiosis occurred only once in evo-

lutionary history (2), many features of mitochondrial biologyare conserved across wide phylogenetic gaps. With respect toprotein import into mitochondria, it is observed that whereaspore or channel-forming subunits of the membrane-boundtranslocases are well conserved, variability is often seen withother components of the import apparatus (20, 21). Thus, com-parison of the plant import apparatus to that of yeast (andmammalian) systems reveals that although Tom40, Sam50,Tim17 and -23, Tim22, and Oxa1p are well conserved, othercomponents differ to varying extents. Thus, none of the threefunctionally characterized protein import receptors of plantmitochondria is orthologous to yeast or mammalian proteinimport receptors (22). Furthermore, the mitochondrial pro-cessing peptidase is membrane-bound in plants compared withmatrix located in yeast (23). In contrast, the presequencedegrading peptidase is located in the matrix in plants, whereasits orthologous counterpart in yeast is located in the intermem-brane space (24, 25).To date no studies have been carried out on components of

the MIA import pathway in plants. As an essential pathwayin yeast, it might be expected to function in a similar manner inplants. We have analyzed the function of Mia40 and Erv1 inArabidopsis thaliana with the aim of defining the essentialcomponents of theMIA pathway, their location, and the effectsof inactivation.

EXPERIMENTAL PROCEDURES

cDNA Clones and Constructs—The chromosomal loci forAtMia40 (At5g23395) and AtErv1 (At1g49880) have been pre-viously identified (26). TheOryza sativa (rice)Mia40was iden-tified as Os04g44550 by using AtMia40 to search the ricegenome (27). cDNAs of AtMia40, AtErv1, and OsMia40 werecloned using gateway cloning (Invitrogen) into vectors express-ing GFP as either N- or C-terminal fusions under the controlof a constitutive promoter (28). The human Mia40 cDNA(AAH33775) was cloned as both N- and C-terminal fusionswithGFP into pcDNA3 (Invitrogen) under the control of a con-stitutive promoter using standard cloning techniques (29, 30).The full cDNA of A. thaliana Tim9 (At3g46560), copper chap-erone for CSD1 (Ccs1, At1g12520), copper/zinc superoxidedismutase 1 (CSD1, At1g08830), copper/zinc superoxide dis-mutase 3 (CSD3, At5g18100), and carbonic anhydrase 2 (CA2,At1g47260) were cloned into pDest14 (Invitrogen) using gate-way-cloning techniques (Invitrogen) for in vitro transcriptionand translation. The cDNA clones encoding the following pro-teins have been described previously: AOX (X68702) (31), Pic(ABO16064) (32, 33), and NDC1 (At5g08740) (34).GFP Subcellular Localizations—To determine the subcellu-

lar localization of AtMia40 and OsMia40 A. thaliana cell cul-ture was transformed by biolistic transformation as previouslydescribed (28). GFP and RFP expression and targeting werevisualized using a BX61 Olympus microscope (Olympus) usingexcitation wavelengths of 460/480 nm (GFP) and 535/555 nm

(RFP) and emission wavelengths of 495–540 nm (GFP) and570–625 nm (RFP). Subsequent images were captured usingCell imaging software as previously described (28). For theHsMia40, 143B osteosarcoma cells were plated onto 13-mmdiameter glass coverslips and allowed to attach overnight.Cells were transfected using FuGENEHDwith both GFP andRFP plasmids for 48 h and washed with Tris-buffered saline(5 mM Tris/HCl (pH 7.4), 20 mM NaCl). Cells were mountedin DABCO (1,4-diazabicyclo(2.2.2)octane)/polyvinyl alco-hol medium. Images were acquired using an Olympus DP70fluorescent inverted microscope as previously described (29).T-DNA Insertion Lines—The following T-DNA insertion

lines were obtained from the SALK collection (35) and geno-typed by PCR to confirm homozygosity for the T-DNA in-sert: AtMia40 (At5g23395): SALK_044358 and AtErv1(At1g498890): SALK_110883, SALK_131166, SALK_001649.Organelle Purification—Mitochondria for in vitro import

experiments were harvested from 20 g (fresh weight) of 14-day-old A. thaliana seedlings grown in liquid culture as previouslydescribed (22) with the BSA omitted from the last two washsteps. Typically 2–3mg ofmitochondrial protein was obtained.For immunodetection assays, highly purifiedmitochondria andperoxisomes were purified from 7-day-old A. thaliana cell sus-pension using free flow electrophoresis as described by (36).In Vitro Import Studies—[35S]Met-labeled precursor pro-

teins were synthesized using rabbit reticulocyte TNT in vitrotranscription/translation lysate (Promega, Melbourne, Austra-lia) as described previously (37). The use of equivalent quanti-ties of mitochondria from different genotypes in import reac-tions was ensured by triplicate measurement of proteinconcentration with the Coomassie protein assay reagent(Thermo Scientific, Rockford, IL). Time course analysis of pre-cursor protein import into intact mitochondria isolated fromwild type (Col-0) or mutant plants was performed as describedpreviously (22, 37). Proteinase K protected, mature, radiola-beled protein was quantified at each time point and normalizedto the highest time point measurement for replicate experi-ments (22).BN-PAGE Imports—For in vitro imports analyzed on BN-

PAGE gels, import assays were performed as for SDS-PAGEexcept 250 �g of mitochondria were used per time point. Afterthe import was carried out for the required time, mitochondriawere pelleted at 20,000 � g for 5 min and then subjected toBN-PAGE according to the method described by Jansch et al.(38). Mitochondrial proteins (250 �g) were solubilized with 5%(w/v) digitonin in a buffer containing 30 mM HEPES, 150 mM

potassium acetate, and 10% (v/v) glycerol (pH 7.4) and incu-bated on ice for 15min. Samples were centrifuged for 20min at15,000 � g, and Serva Blue G (0.2% (v/v) final) was added to thesupernatant. Samples were loaded onto a 4.5–16% (v/v) gradi-ent gel. After migration, gels were fixed in 40% (v/v) methanol,10% (v/v) acetic acid, dried, and exposed as per SDS-PAGE gels.Immunodetection of Proteins—Mitochondria and peroxi-

somes (25 �g) were resolved by SDS-PAGE, transferred toHybond-C extra nitrocellulose membrane, and immunode-tected as previously outlined (39). To generate antibodiesto AtMia40, AtErv1, AtTim23-2 (At1g72750), AtSam50(At3g11070), and the Rieske iron sulfur protein (RISP,

Mia40 Functions in Protein Assembly in Arabidopsis

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At5g13430), recombinant proteins containing the full-lengthAtMia40 and AtErv1, amino acids 31–144 for AtTim23-2, thefirst 200 amino acids of AtSam50, and amino acids 21–151 ofRieske iron sulfur protein fused to an N-terminal His6 affinitypurification tag were expressed in Escherichia coli strain BL21(DE3)pLys (Stratagene, La Jolla, CA). The recombinant proteinwas purified by denaturing immobilizedmetal affinity chroma-tography (IMAC) using the Bio-Rad Profinia protein purifica-tion system. The resultant eluate was separated by SDS-PAGE,and the recombinant protein was extracted using a Bio-RadModel 422 Electro-Eluter. Buffer exchange was performedusing an Amicon Ultracel 5K centrifugal filter device (Milli-pore, Sydney, Australia) such that the antigen was re-sus-pended in PBS solution, recovering a total of 1 mg (AtErv1), 2mg (AtMia40), 2mg (AtTim23-2), 3mg (AtSam50), and 3mgofRieske iron sulfur protein for inoculation. Four separate doseswere administered to a rabbit at regular intervals over a3-month period using standard protocols and Freund’s com-plete adjuvant solution (40). Other antibodies used in this studyhave been described previously: 3-ketoacyl-CoA thiolase (Kat2)(41), Tim17-2 (42), Tom20-2, andTom20-4 (22), Tom20-3, andTom40 (28), coxII, ATP synthase, Ccs1, and CSD1 wereobtained from Agrisera (Vannas), voltage-dependent anionchannel protein (VDAC; PM035) andE1� of pyruvate dehydro-genase (PM030) were obtained from Dr. Tom Elthon (Univer-sity of Nebraska, Lincoln, NE), alternative oxidase (43), andNAD9 (44). The altered inflorescence meristem 1 (Aim1) anti-body was obtained from Dr. Douglas Muench (University ofCalgary).Complex I Activity Assays—Complex I activity wasmeasured

on 140 �g of mitochondria in 1 ml of respiration buffer (0.3 M

sucrose, 5mMKH2PO4, 10mMTES, 10mMNaCl, 2mMMgSO4,and 0.1% (w/v) BSA (pH 6.8)) obtained after freeze-thawing ofthe mitochondria using a Clark-type oxygen electrode (Han-satch Instrument). 1 mM deamino-NADH was added to thechamber, oxygen consumptionwasmeasured, and 2mMCCCPwas then added to ensure mitochondria were uncoupled. Rote-none (5 mM) was then added to inhibit complex I and to mea-sure the rate of oxygen consumption of the alternative NADHdehydrogenases. Finally 0.1 mM of KCN was used to terminatethe reaction. The activity of complex I was defined as therotenone-sensitive rate of oxygen consumption, determinedby subtracting the rate of oxygen consumption of the alter-native NADH dehydrogenases from the total oxygen rateconsumption.Global Transcript Analysis—Analysis of the changes in tran-

script abundance between Col-0 and �mia40 plants in 18-day-old A. thaliana seedlings was performed using AffymetrixGeneChipTM A. thaliana ATH1 Genome Arrays (Affymetrix,Santa Clara, CA). Green tissue from three seedlings was pooledfor each biological replicate; Col-0 and �mia40 tissue sampleswere collected in biological triplicate. For each replicate, totalRNA was isolated from the leaves using the RNeasy Plant MiniProtocol (Qiagen, Clifton Hill, Australia) and quality-verifiedusing a Bioanalyzer (Agilent Technologies, Palo Alto, CA), andspectrophotometric analysis was carried out to determine theA260:A280 and A260:A230 ratios. Preparation of labeled aRNAfrom 500 ng of total RNA (3� IVT Express kit, Affymetrix) and

target hybridization as well as washing, staining, and scanningof the arrays was carried out exactly as described in theAffymetrix GeneChipTM Expression Analysis Technical Man-ual using an Affymetrix GeneChip Hybridization Oven 640, anAffymetrix Fluidics Station 450, and an GeneChip Scanner3000 7G at the appropriate steps.Statistical Analysis—Data quality was assessed using GCOS

1.4 before CEL files were exported into AVADIS Prophetic(Version 4.3, Strand Genomics, San Francisco, CA) and PartekGenomics Suite software, Version 6.3 (Partek, St. Louis, MO)for further analysis. MAS5 normalization algorithms were car-ried out to generate present/absent calls across the arrays. Onlythose probe sets that were called present in at least two of threereplicates in at least one genotype were included for furtheranalysis. Ambiguous probe sets and bacterial controls wereremoved, resulting in a final data set of 12,551 probe identifiers.CEL files were also subjected to guanosine cytosine robustmulti-array average normalization. Correlation plots wereexamined between all arrays using the scatter plot function; inall cases r � 0.97 (data not shown). Guanosine cytosine robustmulti-array average-normalized gene expression values wereanalyzed to identify differentially expressed genes by a regular-ized t test based on a Bayesian statistical framework using thesoftware program Cyber-T (45). Cyber-T employs a mixturemodel-based methods described by Allison et al. (46) for thecomputation of the global false positive and false negative levelsinherent in a DNA microarray experiment. The rates of falsepositives and false negatives as well as true positives and truenegatives at any given p value threshold are estimated, i.e. aposterior probability of differential expression (PPDE) (p) valuefor each genemeasurement and a PPDE (�p) value at any givenp value threshold based on the experiment-wide global falsepositive level and the p value exhibited by that gene. There were322 unique transcripts that were identified as significantly dif-ferentially expressed in the �mia40 plants compared withCol-0 after false discovery rate correction at PPDE (�p) �0.95(95% confidence interval) (supplemental Table 2). Functionalcategorization using GO-biological process annotations wasperformed on the total present set (12,551 transcripts) alongwith the 322 transcripts defined as differentially expressed(either positively or negatively). GO annotations (biologicalprocess) were obtained from the TAIR webpage. To determineany changes in distributions of different cellular locations, tran-scripts in the total present set and differentially expressed setwere annotated based on their subcellular location: plastid,mitochondrial, peroxisomal, or other. Lists of genes encodingplastid and peroxisomal proteins were generated using theSUBA data base and included genes based on experimentaldetermination (found by mass spectrometry or GFP profiling).The list ofmitochondrial proteins used here has been describedpreviously (47). The percentage distribution of each categorywas compared with that of the total present set using a �2 test,and percentile distributions were considered to be significantlydifferent at a 98% confidence interval. To gain a qualitativeoverview of changes in transcript abundance for�mia40 plantscompared with Col-0, the MapMan software was used (48).Only transcripts with significant changes after false discoveryrate were displayed.



