Pathfinders and trailblazers: a prokaryotic targeting system for transport of folded proteins

M I N I R E V I E W

Path¢ndersandtrailblazers:a prokaryotic targetingsystemfortransportoffoldedproteinsFrank Sargent1, Ben C. Berks2 & Tracy Palmer1,3

1School of Biological Sciences, University of East Anglia, Norwich, UK; 2Department of Biochemistry, University of Oxford, Oxford, UK; and 3Department

of Molecular Microbiology, John Innes Centre, Norwich, UK

Correspondence: Frank Sargent, School of

Biological Sciences, University of East Anglia,

Norwich NR4 7TJ, UK. Tel.: 144 (0) 1603

592889; fax 144 (0) 1603 592250; e-mail:

[email protected]

Received 3 October 2005; revised 7 November

2005; accepted 8 November 2005.

First published online December 2005.

doi:10.1111/j.1574-6968.2005.00049.x

Editor: Ian Henderson

Keywords

protein transport; Tat pathway; twin-arginine

signal peptide; chaperones; macromolecular

complexes; membrane proteins.

Abstract

The twin-arginine (Tat) protein translocase is a highly unusual protein transport

machine that is dedicated to the movement of folded proteins across the bacterial

cytoplasmic membrane. Proteins are targeted to the Tat pathway by means of N-

terminal signal peptides harbouring a distinctive twin-arginine motif. In this

minireview, we describe our current knowledge of the Tat system, paying particular

attention to the function of the TatA protein and to the often overlooked step of

signal peptide cleavage.

Introduction

The transport of proteins across cell membranes is an

essential feature of all living organisms. Indeed, even the

smallest bacterial genomes have genes encoding protein

transport components. The general principles of protein

translocation are the presence of membrane-bound trans-

port machine through which proteins are translocated, and

a targeting signal on the substrate to allow specific recogni-

tion by the transporter. Usually, but not always, the targeting

signal is located at the extreme N-terminus of the substrate,

and often it is cleaved off during the transport process by a

specific peptidase. This minireview deals with an unusual

protein transport system termed the twin-arginine protein

transport (Tat) pathway.

The twin-arginine translocation system

The Tat pathway is found in the cytoplasmic membranes of

many bacteria and archaea (and in the thylakoid membranes

of their chloroplast descendants) and its most remarkable

feature is its ability to transport prefolded, and often

oligomeric, proteins across ionically sealed membranes. In

these biological systems, the protein substrates are usually

synthesized with, or associated with folded partner proteins

that possess, distinctive N-terminal signal peptides that bear

a common amino-acid sequence motif. The so-called ‘twin-

arginine’ motif has a consensus sequence of SRRxFLK where

the arginine residues are almost invariant and essential for

efficient protein targeting. Twin-arginine signal peptides

target precursors to the membrane-bound Tat transport

apparatus that utilizes the proton-motive force to drive

protein transport (for a recent comprehensive review, see

Berks et al., 2003).

The Tat system has been found to be an important

virulence factor in plant and animal bacterial pathogens

(e.g. Ochsner et al., 2002; Ding & Christie, 2003). As the Tat

translocase is apparently not a feature of human or other

animal physiologies it has attracted interest from the bio-

medical sector as a possible target for novel drug develop-

ment. In addition, the remarkable ability of the Tat system to

move folded proteins across biological membranes has not

gone unnoticed by the biotechnology industry. For example,

the Tat system has recently been exploited in a new phage

display system (Paschke & Hoehne, 2005). Moreover, the Tat

system appears to have an intrinsic ‘quality control’ activity

FEMS Microbiol Lett 254 (2006) 198–207c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

that prevents transport of unfolded polypeptides (Musser &

Theg, 2000; DeLisa et al., 2003). Harnessing this innate

ability to reject immature or incorrectly assembled protein

and actively select for folded substrates would undoubtedly

increase the efficiency of large-scale protein production

protocols.

TheparadigmEscherichia colimodel

In the model Gram-negative eubacterium Escherichia coli,

four genes – tatA, tatB, tatC and tatE – have been identified

that are involved in the Tat transport process (Bogsch et al.,

1998; Sargent et al., 1998; 1999; Weiner et al., 1998). The

tatABC genes are organized in an operon and tatE is located

elsewhere on the chromosome where it encodes a structural

and functional homologue of the tatA gene product

(Sargent et al., 1998). Each gene encodes an integral inner

membrane protein where TatA/E (�9 kDa each) and TatB

(18 kDa) are predicted to be structurally related, each

comprising a single N-terminal transmembrane a-helix with

a predicted N-terminus outside/C-terminus inside topology

(Settles et al., 1997; Chanal et al., 1998). The TatC protein

has six transmembrane helices, with both termini at the

cytoplasmic side of the membrane (Drew et al., 2002;

Behrendt et al., 2004; Ki et al., 2004), and is the largest

(around 30 kDa) of the known Tat system components.

