RAB GTPASE-ACTIVATING PROTEINS

AT THE GOLGI:ENDOSOME INTERFACE

A DISSERTATION

SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY

AND THE COMMITTEE ON GRADUATE STUDIES

OF STANFORD UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Ryan Michael Nottingham

May 2010

http://creativecommons.org/licenses/by-nc/3.0/us/

This dissertation is online at: http://purl.stanford.edu/dv301zj4923

© 2010 by Ryan Michael Nottingham. All Rights Reserved.

Re-distributed by Stanford University under license with the author.

This work is licensed under a Creative Commons Attribution-Noncommercial 3.0 United States License.

ii

I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.

Suzanne Pfeffer, Primary Adviser

I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.

Gilbert Chu

I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.

Pehr Harbury

Approved for the Stanford University Committee on Graduate Studies.

Patricia J. Gumport, Vice Provost Graduate Education

This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file inUniversity Archives.

iii

iv

ABSTRACT

Rab GTPases are master regulators of membrane trafficking in eukaryotic

cells. With GTP bound, they regulate trafficking by recruiting effectors to

specific membrane-bound compartments. Rab proteins are themselves

regulated by additional factors that mediate their proper localization as well as

their bound nucleotide state. GTPase-activating proteins (GAPs) stimulate a

Rabʼs intrinsic rate of GTP hydrolysis, thus inactivating the Rab by converting

bound GTP to GDP. Regulation of Rab proteins links the formation and

breakdown of sequential, Rab-regulated membrane domains in the secretory

and endocytic pathways. The first chapter introduces the function of

RabGTPases in membrane trafficking and the role of GAPs in regulating Rab

function.

The second chapter of this thesis presents the characterization of a novel

RabGAP, RUTBC1. This protein was identified through a yeast two hybrid

screen for RabGAPs that interact with Rab9, the key regulator of mannose 6-

phosphate receptor (MPR) recycling from late endosomes to the trans Golgi

network. RUTBC1 binds Rab9 in vitro and in cells through interactions with its

N-terminus. Overexpression of RUTBC1 only slightly disrupts MPR trafficking

and RUTBC1 does not function as a GAP for Rab9. An in vitro biochemical

screen of 32 mammalian Rab GTPases revealed that RUTBC1 has GAP

activity toward Rab33b and Rab32 that is catalyzed by its conserved TBC

v

domain. These data suggest that RUTBC1 might function to link inactivation of

these Rabs in relation to a Rab9 microdomain, in support of the existence of a

Rab cascade at the interface between the Golgi apparatus and endosomes.

The third chapter describes the functional role of RUTBC1 in cultured cells.

Depletion of RUTBC1 unexpectedly leads to concomitant depletion of

Atg16L1. Atg16L1 has an established and essential role in macroautophagy,

a highly conserved cellular recycling process. Overexpression of Atg16L1

causes the formation of large puncta in the cytoplasm, which are also labeled

by endogenous RUTBC1 and may represent autophagosomes. Atg16L1 is a

known Rab33b effector, suggesting that Rab33b, RUTBC1 and Atg16L1

function together to regulate autophagosome formation.

The fourth chapter describes another TBC-domain containing protein,

RUTBC2. This protein is highly related to RUTBC1 and also binds specifically

to Rab9. In vitro biochemical screening for RUTBC2ʼs Rab substrates showed

that RUTBC2 had highest GAP activity toward Rab34 and Rab36, two very

similar Rabs thought to play a role in secretion. The difference in substrate

specificity between RUTBC1 and RUTBC2 further exemplifies the highly

complex integration of diverse membrane trafficking pathways in mammalian

cells.

vi

ACKNOWLEDGEMENTS

Above all, I would like to thank my research advisor, Suzanne Pfeffer. During

my time at Stanford, I have learned so much from her: not only how to think

about science, but just as importantly, how to communicate the results. Most

of all though, I appreciate the independence she afforded me and moreover,

the patience that resulted from it. As a mentor, she helped me to realize that

nothing is impossible despite the obstacles and adversity one often

encounters in research.

Secondly, I would like to thank the other members of my committee, Gil Chu

and Pehr Harbury, for their advice and constructive criticism throughout my

time. Always helpful, I wished I had sought their advice more often than I did.

The Biochemistry Department deserves credit as well for making research

here easier through the great atmosphere maintained by the students and

postdocs. My collaborators in this work also deserve special mention: Francis

Barr and his laboratory as well as David Lambright and his laboratory. Without

their help, this thesis would have taken unimaginably longer. I also want to

thank my undergraduate advisor, Dorothy Shippen, for taking a chance on a

directionless undergrad and introducing me to the life of research.