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RESULTS

Erv1 Is an Essential Protein in A. thaliana, but Mia40 IsDispensable—To determine the role of Mia40 and Erv1 inA. thaliana, T-DNA insertion lines that disrupt the genes and,thus, do not produce functional proteins were screened. In thecase of Mia40, one line with a T-DNA insert that disrupted thegene was obtained from screening all lines annotated as havinginsertions in Mia40 (�mia40) (Fig. 1A). The absence of theproteinwas confirmedbyWestern blot analysis against purifiedmitochondria from Col-0 and �mia40 plants (see Fig. 3A). Thelack of Mia40 had no discernable effect on growth (Fig. 1A),with normal seed set production observed compared withCol-0 growth under identical conditions. Even though a singleinsertional mutant could only be obtained for Mia40, theabsence of the protein was confirmed byWestern blotting, andit is the lack of expected phenotype that is reported. Further-

more, backcrossing and Southern blot analysis indicate a singleinsertion in the line characterized.In contrast, despite screening three T-DNA lines for Erv1, no

homozygous T-DNA-inactivated lines for Erv1 could beobtained. In addition to back-crossing the T-DNA lines toremove any other T-DNA inserts or mutations that may bepresent, analysis of the seed from self- fertilized heterozygousplants (Erv1/erv1-T-DNA) consistently resulted in 25% of theseed being aborted for all lines analyzed (Fig. 1B). This is con-sistent with a lethal phenotype due to the absence of a func-tional gene encoding this protein.The lethality of �erv1 was not surprising as it is an essential

protein in yeast. In yeast it is required for the import and assem-bly of small intermembrane space proteins (16, 49), specificallythe small Tims that in turn are required for the import of carrierproteins to the inner membrane. They also play a role in �-bar-rel protein assembly in the outer membrane (3, 50). Therefore,a similar phenotype would be predicted for �mia40 plantsbased on extensive studies in yeast andmammalian systems (12,15, 51, 52). Examination of the sequences for both predictedproteins in A. thaliana revealed that they differed comparedwith their yeast orthologues. In the case ofA. thalianaMia40 itwas a much smaller protein of 162 amino acids (supplementalFig. 1A). ThemammalianMia40 protein is also shorter than theyeast orthologue, and the smaller “core” conserved region ofMia40 has been shown to be functional in the disulfide relaysystem (53). The only noticeable “unique”feature of theA. thaliana Mia40 was the presence of a putative peroxisomalPTS1 targeting signal, SKL, at theC terminus (54). Examinationof the A. thaliana Erv1 protein sequence revealed that it dif-fered in its arrangement of cysteine pairs to that of yeast andhumans (supplemental Fig. 1, B and C). In all Erv1 sequencesanalyzed to date two pairs of cysteinemotifs are conserved in allorganisms, the CXXC and CX16C motifs. In regard to the thirdcysteine pair, yeast and human Erv1 proteins have an N-termi-nal CXXCmotif, whereas A. thaliana has a C-terminal-locatedCXXXXC motif (Fig. 1C) (55). This cysteine motif shows dis-tinct similarities to the yeast endoplasmic reticulum Erv2p (56)and the trypansome Erv1, both of which also have their thirdcysteine pair at theC terminus (Fig. 1C) (57). Phylogenetic anal-ysis of all Erv1 and Mia40 protein sequences revealed that theplant proteins formed distinct groups (supplemental Fig. 2),noticeably the trypanosome Erv1 branches closest to the plantgroup, this group being the only group containing the thirdcysteine pair at the C-terminal end of the protein (Fig. 1C, sup-plemental Fig. 2).Mia40 Is Targeted to Mitochondria and Peroxisomes in

Plants—We fused A. thaliana Mia40 to GFP at its N and Ctermini to determine whether the predicted peroxisomal PTS1targeting signal was functional. When GFP was fused to the Nterminus, a pattern identical to peroxisomal-targeted RFP wasseen (Fig. 2A,GFP-AtMIA40). AsMia40 has been characterizedas an exclusively mitochondrial protein in yeast and mamma-lian systems (organisms that also contain peroxisomes), it wasinvestigated if the peroxisomal targeting ability of A. thalianaMia40 was a general feature of plant Mia40 proteins by testingthe targeting of the riceMia40. A similar result was observed in

FIGURE 1. T-DNA insertional inactivation of A. thaliana Mia40 and Erv1.A, shown is a picture of 25-day-old Col-0 and �mia40 A. thaliana plants.B, shown is a representative picture of seeds from Col-0 and �erv1 (heterozy-gous) self-fertilized plants. Arrows indicate aborted seed. C, shown is a sche-matic diagram of Erv1 proteins from different organisms. The gray regionrepresents the most conserved region between different Erv1 sequencescontaining the redox center, the FAD binding site, and two conserved cys-teine domains (CXXC and CX16C ). The location of the final cysteine pair differsbetween organisms. Yeast Erv1 and human Erv1 have it at the N terminus, andthose for yeast Erv2, A. thaliana, and trypanosomes are at the C terminus.Yeast, S. cerevisiae; human, Homo sapiens; Arabidopsis, A. thaliana; trypano-somes, Trypanosoma brucei.



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that rice Mia40 was targeted to peroxisomes when GFP wasplaced at its N terminus (Fig. 2A, GFP-OsMIA40).When GFP was fused to the C terminus of both A. thaliana

and rice Mia40, a mitochondrial pattern of florescence wasobserved (Fig. 2A, top panel). Noticeably, the fluorescence pro-duced ring like structures, which we have previously observedfor mitochondrial proteins linked to GFP (22). They may rep-resent GFP that has been targeted to mitochondria but nottranslocated across the outer membrane, possibly due to thelack of any pulling force by an intermembrane space protein

such as Mia40. High backgroundfluorescence in the cell cytosol wasalways evident with both A. thali-ana and rice Mia40 with GFP fusedto the C terminus despite testing inseveral cell types including onion,A. thaliana leaf, and root (data notshown). This high background fluo-rescence is not observed for a vari-ety of other mitochondrial targetingsignals and is unlikely to be a tech-nical limitation of the system (28,39). Additionally, the subcellularlocalization of Mia40 to both mito-chondria and peroxisomes was con-firmed using Western blotting onhighly purified mitochondrial andperoxisomal fractions using anantibody raised against A. thalianaMia40 (Fig. 2B). This antibodydetected a specific protein in bothhighly purified mitochondria andperoxisomes purified by free flowelectrophoresis (36). No Tim17-2protein or E1�-subunit of pyruvatedehydrogenease (E1�-PDH)wasde-tected in peroxisomes, indicatingthe purity of the peroxisomal frac-tions, thus verifying this novel per-oxisomal location for Mia40 (Fig.2B). The absence of significantperoxisome contamination in themitochondrial fraction was con-firmed using two peroxisomalmarkers, Kat2 and Aim1. Bothmarkers show that the mitochon-drial fraction was free of anysignificant peroxisome contami-nation. As A. thaliana Mia40 ap-peared to target GFP poorly tomitochondria, we investigated theimport of A. thaliana Mia40 intoisolated mitochondria (Fig. 2C).A. thaliana Mia40 was importedinto a protease-protected locationin a membrane potential-indepen-dent manner (Fig. 2C, lanes 1–5),consistent with a location in the

intermembrane space. Rupture of the outer membranebefore adding protease resulted in digestion of importedMia40 (Fig. 2C, lanes 6–9). Import of A. thaliana Tim23 as acontrol verified that the membrane potential was collapsedas import of Tim23 was inhibited in the presence of valino-mycin (Fig. 2C, lanes 1–5) and that the inner membrane wasintact, as evidenced by the presence of a characteristic mem-brane-protected fragment of Tim23 upon the addition ofprotease when the outer membrane was ruptured (Fig. 2C,lanes 6–10).

FIGURE 2. Determination of the targeting ability of Mia40 using GFP tagging, Western blots, and in vitroimport assays. A, the localization of A. thaliana, rice, and human Mia40 was determined by fusing them to GFPat their N and C termini. A. thaliana and rice Mia40 GFP plasmids were transformed into A. thaliana suspensioncells, and human Mia40 GFP plasmids were transfected into 143B osteosarcoma cells. For the A. thalianasuspension cell cultures, the RFP was targeted to mitochondria using the mitochondrial alternative oxidasetargeting signal as a mitochondrial marker and RFP targeted to peroxisomes using a C-terminal SRL sequenceas a peroxisomal marker. For the 143B osteosarcoma cells, RFP was targeted to mitochondria using the mito-chondrial targeting signal from yeast cytochrome c oxidase 4 as a mitochondrial marker and RFP targeted toperoxisomes using a C-terminal SRL sequence as peroxisomal marker. B, Western blot analysis is shown ofisolated mitochondria and peroxisomes with antibodies raised against A. thaliana Mia40, two peroxisomalmarkers, Kat2 and AIM1, and two mitochondrial markers, Tim17-2 and E1� of pyruvate dehydrogenase (E1�-PDH). C, in vitro import of radiolabeled Mia40 into isolated mitochondria is shown. Lane 1, precursor proteinalone. Lane 2, precursor protein incubated with mitochondria under the conditions that support import intomitochondria. Lane 3, as lane 2 with proteinase K added after the incubation of precursor with mitochondria.Lanes 4 and 5, as lanes 2 and 3 with valinomycin added to the import assay before the addition of precursorprotein. Lanes 6 –9, as lanes 2–5 except that the mitochondrial outer membrane was ruptured after the incu-bation period with precursor protein but before the addition of proteinase K. Mit, mitochondria; Mit-OM,mitochondria with the outer membrane ruptured; PK, proteinase K; Val, valinomycin; p, precursor protein band;m*, inner membrane protected fragment of Tim23.



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Analysis of humanMia40 showed that it was only targeted tomitochondria when GFP was fused to the C-terminal end ofhuman Mia40 (Fig. 2A, HsMIA40 GFP). Fusing GFP to theN-terminal end of human Mia40 resulted in no targeting ofGFP as evidence by fluorescence throughout the cytoplasm(Fig. 2A,GFPHsMIA40). Notably, analysis of the targeting abil-ity of A. thaliana Erv1 revealed that it is only targeted to mito-chondria (data not shown).

Cellular Affects of Inactivation of�mia40 inA. thaliana—AsA. thali-ana Mia40 is not an essential pro-tein in A. thaliana, we investigatedthe abundance of variousmitochon-drial and peroxisomal proteins togain insight into the function(s) ofMia40 (Fig. 3). Analysis of the abun-dance of a variety of protein importcomponents in �mia40 plants re-vealed that Erv1 and Tim23-2 hadincreased in abundance, but manyother components were unchanged(Fig. 3A). Examination of a variety ofother mitochondrial proteins re-vealed that although many wereessentially unchanged in abun-dance, there were notable excep-tions (Fig. 3B). First, there was a lackof CSD1, the intermembrane spacelocated copper/zinc superoxide dis-mutase (58, 59), and the chaperoneprotein associatedwithCSD1, Ccs1,which plays a role in inserting cop-per into the active site of CSD1 (58,59). The Nad9 subunit of complex Ialso decreased in abundance signif-icantly (40%). The reduced amountof Nad9 on SDS-PAGE gels wasconfirmed by BN-PAGE (Fig. 3C),which indicated that the reductionof the amount of Nad9was linked toa reduction in the amount of com-plex I in�mia40mitochondria. Thecapacity of complex I was measuredusing deamino NADH with mito-chondria that had been subjected tofreezing and thawing to allowdeamino-NADH access to the ma-trix side of the inner membrane inthe presence of CCCP to ensureunrestricted flow of electrons. Therotenone-sensitive deamino NADH-dependent oxygen consumption was13.9nmol ofO2/min/mgof protein inmitochondria from wild type plantscompared with 6.1 nmol of O2/min/mg of protein in mitochondriafrom �mia40 plants. This indicatedthat complex 1 capacity was reduced

by 44% in mitochondria from �mia40 plants, consistent with thepercentage reduction of the Nad9 protein from complex I.The availability of antibodies to peroxisomal proteins in plants

is limited.NeverthelessWesternblot analysis using three antibod-ies revealed that two proteinswere altered in abundance (Fig. 3D).Although levels of Kat2were unchanged in abundance, CSD3wascompletely absent in peroxisomes from �mia40 plants, whereasAim1 was reduced in abundance by 25%.

FIGURE 3. Western blot analysis to determine the affect of deleting A. thaliana Mia40 on mitochondrialand peroxisomal proteins and blue native gel analysis of �mia40 mitochondria. A, Western blot analysisof mitochondria isolated from Col-0 and �mia40 plants with a variety of antibodies raised against mitochon-drial import components is shown. Numbers on the side represent the relative abundance of the protein in the�mia40 mitochondria expressed relative to Col-0 (1); S.E. is also shown for three biological replicates. B, West-ern blot analysis of mitochondrial proteins is not involved in protein import. VDAC, voltage-dependent anionchannel protein; AOX, alternative oxidase; E1�-PDH, E1�-subunit of pyruvate dehydrogenease; RISP, Rieskeiron sulfur protein. C, blue native gel analysis of Col-0 and �mia40 mitochondria is shown. Mitochondrialmembrane complexes were separated by BN-PAGE and transferred onto a PVDF membrane. Membranes werestained in Coomassie and probed with antibodies against Tom40 and NAD9. D, shown is Western blot analysisof peroxisomes isolated from Col-0 and �mia40 plants with a selection of antibodies raised against peroxi-somal proteins.