These three classes of relatively small membrane proteins

form at least two types of very high molecular mass

complexes in the E. coli cytoplasmic membrane. From

detergent-solubilized membranes, the TatA protein can be

purified as a large, heterogeneous complex of up to 600 kDa

(Gohlke et al., 2005). When the known Tat components are

overexpressed, this TatA complex also contains small

amounts of TatB (Sargent et al., 2001; de Leeuw et al., 2002;

Porcelli et al., 2002; Oates et al., 2005). In resting mem-

branes, all of the TatC protein is found in a second large

complex, again between 350 and 600 kDa, in an equimolar

ratio with most of the TatB protein and a very low and

variable amount of TatA (Bolhuis et al., 2001; de Leeuw

et al., 2002). For clarity, this Tat(A)BC complex will be

termed here simply the ‘TatBC complex’.

The TatBC complex appears as an oval structure when

viewed in projection by negative stain electron microscopy

(Oates et al., 2003). Current evidence suggests that TatBC is

the initial site of interaction for twin-arginine signal pep-

tides (Cline & Mori, 2001; Alami et al., 2003) and that TatC

bears a specific recognition pocket for the distinctive twin-

arginine motif itself (Alami et al., 2003). Once the TatBC

complex has recognized and bound a Tat substrate protein

the TatA complex can be recruited in a process that is

dependent upon the transmembrane proton electrochemical

gradient (Mori & Cline, 2002; Alami et al., 2003) It is

thought that the TatA complex forms the protein conduct-

ing channel through which the substrate must pass. The

dynamic formation of a TatBC::substrate::TatA supercom-

plex is transient and rapidly dissipates once the substrate

had been transported (Mori & Cline, 2002). There is some

evidence to suggest that the protein transport step is also

dependent upon the transmembrane proton electrochemical

and that each translocation cycle may be accompanied by

the retrograde transport of thousands of protons (Alder &

Theg, 2003).

TheTatAprotein

The TatA protein is the most abundant of all the Tat

components and probably performs the critical role of

forming the protein-conducting channel. When overpro-

duced, the E. coli TatA protein assembles in the membrane as

bundles of at least homotrimers or homotetramers

(de Leeuw et al., 2001). This oligomerization is a property

conferred by the transmembrane helix as its removal results

in the recovery of protein monomers (Porcelli et al., 2002)

The bundles of TatA associate further, possibly sequentially,

into large structures ranging from 450 to 750 kDa in size –

depicted schematically in Fig. 1 (Sargent et al., 2001; Porcelli

et al., 2002; Oates et al., 2005). Low-resolution 3D structures

determined by single particle electron microscopy show that

TatA complexes of different sizes are similar in shape, each

resembling thick-walled rings with an asymmetric ‘lid’ at

one end (Gohlke et al., 2005). Crucially, the internal cavities

of the rings of increasing molecular mass have increasing

volume. This suggests that specific TatA channels are ‘se-

lected’ by TatBC depending on the physical dimensions of

the substrate to be transported. Whether an appropriately

sized TatA channel is selected on a trial-and-error basis or by

active recruitment of TatA protomers remains to be deter-

mined.

The E. coli TatA protein is only 89 amino acids in length;

however, dissecting the structure and function of this small

protein has proven less than straight forward. Genetic

analysis has shown that the N-terminal transmembrane

helix (residues 1–21) is essential for TatA activity (de Leeuw

et al., 2001), but that up to 40 amino acids can be trimmed

from the C-terminus of the protein with no serious detri-

mental effects on in vivo Tat protein transport (Lee et al.,

2002). This suggests the minimal functional TatA protein is

around 50 amino acids in length. However, it seems the

composition of amino acids that follow the initial 50 is

important since a fusion of E. coli alkaline phosphatase

(PhoA) at position 53 completely blocked TatA activity

(Gouffi et al., 2004), although it should be considered that

the presence of a relatively large PhoA protein at this

position may sterically hinder TatA function. More interest-

ingly, perhaps, analysis of this same fusion suggested that

this region of TatA was exposed to the periplasmic face of the

FEMS Microbiol Lett 254 (2006) 198–207 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