Next, I want to express my thanks to the past and present members of the

Pfeffer Lab. What a ride! I am glad that I have met each of you – it is hard to

vii

imagine going through the ups and downs of graduate school with any other

group of people. I especially want to thank Maïka Deffieu for all of her helpful

discussions about autophagy (and for being a great friend!). I also especially

want to thank the postdocs who were here when I joined the lab (Pfeffer One!):

Dikran Aivazian, Leo Serrano, Ian Ganley, Sridevi Khambhanpaty and Monica

Calero. They made the lab a fun and joyful place…it was “beyond dreams.”

To my fellow Pfeffer Lab graduate students – I have never enjoyed discussing

science, music, religion and other esoterica more. A big thanks to Garret

Hayes, Eric Espinosa, Peter Lee and Frank Brown for being great friends as

well as great colleagues. Garret and Peter put up with me as bay mates and

were always great sounding boards for ideas about anything. Eric and Frank

were essentially my bay mates – I spent as much time in B455 as in B457 –

and showed me that “you, too, can get out of bed in the morning!”

Finally, I express my love and thanks to the people who have listened to whiny

phone calls and e-mails, trekked out to visit me, moved me half-way across

the country and never ever stopped being my biggest boosters and

supporters. Thank you to my friends in Texas and California. To my parents,

Mike and Patty, my brothers Dean and Sean, and my sister Erin, I love you

and none of this would have been possible without you.

viii

TABLE OF CONTENTS

1) Introduction 1

Rab GTPases 2

Rab Localization and Microdomains 5

The Rab Cycle 9

Rab GTPase-Activating Proteins 15

GAPs and GEFs: Defining Boundaries 28

References 34

Table 54

2) RUTBC1: a novel Rab9 effector that activates GTP hydrolysis by

Rab33B and Rab32 58

Abstract 59

Introduction 60

Methods 63

Results 70

Discussion 77

References 82

Figure Legends 88

Figures 91

ix

3) Interaction of RUTBC1, a Rab33B GAP, with the Rab33B effector,

Atg16L1 97

Abstract 98

Introduction 99

Methods 103

Results 106

Discussion 109

References 111

Figure Legends 114

Figures 116

4) Characterization of Rab substrates and binding

partners of RUTBC2 122

Abstract 123

Introduction 124

Methods 127

Results 133

Discussion 138

References 141

Figure Legends 145

Figures 148

5) Summary and Future Perspectives 153

x

LIST OF TABLES

Introduction

Table I. Summary of mammalian Rab GTPase-activating proteins 54

xi

LIST OF FIGURES

Chapter 2

Figure 1: RUTBC1 interacts with Rab9 91

Figure 2: RUTBC1 is an effector of Rab9 92

Figure 3: RUTBC1 binds to, but is not a GAP for Rab9 in cells 93

Figure 4: RUTBC1 TBC domain has GAP activity toward 94

Rab33B and Rab32 in vitro

Figure 5: RUTBC1 TBC domain stimulates GTP hydrolysis 95

Chapter 3

Figure 1: Domain architecture of RUTBC1 and Atg16L1 116

Figure 2: RUTBC1 solubilizes Atg16L1 from the Golgi 117

Figure 3: RUTBC1 and Atg16L1 interact in cells 118

Figure 4: RUTBC1 localizes to Atg16L1-positive puncta 120

Chapter 4

Figure 1: RUTBC2 interacts with Rab9 148

Figure 2: RUTBC2 is an effector of Rab9 149

Figure 3: RUTBC2 binds to, but is not a GAP for Rab9 in cells 150

Figure 4: RUTBC2 TBC domain has GAP activity toward 151

Rab36 and Rab34 in vitro

Figure 5: RUTBC2 is stably associated with membranes 152

in SK-N-SH cells

1

INTRODUCTION

Eukaryotic cells contain membrane bound compartments that segregate

different biochemical functions from each other and from those occurring in the

cytoplasm. Organelles are covered with different sets of proteins and lipids

that distinguish different compartments. The maintenance of individual

compartment identity and the transfer of protein and membrane between

compartments occur through molecular events collectively termed membrane

trafficking.

The molecules that provide specificity for each trafficking event, as well as

those that remodel membrane during vesicle budding and fusion, have been

investigated actively over the last thirty years. A fundamental question in the

field is how these organelles remain distinct despite the constant flux of

membrane and protein trafficked throughout the cell. This chapter focuses on

the master regulators of trafficking events, the Rab GTPases. I will discuss the

roles played by Rab proteins and their regulators, with emphasis on Rab

GTPase-activating proteins (RabGAPs), in giving compartments specific

identity through formation, maintenance and breakdown of membrane

microdomains.