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Analysis of Protein Import into Mitochondria of �mia40Plants—To investigate if the changes observed in the amount ofmitochondrial proteins were due to changes in the rate ofimport or stability of imported proteins, in vitro import assayswere carried out with a number of mitochondrial proteins intomitochondria isolated from wild type and �mia40 plants (Fig.4). A variety of precursor proteins were used; the alternativeoxidase, alternative NAD(P)H dehydrogenase (NDC1), a sub-unit of complex 1 that has been labeled as CA2 (60, 61) alongwith CSD1 and Ccs1. Tim9 was used to test the uptake of smallTim proteins into the intermembrane space (17). Finally, weanalyzed the import of the phosphate carrier (Pic), which isimported via the carrier import pathway utilizing the small Timproteins of the intermembrane space (3, 4). Although theuptake of alternative oxidase and NDC1 was reduced to �50%compared with wild type, the import of CSD1 and CA2 wasunaffected, with the import of the former slightly higher in

mitochondria from �mia40 plants.Import of Ccs1 was reduced by 20%in mitochondria from �mia40plants compared with wild type.Thus, although the abundance ofsome proteins was reduced, this didnot result from alternations the rateof import. Note that as Nad9 is amitochondrially encoded subunit inA. thaliana (62), its rate of importcould not be tested.The import of both Tim9 and

Pic were unaffected (Fig. 4, A andB). The latter two proteins wouldbe expected to be affected by�mia40 A. thaliana mitochondriaas Tim9 is a direct substrate ofMia40 in yeast and the import ofcarrier proteins (Pic) depends onthe function of small Tim pro-teins. It has been previously dem-onstrated in plants that smallintermembrane space proteins areinvolved in the import of carrierproteins (22). Thus, the lack of aphenotype for �mia40 plants andthe lack of an effect on proteinimport via the carrier importpathway suggests that Mia40 inA. thaliana does not play an essen-tial role in the disulfide relay systemfor small Tim proteins as observedin yeast.The reduction in the amounts of

the proteins outlined above can beexplained by either a failure to cor-rectly assemble these proteins, al-tered expression of the genes thatencode these proteins, or a combi-nation of both. To investigate bothof these possibilities, the assembly

of proteins into mitochondria after import was investigated(Fig. 4C), and alterations in transcript abundance were ana-lyzed (Fig. 5).To investigate the assembly of protein after import, the

assembly of a complex I protein was analyzed. Assembly ofimported CA2 into complex I was investigated using BN-PAGE to assess the location of newly imported radiolabeledproteins, as previously carried out for a number of studiesinvestigating assembly into multisubunit protein complexesin mitochondria (63, 64). It was evident that incorporation ofCA2 into complex I, especially the supercomplex of complexI and III, was reduced in mitochondria from �mia40 plantscompared with wild type plants (Fig. 4C). In addition toobserving reduced radiolabeling into this higher molecularmass complex, the intensity of radiolabeling at the lowerregions of the gel was also altered (Fig. 4C). Although theintensity of radiolabeling at the lower regions was higher in

FIGURE 4. In vitro uptake assays into mitochondria isolated from Col-0 and �mia40 plants. A, import of thealternative oxidase (mitochondrial (m) and (p) AOX), alternative NAD(P)H dehydrogenase (NDC1), Tim9, phos-phate translocator (Pic), copper/zinc superoxide dismutase 1 (CSD1), Ccs1, and CA2 precursor proteins intoisolated mitochondria is shown. p, precursor protein; m, mature protein, where indicated. B, shown is quanti-fication of the rate of import of the various precursor proteins into mitochondria. The amounts of import intoCol-0 mitochondria after 20 min was set to 1, and all other values are expressed in a relative manner. C, importof the complex I subunit CA2 into Col-0 and �mia40 mitochondria and analyzed by BN-PAGE is shown.



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mitochondria from �mia40 plants compared with wild typeat 10 and 30 min, it was substantially decreased at 120 and180 min. This suggests that it is the assembly of subunits intocomplex I that is affected in mitochondria from �mia40plants compared with wild type plants. Furthermore, theunassembled, imported, radiolabeled CA2 appeared to bedegraded in mitochondria from �mia40 plants as evidencedby the decrease in the intensity of lower molecular massproducts with time (Fig. 4C).

To gain further insight into the effects of deleting Mia40,the transcriptome of �mia40was compared with that of wildtype plants. 322 transcripts were altered in abundance in the�mia40 plants and were significantly overrepresented in theGO annotation-response to stress (biological processesfunctional categories), representing 8.4% of the changesobserved compared with 5.7% in the genome. Furthermore,analysis of the predicted location of the proteins of geneswhose transcripts changed in abundance revealed that pro-teins predicted to be located in peroxisomes were signifi-cantly overrepresented by 6.8% compared with the 1.5%observed in the genome (Fig. 5A). A MapMan overview ofdifferentially expressed genes revealed that transcriptsinvolved in responses to biotic stresses were overrepre-sented, including four superoxide dismutase genes (threecopper zinc and one manganese) (Fig. 5B). Thus, overall per-oxisomal function seemed to be affected to a greater extentthan mitochondrial function at a transcript level. Specifi-cally, transcripts from genes encoding CSD1, CSD2, and

Ccs1 were decreased in abundanceby more than 2-fold, with signifi-cant decreases also observed forCA1 (reduced �1.9-fold). Thiscorrelated with the decreasein protein observed by Westernblotting.

DISCUSSION

Characterization of the functionsof Mia40 in A. thaliana reveal con-served and novel functions com-pared with studies in yeast. Theabsence of CSD1 and Ccs1 in mito-chondria from �mia40 plantswould be predicted from studies inyeast, and thus, this appears to be aconserved function of Mia40 acrosswide phylogenetic gaps. However,the decrease in amount and activityof complex I by �40% represents anadditional role for Mia40 that hasnot been previously reported. Addi-tionally, Mia40 does not play anessential role in the import and/orassembly of small Tim proteins intoA. thaliana mitochondria, as evi-denced by the fact that the carrierimport pathway operated normally.Mia40 has also taken on additional

novel roles in A. thaliana; it is also located in peroxisomeswhere it is required for the assembly of CSD3 and affects theabundance of a protein (Aim1) associated with �-oxidation offatty acids. The changes in transcript abundance for manygenes encoding peroxisomal proteins suggest that many moreproteins are likely to be affected. Finally the absence of Mia40results in an alteration of the basal transcriptome in A. thali-ana. Global transcript analysis illustrated that for many of theproteins that decreased in abundance in Western blots, tran-script abundances also decreased, suggesting that not only isMia40 required for their assembly and stability, but that in itsabsence signals from the organelles result in these pathwaysbeing down-regulated.The functions of Mia40 in A. thaliana differ considerably

compared with those observed in yeast. Our results indicatethat it is possible for themitochondrial disulfide relay system tofunction without Mia40 in A. thaliana. This is consistent withthe findings in trypanosomes, which also lack aMia40 gene butstill successfully import small Tim proteins into the intermem-brane space (65). The different arrangement of cysteine pairs inErv1 in A. thaliana (plants) and trypanosomes (57) may allowErv1 in these organisms to function alone as a replacement oftheMia40/Erv1 system in yeast. Another possibility is that Erv1functions in another, as yet unknown, pathway or with otheruncharacterized components. Notably, the A. thaliana Erv1protein could not complement a yeast erv1 mutant, indicatingthat it functions differently (supplemental Fig. 3), as observedpreviously (55). Furthermore, the amount of Erv1 significantly

Plas d: 9.62%Mitochondria: 6.39%Peroxisome: 1.56%other cellular loca on: 82.43%

Plas d: 9.63%Mitochondria: 7.14%Peroxisome: 6.83%other cellular loca on: 76.40%

Transcripts ∆mia40 - 322 transcripts

FIGURE 5. Analysis of the changes in transcriptome of A. thaliana �mia40 plants. A, shown is the propor-tion of transcripts encoding proteins located in mitochondria, plastids, and peroxisomes in Col-0 and �mia40plants. B, shown is a MapMan visualization of changes in transcript abundance of genes encoding proteinsinvolved in stress and redox metabolism. FDR, false discovery rate; PPDE, posterior probability of differentialexpression.



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increased in A. thaliana �mia40 plants, indicating thatalthoughA. thalianaMia40may not be an essential componentfor the Erv1 disulfide relay pathway in A. thaliana mitochon-dria, it may still participate in the process. Finally, the increasein Erv1 did not compensate for all of the functions of Mia40 inmitochondria from A. thaliana as both CSD1 and Ccs1 wereabsent in �mia40 A. thaliana plants, in addition to the reduc-tion in the amount and activity of complex I.The reasons for this difference in the MIA pathway between

yeast and plants is unclear, but in plants it may relate to the factthat the intermembrane space is also the location of ascorbatebiosynthesis (26), and ascorbate can participate in oxidativeprotein folding (66). No change in total ascorbate was detectedfor �mia40 plants (supplemental Fig. 4). Thus, either the plantErv1 protein can function alone in the disulfide relay system, orif Mia40 is present, it is not essential and can be compensatedfor by an increase in the abundance of Erv1. It cannot be ruledout that other components that may be unique to the inter-membrane space in plants replace the function of Mia40. Notethat a calculation of the redox potentials of components of themitochondrial disulfide relay system suggest that shuttlingelectrons from a protein substrate (redox potential �310 to�340mV) viaMia40 (redox potential�200mV) to Erv1 (redoxpotential�320mV) presents a thermodynamically unfavorablereaction thatmay be overcome by a variety ofmechanisms (57).Thus, the absence of Mia40 in this system does not present anymechanistic barrier to the operation of a disulfide relay.Mia40 acts as a receptor for proteins imported in the inter-

membrane space in yeast (3). In A. thaliana (and by inferenceplants in general) the outermembrane receptor componentsdiffer from the yeast andmammalian systems. The plant outer-membrane Tom20 and OM64 proteins are not orthologous toyeast Tom20 and Tom70 (22, 67–69). Although A. thalianaMia40may be orthologous to yeast Mia40, it differs in function(and location). Recently, in yeast it has been shown that Mia40binds a specific nine-amino acid motif, intermembrane space-targeting signal (ITS) (70). A difference in this intermembranespace-targeting signal sequencemay represent a differentmodeof import for plant intermembrane space proteins comparedwith yeast. However, when comparing the A. thaliana inter-membrane space-targeting signal sequences to yeast, they showa high degree of similarity with at least one of the two crucialamino acids being identical across both species (70) (data notshown). This demonstrates that the binding region in the sub-strates ofMia40 is the same in yeast andA. thaliana, thus, indi-cating that this is unlikely to be the reason for the differentfunctions between yeast and A. thalianaMia40 proteins.Although there was no deleterious phenotype associated

with the deletion of Mia40, there were a number of changes inprotein abundance in mitochondria and peroxisomes and areduction in the import of some precursor proteins via the gen-eral import pathway. The abundances of Erv1 and Tim23-2were increased, but no other significant changes in componentsof the mitochondrial protein import apparatus were observed.Some components showed a slight decrease, such as Tom20-2,Tom20-3, andTom20-4 and the cytochromebc1 complex, indi-cated by the reduction in the Rieske FeS protein. However, asinactivation of two of three Tom20 protein isoforms inA. thali-

ana does not greatly affect protein import via the generalimport pathway (22), these changes in Tom20 isoforms aloneare unlikely to account for the observed changes in proteinimport.A decrease in the Nad9 subunit of Complex I of the respira-

tory chain and a parallel decrease in complex I activity wasobserved in �mia40 plants. The abundance of the other respi-ratory complexes did not differ from mitochondria from wildtype plants. Additionally, no change was observed for the alter-native oxidase at a protein or transcript level, indicating nogeneral mitochondrial stress as a result of deletion of Mia40(47). Finally,�mia40 plants did not display any of the growth ordevelopmental abnormalities associated with the absence ofcomplex I that have been previously characterized in A. thali-ana and tobacco (71, 72). As the import ability of a variety ofproteins was largely unaffected, including several complex Isubunits (CA2 and NDUFS8) (60), the reduction in the rate ofprotein import for some proteins is unlikely to be the cause forthe changes in protein abundances observed. Previously wehave characterized A. thaliana plants that have all functionalTom20 receptor components inactivated. Although import viathe general import pathwaywas decreased by 80%, therewas nodetectable affects on plant growth or abundance of complex I orother respiratory chain complexes (22). Thus, the rate of pro-tein import does not affect respiratory complex abundance inmitochondria in A. thaliana.A study analyzing the phylogenetic distribution of proteins

that are substrates for the intermembrane disulfide relay sys-tem revealed either two CX3C or two CX9C motifs with sizesbetween 9 and 18 kDa. From this study it was concluded thatthese proteins were an ancient family that are widespread invarious eukaryotic lineages (74). The functions ofmany of theseproteins (cmc1, cox17, cox19, pet 191, and som1) have beeninvestigated in yeast and are associated with cytochrome c oxi-dase assembly and to a lesser extent in the cytochrome bc1complex (12, 74). This is in contrast to what was observed herewhere cytochrome oxidasewas unaffected, and the cytochromebc1 complex was minimally affected. However, as yeast (S. cer-evisiae) lack complex I, any role for Mia40 in complex I assem-bly (or stability) would not be uncovered using this system. Ithas been reported that mutations in the human orthologue ofErv1, GFER (growth factor, augmenter of liver regeneration)results in a reduction in activity of complex I, II, and IV activity(52). However in this study, although the absence of Mia40results in an absence of Ccs1 in mitochondria, there does notappear to be any affect on assembly of cytochrome oxidase thatmight be associated with altered copper homeostasis and/orinsertion into cytochrome oxidase.The absence ofMia40 inA. thaliana also resulted in an alter-

ation of the transcriptome. The majority of the changesobserved were decreases in transcript abundance. Genesencoding peroxisomal proteins were disproportionally affectedcompared with those encoding mitochondrial or plastid pro-teins. This is consistent with the targeting and accumulation ofMia40 to peroxisomes. Several transcripts encoding peroxiso-mal proteins were down-regulated in�mia40. Two short chaindehydrogenase/reductase (SDRa and SDRb) and Kat2 areinvolved in �-oxidation (75). The peroxisomal �-oxidation



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pathway in plants metabolizes saturated long chain fatty acidsto supply energy during germination and seedling establish-ment (76). �-Oxidation not only metabolizes fatty acids butother substrates such as unsaturated fatty acids and hormoneprecursorsmay also be fed into this pathway (76, 77). It has beenproposed that short chain dehydrogenase/reductase proteins inA. thaliana play an important role in this alternate form of the�-oxidation pathway in hormone metabolism, which alsorequires Kat2 to provide the thiolase activity (75). This suggeststhat Mia40 plays some role in fatty acid metabolism in peroxi-somes, consistent with the decrease observed in Aim1 proteinabundance.Another interesting transcript, which decreased in abun-

dance in �mia40, was the homolog of the yeast Ccs1 (copperchaperone for superoxide dismutase 1 (Sod1)), AtCcs1. Mia40in yeast has been shown to be required for the biogenesis ofCcs1 and Sod1 in the intermembrane space of mitochondria(73). In A. thaliana there are three Sod1-like proteins and oneCcs1-like protein. The three Sod1 proteins are located in plas-tids (CSD2), the cytosol (CSD1), and peroxisomes (CSD3), andall three have been presumed to be dependent on the one Ccs1protein for proper function (58). Although no significantdecreases were observed in the import of Ccs1 and CSD1 intomitochondria from�mia40 plants, the lack of these proteins inmitochondria isolated from �mia40 plants indicates thatalthough Erv1 may be sufficient for import of these proteins, itcannot fulfill the role of Mia40 in assembly of Ccs1 and/orCSD1. Likewise, the absence of CSD3 in peroxisomes suggeststhatMia40 is required for the assembly of Ccs1 in peroxisomes.Western blot analysis failed to detect Ccs1 in peroxisomes inA. thaliana, even from wild type plants, suggesting it is presentin very low abundance.