199Pathfinders and trailblazers

membrane (Gouffi et al., 2004). Additional PhoA fusions to

TatA at positions 38, 40 and 51 confirmed a periplasmic

localization for this part of the protein. The positions of

these fusions are marked on a cartoon of the TatA protein

shown in Fig. 2. These data were surprising as biochemical

analysis suggested the TatA C-terminus was located in the

cytoplasm and there was no indication of a second trans-

membrane helix in TatA (Porcelli et al., 2002). Paradoxically,

fusions of UidA (b-glucuronidase) to TatA at position 53

implied the opposite topology to the PhoA fusion with the

C-terminal domain located in the cytoplasm (Gouffi et al.,

2004). Taken together, this raises the possibility of either a

static dual topology or a dynamic flipping of the C-terminal

domain of TatA through the membrane under some cir-

cumstances. TatA is predicted to possess a short amphi-

pathic helix close to the N-terminal transmembrane domain

and it is possible this could insert in the membrane to

generate a helical ‘hairpin’-shaped membrane protein (de-

picted schematically in Fig. 1). Indeed insertion of this

isolated domain into lipid monolayers has been observed

(Porcelli et al., 2002). The amphipathic nature of the hairpin

could provide a hydrophilic lining to the protein-conduct-

ing channel. Alternatively, the insertion of the amphipathic

helix through the membrane could provide the driving force

for the transport process itself.

The low-resolution 3D density maps of isolated E. coli

TatA reported by Gohlke et al. (2005) show that the TatA

complexes are annular, approximately 50 A deep (enough to

span the bilayer) and�30 A wide. The 3D density contribut-

ing to the width of the ring ‘walls’ is such that at least one

and possibly two transmembrane helices could be accom-

modated. This could simply be indicative of the clustering of

dimers and trimers of TatA around their hydrophobic

transmembrane segments, or it could point to the hairpin

topology predicted by genetic analysis (represented in

cartoon form in Fig. 1). Indeed, it is notable that the ring

formed by two-helix hairpin subunit c of yeast ATP synthase

is of a similar width to the TatA ring (Stock et al., 1999).

The putative periplasmically exposed extreme C-terminus

of the amphipathic helix of E. coli TatA is of key functional

significance. As shown in Fig. 2, amino acid F39 is located in

this region and has been shown to be critical for TatA

function (Hicks et al., 2003, 2005; Barrett & Robinson,

2005). A genetic screen based on the Tat-dependent export

of chloramphenicol acetyltransferase suggested that only

aromatic side-chains (Phe, Trp, Tyr) could be tolerated at

position 39 and all other substitutions resulted in an inactive

Tat translocase (Hicks et al., 2005). Interestingly, the TatA

F39A variant conferred a dominant-negative (i.e. tat�)

phenotype on wild-type E. coli (Hicks et al., 2003, 2005)

The reason for this remains unclear. One possibility is that

the F39A variant protein is ‘locked’ in the hairpin orienta-

tion. However, it has been observed that, while F39A TatA

can still form minimal trimers in the membrane (Hicks

et al., 2003), Blue-native polyacrylamide gel electrophoresis

(Barrett et al., 2005) and affinity chromatography (McDevitt

et al., 2005) suggest a portion of F39A TatA fails to

oligomerize into large oligomeric ring structures. If the

hairpin structure is essential for TatA ring formation, then

these unusually small TatA complexes could represent a

population that is ‘locked’ in the cytoplasmic amphipathic

helix orientation.

Integral membrane proteins with the structure and topol-

ogy of TatA are relatively rare in prokaryotes. In E. coli, the

closest native membrane proteins with a similar structure

are the b and c subunits of the F1Fo ATP synthase. Subunit b

(encoded by the uncF/atpF gene) comprises a single trans-

membrane span with an N-periplasm/C-cytoplasm orienta-

tion and subunit c (encoded by the uncE/atpE gene) is a

helical hairpin with N-periplasm/C-periplasm topology

(both depicted in Fig. 1). Membrane integration of subunit

c is mediated solely via the YidC membrane protein ‘in-

sertase’ system and completely by-passes the more usual

Sec-pathway for biogenesis of integral membrane proteins

Periplasm

Membrane

Cytoplasm

Periplasm

Membrane

Cytoplasm

(a) (b) (c)

(d) (e) (f) (g)