2

Rab GTPases

Rab GTPases are members of the Ras-like GTPase superfamily – a large

group of proteins of approximately 25kDa that derive their function from their

ability to both bind and hydrolyze GTP. These so-called ʻGʼ proteins act as

switches governed by the identity of the bound nucleotide; this is a

consequence of a conformational change between active GTP- and inactive

GDP-bound states (Colicelli, 2004). Exchange of GDP for GTP leads to

changes in two regions of G proteins, called switch I and switch II, that form

hydrogen bond contacts with the phosphate groups of GTP. Hydrolysis of GTP

leads to the loss of these hydrogen bonds and a subsequent conformational

change back to the GDP-bound structure. This difference in conformation

allows “effector” proteins that bind small GTPases in their active state to

discriminate between the GTP- and GDP-bound forms and thus regulate

cellular processes. The superfamily is divided into five major subfamilies

including Ras, Rho, Ran, Arf and Rab. The last two subfamilies are critically

important in the regulation of membrane trafficking. Arf proteins (and the

related Arls) play a role in coat recruitment to vesicle budding sites, notably at

the Golgi and plasma membrane as well as in cytoskeletal dynamics

(Gillingham and Munro, 2007). Rab proteins, the largest subfamily, participate

in all steps of membrane trafficking including vesicle formation, motility,

tethering and docking and fusion (Segev, 2001; Zerial and McBride, 2001;

Stenmark, 2009).

3

The first members of the Rab family were discovered in S. cerevisiae. Sec4

was first identified in a screen for secretion mutants (Novick et al., 1980).

Yeast blocked in secretion become more dense than normal cells due to the

accumulation of dense secretory vesicles and other membranes. This property

allowed Schekman and coworkers to select for mutant cells by density

centrifugation. They discovered twenty-three complementation groups of

secretion mutants, many of which are well studied today. Before the product of

the SEC4 gene was identified, another gene named YPT1 was characterized

as an open reading frame between the tubulin and actin genes that had high

homology to the Ras oncogenes (Gallwitz et al., 1983). Both SEC4 and YPT1

are essential genes and YPT1 could not rescue a double deletion of

RAS1/RAS2 in yeast, suggesting that Ras-like GTPases performed a diverse

set of functions (Salminen and Novick, 1987; Schmitt et al., 1986). Soon after,

the SEC4 gene product was discovered to have homology to Ras and it was

even more closely related to YPT1; its overexpression could also suppress the

phenotypes of many of the late acting SEC mutants (Salminen and Novick,

1987). A ypt1 conditional-lethal mutant showed defects in protein secretion

(but at an earlier step than SEC4) and improper membrane growth (Segev et

al., 1988). Studies of these two proteins indicated that a novel family of

GTPases controlled membrane dynamics in cells. Later work in mammalian

cells identified four additional genes in a screen of cDNAs from rat brain with

oligonucleotides specific for Ras-like proteins (Touchot et al., 1987). This is

4

the genesis of the term Rab for “Ras-like GTPase from rat brain.” These genes

were quickly determined to have homology to YPT1 and SEC4 (Zahouri et al.,

1989). The remarkable discovery that mouse Rab1 could functionally replace

Ypt1 in yeast suggested that secretion (and likely other membrane trafficking

events) were regulated by conserved machineries (Haubruck et al., 1989).

This was consistent with contemporary findings showing homology between

the SEC18 gene product and NSF (N-ethylmaleimide sensitive factor), an

AAA+ ATPase required for vesicle fusion (Wilson et al., 1989). From eleven

members in yeast to over seventy members in mammalian cells, the Rab

subfamily is the largest group in the Ras-like GTPase superfamily (Pereira-

Leal and Seabra, 2001).

Rabs are composed of two different domains: a G domain that binds and

hydrolyzes guanine nucleotides and a C-terminal, so-called “hypervariable”

domain. The G domain is a typical nucleotide binding fold consisting of a

series of beta strands surrounded by alpha helices and all five conserved G

protein motifs are present in Rabs (Bourne et al., 1991, Colicelli, 2004). The

hypervariable domain is unique to the Rab subfamily of Ras-like small

GTPases. It is thought to have an irregular structure (Ostermeier and Brunger,

1999) and it plays a role in Rab localization (Chavrier et al., 1991; Aivazian et

al., 2006). Rab proteins associate with membranes by the addition of at least

one, but usually two, C-terminal cysteine-linked geranylgeranyl groups

5

(Khosravi-Far et al., 1991). Mutation of both cysteine residues abolishes Rab

membrane association and interferes with their functions in cells (Molenaar et

al., 1988; Walworth et al., 1989). Stable membrane association is thus

essential for Rabs to function as membrane organizers.

Rab Localization and Microdomains

One of the most striking discoveries in membrane trafficking was that each

organelle contains a specific set of Rab proteins (Chavrier et al., 1990). Zerial

and co-workers isolated eleven cDNA clones from Madin-Derby canine kidney

cells that had high homology to SEC4 and YPT1. Immunofluorescence and

electron microscopy showed that three of these Rabs were differentially

localized to organelles throughout the cell: e.g., Rab2 appeared to be on a

compartment intermediate between the endoplasmic reticulum (ER) and Golgi

apparatus, while Rab5 was localized to the plasma membrane and structures

in the cytoplasm (Chavrier et al. 1990). With over 70 Rabs in humans and at

least 40 expressed in one cell type alone (Nguyen et al., 2009), Rabs provide

both specificity and irreversibility to the organization of the diverse array of

membrane trafficking events.