Acknowledgments—We thank Prof. Harvey Millar and Dr. HolgerEubel for assistance with purification of peroxisomes.

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Chapter 7 The mitochondrial protein import apparatus of plants

99

Chapter 7

The mitochondrial protein import apparatus of plants

Chapter 7 The mitochondrial protein import apparatus of plants

100

Foreword to Study VI Study IV identified OM64 as a novel plant mitochondrial import component and

study V demonstrated that the function of Mia40 was different in Arabidopsis compared

with other organisms. This study aimed to determine if the components of the

mitochondrial import apparatus differed within and between plant species and non-plant

species (eg: Saccharomyces cerevisiae and Homo sapiens). This study focused on

identifying all the known mitochondrial import components in a variety of plant

species, including Chlamydomonas reinhartdii, Ectocarpusu siliculosus,

Cyanidioschyzon merolae, Physcomitrella patens, Arabidopsis thaliana, and Oryza

sativa.

The results of this study demonstrates that the core channel forming subunits of

the mitochondrial outer membrane (Tom40 and Sam50) were conserved between all

plant groups and other eukaryotes. However, the receptor components of green plants

(Chlamydomonas reinhartdii, Physcomitrella patens, Arabidopsis thaliana, and Oryza

sativa), in particular, Tom20, were not orthologous to those of yeast and humans, but

are specific to the plant lineage. It was also found that the red algae Cyanidioschyzon

merolae, contains a full Tom22 receptor subunit of the TOM complex, which is

orthologous to the Tom22 receptor of yeast. This is in contrast to the green plant

lineage, where the cytosolic receptor domain of Tom22 has been lost, suggesting that

this loss occurred after the divergence of green plants from red algae. Furthermore, the

receptors of plant mitochondrial import apparatus displayed differences between the

various plant species. Specifically, distinct motifs were present in the receptor-binding

domain of plant Metaxin, which is absent in red algae. Also, the presence of OM64 on

the outer membrane of mitochondria, was found to be only present in the higher plants

Arabidopsis and rice, suggesting it is a recent addition to mitochondrial import

components.

Findings from this study has led to the proposal that the observed functional

divergences are due to the selective pressure to sort proteins between mitochondria and

chloroplasts in plants, resulting in the differences in protein receptor components seen

between plant groups and other eukaryotes.

RESEARCH ARTICLE Open Access

An in silico analysis of the mitochondrialprotein import apparatus of plantsChris Carrie, Monika W Murcha, James Whelan*

Abstract

Background: An in silico analysis of the mitochondrial protein import apparatus from a variety of species; includingChlamydomonas reinhardtii, Chlorella variabilis, Ectocarpus siliculosus, Cyanidioschyzon merolae, Physcomitrella patens,Selaginella moellendorffii, Picea glauca, Oryza sativa and Arabidopsis thaliana was undertaken to determine ifcomponents differed within and between plant and non-plant species.

Results: The channel forming subunits of the outer membrane components Tom40 and Sam50 are conservedbetween plant groups and other eukaryotes. In contrast, the receptor component(s) in green plants, particularlyTom20, (C. reinhardtii, C. variabilis, P. patens, S. moellendorffii, P. glauca, O. sativa and A. thaliana) are specific to thislineage. Red algae contain a Tom22 receptor that is orthologous to yeast Tom22. Furthermore, plant mitochondrialreceptors display differences between various plant lineages. These are evidenced by distinctive motifs in all plantMetaxins, which are absent in red algae, and the presence of the outer membrane receptor OM64 in Angiosperms(rice and Arabidopsis), but not in lycophytes (S. moellendorffii) and gymnosperms (P. glauca). Furthermore, althoughthe intermembrane space receptor Mia40 is conserved across a wide phylogenetic range, its function differsbetween lineages. In all plant lineages, Tim17 contains a C-terminal extension, which may act as a receptorcomponent for the import of nucleic acids into plant mitochondria.

Conclusions: It is proposed that the observed functional divergences are due to the selective pressure to sortproteins between mitochondria and chloroplasts, resulting in differences in protein receptor components betweenplant groups and other organisms. Additionally, diversity of receptor components is observed within the plantkingdom. Even when receptor components are orthologous across plant and non-plant species, it appears that thefunctions of these have expanded or diverged in a lineage specific manner.

BackgroundThe endosymbiotic event giving rise to the origin ofmitochondria is thought to have occurred 1 to 2 billionyears ago [1,2]. Details of the conditions that favouredthis event and the exact identity of the host cell thatengulfed the a-proteobacterial cell are still unclear. Ithas been proposed that the endosymbiosis that gave riseto mitochondria occurred under anaerobic conditions,followed by early diversification of eukaryotic cells [3].For plastids, an endosymbiotic event occurred ~1 billionyears ago when a heterocyst forming cyanobacteriumwas engulfed [4,5]. Over time the loss and/or transfer ofgenes and genomes from the endosymbionts to the hostcell nucleus has resulted in the formation of organelles

with limited coding capacity [6-8]. The majority of pro-teins located in mitochondria and plastids are encodedby nuclear located genes, translated in the cytosol andimported into these organelles. Notably, the proteomesof both mitochondria and chloroplasts are derived froma variety of sources and are not simply a subset of theproteins derived from the ancestral endosymbiont [9]. Inthe most extreme cases, it is thought that all genes thatwere present in the endosymbiont have been lost, result-ing in specialized organelles such as hydrogenosomesand mitosomes [10].Although mitochondria have a single origin there is

variation observed between different mitochondria pre-sent in the major branches of life [10]. Mitochondria inplants contain many unique features compared to theirfungal or animal counterparts. These include a largergenome, ranging from 200 Kb to 2000 Kb in size [11],

* Correspondence: [email protected] Research Council Centre of Excellence in Plant Energy Biology,University of Western Australia, 35 Stirling, Crawley 6009, WA, Australia

Carrie et al. BMC Plant Biology 2010, 10:249http://www.biomedcentral.com/1471-2229/10/249

© 2010 Carrie et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

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extensive cis and trans splicing of introns, [12], relativelyslow rates of mutations [13,14], extensive editing ofmRNA [15] and incorporation of foreign DNA [16].Another notable feature is the presence of a branchedrespiratory chain [17]. Although fungi contain alterna-tive NAD(P)H dehydrogenases and an alternative oxi-dase, these are usually only expressed under conditionswhere the cytochrome chain is inhibited [17]. In con-trast, plant mitochondria contain components of thealternative respiratory pathways which exhibit both con-stitutive and stress induced expression [18]. Further-more, mitochondria of plants and animals havediversified in a lineage specific manner to include orexclude various biochemical pathways, such as theb-oxidation of fatty acids that occurs in peroxisomes inplants and mitochondria in animals [19].In plants, the presence of plastids in cells also adds to

the complexity of protein sorting required to avoidmis-targeting of proteins to organelles. Plastidic andmitochondrial targeting signals, referred to as transitpeptides and presequences respectively, are typicallylocated at the N-terminal end of the protein and areenriched in positively charged residues such as lysineand arginine [20]. It is not known how mis-sorting ofproteins is prevented between plastids and mitochon-dria. A combination of the predicted ability of transitpeptides and presequences to form different secondarystructures, the proposed presence of cytosolic targetingfactors and even targeting of mRNA to the surface oforganelles, may all combine to achieve the observed spe-cificity of protein targeting [21,22]. There is a mechanis-tic difference between recognition of targeting signals bypreprotein receptor proteins in plastids and mitochon-dria, the former involving a GTP/GDP cycle while noenergy requirement is observed for receptor binding inmitochondria. This mechanism among others may con-tribute to the specificity of targeting signal recognitionat the surface of each organelle [23,24].Our knowledge of the mitochondrial protein import

apparatus in plants, both experimental and predicted, islargely derived from studies in Arabidopsis, and to a les-ser extent from Solanum tuberosum (potato). Purifica-tion of the translocase of the outer membrane (TOM)complex from both Arabidopsis and potato revealedthat Tom40 and Tom7 are orthologous with those fromyeast, while Tom20 is not orthologous to yeast or mam-malian proteins [25-27]. The other import receptorscharacterized in yeast (and mammals), Tom70 andTom22, appear to be absent [28,29]. It has been shownthat plant Tom9 is the most likely equivalent to yeastTom22, but lacks the cytosolic receptor domain [30].The mitochondrial processing peptidase has been puri-fied from potato and shown to be integrated into thecytochrome bc1 complex [31,32]. This is also the case in

lower plants examined both in the elkhorn fern Platy-cerium bifurcatum and the field horsetail Equisetumarvense [33]. Biochemical purification of the prese-quence degradation peptidase (PreP) has shown that itis a dual targeted protein and that it is a zinc metallo-protease [34]. Biochemical studies have shown thatsmall intermembrane space proteins also mediate mito-chondrial carrier protein import in potato mitochondria.In addition, the plant TIM17:23 complex differs to thatin yeast in that the Tim17 in Arabidopsis contains a C-terminal extension that must be removed before it cancomplement a tim17 mutant in yeast [35,36].However, there are limited studies on the nature of

the mitochondrial protein import apparatus from otherplants, ranging from single celled algae to monocots.Thus, in order to gain a better overview of the proteinimport apparatus in plants, compared to fungal and ani-mal counterparts, an in silico analysis of these compo-nents was carried out. This was based on the fact thatcomplete genome sequences now exist for the singlecelled green algae, Chlamydomonas reinhardtii (Chloro-phyte) [37] and Chlorella variabilis, an intracellular sin-gle celled green algae photosynthetic symbiont inParamecium bursaria [38], a moss, Physcomitrellapatens (Bryophyte) [39], Selaginella moellendorffii, anancient vascular plant [40], and higher plants Oryzasativa [41], Arabidopsis thaliana [42] and Picea glauca[43] (Spermatophytes) (Figure 1). We have also includedanalysis from brown algae, Ectocarpus siliculosus(Phaeophyceae) [44] and the red algae, Cyanidioschyzonmerolae (Rhodophyta) [45] (Figure 1). Red algae repre-sent a cell lineage with a primary plastid endosymbiosisthat is proposed to have been derived from the sameevent that gave rise to the plastids in green plants, butdiverged from the green plant lineage early after thisendosymbiotic event [46]. Brown algae have obtainedtheir plastids via a secondary endosymbiosis, and con-tain four plastid envelope membranes. Thus, plastid pro-teins are first targeted to the outer membrane via ahydrophobic signal sequence and secondary targetingsignals mediate uptake into plastids [47].