CC

C

C

C

C C

C C

Fig. 1. Oligomerization of twin arginine (Tat) TatA and membrane

proteins with similar topologies. In the upper panel, cartoon representa-

tions of (a) monomeric Escherichia coli TatA in the hairpin orientation

described by Gouffi et al. (2004), (b) the membrane-bound TatA bundle

identified by chemical crosslinking (de Leeuw et al., 2001), and (c) the

oligomerization of TatA to form a thick-walled annulus that could act as

a protein-conducting channel (Gohlke et al., 2005) are shown. In the

lower panel, cartoon representations of small membrane-bound proteins

from E. coli each with N-terminal a-helices displaying N-out/C-in topol-

ogies are shown. In blue is the b subunit (d) and in yellow is the c subunit

(e) of the E. coli F1Fo ATP synthase. In green is the TatA protein adopting

the C-in orientation (f), and in purple is the TatB protein (g). The locations

of the extreme C-termini are indicated by the letter C. Tat, twin-arginine

protein transport.


200 F. Sargent et al.

(Yi et al., 2003). Biosynthesis of subunit b requires YidC at a

late stage, but targeting and translocation is Sec-dependent

(Yi et al., 2004). From the biosynthesis of these structurally

related membrane proteins it could be presumed that E. coli

TatA would require some YidC involvement in its biosynth-

esis. However, it would be interesting to establish experi-

mentally the mode of E. coli TatA membrane integration

especially as membrane targeting of the thylakoidal TatA

homolog Tha4 has been shown to be both Sec- and YidC-

independent (Fincher et al., 2003) and also because a

portion of cellular TatA has been reported to be water-

soluble in some other bacterial systems (e.g. Pop et al.,

2003).

Nothing is known about how the Tat translocase couples

the proton electrochemical gradient first to the assembly of

the complete Tat translocase, and then to the protein

transport step. This is a gap in our knowledge and

fundamental to understanding the Tat mechanism at the

molecular level. While TatA could simply form a passive

channel for protein transport, it is possible that the TatA

ring has a much more proactive role in the export process.

Returning again to subunit c of ATP synthase, the oligo-

meric state of subunit c has further resonances with the TatA

protein as these hairpins also assemble into ring-like com-

plexes. The subunit c hairpins contain essential aspartate

residues that are involved in the proton translocation event

(Stock et al., 1999). If TatA was similarly involved in sensing

or transducing the proton motive force, it is worth noting

that the TatA amphipathic helix contains a well-conserved

acid residue (D31) that is essential for protein transport

activity (Hicks et al., 2005) and would be a good candidate

for a proton ligand in the hairpin conformation (Fig. 2).

Moreover, the c-ring transduces the energy of the proton

electrochemical gradient into physical work – proton trans-

location causes the c-ring to rotate in the lipid bilayer, which

in turn drives protein conformation changes elsewhere in

TMH

APH

COOH

COOH

Periplasm

Cytoplasm

Me

(a) (b)

Fig. 2. The dual membrane topologies of the twin-arginine (Tat) TatA protein. Primary structure and predicted topologies of the Escherichia coli TatA

protein. (a) TatA with a single N-terminal transmembrane a-helix (TMH) followed by an amphipathic a-helix (APH) at the cytoplasmic side of the

membrane. (b) An identical TatA molecule but this time with the amphipathic a-helix acting as a second transmembrane a-helix thus generating a helical

hairpin structure (indicated by the curved arrow). Amino acids are indicated by their single-letter code and the carboxy termini are marked. Red amino

acids are those shown by Blaudeck et al. (2005) that, when substituted by other side-chains, allow compensation by variant TatA for the absence of TatB.

Blue amino acids indicate the locations of PhoA fusion-junctions used by Gouffi et al. (2004) to assess the in vivo topology of TatA. The residues

highlighted in pink (D31) and black (F39) are key side-chains implicated in TatA biological activity (Hicks et al., 2003, 2005; Barrett & Robinson, 2005)

and the dumb-bell indicates the point of maximum truncation from the C-terminus that can be tolerated without loss of TatA function (Lee et al., 2002).

Tat, twin-arginine protein transport.



the complex (Stock et al., 1999). Indeed, membrane protein

rotation is also associated with proton translocation during

flagella movement (Berg, 2003). Given the topology of the

TatA monomer, together with its c-ring-like oligomerization

in the membrane, and the requirement for proton gradient

dissipation during Tat transport, one possibility is that the

TatA ring could also revolve in the bilayer during the

translocation process.