The determinants of Rab localization are rather complex. Experiments using

chimeras of Rab5 and Rab7 led to the initial conclusion that the hypervariable

domain itself was sufficient to localize a Rab (Chavrier et al., 1991). Rab5

6

bearing the C-terminus of Rab7 seemed sufficient to re-localize Rab5 from

early endosomes to late endosomes. The C-termini of both Rab5 and Rab7

were also able to shift Rab2, normally on the Golgi, to early or late

endosomes, respectively (Chavrier et al., 1991). Similar experiments using

chimeras of Rab9, Rab5 and Rab1 revealed a more elaborate mechanism for

Rab localization. Pfeffer and co-workers showed that Rab localization was

dependent on effector binding and not simply the hypervariable domain

(Aivazian et al., 2006). They showed that re-localization of Rab5 bearing the

Rab9 hypervariable domain was dependent on the Rab9 effector TIP47 (Tail

Interacting Protein of 47kDa). Mutations in TIP47 that abrogate Rab9 binding

also failed to re-localize chimeric Rab5 (Aivazian et al., 2006). Thus, Rab

localization is dependent on both prenylation and effector binding to recruit

Rabs to the correct membrane. In several cases, the hypervariable domain is

important for effector binding, which may explain Chavrier et al.ʼs original

findings.

Rabs further define subdomains on organelles called microdomains (Zerial

and McBride, 2001). They provide identity to the membrane they are localized

to by specifically concentrating their effector proteins at these sites (Pfeffer,

2001). Effector proteins can include integral membrane proteins or soluble

proteins recruited to membranes by the Rab; moreover Rabs recruit lipid

modifying enzymes such as phosphoinositide kinases (Christoforidis et al.

7

1999a), further adding specificity by enriching for particular lipids. As an

example, Rab9 helps to define a microdomain on late endosomes separate

from Rab7 (Lombardi et al., 1993; Barbero et al., 2002). Rab7 functions in the

conversion of early endosomes into late endosomes and their eventual

maturation into lysosomes (Zhang et al., 2009). Mannose 6-phosphate

receptors (MPRs) deliver newly synthesized acid hydrolases to pre-lysosomal

compartments (Ghosh et al., 2003). There, the hydrolases dissociate from the

receptor and eventually localize to the lysosome, while MPRs must be

recycled back to the trans Golgi network (TGN) for another round of delivery.

Rab9 is essential for this recycling step and facilitates retrieval of MPRs

through its effector, TIP47. TIP47 binds to the cytoplasmic domains of MPRs

(Diaz and Pfeffer, 1998) The affinity of TIP47 for MPR cytoplasmic domains is

~1µM (Krise et al., 2000). In the presence of Rab9, the affinity of TIP47 for

MPR cytoplasmic tail is increased to ~300nM (Carroll et al. 2001). Thus, active

Rab9 works with its effector, TIP47, to segregate MPRs into a domain on late

endosomes that prevents them from being sorted to the lysosome.

Perhaps an even more elaborate example of Rab-mediated microdomain

formation comes from studies of Rab5. Rab5 and Rab4 are both found on

early endosomes (van der Sluijs et al., 1991; Bucci et al. 1992) but in distinct

domains. Expression of fluorescently tagged Rab5 and Rab4 showed that

endocytosed transferrin first entered a Rab5 domain on early endosomes and

8

then entered a Rab4 domain (Sönnichsen et al., 2000). This is consistent with

the function of both Rabs: Rab4 regulates recycling of transferrin receptor

back to the plasma membrane (van der Sluijs et al., 1991) while Rab5

functions in homotypic fusion of endosomes and clathrin-coated vesicles

through interaction with at least twenty different effectors (Christoforidis et al.,

1999b). While distinct, the Rab4 and Rab5 are coupled to each other. Each

Rab has a diverse set of effectors while effectors themselves generally only

bind to one or a few Rabs. Rabaptin-5 and Rabenosyn 5 bind both Rab5 and

Rab4 through distinct regions and might function to link these microdomains

together on the surface of early endosomes (Vitale et al., 1998; de Renzis et

al., 2002). Additional examples of “microdomain tethers” are likely to exist,

particularly in the Golgi stack. In a small number of cases where multiple Rabs

bind to a particular effector (Hayes et al., 2009; Sinka et al., 2008), these

interactions may be a dynamic but regulated way of keeping certain

microdomains linked in order to form some higher order structure or function,

e.g. an intact Golgi ribbon.