Results and DiscussionTranslocase of the Outer Membrane (TOM)The TOM complex represents the gateway into mito-chondria, through which almost all mitochondrial pro-teins pass (exceptions include Fis1 [48]). It has beencharacterized from yeast, Neurospora, mammals andplants, in particular Arabidopsis. In addition to beingpurified from Arabidopsis, functional studies on theTom20 receptor components show that all three iso-forms can be deleted, resulting in a reduced rate ofimport for several precursor proteins, but no deleteriousphenotypic lesions. The complex typically contains 7


Page 2 of 15

subunits, Tom70, 40, 22, 20 7, 6, and 5, but in Arabi-dopsis Tom22 is replaced by Tom9 and no orthologueto Tom70 can be identified. Arabidopsis contain a pro-tein termed OM64 that is not present in yeast or mam-mals, which appears to play a role as an importreceptor.The TOM complex fulfills the vital function of specifi-

cally recognizing mitochondrial proteins from the poolof all proteins synthesized in the cytosol. Of the sevencomponents characterized biochemically to be presentin the TOM complex from yeast, only Tom40 is con-served between yeast, mammals and plants (Figure 2,Additional file 1). While Tom40 is a b-barrel protein,there is no significant sequence similarity with bacterialb-barrel proteins [30], nonetheless, hidden Markovmodel searches define this as a universal component ofall mitochondria, including mitosomes in Entamoebahistolytica and Giardia intestinalis [49,50].Tom22 has been shown to fulfill a central receptor

role in yeast, and insertional inactivation in yeast resultsin a strong impairment of mitochondrial biogenesis,compared to the other two preprotein receptors charac-terized, Tom20 and Tom70 [51]. Searching the genomeof the red algae C. merolae for Tom22-like proteinsidentified a protein with a predicted molecular mass of20 kDa. This protein displays sequence identity and asimilar domain structure to the yeast Tom22 (Figure 2,Figure 3b). Thus, the TOM complex of C. merolaeappears to be similar to that of D. discoideum, in that itcontains a single receptor Tom22-like protein [49]. Incontrast, green plants and E. siliculosus do not contain a

Tom22 protein. Rather, they contain a Tom9 proteindomain component (Figure 2, Additional file 1). PlantTom9 is predicted to be structurally similar to yeast andmammalian Tom22, except that it lacks the cytosolicreceptor domain [30]. Thus, Tom22 has either lost thereceptor domain to form Tom9 or been replaced by adifferent protein. Irrespective of the mechanisms bywhich Tom9 arose, it appears that green plants havelost the Tom22 receptor. The presence of the Tom22receptor component in C. merolae and D. discoideumsuggests that it represented a universal mitochondrialreceptor component prior to the divergence event thatgave rise to plants verse animals and fungi.None of the receptor proteins characterized in yeast

or mammalian systems, Tom20, Tom70 and Tom22, arepresent in green plants [52,53] (Figure 2). The evolu-tionary situation for Tom22 is outlined above, andalthough a Tom20 receptor protein is present in plants,it represents a case of convergent evolution that hasbeen previously well described [27,54,55], thus, plantand yeast Tom20 proteins are not orthologous (Figure 2,Additional file 1). The third receptor component,Tom70, is only present in animals and fungi [45] (Fig-ure 1). Tom70 is not present in any green plant genome[29], a variety of searches in this study failed to detectany Tom70 like sequences in the green plant genomesinterrogated. However, in the genome of the brownalgae E. siliculosus, a protein with a similar domainstructure to Tom70 was identified (Figure 3C). ThisTom70 like protein contains an N-terminal transmem-brane domain and 11 Tetratricopeptide repeat (TPR)

Homo sapiens

Saccharomyces cerevisiae

Oryza sativa

Arabidopsis thaliana

Physcomitrella patensChlamydomonas reinhardtii

Dictyostelium discoideum

Cyanidioschyzon merolae

Ectocarpus siliculosus

Picea glaucaSelaginella moellendorffii

Chlorella variabilis

02004006008001000120014001600Million years ago

Opisthokonts

Amoebozoa

Green Plants

Red algae

Brown algae

Figure 1 Overview of the evolutionary relationship of organisms used in this study. The taxonomy database at NCBI was used to draw aphylogenetic tree, which was visualized using PHY-PI [91]. The timeline is based upon [57].


Page 3 of 15

OM

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B)

OM

Ectocarpus

Yeast

70

40 7

9

Cyanidioschyzon

40

22

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5 7 6

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Chlorella

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Chlamydomonas Physcomitrella

Selaginella

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RiceGlycine

ZeaPoplar

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64

9

Picea

Figure 2 Diversity of the TOM complex in plants. A) Schematic diagrams of the TOM complex from a selection of plant species using theTOM complex from yeast as a reference. B) A table depicting components of the TOM complex in a variety of organisms. The pink color refersto proteins that are conserved across all organisms and likely have a common ancestor. The lime green colored proteins are specific to the plantlineage. The pale green proteins are proteins that have an unknown origin. Yeast - Saccharomyces cerevisiae, Ectocarpus - Ectocarpus siliculosus(Es), Cyanidioschyzon - Cyanidioschyzon merolae (Cm), Chlorella - Chlorella variabilis (Cv), Picea - Picea glauca (Pg), Selaginella - Selaginellamoellendorffii (Sm), Chlamydomonas - Chlamydomonas reinhardtii (Cr), Physcomitrella - Physcomitrella patens (Pp), Rice - Oryza sativa (Os),Arabidopsis - Arabidopsis thaliana (At), Human - Homo sapiens, Poplar -Populus tricocarpa (Pt), Glycine - Glycine max (Gm) Zea - Zea mays (Zm).


Page 4 of 15

A)

B)

C)

617 AAScTom70 321 4 8765 11109TM

603 AAAtOM64 Amidase 321TM

739 AAEsTom70 321 4 8765 11109TM

AtmtO

M64

PtmtOM64VvmtOM64GmmtOM64-1GmmtOM64-2

GmmtOM64-3

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GmToc64-1GmToc64-2

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64PtTo

c64-

1

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oc64

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0.2

Mitochondrial Chloroplastic

Figure 3 Analysis of components of the plant TOM complex. A) Phylogenetic tree of the chloroplastic Toc64 and mitochondrial OM64sequences from plants. Only sequences which showed the characteristic domain structure of Toc64, an N-terminal transmembrane domainfollowed by an amidase like domain with 3 TPR repeats at the C-terminus, were used for phylogenetic analyses. No such proteins wereidentified in Ectocarpus siliculosus, Cyanidioschyzon merolae, Chlamydomonas reinhardtii and Chlorella variabilis. For most plant species a clearToc64 and mtOM64 homologue can be identified however only 1 sequence can be identified in Physcomitrella patens, Selaginella moellendorffiiand Picea glauca which all branch closest to the Toc64 chloroplastic proteins. B) Sequence alignment of the Saccharomyces cerevisiae Tom22with the Arabidopsis thaliana Tom9 and Cyanidioschyzon merloae Tom22. While most plants contain only the Arabidopsis Tom9 like proteinCyanidioscyzon contains a full Tom22 receptor, which shows a high similarity with the yeast Tom22. C) The domain organization of the yeastTom70, Arabidopsis mtOM64 and an EsTom70 like protein. TM - transmembrane domain and the numbers correspond to the TPR repeats. At -Arabidopsis thaliana, Vv - Vitis vinifera, Gm - Glycine max, Pt - Populus tricocarpa, Os - Oryza sativa, Pp - Physcomitrella patens, Zm - Zea mays, Sc -Saccharomyces cerevisiae, Cm - Cyanidioschyzon merolae, Es - Ectocarpius siliculosus, Sm - Selaginella moellendorffii and Pg - Picea glauca.


Page 5 of 15

motifs similar to yeast Tom70 [29]. However, the levelof sequence identity is low (20%), and it is unclear ifthis protein represents a Tom70 orthologue.A protein with a predicted molecular mass of 64 kDa

(OM64) is found on the outer membrane of mitochon-dria in Arabidopsis, displaying ~70% sequence identitywith the Toc64 protein (translocase of the outer envel-ope of chloroplasts) from plastids [56] (Figure 3A). Inplant mitochondria, this protein has been shown to beinvolved in the import of some precursor proteins [53].Analysis of various plant genomes reveals that OM64appears to be present only in a sub-set of vascularplants and is absent in P. glauca and S. moellendorffii,as well as lower plant groups represented by C. rein-hardtii, C. variabilis, P. patens, E. siliculosus andC. merolae (Figure 2A). Tom7 represents an interestingcase in that it is absent in C. merolae, but present in allother plants and eukaryotes (Figure 2). TBlastx and hid-den Markov model based searches of all red algae gen-omes available failed to find this component [50]. Thuseven if it was not annotated in the genome sequence ofC. merolae these searches should detect its presence.However, it cannot be ruled out that it may have beenmissed in the sequencing and/or assembly of theC. merolae genome. Tom5 and Tom6, proteins ofapproximately 50 amino acids long, were not detectedin C. variabilis, using either plant or yeast interrogationsequences in searches. However, the small size of Tom5and 6 proteins means that it is difficult to define theirevolutionary relationship across wide phylogenetic gaps.Tom7, on the other hand appears to be orthologousacross all groups, with the exception that it cannot befound in C. merolae. Thus suggesting that the smallTOM proteins may be lineage specific, as is the case ofthe Tom20 receptor.It is evident that the TOM complex of plants displays

diversity with respect to the receptor components pre-sent. While Tom40 is universally present, the presenceof Tom20 is only evident in green plants, Tom70 is onlypresent in E. siliculosus, and OM64 appears to havearisen by a relatively recent evolutionary event as it isonly present in a variety of monocot and dicot plantsexamined and could not be detected in P. glauca andS. moellendorffii (Figure 2A and 3A). The brown algaeE. siliculosus, contains a Tom70 type receptor. As thereis no Tom70 like sequences in green plants [29], theTom70 type receptor was either derived from the speci-fic host in the symbiosis that led to the formation ofbrown algae, or alternatively, it may represent a case ofconvergent evolution, as has been observed betweengreen plants and Opisthokonts for the Tom20 receptor[27,55].An analysis of the mitochondrial protein import appa-

ratus in a variety of plants reveals that C. merolae

clearly contains a Tom22 type receptor in contrast to allother plant lineages. Thus, this component may eitherhave been lost from brown algae and green plantlineages or the presence of a Tom22 type receptor inC. merolae represents another case of convergent evolu-tion. As brown algae are proposed to have been derivedfrom red algae, after the latter branched from greenplants [57], the Tom22 receptor would have to be lostindependently in green plants and brown algae. How-ever, caution needs to be exercised, as the sequence ofE. siliculosus may not be fully representative of allbrown algae.The question of how red algae solve the sorting pro-

blem between plastids and mitochondria may relate tothe binding substrates of the receptors, that is, the tar-geting signals. Analysis of plastid targeting signals fromall plant lineages reveals that red algae (and the otherprimary plant lineage, glaucophytes) contain a phenyla-lanine residue within a few amino acids of theN-terminus, which is in a hydrophobic context [58].This ‘ancestral’ plastid targeting motif is not present inplastid targeting signals in green plants [58], and thusthe differentiation of plastid and mitochondrial targetingsignals in green plants differs to red algae. In red algae,the “phenylalanine containing” transit peptide may serveas a means for mitochondria and plastid targeting sig-nals to be recognized or rejected by plastidic or mito-chondrial receptors respectively.

Sorting and Assembly Machinery of the OuterMitochondrial Membrane (SAM)The SAM complex is required for the insertion ofb-barrel and a-helical proteins into the outer membrane[52]. The insertion of b-barrel proteins into the mito-chondrial outer membrane is conserved from bacteria tomitochondria and plastids, where Omp85, Sam50 andToc75 are orthologous b-barrel proteins that are essen-tial for this process [59,60]. However, apart from thiscentral component, there are no other conserved com-ponents identified for the insertion of b-barrel proteinsinto membranes from bacteria to mitochondria andplastids (Figure 4A and 4B). In yeast, four additionalcomponents are involved; Sam35, Sam37, Mdm10 andMim1, with Sam35 representing an essential component.As the SAM complex has not been biochemically char-acterised from mammalian or plant systems, any addi-tional components are unknown in these systems. Thegenome of D. discoideum has a gene encoding Sam50,but lacks the other components identified in yeast. AsD. discoideum is an amoeba that diverged from Opistho-konts after this lineage had split from plants, this sug-gests that the additional components observed in yeastarose after the lineage divergence of plants from othergroups. Although additional components are likely to be


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B)

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Figure 4 The SAM complex of plants. A) Schematic representation of the SAM complexes found in yeast and plants. B) Table indicating thepresence or absence of a component of the SAM complexes found in plants. C) Representation of the different domains of yeast Sam37,Human Metaxin and Arabidopsis metaxin proteins. All three share similar Glutathione S-transferase (GST) domains. However the location of thetransmembrane domains (TM) differs. A motif analysis search of plant metaxin sequences identified Motif 1 to regions between amino acids 37and 79 and motif 2 between amino acids 104 and 155 in Arabidopsis. These motifs appear only in plant like Metaxins and near a regionrequired for protein binding. Colors and abbreviations are the same as Figure 2.


Page 7 of 15

present in the SAM complexes from plants, they areunlikely to be orthologous to the components in yeast.In Arabidopsis, a protein called Metaxin has been

shown to be involved in the import of b-barrel proteinsinto the outer membrane. The deletion of Metaxin isnot lethal in Arabidopsis, although plants are sterile andgrow poorly [53]. Deletion of Metaxin results in a largeup-regulation of transcript abundance for genes encod-ing the mitochondrial b-barrel proteins porin andTom40, with an accumulation of porin observed in thecytosol, indicating that Metaxin plays a role in the inser-tion of b-barrel proteins in plants. Mammalian genomescontain two genes encoding Metaxin, in fact the plantMetaxin protein was identified using blast searches ofthe mammalian Metaxin protein [53] (Figure 4C). Mam-malian Metaxin has also been shown to be involved inthe import of b-barrel proteins into the outer membraneof mitochondria [61]. Mammalian Metaxin does identifywith Sam37 in a blast search, although the sequenceidentity is very low (Additional file 1). A number of fea-tures distinguish plant and animal Metaxins fromSam37 in yeast. Firstly, human and Arabidopsis Metax-ins are anchored to the outer membrane in the oppositeorientation compared to Sam37 (Figure 4C). YeastSam37 is anchored to the mitochondrial outer mem-brane by an N-terminal transmembrane domain,whereas human and Arabidopsis Metaxins containC-terminal transmembrane domains. Secondly, humanand Arabidopsis Metaxins contain conserved glutathioneS-transferase (GST) domains (Figure 4C). Plant and ani-mal Metaxins are distinguished by the fact that Metaxinis not found in a complex with human Sam50 [61],whereas plant Metaxin is in a complex with plantSam50 (Duncan and Whelan - unpublished data). InArabidopsis Metaxin there are two conserved motifs ina region critical for binding that are only found in planttype Metaxins (Figure 4C motif 1 and 2). It is also ofinterest to note that while Trypanosomes do contain aMetaxin like protein [62], there are no Metaxin orSam37 like proteins identified in D. discoideum [49] orC. merolae in this study. The presence of a Metaxinprotein in E. siliculosus may be derived from the hostcell. Thus, plant and animal Metaxins may be ortholo-gous, but functions are likely to have diverged overtime. Biochemical characterization of the plant SAMcomplex would provide information on the accessoryproteins of this complex and provide a clearer picture ofthe evolutionary nature of the accessory subunits in thiscomplex.