TheTatBenigma

Some bacteria, including Bacillus subtilis, have been shown

to express an active Tat translocation system that comprises

only TatA and TatC and many more Gram-positive prokar-

yotes, for example Staphylococcus aureus, have genomes that

are completely devoid of a tatB homologue (Pop et al., 2002;

Jongbloed et al., 2004). However, for bacteria that do

produce an obvious TatB protein, e.g. E. coli, disruption of

tatB alone is sufficient to block transport of native Tat

substrates (Weiner et al., 1998; Sargent et al., 1999). How is

it possible that TatB can be either utterly essential or utterly

redundant in different biological systems?

Primary sequence analysis shows that E. coli TatB shares

limited, but significant, sequence similarity with E. coli TatA

and probably arose from a common ancestor (Chanal et al.,

1998; Yen et al., 2002). Both possess a transmembrane a-

helix at their extreme N-terminus with an N-periplasm

orientation, followed by an amphipathic a-helix (Fig. 1). In

the case of TatB, however, the amphipathic helix is longer

than that of the TatA protein and is probably not exposed to

the periplasm under any circumstances (Bolhuis et al.,

2001). Finally, like TatA, the extreme C-terminal region of

TatB is predicted to be unstructured and is not essential for

successful Tat translocation (Lee et al., 2002).

Recently, an elegant study using a facile, efficient, and

sensitive in vivo Tat transport screen has shed light on the

molecular basis of what defines a TatA and a TatB protein at

the molecular level (Blaudeck et al., 2005). Although a native

Sec substrate, the maltose-binding protein (MalE) is an

exclusive Tat substrate when fused to the E. coli TorA twin-

arginine signal peptide and as a result tat1 and tat� E. coli

cells can be easily distinguished on pH-sensitive indicator

plates. Interestingly, although unable to export native Tat

substrates, a DtatB strain could export very low, but none-

theless detectable levels of the MalE reporter. Remarkably,

random mutagenesis and screening identified a number of

modified tatA genes that could complement the DtatB

phenotype and thus allow export of a native Tat substrate.

In four out of five cases, single amino-acid substitutions

within the first six residues of TatA were sufficient to confer

significant TatB-like activity on the variant protein (marked

on a cartoon of TatA in Fig. 2). Each modified protein was

bifunctional since each also retained its original TatA

activity (Blaudeck et al., 2005). This work suggests strongly

that the biological activity of TatA and TatB has been

condensed into one protein in those systems that do not

encode an obvious TatB protein. In addition these observa-

tions clearly imply that the long amphipathic helix and C-

terminus of native TatB is not essential for its interaction

with TatC. The channel-forming and TatC-binding activities

of the bifunctional variant TatA proteins can be assumed

from this successful in vivo study, however swapping of the

entire TatA transmembrane helix for that of TatB led to a

completely inactive chimera suggesting bonafide TatB pro-

teins have lost the ability to form active protein-conducting

channels (Lee et al., 2002).

TheTatCprotein

TatC is the largest and most highly conserved component of

the Tat machinery. The initial controversy regarding the

topology of TatC (Gouffi et al., 2002) is now apparently

resolved, with two groups using independent methodologies

to show that the protein has six transmembrane domains

(Behrendt et al., 2004; Ki et al., 2004). Other than forming a

tight complex with TatB (Bolhuis et al., 2001) and recogni-

tion of the twin-arginine motif of the Tat signal peptide

(Alami et al., 2003), our current knowledge of TatC struc-

ture and function is surprisingly rudimentary.

Twin-arginine signal peptidesandsignalpeptide cleavage

Twin-arginine signal peptides are essential for protein

transport on the Tat pathway. Unlike the Sec pathway, where

mutations that negate the requirement for Sec signal pep-

tides during translocation have been isolated, all proteins or

complexes of proteins destined for Tat export must be

covalently attached to one of these specialized N-terminal

twin-arginine signal peptides. In common with Sec signal

peptides, Tat targeting signals exhibit a tripartite structure

comprising a polar ‘n-region’, relatively hydrophobic ‘h-

region’, and a polar ‘c-region’. The conserved SRRxFLK

twin-arginine motif is always located at the boundary

between the n- and h-region (Berks, 1996). Between sub-

strates, the n-regions of Tat signal peptides can vary greatly

in size and amino-acid composition. Indeed the precise

physiological role of the Tat signal peptide n-region is yet

to be definitively described. Tat signal peptide c-regions

often contain proline residues, a positive charge, and an

A�A cleavage site. The role of the positive charge has been

well studied and has been dubbed the ‘Sec-avoidance’ motif

since it apparently prevents mistargeting of Tat signal

peptides to the Sec pathway (Bogsch et al., 1997). The role

of the proline residue is unknown, but is always located

between the h-region and the A�A motif and could act as a



‘helix breaker’ to facilitate peptidase recognition of the

cleavage site.