Rab5 microdomains seem to assemble cooperatively (Zerial and McBride,

2001). Rab5 binds to Rabaptin-5, which in turn, also forms a complex with

Rabex-5, a guanine nucleotide exchange factor or GEF (Stenmark et al., 1995;

Horiuchi et al., 1997). Thus, Rab activation is coupled to effector binding: in

this manner, a feedback loop exists where increasing amounts of active Rab5

9

exist on endosomes. Rab5 then recruits Early Endosome Antigen 1 (EEA1), a

tethering factor required for endosome fusion that also has a FYVE domain (a

binding domain for phosphatidylinositol-3-phosphate (PIP3)) and both Rab5

binding and PIP3 binding are required to recruit EEA1 to endosomes

(Christoforidis et al., 1999b; Stenmark et al., 1996; Simonsen et al., 1998).

Remarkably, Rab5 also binds Vps34, a phosphoinositide kinase that

generates PIP3 on early endosomes (Christoforidis et al., 1999a). Working

together with its effectors, Rab5 catalyzes the formation of this specialized

membrane domain. On other early endosomes, Rab5 also catalyzes a distinct

domain that contains APPL but not EEA1 (Miaczynska et al., 2004). What

causes microdomains to not simply diffuse apart in the plane of the

membrane? One model posits that oligomerization of effectors coupled with

lipid enrichment may stabilize a microdomain until GTP hydrolysis initiates its

dismantling (Rybin et al., 1996; Zerial and McBride, 2001).

The Rab Cycle

Key to proper microdomain formation, and therefore membrane trafficking, is

the regulation of active Rab localization. Rab localization is intimately tied to

two different cycles that generally correlate with each other: a cycle of

nucleotide binding and a cycle of membrane association. Cytosolic Rabs are

exclusively GDP-bound, and are activated after delivery to the membrane.

Membranes can contain both GTP- and GDP-bound Rabs, but only GDP Rabs

10

can be removed from membranes. These cycles provide multiple points of

regulation to ensure well organized membrane trafficking.

Newly synthesized, GDP-bearing Rabs are bound by Rab Escort Protein or

REP (Andres et al., 1993). REP presents the new Rab to the RabGGTase

prenylating enzyme, which utilizes only the REP:Rab complex as substrate

(Andres et al., 1993). RabGGTase has little affinity for Rab proteins on their

own and requires the presence of REP for efficient prenylation (Alexandrov et

al., 1999). The REP:Rab complex then dissociates from RabGGTase; after

prenylation, a prenyl group-triggered conformational change in REP disrupts

the REP:RabGGTase binding site (Rak et al., 2004). Another protein, GDP-

dissociation inhibitor or GDI, also delivers prenylated Rabs to membranes but

has the added function of being able to extract Rab proteins from membranes

as well (Sasaki et al., 1990, Araki et al., 1990). GDI binds only to prenylated

Rabs that are also bound to GDP (Shapiro and Pfeffer, 1995; Rak et al.,

2003). Both REP and GDI bind to the switch I and switch II regions of Rab

proteins, which explains the requirement for GDP-bound Rabs as well as the

name GDI. GDI is able to extract GDP-bound Rabs from membranes while

REP is poor at this function. This is explained by the binding specificities for

prenylated and unprenylated Rabs. REP has high affinity for the Rab itself

since it can bind either form of the Rab, while GDI has high affinity specifically

for prenylated Rabs. Thus, REP likely cannot overcome the energy barrier

11

required to extract the prenyl groups from the membrane, even though it

seems to be able to keep the prenyl groups from aggregating before

membrane delivery (Goody et al., 2005). Interestingly, the REP binding site

for RabGGTase has the highest sequence and structural homology to GDI

(Pylypenko et al., 2003). The difference in binding arises from the presence of

two REP residues, a phenylalanine and an arginine, found in REP that are

absent from GDI (Alory and Balch, 2000).

GDI:Rab complexes contain all information necessary to deliver Rabs to the

correct membrane, and nucleotide exchange occurs after association with

membranes (Soldati et al., 1994; Ullrich et al., 1994). Because of GDIʼs high

affinity for prenylated Rabs as well as the inability of some GEFs to use

GDI:Rab complexes as substrates, an enzymatic activity was hypothesized to

exist that catalyzed the release of Rabs from GDI (“GDF”; Dirac-Svejstrup et

al., 1997). Pfeffer and co-workers used reconstituted GDI:prenyl-Rab

complexes as substrates to probe purified late endosome membranes for this

activity. Rabs in complex with GDI do not exchange nucleotide (Sasaki et al.,

1993). Displacement of GDI would permit nucleotide exchange at either the

intrinsic or GEF-stimulated rate. Protease sensitive GDF activity was detected

in membranes. Intriguingly, this crude GDF was able to dissociate endosomal

Rab:GDI complexes but not those associated with the secretory pathway

(Dirac-Svejstrup et al., 1997). Yip3/PRA1 is the only mammalian protein

12

identified to date that possesses GDF activity and it too, is specific for

endosomal Rabs (Sivars et al., 2003). Yip3/PRA1 belongs to a conserved

family of integral membrane proteins from yeast to humans that interact with

Rabs, have distinct localizations, and at least one member has low affinity for

GDI (Yang et al., 1998; Abdul-Ghani et al., 2001; Hutt et al., 2000). These

properties make Yip/PRA proteins excellent candidates to be GDFs.