Intermembrane space - Mitochondrial intermembranespace import and Assembly (MIA) and Tiny TIMsThe intermembrane space contains two sets of proteinsthat are essential for cell viability in yeast. The tiny TIM

proteins 8, 9, 10 and 13 appear to be present in a widevariety of eukaryotes (Figure 5A). They play an essentialrole in the import of carrier proteins into the innermembrane and also the assembly of b-barrel proteinsinto the outer membrane [52]. It has been proposedthat they arose from an ancestral protein present in theoriginal host that housed the mitochondrial endosym-biont [63]. There are eukaryotes that lack the smallTims (Trichomonas vaginalis and Encephalitozoon cuni-culi) or only contain one small Tim protein (Cryptospor-idium hominis) [64,65], indicating that they are notabsolutely essential, even though these organisms con-tain carrier type proteins on the inner membrane thatshould require these components for import. Thus, thelack of small Tims is likely to be a derived situationassociated with the presence of highly modified mito-chondria (i.e. mitosomes) in these organisms.The MIA pathway is the most recently described

import pathway for mitochondrial proteins. It consistsof two essential proteins in yeast, Mia40 and Erv1,which catalyse the oxidative folding of proteins whenthey enter the intermembrane space. Substrates of thispathway are proteins that contain conserved cysteineresidues that undergo oxidative protein folding in theintermembrane space. Both Mia40 and Erv1 are essen-tial proteins in yeast, with Mia40 proposed to act as theintermembrane space receptor for proteins [66]. Whilstdetailed structural and mechanistic analysis has beencarried out on this system in yeast [67], little is knownabout the components in other organisms. Interestinglythe apparent lack of a gene encoding Mia40 in trypano-somes suggests that this pathway may display variationsbetween species [68].For the MIA machinery, orthologues of Mia40 and

Erv1 are present in yeast, humans and plants (Figure5A). Although Hot13 has been reported to be wide-spread in eukaryotes, analysis of the proteins identifiedindicates it is ~600 amino acids long and most likely atranscription factor in green plants. Brown algae containa protein similar to yeast Hot13 (Figure 5A, Additionalfile 1). However, given the small size of this proteinwith conserved metal domains it is unclear if it is ortho-logous to the yeast protein. Although plant Erv1 andMia40 are orthologous to their yeast counterparts, theprimary structure of the protein differs (Figure 5B), sug-gesting possible mechanistic differences. Deletion ofMia40 in Arabidopsis is not lethal, and in fact normalgrowth and development are observed [69]. Erv1 isessential in Arabidopsis and analysis of the primarysequence indicates that the arrangement of cysteines dif-fers to that in yeast (and humans). Arabidopsis Erv1 issimilar to that found in Trypanosoma brucei and a pro-tein called Erv2 that is located in the endoplasmic reti-culum of yeast (which operates without a Mia40 like


Page 8 of 15

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B) Yeast Erv2p (196 AA)

Yeast Erv1p (189 AA)

Human Erv1 (205 AA)

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CXXC C-16-CFAD

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CXXC C-16-CFAD

Figure 5 Components of the MIA and IMS protein import apparatus of plants. A) A table displaying the components of the small Timproteins and MIA pathway components of plants. B) Schematic diagram of the different Erv1 sequences found in different organisms. The greyregion represents the most conserved region between different Erv1 sequences containing the redox centre, the FAD binding site and twoconserved cysteine domains (CXXC and C-16-C). The location of the third cysteine pair differs between organisms which seems to be dependenton either a Mia40 protein being present or whether that Mia40 is essential or not. Abbreviations are the same as Figures 2 and 4.


Page 9 of 15

protein) (Figure 5B). Given that the import of the smallTim proteins and carrier proteins is normal in Arabi-dopsis plants that lack Mia40 [69], mechanistically Erv1can function without Mia40 in oxidative protein foldingin the intermembrane space. Analysis of the genome ofD. discoideum indicates that Mia40 is present [68]. AsMia40 is absent in Trypanosoma brucei, Encephalitozooncuniculi [65,68], and the brown algae E. siliculosus(Figure 5A, Additional file 1), this suggests that the pre-sence of Mia40 in plants is a primitive situation, but itsfunction(s) differ in various lineages.

Translocases of the inner membrane (TIMS)The inner mitochondrial membrane contains two trans-locases, the TIM17:23 complex that is responsible forthe import of proteins via the general import pathway,and the TIM22 complex, that is responsible for theimport of carrier proteins into the inner membrane. TheTIM17:23 complex is responsible for the import of pro-teins that contain N-terminal targeting signals into oracross the inner mitochondrial membrane [23]. Thiscomplex contains 9 components in yeast and several ofthe components are essential in yeast [52]. Tim23 formsa presequence and voltage sensitive channel [70], whileTim17 plays a crucial role in voltage sensing [71,72].The TIM17:23 complex can be divided into the PAMcomplex, the presequence assisted motor consisting offive subunits (Tim44, HSP70, Pam 16, 17 and 18) andthe membrane components of Tim17, 23, 21 and 50.The TIM22 translocase is responsible for the import ofproteins that contain internal targeting signals and con-tain multiple (4 or 6) transmembrane spanning regionsinto the inner membrane [23]. In contrast to theTIM17:23 complex, the mechanistic details of how itoperates are not yet fully understood. However Tim22has been shown to have channel activity that is onlyactive in the presence of a substrate protein [73]. Inyeast it contains three accessory proteins, Tim54, 18and 12 [52]. However no details on the composition ofthis complex in other organisms have been reported.In contrast to the TOM complex on the outer mem-

brane, eight of the nine components of the Tim17:23complex are conserved between yeast, humans andplants (the only difference being that yeast contain aPam17 protein not present in humans and plants)(Figure 6A, Additional file 1). It has been previouslyproposed that the channel forming subunits of this com-plex, Tim23 and Tim17, are derived from amino acidtransporters in bacteria, specifically LivH, and defined afamily of proteins termed PReprotein and Amino acidTransporters (PRAT) [74]. It seems that originally therewas one PRAT type protein that subsequently divergedto give rise to the three different PRAT proteins typi-cally found in mitochondria [75]. In some organisms a

single PRAT protein exists, which is likely a derivedcondition where the other PRAT proteins have beenlost [65]. Additionally, not all subunits of the TIM17:23complex are observed in all organisms, i.e. the absenceof Tim50 in D. discoideum [49], suggests that accessorysubunits can be lost.In the case of the TIM22 complex, the translocase

responsible for the import of metabolite carriers, ormultiple spanning proteins of the inner membrane,including the PRAT proteins themselves, only theTim22 component is conserved. In fact Tim18 andTim54, along with Tim12, are only found in yeast andnot in other organisms including plants. Thus, the addi-tional components of this translocase are yet to be char-acterized in other organisms.Although the TIMs seem to be better conserved in

terms of orthology compared to the TOM complex,there are notable differences in plants. Firstly, the familyof PRAT proteins has greatly expanded in plants com-pared to yeast and mammals. In Arabidopsis there are17 members, rice has greater than 24 members andexamination of C. reinhardtii and P. patens reveal 5 and21 members respectively. Some of these PRAT proteinsare located in plastids, while others are found in mito-chondria [76]. In addition to the greater number ofPRAT proteins in plant genomes, Tim17 in plants variesin size from 133 amino acids to 252 amino acids, thedifference to yeast Tim17 of 158 amino acids is at theC-terminal end of the protein (Additional file 1).A C-terminal extension is found on Tim17 proteinsfrom C. merolae through to Arabidopsis. It has beenshown in Arabidopsis that this C-terminal extension isexposed on the outer surface of the outer membrane[36] and Arabidopsis Tim17 can only complement ayeast Tim17 mutant if this extension is removed [36].In order to investigate possible function(s) of the

Tim17 C-terminal extension in plants we conducted amotif search on all the identified Tim17 extensions.A distinct motif was detected (Figure 6C). Using thismotif in blast searches identifies a number of differentnucleic acid binding proteins (Additional file 2). Thissuggests that Tim17 in plants may be able to bind RNAand/or DNA. As plant mitochondria import tRNAs andhave recently been shown to bind mRNA [22,77], sug-gests a possible role for Tim17 in the binding and/orimport of nucleic acids into plant mitochondria.

Mitochondrial processing peptidase(s)Mitochondria require a number of peptidases to removethe targeting signals from proteins before or after theyare assembled into functional protein complexes. Thesepeptidases range from activities that remove the target-ing signals, such as mitochondrial processing peptidase(MPP) and intermediate processing peptidase (IMP)


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AtTim17-2 243 Amino acidsM1TM TM TM TM

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Figure 6 The TIM17:23 and TIM22 complexes of plants. A) A schematic representation of the TIM17:23 complex from yeast and plants.B) A table displaying the components of the TIM17:23 and TIM22 complexes in all plant species analysed. C) A diagram showing ArabidopsisTim17-2 that contains four transmembrane domains (TM) and an extra C-terminal extension. This C-terminal extension is found in all higherplant species and motif analysis of the C-terminal extension identified to highly conserved regions of which both are related to nucleic acidbinding proteins. Colors and abbreviations are the same as Figures 2 and 4.


Page 11 of 15

[23], to the removal of a single amino acid from pro-teins that have already had the targeting signal removed[78], to the presequence degrading peptidase (PreP) thatdegrades the targeting signals once they have beenremoved [34].Plant mitochondria contain a number of orthologous

proteins in comparison with the various processing pep-tidases of yeast mitochondria (Additional file 1). At asequence level the processing peptidases found in plantmitochondria look similar to those of yeast, however,functional investigations have revealed a number of dif-ferences. One major difference between yeast and plantsis the location of the mitochondrial processing peptidase(MPP). In yeast both a and b MPP subunits are locatedin the matrix, however, in plants it has been demon-strated that they are integrated into the cytochrome bc1complex located in the inner membrane [31,32]. Addi-tionally, the presequence degrading peptidase of yeast islocated in the intermembrane space whereas in plants itis located in the matrix [79,80]. Interestingly, the prese-quence degrading peptidase of plant mitochondria isalso dual targeted to plastids where it degrades plastidtargeting signals [79].In terms of the processing site recognition by MPP in

plants, it has been reported that the majority of plantmitochondrial presequences fall into 2 classes. In class 1the processing signal is a -2 Arg residue while in class 2presequences the signal is a -3 Arg residue [20,81], simi-lar to what has been reported for yeast and mammals[23]. However, it has been demonstrated that in factthere is a second processing step in yeast [82], a novelpeptidase called intermediate cleavage peptidase (Icp55)was found to process mitochondrial presequences afterMPP, cleaving only 1 amino acid from the N-terminus,turning the proposed -2 cleavage signal into a -3 clea-vage signal [78]. It is tempting to speculate that asplants contain an orthologue of Icp55 that the samecleavage is occurring, however this awaits experimentalconfirmation. Despite the orthology between many plantpeptidases and those in other organisms it is still neces-sary to define their specific functions in plants. It hasbeen demonstrated that the plant orthologue of therhomboid protease from yeast does not carry out thesame processing roles/activities in plants such as Arabi-dopsis [83].

ConclusionsThe plant mitochondrial import apparatus displaysmany differences compared to other non-plant organ-isms and between plant groups. The TOM complex inplants displays the most variability in that as many asfive different TOM complexes exist in plants when redalgae, brown algae and green plants are considered.Even in the green plant lineage variation is observed

with OM64 only being present in monocot and dicotplants. While the composition of the other protein com-plexes may appear more conserved, the lack of biochem-ical characterization of these complexes in any plantgroup means that the presence of plant specific acces-sory subunits in various lineages cannot be judged.Additionally for some proteins, such as Mia40 andTim17, functions have expanded in plants compared tothose characterized in yeast.