One of the most heavily exploited bacterial twin-arginine

signal peptides is that of the trimethylamine N-oxide

reductase (TorA) from E. coli. The TorA signal peptide is 39

amino acids long and endures a complicated pre-export

activity before contact with the Tat machinery. Possibly to

prevent premature interactions between the TorA signal

peptide and the Tat system, the TorA signal peptide is

initially bound tightly by a dedicated cytoplasmic chaperone

(TorD) (Jack et al., 2004). Recognition and binding of the

TorA signal peptide by TorD is not influenced by the

passenger protein to which the peptide is attached. For

example TorD has been reported to interact with TorA signal

peptide fusions to a hydrogenase subunit and an adenylate

cyclase domain in vivo, and with passenger-less synthetic

peptides in vitro (Jack et al., 2004; Hatzixanthis et al., 2005).

Although the interaction between TorD and the TorA signal

peptide is exquisitely specific, and therefore probably not a

general feature of all traffic on the Tat pathway, database

searches identify over 300 TorD-family gene products de-

monstrating that this type of signal peptide binding protein

is widespread in nature. Interestingly it has recently been

demonstrated that coexpression of TorD with a TorA signal

peptide-GFP construct markedly enhanced export of the

fusion protein (Li et al., 2005). If these observations hold

true for other fusion proteins it suggests that coexpression of

a cognate chaperone protein may be an important trick to

enhance yields of industrially important proteins routed

through the Tat pathway.

Based on recent studies, we propose that twin-arginine

signal peptides be divided into two classes (Table 1): ‘Class 1’

twin-arginine signal peptides would be those that are

associated with complex cofactor-containing proteins, such

as the TorA signal peptide described above, and have dual

functionality. These peptides operate both as the principle

binding sites for biosynthetic chaperones before export

(Jack et al., 2004; Hatzixanthis et al., 2005), and, through

their affinity for the Tat translocase (Jong et al., 2004), as

bonafide Tat export signals. A recent estimate suggests that

more than one third of E. coli Tat signal peptides would fall

into this category (Berks et al., 2005). ‘Class 2’ twin-arginine

signal peptides would lack any known or predicted chaper-

one binding function and would therefore be solely involved

in recognizing the Tat transport apparatus and triggering the

protein export event. Likely candidates for Class 2 signal

peptides include that of the monomeric and cofactor-less

protein SufI from E. coli and that of B. subtilis PhoD. Such is

Table 1. Examples of prokaryotic twin-arginine (Tat) signal peptides

Prokaryote Protein Signal peptide Tat� Cleavagew

Class 1 Tat signals

Escherichia coli TorA MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA�AQA Yes SP-I

Escherichia coli DmsA MKTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTLPFSRIAHA�VDS Yes SP-I

Class 2 Tat signals

Escherichia coli SufI MSLSRRQFIQASGIALCAGAVPLKASA�AGQ Yes SP-I

Bacillus subtilis PhoD MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLSLGLTIAQSVGAFEVNA�APN Yes SP-I

Putative lipoproteins

Ralstonia solanacearum Rsc2274 MTPFLDTIRAGRRRWLAVAAGFGLALALSA�CAV Possible SP-II

Ralstonia metallidurans Rmet1154 MNRMTTRMGRVMAWCGALGAALWLAG�CAV Unlikely SP-II

Mycobacterium leprae ML1427 MHGKLFGRRSLLRGAGALTAAALAPGAVG�CSS Possible SP-II

Mycobacterium tuberculosis Rv2041c MVNKPFERRSLLRGAGALTAASLAPWAAG�CAA Possible SP-II

Streptomyces coelicolor Sco0273 MRPRHLRAGAALLCLAALTTLVS�CGS Unlikely SP-II

Arthrobacter sp FB24 Arth1367 MTGATNGHLREITRRTALGALGAGIIGATVAS�WPR Possible SP-I

Streptomyces coelicolor Sco4884 MRRTSRLIRVAVGVASLALAATA�CGG Possible SP-II

Streptomyces coelicolor Sco4885 MRRISRITVAGAATASLALALAA�CGG Possible SP-II

Ralstonia solanacearum Rsc2818 MTVMTTTRGARRALVPLALAATLALTA�CGH Possible SP-II

Mycobacterium leprae ML1116 MRLSVRGRRSVFAGVAVLVSAALVVTG�CSR Possible SP-II

Mycobacterium tuberculosis LprC MRRVLVGAAALITALLVLTG�CTK Possible SP-II

Nocardia farcinica Nfa50790 MRGGALMGRVPGRRAAALSGAVLALVAMVAG�CGR Possible SP-II

A selection of twin-arginine signal peptides sorted into Class 1 (dual function; targeting and biosynthesis), Class 2 (mono function; targeting only), and