GDI displacement and subsequent insertion of the prenyl groups into

membranes are still poorly understood processes. After membrane

attachment, Rabs undergo nucleotide exchange, replacing GDP with GTP

(Soldati et al., 1994). This step is necessary to prevent solubilization of the

newly-delivered Rab by GDI. In vitro, GDP dissociation is the rate-limiting step

of the nucleotide cycle of GTPases. Rabs also have slow, intrinsic rates of

GDP dissociation, and so another enzymatic activity called a guanine

nucleotide exchange factor (GEF) is required to catalyze this process. It has

recently been proposed that GEF activity alone might be sufficient for GDI

displacement (Schoebel et al., 2009; Suh et al., 2010). This model is based on

studies of the Leishmania DrrA/SidM protein that was identified initially as a

protein containing both GDF and GEF activity (Ingmundson et al., 2007;

Machner and Isberg, 2007). DrrA is a potent GEF for Rab1 with a Kd of ~2pM

for nucleotide-free Rab1, which led the authors to argue that GDI displacement

is due to GEF activity alone (Schoebel et al 2009; Suh et al 2010). Because

13

pathogens probably have evolved highly efficient infection processes, it is

unclear if endogenous GEFs can apparently displace GDI as DrrA can.

Indeed, Transport protein particle complex I (TRAPPI), an endogenous GEF

for Ypt1 in yeast, has a much weaker Kd for nucleotide-free Ypt1 (~200nM),

so they may be unable to drive a GDF reaction (Chin et al., 2009).

The basic mechanism for GEF-catalyzed nucleotide exchange is very similar

among all Ras-like GTPases (Itzen et al., 2007). GEFs disrupt the nucleotide

binding site by driving out the phosphate groups of GDP (Bos et al., 2007).

This causes a conformational change in the switch I region that is incompatible

with nucleotide binding. GEF dissociates when GTP binds. GEFs do not have

specificity for one nucleotide over the other; nucleotide exchange is driven by

the almost 10-fold higher cellular levels of GTP over GDP (Goody and

Hoffman-Goody, 2002). This allosteric competition between nucleotide and

GEF binding is near universal among the Ras-like GTPase superfamily (Bos et

al., 2007). Currently, RabGEFs are poorly characterized: unlike other

regulatory proteins there is no common RabGEF domain. Vps9 domain-

containing proteins catalyze nucleotide exchange on Rab5 subfamily members

through a helical bundle (Delprato et al., 2004). Sec2 utilizes a coiled-coil

domain to catalyze nucleotide exchange on Sec4 (Dong et al., 2007). TRAPPI

consists of multiple subunits, most of which are required for GEF activity on

Ypt1 (Wang et al., 2000; Cai et al., 2008). In this case, multiple subunits form

14

the interface for Rab binding. Other work has shown that addition of three

further subunits changes both the localization and substrate specificity of

TRAPP. This complex is then called TRAPPII and possesses GEF activity on

Ypt31/32 (Jones et al., 2000; Morozova et al., 2006).

At this part of the cycle, Rabs are both membrane associated and bound to

GTP. This is the fully active state of the Rab and the state to which numerous

and diverse effector proteins bind. Since the membrane associated, GTP-

bound Rab gives a portion of membrane specific identity, inactivation of Rab

proteins is of critical importance to coordination and maintenance of organelle

function. Rabs that are mis-localized would recruit effectors to the wrong

compartment leading to establishment of “ectopic” microdomains. Rabs are

inactivated by hydrolysis of GTP to GDP and inorganic phosphate. Like all

GTPases, Rabs possess a slow, intrinsic rate of hydrolysis (Colicelli, 2004).

This presents another opportunity for regulation. Cells have evolved GTPase-

activating proteins (GAPs) to rapidly terminate active signaling. GAPs can

stimulate the intrinsic rate of hydrolysis by several orders of magnitude (Bos et

al., 2007). The discovery of RabGAPs as well as their proposed biochemical

mechanism of stimulation will be discussed below.