MethodsThe protein sequences for all of the known mitochon-drial protein import components from Saccharomycescerevisiae (Tom20, Tom70, Tom71, Tom40, Tom22,Tom5, Tom6, Tom7, Sam50, Sam37, Sam35, Mdm10,Mim1, Mia40, Erv1, Hot13, Tim9, Tim10, Tim8, Tim13,Tim12, Tim22, Tim54, Tim18, Tim23, Tim17, Tim50,Tim21, mtHsp70, Mge1, Tim44, Pam18, Mdj2, Pam16,Pam17, MPPa, MPPb, Oct1, Imp1, Imp2, Som1, Yta12,Yta10, Yme1, Mgr1, Mgr3, Pcp1, Icp55, Oxa1, Mba1,Cox18, Pnt1, Mss2, Mdj1, Hsp60, Hsp10, Hsp78 andZim17) were downloaded from the NCBI protein data-base (http://www.ncbi.nlm.nih.gov/protein/). TheMetaxin protein sequences were also obtained fromHomo sapiens. Using the above protein sequences Blastp[84] searches of the protein sequences from Physcomi-trella patens, Selaginella moellendorffii, Chlamydomonasreinhardtii, Arabidopsis thaliana, Oryza sativa, Zeamays, Vitis vinifera, Glycine max and Populus tricocarpawere performed using the Phytozome (http://www.phyto-zome.net) database. Blastp [84] searches of Cyanidioschy-zon merolae were performed using the Cyanidioschyzonmerolae genome project website (http://merolae.biol.s.u-tokyo.ac.jp/). Blastp [84] searches of Ectocarpus siliculo-sus were performed at the Bioinformatics online genomeannotation system website (http://bioinformatics.psb.ugent.be/webtools/bogas/overview/Ectsi). Blastp [84]searches of Chlorella variabilis NC64A genome [38] wasperformed at the Chlorella genome website (http://gen-ome.jgi-psf.org/ChlNC64A_1/ChlNC64A_1.home.html).To identify mitochondrial import components of Piceaglauca tblastn [84] searches were carried on ESTsequences [43] at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).All multiple sequence alignments were carried out

using MAFFT [85] and visualized using Multiple alignshow (http://www.bioinformatics.org/sms/multi_align.html). The program IQPNNI [86] was used to recon-struct a maximum likelihood phylogeny assuming theWhelan and Goldman model [87]. Phylogenetic treeswere finally visualized using the program Geneious(http://www.geneious.com).TMpred (http://www.ch.embnet.org/software/TMPRED_

form.html), TMHMM (http://www.cbs.dtu.dk/services/


Page 12 of 15

http://www.ncbi.nlm.nih.gov/protein/

http://www.phytozome.net

http://www.phytozome.net

http://merolae.biol.s.u-tokyo.ac.jp/

http://merolae.biol.s.u-tokyo.ac.jp/

http://bioinformatics.psb.ugent.be/webtools/bogas/overview/Ectsi

http://bioinformatics.psb.ugent.be/webtools/bogas/overview/Ectsi

http://genome.jgi-psf.org/ChlNC64A_1/ChlNC64A_1.home.html

http://genome.jgi-psf.org/ChlNC64A_1/ChlNC64A_1.home.html

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://www.bioinformatics.org/sms/multi_align.html

http://www.bioinformatics.org/sms/multi_align.html

http://www.geneious.com

http://www.ch.embnet.org/software/TMPRED_form.html

http://www.ch.embnet.org/software/TMPRED_form.html

http://www.cbs.dtu.dk/services/TMHMM/

TMHMM/), and DAS (http://www.sbc.su.se/~miklos/DAS/) [88] were used in the prediction of transmem-brane regions. TPR repeats were predicted usingTPRpred (http://toolkit.tuebingen.mpg.de/tprpred) [89].Motif analysis was performed using MEME (http://meme.nbcr.net/meme4_4_0/cgi-bin/meme.cgi) usingdefault parameters for all plant like Metaxin sequencesand sequences of the plant Tim17 extensions [90].

Additional material

Additional file 1: Supplementary table 1. The mitochondrial importmachinery of plants.

Additional file 2: Supplementary table 2. The top 50 proteinsidentified using the conserved motifs on the C-terminal of plantTim17 in a Blastp search.

AbbreviationsErv1: Essential for respiration and vegetative growth 1; FAD: Flavin adeninedinucleotide; GDP: Guanosine diphosphate; GTP: Guanosine diphosphate;GST: Glutathione S-transferase; Hot13: Helper of Tim protein 13; Icp55:Intermediate cleavage peptidase of 55 kDa; Mdm10: Mitochondriadistribution and morphology protein 10; MIA: Mitochondrial import andassembly; Mim1: Mitochondrial import 1; MPP: Mitochondrial processingpeptidase; OM64: Mitochondrial outer membrane protein of 64 kDa; Omp85:Outer membrane protein of 85 kDa; PRAT: Preprotein and amino acidtransporter; TIM: Translocase of the inner membrane; TOC: Translocase of theouter envelope of chloroplasts; TOM: Translocase of the outer membrane;TPR: Tetratricopeptide repeat; SAM: Sorting and assembly machinery.

AcknowledgementsThis work was supported by an Australian Research Council Centre ofExcellence Grant CEO561495.

Authors’ contributionsCC carried out the data analysis with the help of MM. JW oversaw theanalysis, design and implementation. CC, MM and JW drafted themanuscript. All authors read and approved final manuscript.

Received: 26 August 2010 Accepted: 16 November 2010Published: 16 November 2010

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http://www.cbs.dtu.dk/services/TMHMM/

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doi:10.1186/1471-2229-10-249Cite this article as: Carrie et al.: An in silico analysis of the mitochondrialprotein import apparatus of plants. BMC Plant Biology 2010 10:249.

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Chapter 8 General discussion

116

Chapter 8

General discussion


117

General discussion Dual targeting in plants was first uncovered in 1995 and now more than

50 proteins are known to be dual targeted to mitochondria and plastids in Arabidopsis

(Creissen et al., 1995; Carrie et al., 2009). While the vast majority of plant dual targeted

proteins are targeted to mitochondria and chloroplasts, an increasing number are being

discovered in other organelles; chloroplasts and nucleus (Schwacke et al., 2007),

chloroplasts and peroxisomes (Reumann et al., 2007; Sapir-Mir et al., 2008),

chloroplasts and endoplasmic reticulum (Levitan et al., 2005), chloroplasts and cytosol

(Thatcher et al., 2007), and mitochondria and peroxisomes (Studies II and III). The

functions of dual targeted proteins in plants also vary greatly; from tRNA synthetases

(Duchene et al., 2005), DNA polymerases (Christensen et al., 2005), peptidases

(Bhushan et al., 2003) and antioxidant defence proteins (Chew et al., 2003). However

the mechanisms and reasons for dual targeting are yet to be fully understood.

7.1 Defining dual targeted proteins: targeting vs accumulation studies

Our understanding of the reasons and mechanisms of protein dual targeting has

been limited by the relatively slow pace at which dual targeted proteins have been

characterised. The main reason for the slow pace at which dual targeted proteins have

been identified, is that demonstrating that a protein is dual targeted is more difficult than

defining a single location for a protein. Consequently, most dual targeted proteins have

been uncovered in small or single protein studies. One exception is a study by (Duchene

et al., 2005), which characterised a large number of Arabidopsis tRNA synthetases as

dual targeted proteins. In fact, several proteins defined as dual-targeted proteins in this

thesis had been previously defined as targeted to a single location (Study II and III).

Thus the burden to identify dual targeting is greater than compared to single location

proteins.

The location of a protein in a cell can be assessed in a number of ways,

including computational predictions, targeting uptake assays, and accumulation studies

(Millar et al., 2009). Each approach has strengths and weaknesses, and to accurately

determine if a protein is located in one or more locations, the use of multiple lines of

evidence is necessary. Several prediction programs are now available to predict a

proteins targeting ability (Claros and Vincens, 1996; Hua and Sun, 2001; Bannai et al.,

2002; Emanuelsson et al., 2003; Guda et al., 2004; Small et al., 2004; Nair and Rost,

2005; Hoglund et al., 2006; Emanuelsson et al., 2007; Horton et al., 2007). These


118

programs yield varying results depending on the type of algorithms used (weight

matrix, neural networks, hidden markov models, and support vector machines), the set

of protein sequences used in training the program, the range of locations predicted, the

predicted hydrophobicity of the input sequence, and the significance level used as a cut-

off (Emanuelsson et al., 2007). In terms of dual targeted proteins, the current batch of

programs are only a guide at best, especially for proteins targeted to both mitochondria

and chloroplasts. Due to the similarity of the targeting signals between these two

organelles and given that most programs are not trained with dual targeted proteins,

predictions on dual targeted proteins have varying levels of accuracy. However, in

recent years, there have been several attempts to predict dual targeted proteins in plants

(Schwacke et al., 2007; Mitschke et al., 2009). The accuracy of these predictions are

still quite limited, and more experimental evidence is required to accurately assess the

quality of these predictions. Never the less, predictions, do offer a number of

advantages, including; speed and ease of use, unbiased by the user, and predictions form

a platform to establish new lines of experimental inquiry. For example, three NAD(P)H

dehydrogenases that were previously experimentally defined as mitochondrial

(Michalecka et al., 2003), were predicted to also be peroxisomal, and subsequent

experimental testing confirmed them to be dual targeted to both mitochondria and

peroxisomes (Study III). Thus, predictions can be a valuable tool contributing to the

identification of dual targeted proteins.

Most experimental testing of protein targeting is conducted using either in vitro

or in vivo targeting uptake assays. In vitro import assays are generally performed by

purifying the organelle of interest, and testing whether the protein of interest enters the

isolated organelle in solution. Import is then assessed by observing if the incorporated

protein is protected from an externally added protease. In vitro import assays typically

have a low false positive rate as generally only the proteins found in an organelle are

imported in vitro. However, in vitro import assays are only performed with one

organelle, which is vastly different from the actual cellular landscape that contains

several organelles. Thus using this technique it may never be known whether a protein

is targeted to multiple organelles. A good example of this is NDC1, which was

previously only imported into mitochondria and subsequently assigned as having a

solely mitochondrial location (Michalecka et al., 2003). However, work in study III

revealed that it could also be imported and targeted to chloroplasts. Therefore, while in

vitro import assays work well for single organelle localised proteins, they fail to


119

reproduce the complex intracellular environment and thus cannot reveal the targeting

preference between multiple organelles.

The use of GFP and its variants as passenger proteins for in vivo targeting

studies has become routine in recent years. The main advantage of in vivo targeting

assays is that the cellular environment is maintained, and thus protein uptake into all

organelles is tested simultaneously. However, the primary source of error in in vivo

targeting experiments relates to the position of the passenger protein (e.g. GFP). Placing

the passenger protein at the wrong location or using only part of the protein of interest

are common mistakes in targeting assays (Millar et al., 2006). It is common practice

when determining protein targeting, to attach only the N-terminal (50 – 100 amino

acids) to the passenger protein. Notably, this ignores targeting signals at the C-terminus

and throughout the protein. The mitochondrial NAD(P)H dehydrogenases are good

examples of this practice, as previous targeting studies using the N-terminus assigned a

mitochondrial only location (Michalecka et al., 2003). The peroxisomal targeting

signals at the C-terminus of the these three proteins were missed, and thus this

technique failed to identify the peroxisomal location (Study III). Also, when the full

protein sequence of NDC1 was fused to GFP, it was shown to be dual targeted to both

chloroplasts and mitochondria (Study III). There are now several studies showing that

for dual targeted proteins, the nature of the passenger protein is important, and that

targeting signals that support dual targeting of one passenger protein do not support the

dual targeting of alternative passenger proteins (Chew et al., 2003).

As with in vitro import assays, there are also important considerations required

for in vivo targeting studies. The first relates to the translation product(s) used in the

targeting assay. It has become apparent that alternative splicing can produce proteins

with different targeting signals, e.g. AP2 (Study II), isoaspartylmethyltransferase 2

(Dinkins et al., 2008) and glutathione S-transferase F8 (Thatcher et al., 2007).

Therefore, where possible, it is imperative to analyse all possible alternative transcripts

or gene models for a protein, in order to assess possible targeting to different locations.

The second consideration in targeting studies is the type of tissue used for the assay. In

study II, it was demonstrated that the amount of dual targeting of a protein was different

in different tissue types. In some cases, the GFP labelling of mitochondria and

chloroplasts were equal, but for other proteins it was seen that the chloroplast signal

dominated the mitochondria signal in Arabidopsis cell culture. However, when the same


120

protein was analysed in onion epidermal cells, the intensity of fluorescence changed

back to be almost equal between organelles (Study II). This is consistent with a previous

study, which reported that the dual targeting of the protein sigma factor 2B was only

observed in one cell type (Beardslee et al., 2002). It has also been reported that GFP

fluorescence intensity differs between replicate experiments performed on dual targeted

proteins (Pujol et al., 2007). Thus, attention to the details of what is tested and where,

should be considered in the design and interpretion of in vivo targeting studies.

The ability of a protein to target a specific location does not necessarily lead to

accumulation within that location. It has been evidenced that a number of proteins are

first targeted to one location (e.g. endoplasmic reticulum) before being transported to a

second and final organelle (Tabak et al., 2003; Villarejo et al., 2005). The use of

chimeric constructs in targeting studies may therefore mask secondary sorting signals

and thus incorrectly determine location. Immunoblotting, immunolocalization and

activity assays are common approaches used to define the location or accumulation of

proteins. These assays are only informative when the assay is specific, which is

generally not the case in activity based assays, and varies with different antibodies as

specificity of antibodies can be difficult to demonstrate. Hence this lack of specificity

has led to the use of peptide mass spectrometry approaches to define the location of

large numbers of proteins.