those associated with putative or known lipoproteins. The twin-arginines of the canonical motif are shown in bold, as are the proline residues often

found immediately following the hydrophobic h-regions (shown in italics) are also in bold. The signal peptides of the putative lipoproteins are grouped

together according to sequence homology between their cognate passenger proteins and the lipid attachment sites are shown by bold underlined C’s.�Indicates whether the signal peptide directs its passenger to the Tat translocasewIndicates whether the protein is known or predicted to be processed by signal peptidase I (which recognises an A�A�motif immediately N-terminal to

the cleavage site denoted by �), or by signal peptidase II (which recognises a ‘LIPO box’ designated [L/I/G/A][A/G/S] � C around the cleavage site denoted

by �).

Tat, twin-arginine protein transport.



the fast pace of research in this area, it should be noted that

many current apparent Class 2 peptides may need to be

reclassified as dual function Class 1 Tat peptides as further

chaperones are unearthed.

Although Tat signal peptides are often predicted to adopt

a-helical conformations, in practice these peptides appear to

be highly dynamic entities. Nuclear magnetic resonance

experiments have clearly shown that the twin-arginine signal

peptide on the Allochromatium vinosum High Potential Iron

Protein precursor is unstructured in aqueous solution

(Kipping et al., 2003). Likewise, circular dichroism studies

of a synthetic signal peptide based on the E. coli SufI protein

also point to a random coil conformation in aqueous

solution. Upon switching to more hydrophobic environ-

ment, however, the synthetic peptide showed clear signs of

adopting an a-helical conformation (San Miguel et al.,

2003). It is possible, therefore that lipid-embedded Tat

signal peptides could form helical structures. The crystal

structure of the cytochrome b6f complex from the cyano-

bacterium Mastigocladus laminosus demonstrates that this is

indeed the case. The Rieske Fe–S protein subunit of this

complex contains an uncleaved twin-arginine signal peptide

which serves as a signal-anchor. The Rieske signal-anchor is

located on the periphery of the cytochrome b6f complex and

would normally be exposed to the hydrophobic lipid

environment. The h-region of the Rieske signal-anchor is

clearly a-helical in the crystal structure (Kurisu et al., 2003).

Sequence, mutagenic, and structural analysis suggests that

the analogous Rieske proteins found in the cytochrome bc1

and b6f complexes of many bacteria also remain uncleaved

after the targeting and transport process is complete.

Indeed, the confirmed existence of signal-anchored Tat

substrates may also give some insight into the mechanism

of one of the most common and fundamental, but also one

of the most frequently overlooked, events in Tat transport –

signal peptide cleavage.

Most bacterial twin-arginine signal peptides contain a

conserved A�A amino-acid motif (or an acceptable varia-

tion thereof) within the c-region and this is widely accepted

as the recognition site for Type I signal peptidases (LepB in

E. coli). Uncleaved signal-anchored Rieske proteins lack an

obvious A�A motif suggesting that LepB-type enzymes are

indeed responsible for removal of Tat signal peptides. More-

over, the use of an unusual b-lactam drug in E. coli,

previously shown to irreversibly inhibit LepB activity, de-

monstrated that this enzyme probably does remove the

twin-arginine signal peptide of SufI during export (Yahr &

Wickner, 2001). There is, of course, an argument to be made

that some other protease could also have been inhibited by

this compound in E. coli. However, inspection of the E. coli

K-12 genome does not provide any obvious clues to the

existence of orthologous LepB-like proteases. The 3D struc-

ture of a soluble fragment of E. coli LepB has been solved.

This soluble globular domain harbours the serine protease

activity and is normally anchored to the periplasmic face of

the cytoplasmic membrane by two N-terminal transmem-

brane helices. The structure provided vital clues to the

action of LepB as the active site was found to be located on

the flat underside of the protease domain predicted to be

located on the surface of the lipid bilayer in contact with the

headgroups themselves (Paetzel et al., 1998). As a result,

LepB-dependent proteolysis of cytoplasmically located pre-

cursors would be impossible and processing of soluble

precursors in the periplasm after transport would be diffi-

cult. It also seems unlikely that LepB would make contact

with the Tat channel during protein transport. To date, a

signal peptidase has never copurified with any Tat compo-

nents, or indeed with the Sec machinery. Therefore it seems

reasonable that signal peptide cleavage can only efficiently

occur if the A�A motif is correctly oriented in the lipid

bilayer. Most importantly, this assertion suggests a process

that happens after Tat transport is complete.