At the end of the cycle Rabs are again bound to GDP; effectors have much

lower affinity for their cognate, GDP-Rabs and the microdomain begins to

15

break down. Some Rabs may be simply re-activated by GEFs still present in

the microdomain; others may be extracted by GDI into the cytosol before a

GEF can act. GDI then recycles the Rab back to its donor compartment for re-

delivery to membranes. This recycling step is not essential for some Rab-

mediated events -- Ypt1 and Sec4 modified at their C-termini with permanent

membrane anchors were only slightly less efficient at growth and secretion

(Ossig et al., 1995). Presumably the lowered efficiency reflected the need to

synthesize new Rab proteins, as the permanently membrane anchored Rabs

might diffuse throughout the membrane system or eventually be degraded.

Rab GTPase-Activating Proteins

GAPs specific for Rab proteins were first identified in yeast (Strom et al.,

1993). Gallwitz and co-workers overexpressed a library of 2500 multi-copy

plasmids and a single transformant encoded a GTPase-activating activity that

showed highest activity toward Ypt6 (the yeast homolog of Rab6). The clone

was isolated and its identity was confirmed by expression and purification from

E. coli. This protein was termed Gyp6 (GAP for Ypt6). Gyp6 is not an essential

gene and deletion of Gyp6 produced no readily identifiable phenotypes. (Strom

et al., 1993). In vitro, Gyp6 was rather specific in substrate utilization; only

Ypt7 was comparable to Ypt6. Intriguingly, in gyp6∆ cells, Ypt7GAP activity

could still be detected, suggesting that each Rab might have its own GAP,

which could help to organize membrane trafficking events (Strom et al., 1993).

16

Eight yeast RabGAPs were identified: Gyp7 with activity on Ypt7 (Vollmer and

Gallwitz, 1995), Gyp1 with activity on Ypt1 and Sec4 (Du et al., 1998; Vollmer

et al., 1999), Gyp2 and Gyp3 that had broad substrate specificity (Albert and

Gallwitz, 1999), Gyp4 with activity on Sec4, Ypt6, and Ypt7 (Albert and

Gallwitz, 2000), and Gyp5 and Gyp8 which show preferred activation of Ypt1

(De Antoni et al., 2002).

These discoveries were aided by the fact that unlike RabGEFs, RabGAPs

have homology to each other. The Gyp proteins were originally thought to be

structurally unrelated, but sequence alignments proved otherwise. As the

amount of annotated sequences became available, it was noted that Gyp

proteins share a common domain (Neuwald, 1997). This domain was named

TBC for Tre2/Bub2/Cdc16. Tre2 is an oncogene that is formed by the fusion

of a TBC domain and a ubiquitin specific protease domain (Nakamura et al.,

1992). Bub2 and Cdc16 are homologous proteins in bakerʼs and fission yeast,

respectively, that are parts of a two-component GAP that regulates spindle

assembly and septum formation during mitosis through the Ras-like GTPase,

Tem1/Spg1 (Wang et al., 2000; Furge et al., 1998).

The TBC domain has six conserved motifs; the first three are nearly

universally conserved in all members. These “fingerprint” motifs are RxxxW in

motif A; IxxDxxR in motif B; and YxQ in motif C (Neuwald, 1997). Truncation

17

analyses of Gyp1 and Gyp7 confirmed functional homology in the putative

catalytic domain and identified conserved arginine residues in motifs A and B

that are critical for GAP activity (Albert and et al., 1999). This led Gallwitz and

co-workers to conclude that RabGAPs catalyze GTP hydrolysis by a

mechanism similar to GAPs for other Ras-like GTPases.

The crystal structure of Gyp1 showed that the TBC domain consists of 16

alpha helices (Rak et al., 2000). Surprisingly, the overall fold of the Gyp1 TBC

domain showed no similarity to that of other Ras-like GTPase family GAPs,

despite their content of alpha helices (Rak et al., 2000; Scheffzek et al., 1997;

Rittinger et al., 1997). The TBC domain adopts a “V” like shape, where

sequence motif A is in the core of the protein while motifs B and C are located

in the groove inside the “V” (Rak et al., 2000). The key arginine in motif A is

thought to contribute to overall fold stability rather than catalysis, while motifs

B and C define a putative Rab binding site. All RabGAPs have relatively low

affinities for their substrates, analogous to other GAP families (Bos et al.,

2007). This low affinity might be overcome in cells by recruitment of GAPs to

membranes. Gyp1, for example, localizes to the Golgi at steady-state, while

TBC1D20 even has a transmembrane domain (Du and Novick, 2001; Haas et

al., 2007; Sklan et al., 2007).