Subcellular proteomics involves the cataloguing of components of an isolated

organelle after cell disruption. Subcellular proteomics has facilitated the designation of

organelles for over 4600 proteins in Arabidopsis (Heazlewood et al., 2007). However,

proteomics is still largely based on the purification of organelles from plant tissues, and

with the increasing sensitivity of new mass spectrometers, there is increasing

identification of low abundance contaminants. This has been somewhat overcome by

the use of quantitative comparison of organelle protein fractions with different degrees

of enrichment for the specific organelles of interest (Eubel et al., 2008; Huang et al.,

2009). However, many proteins are still described in protein databases as being found in

more than one subcellular location (Heazlewood et al., 2007). These proteins location is

then left unresolved until it is identified as targeted to a specific organelle or found to be

a contamination of an organelle fraction. Kat2 is a good example of a protein defined as

mitochondrial by proteomics but subsequently found to only target peroxisomes in

Arabidopsis (Study I). It has to be noted that subcellular proteomics is also limited, in


121

that certain classes of proteins are rarely detected (e.g. very small, hydrophobic and/or

basic). Another limitation of proteomics is eclipsed distribution, a phenomenon

whereby a protein that is targeted to two locations, is only identified in one, due to the

uneven distribution of the proteins abundance between the two organelles (Regev-

Rudzki and Pines, 2007).

A good example of a protein with an eclipsed distribution is the yeast

aconitase 1 protein, of which 95% is targeted to mitochondria and the remaining 5% is

located within the cytosol (Regev-Rudzki et al., 2005). The challenge in identifying

proteins with eclipsed distribution is the presence of artifacts derived from organelle

leakage or fraction contamination, scenarios commonly present in traditional methods,

such as immunoblots or activity assays. The use of in vivo tagging assays are also

limited, as low abundance proteins may not be detected, as the associated level of

fluorescence may be to low or the passenger protein may affect targeting to the second

location. Hence there is a need for specific assays to determine if a protein shows an

eclipsed distribution (Regev-Rudzki and Pines, 2007). One such assay makes use of

split reporter genes such as GFP (Ozawa et al., 2003; Ozawa et al., 2005) or β-

galactosidase (Karniely et al., 2006). The advantage of this method is that the large

amount of protein in one location does not produce a signal on its own and subsequently

does not mask the signal from the eclipsed location. A second method relies more on

genetics and involves knocking out the gene of interest and replacing it with the protein

only targeted to one of its locations (Regev-Rudzki and Pines, 2007). This not only

demonstrates that the protein is dual targeted, but also permits functional analysis.

In order to reduce the error rate in defining proteins as dual targeted or location

specific, a combination of targeting and accumulation assays are recommended. In

studies focused on small numbers of proteins, a targeting assay such as a fluorescent

tagging should be combined with an accumulation assay on the protein to independently

validate localisation and give complimentary biologically relevant information. In other

words, the experiment must be designed in a manner that allows accurate determination

of dual targeting and relative abundance. With large scale approaches, such as sub-

cellular proteomics, it would be beneficial to know the error rate of defining specific

locations. Often, lists of proteins in publications may have known contaminants

removed, thus full access to the raw data is important. With large scale expression

studies, it is mandatory to deposit raw microarray data files in publicly available


122

databases. This is also necessary for proteomic studies, so that other researchers can

carry out independent analysis. The availability of raw datasets and their independent

analyses will allow localisation errors to be determined and rectified.

7.2 Mechanisms of dual targeting – signals and import receptors

The signals that define a protein as dual targeted have been intensively studied

(Chew et al., 2003; Berglund et al., 2009). There are two different types of signals that

define a protein as dual targeted. Proteins can be dual targeted due to multiple

translation start sites, thus producing two different proteins from a single transcript.

Alternatively, a single protein is produced from a mRNA that is targeted to two or more

organelles. In the instance were a gene produces two (or more) translation products,

either by multiple RNAs or multiple translation start sites, the proteins produced contain

separate targeting signals for each organelle. A protein from a single translation product

can be dual targeted due to an ambiguous targeting signal or two separate targeting

signals in the same protein, or even reverse translocation. In plants, the most studied

dual targeted proteins appear to have ambiguous targeting signals and tend to be

proteins targeted to both mitochondria and chloroplasts. In this case, the ambiguous

targeting signal displays most of the properties of both mitochondrial and chloroplastic

targeting signals; enriched in hydroxylated, hydrophobic, and positively charged amino

acids, and a low abundance of negatively charged residues. Analysis of a variety of

ambiguous targeting signals has shown that deletions in the signal affects targeting to

both organelles, while smaller changes, such as single amino acid substitutions, can

affect targeting to one organelle more than another (Rudhe et al., 2002; Chew et al.,

2003; Berglund et al., 2009). Overall, it has been concluded that the dual targeting

signal is distributed throughout the targeting peptide and, importantly, it cannot be

deduced what defines a signal as a dual targeting signal, compared to a plastid or

mitochondrial specific signal.

Using two distinct signals, several proteins have now been discovered to target

to both the mitochondria and peroxisomes (Study II and III), along with chloroplasts

and peroxisomes (Reumann et al., 2007). This dual targeting is achieved either by an

N-terminal mitochondrial or chloroplastic targeting signal and a C-terminal peroxisomal

targeting signal (Study II and III)(Reumann et al., 2007). This mechanism of dual

targeting raises the question of which signal is recognised, and if ‘competition’ for the

protein occurs between the organelles. Although these questions are unanswered, they


123

raise more questions about the levels of regulation of gene expression, as now

regulation of the targeting between organelles can be an important point of regulation.

One point of regulation has been speculated to involve the import pathways used for the

import of dual targeted proteins into their respective organelles.

Little is known about the role of organelle import receptors in the import of dual

targeted proteins. The dual targeted proteins identified to date are thought to be required

in a number of different plastid types, which suggests they might employ different

receptors to the Toc64 and Toc159 pathways used for photosynthetic proteins (Soll and

Schleiff, 2004). It has also been speculated that because there are four members of the

Toc159 family (Toc159, Toc132, Toc120, and Toc90), one of them may be specialised

in the import of dual targeted proteins (Jarvis, 2008). However, no experimental studies

to date have determined the import pathways or receptors for dual targeted proteins in

plastids. It has been recently demonstrated that the different A-domains of Toc159

receptors regulate their selectivity for precursor binding thus it is possible that this

differential selection may also affect the import of dual targeted proteins (Inoue et al.,

2010).

In the case of mitochondrial receptors involved in the import of dual targeted

proteins, there is some functional/experimental data on the nature of the receptor

proteins involved. In a double knockout of Tom20-2/-3, leaving only one functional

Tom20-4 isoform, it was observed that the import of GR was higher when compared to

wild-type, even though the import of several other proteins was decreased (Study IV).

In the Tom20 triple knockout, the import of GR remained at the same level compared to

wild-type, even though all other precursors were significantly decreased in import

(Study IV). These findings led to the hypothesis that GR and possibly other dual

targeted proteins, might utilize an alternative import receptor, when compared to several

mitochondrial specific proteins. One such hypothesised receptor was OM64, because it

showed a high similarity to the Toc64 receptor from chloroplasts. However, OM64 was

determined to have no specific role in the import of GR and other dual targeted proteins

(Study IV). The only receptor like protein to have a major effect of the import of GR

was Metaxin. In knockouts of Metaxin, GR import was significantly reduced, and in a

competitor import reaction with in vitro synthesised Metaxin, it could compete for the

import of GR (Study IV). However, the role of Metaxin as an import receptor is still

unclear. In mammals, Metaxin is thought to be part of the SAM complex and is


124

involved in the import and assembly of β-barrel proteins. This reduction in β-barrel

import is also seen in Arabidopsis (Study IV) so the deficiencies in GR import could be

a secondary effect. However, the fact that the addition of soluble Metaxin protein was

seen to compete for the import of GR suggests that Metaxin can bind GR. This provides

evidence that dual targeted proteins utilise a different pathway to classical the Tom20

pathwaywhich most mitochondrial proteins utilise.

It is interesting to note that studies on the import components of a number of

plant species suggest that there is no one single plant import apparatus (Study VI).

When the plant mitochondrial import components are compared with yeast and humans,

it is clear that the main translocase subunits (Tom40, Sam50, Tim17:23, and Tim22) are

well conserved throughout all organisms. However, in terms of the receptor subunits

(Tom20, Tom22, and Tom70), there is little or no conservation between yeast and

plants and also very little conservation between different plant species. The plant TOM

complex displays a number of different arrangements in terms of receptors: red algae

only contain a Tom22 like receptor; green algae, moss and lower plants only contain a

Tom20; higher plants such as Arabidopsis and rice contain both Tom20 and OM64

(Study VI). This difference in receptor subunits may be due to the selective pressure of

the cell to differentiate between plastid and mitochondrial targeted proteins. This is

evidenced by the change in plastid targeting signal properties between red algae and

green plants which corresponds to the presence or absence of a full Tom22 receptor. It

has recently been speculated that the dual targeting of a protein is a gain of function

condition derived through gene duplication (Brandao and Silva-Filho., 2010). This

hypothesis is based on the theory that a dual targeted protein started out as a protein

targeted to only one location (mitochondria or chloroplast) and through a gene

duplication event, became subsequently targeted to both organelles and subsequently

lost the single organelle targeted protein (Brandao and Silva-Filho., 2010). If all dual

targeted proteins were once single organelle targeted proteins, it is tempting to speculate

that the evolution of dual targeting goes hand in hand with the diversity of the

mitochondrial import receptors. A better understanding of the evolutionary history of

dual targeted proteins may help to test this theory.

7.3 Reasons for dual targeting

One obvious and outstanding question is why are proteins dual targeted? It is

well known that organelles, such as mitochondria and chloroplasts, share many common


125

enzymatic steps and the majority of these steps are carried out by location specific

isoforms. So what is the role of dual targeted proteins? Dual targeting does not seem to

be a way of limiting gene numbers in the nuclear genome because organelles also

contain location specific isoforms of dual targeted proteins. A good example is

evidenced with organelle RNA polymerases in Arabidopsis, which contains three

isoforms; one mitochondrial, one chloroplastic, and one dual targeted (Hedtke et al.,

1997, 2000). Rice only contains two; one of which is mitochondrial and the other is

chloroplastic (Kusumi et al., 2004). When the same proteins were analysed in the moss,

Physcomitrella, both were found to be dual targeted to mitochondria and chloroplasts

(Richter et al., 2002). However, conflicting views remain on whether these proteins are

truly dual targeted (Kessler et al., 1994). Therefore, it appears that similar proteins are

not always dual targeted in different species of plants, further confounding the reason as

to why some, but not others are dual targeted. In Arabidopsis inactivation of the dual

targeted RNA polymerase leads to alterations of mitochondrial, but not plastidial,

functions (Kuhn et al., 2009). Thus, dual targeting of proteins may allow specialisation

of function for particular proteins.

One reason for dual targeting that has been proposed, is that it may be a form of

intra-organellar communication. Targeting the same protein to two organelles at the

same time means that these organelles are at least capable of carrying out the same

functions in a co-ordinated manner. This is particularly relevant to plant cells, as they

contain two organelles with their own genome, the replication and/or expression of

which may need to be co-ordinated. Another reason for dual targeting is that new

functions and/or roles can be gained for a specific protein. A good example of this is

Mia40 from Arabidopsis, which was shown to target and accumulate in both

peroxisomes and mitochondria (Study V). This was in contrast to yeast and human

Mia40 proteins, which are solely located within the mitochondrial IMS. The role for

Mia40 in both organelles in plants appears to be similar i.e. in the correct folding of

Ccs1 and subsequently, SOD1. However, by dual targeting Mia40 to peroxisomes as

well as mitochondria, plants have gained an extra function for Mia40 compared to yeast

and humans.

In cases where no organelle specific isoform exists, dual targeting appears to

simply coordinate the same function in both organelles. The majority of known dual

targeted proteins from Arabidopsis are involved in replication, transcription and


126

translation. This could simply be because the limited number of dual targeted proteins

identified to date are biased towards these roles. Further analysis of the expression

patterns of known dual targeted proteins has revealed that these genes are relatively

static, displaying similar expression levels in most plant tissues and developmental

stages, suggesting that these proteins perform basic, essential functions required in both

mitochondria and plastids (Carrie et al., 2009).

7.4 Future perspectives

Since the discovery of dual targeting in 1995, there have been substantial

developments, with more than 50 proteins described from various plant species that

target to more than one subcellular location. However, our understanding of how, why,

and when this occurs is still very limited. One of the most compelling questions about

dual targeting concerns how proteins are partitioned to each organelle. It has been

observed with some GFP constructs that in different tissues, some proteins appear to

target different organelles at different levels (Study II). The level of GFP fluorescence

for some dual targeted proteins is brighter in plastids compared to mitochondria, while

for other proteins it is equal. Western blots of dual targeted proteins in Arabidopsis

show that more Mia40 is targeted to mitochondria than peroxisomes, and the vice versa

for NDA1 (Studies III and V). A greater understanding of the mechanisms regulating

this partitioning is needed. In yeast, it has been shown that the level of dual targeting of

fumarase is regulated by the metabolites of the glyoxylate shunt cycle (Regev-Rudzki et

al., 2009). It would be interesting to see if different environmental conditions such as

light intensity, temperature or humidity can affect the partitioning of dual targeted

proteins in plants.

While understanding the regulation of dual targeting in plant cells would be a

major breakthrough, it would still leave one big question. That is, why dual target

proteins in the first place? As discussed above, there are a number of theories about why

but there is no definitive answer as yet. A greater knowledge of the evolutionary history

of dual targeting of proteins, combined with the knowledge of the functions of dual

targeted proteins, especially where location specific isoforms exist, will provide the

necessary steps towards understanding why dual targeting of proteins occurs.

References

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Characterisation of protein dual targeting to energy ... · Characterisation of protein dual targeting to energy organelles in Arabidopsis thaliana Chris Carrie This thesis was submitted