Available evidence points to the initial insertion of the Tat

signal peptide into the Tat translocase in a ‘loop’ or ‘hairpin’

orientation with n-region and passenger domain on the

same side of the membrane (Fincher et al., 1998). Assuming

the signal peptide n-region remains in the cytoplasm

throughout, Tat translocation of the passenger domain

would result in straightening of the signal peptide ‘hairpin’

through the channel and result in a protein with a topology

identical to that of a signal-anchored Rieske-type protein.

The next crucial step is the transfer of the channel-located

signal peptide to the lipid bilayer. It is most likely that the

uncleaved signal peptide would escape laterally through a

‘side gate’ in the Tat channel (the same lateral escape that has

also been postulated for the release of C-terminal trans-

membrane helices of Tat-dependent membrane proteins;

Hatzixanthis et al., 2003). Upon immersion in this highly

hydrophobic environment the h-region would likely spon-

taneously form an a-helix and the protein would take on the

form of a signal anchored Tat substrate within the lipid

bilayer. For signal-anchored Rieske Fe-S proteins this would

be the final step in targeting. Lipid-embedded Tat signals

would be expected to almost spontaneously form into a-

helical structures and this is certainly the case for the Rieske

Fe-S protein, where the structure is known. For all other Tat

substrates, however, we finally have lipid-embedded Tat

signals with A�A motifs correctly located within, or close

to, the headgroups. Cleavage would quickly follow. It is

conceivable, therefore, that the apparently specialized me-

chanism of Tat-dependent transmembrane segment integra-

tion is arguably very intimately linked to the fundamental

process of signal peptide cleavage.

Finally it should be noted that our model of signal

sequence release into the lipid bilayer before cleavage raises

the possibility of cleavage by alternative signal peptidases.



Signal peptidase II specifically cleaves the signal peptides of

exported lipoproteins at a distinctive recognition site called

the ‘LIPO box’, the key feature of which is an invariant

cysteine residue at the 11 position relative to the cleavage

site. In the mature lipoprotein this cysteine residue is

attached to a membrane-associated lipid anchor. The ex-

istence of Tat-dependent lipoproteins has never been ex-

perimentally proven, however some predicted twin-arginine

signal peptides do contain plausible LIPO boxes (Table 1).

Moreover, many of the putative Tat substrates of Halobac-

terium sp. NRC-1 were predicted to be lipoproteins based on

bioinformatic analysis (Rose et al., 2002), and there are also

predictions of lipoproteins in the in silico-generated Tat

proteome of Streptomyces coelicolor (Dilks et al., 2003) – see

also http://www.sas.upenn.edu/�pohlschr/tatprok.html.

However, many predicted Tat-dependent lipoproteins are

either not strictly Tat-dependent, or do not have conserved

LIPO boxes, across closely-related species (Table 1). Thus

the existence of prokaryotic Tat-dependent lipoproteins still

awaits experimental verification.

Concluding remarks

Despite the fact that it is well under a decade since the Tat

pathway was discovered, we have made enormous strides in

our understanding of how this remarkable system recog-

nizes and transports folded proteins. With mounting evi-

dence for a significant role of the Tat pathway in bacterial

pathogenesis and an increasing interest in exploiting the

pathway for biotechnological applications, future develop-

ments in the structure, function and mechanism of Tat-

dependent protein transport are eagerly awaited.

Acknowledgements

We thank our colleagues, past and present, with whom we

have shared ideas the Tat system. Frank Sargent is a Royal

Society University Research Fellow and Tracy Palmer is an

MRC Senior Non Clinical Research Fellow. Research in the

authors’ laboratories has been or is currently supported by

the Biotechnology and Biological Sciences Research Coun-

cil, the Medical Research Council, the Commission of the

European Community, the Leverhulme Trust, the Wellcome

Trust, the Royal Society, the John Innes Centre, the EPA

Cephalosporin Fund, the John and Pamela Salter Charitable

Trust, the University of East Anglia and the University of

Oxford.

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Pathfinders and trailblazers: a prokaryotic targeting system for transport of folded proteins