18

GTPase activation by most Ras-related GAPs involves an arginine “finger”

provided in trans by the GAP and a conserved glutamine provided in cis by the

GTPase. This conserved glutamine mediates GTP hydrolysis by coordination

of a water molecule for nucleophilic attack on the gamma phosphate, both in

the context of the GTPase alone and in complex with its GAP (Wittinghofer et

al., 1997). Binding of the GAP is thought to order the switch II region in order

to favor this alignment. This leads to a shift of negative charge from the

gamma to the beta phosphate; this charge distribution is closer to GDP than to

GTP (Allin et al., 2001). The accumulating negative charge is stabilized by the

guanidinium group of the arginine. This charge compensation by arginine is

thought to reduce the activation energy for breaking the beta-gamma

phosphoanhydride bond (Kotting et al., 2006). It is also thought to promote

the formation of a dissociative transition state with a penta-coordinated

phosphate group (Scheffzek et al., 1998). Co-crystals of Ras:RasGAP and

Rho:RhoGAP revealed that both GTPase:GAP pairs use this mechanism,

despite the absence of primary sequence conservation (Scheffzek et al., 1997;

Rittinger et al., 1997). This basic mechanism has many variations among

different family members of Ras-like GTPases: RanGAP uses an asparagine

to stabilize the glutamine orientation in Ran, while RapGAP uses an

asparagine, but this residue is thought to replace the canonical glutamine in

the active site as Rap lacks the conserved glutamine (Seewald et al., 2002;

Daumke et al., 2004). Sec23, the GAP for Sar1 (part of the COPII coat), also

19

uses an arginine finger but helps to align a histidine in place of the glutamine

in the Sar1 active site (Bi et al., 2002).

The co-crystal structure of Gyp1 in complex with human Rab33b revealed a

new variation in GAP mechanism: both the arginine and the glutamine

residues are provided by the GAP in trans (Pan et al., 2006). The GAPʼs

catalytic B and C motifs that contain the essential arginine and glutamine

residues, respectively, form a loop that extends into the Rab nucleotide-

binding pocket (Pan et al., 2006). The structure also confirmed that the Rab

does bind the GAP in the V-shaped groove via multiple α-helices from the

GAP (Pan et al., 2006). These helices interact primarily with both switch

regions and the P-loop, which helps explain why GAPs interact preferentially

with GTP-bound Rabs and suggests a possible mechanism for substrate

selectivity. Highly variable surfaces can exist in the switch regions due to the

varying conformations of three invariant hydrophobic residues (Merithew et al.,

2001). GAPs (as well as effectors) may use this surface to distinguish

between Rab substrates (Pereira-Leal and Seabra, 2000). This structural

work also helped to explain observations from numerous groups that GAP

activities in cytosol also stimulated the GTPase activity of Rab “Q-L” mutants

that lack the conserved G3 glutamine residue.

20

Puzzlingly, many purified, truncated yeast RabGAPs showed broad substrate

reactivity in vitro (Albert and Gallwitz, 1999; Will and Gallwitz, 2001). These

authors used truncated forms initially because of technical difficulties in

producing recombinant full-length protein. Rabs regulate specific trafficking

events and promiscuous GAPs might disrupt membrane identity. Data from

mammalian cells suggest that this apparent promiscuity is due to deleted

regions being necessary for discriminating between Rab substrates. Barr and

co-workers demonstrated that GAPCenA truncations decreased substrate

specificity -- full-length GAPCenA stimulated Rab4 exclusively (Fuchs et al.,

2007). Strict specificity has also been observed for other mammalian GAPs

including TBC1D30, which can even discriminate between closely related

Rab8 isoforms (Yoshimura et al., 2007).

Why do both Bub2 and Cdc16 require adaptors to function as GAPs for

Tem1/Spg1? Both contain the first three “fingerprint” motifs but neither has

the last three alpha helices (14-16) (Rak et al., 2000). Helix 15 truncations of

Gyp1 and Gyp7 lack GAP activity (Albert et al., 1999). Perhaps in a ternary

complex, the adaptor proteins provide the last three helices to complete the

TBC domain fold (Rak et al., 2000). Alternatively, this adaptor might change

the conformation of Tem1/Spg1 to one that promotes stimulation by

Bub2/Cdc16 (Geymonat et al., 2002). Crystal structures of Bub2/Cdc16

complexed with Tem1/Spg1 and their adaptors will resolve this puzzle.

21

While the biochemistry of RabGAPs has been studied extensively, their

functions in cells remain somewhat elusive. For Rab proteins, the functional

ramifications of GTP hydrolysis were initially somewhat controversial. In 1988,

Henry Bourne proposed that the secretory GTPases would not function like

Ras, where GTP hydrolysis attenuates a signal but does not affect protein

function. Instead, he proposed that they would function like elongation factors

where a cycle of nucleotide binding and hydrolysis was essential to ensure the

continued growth of the nascent polypeptide chain (Bourne, 1988). He

reasoned that secretion (and other transport events) might use nucleotide

hydrolysis to ensure irreversibility of a trafficking event.

Studies of Ras have greatly aided functional biochemical studies of Rab

proteins. Mutation of the conserved G3 motif glutamine of Ras-like GTPases

(often to leucine but also alanine) creates a protein deficient in hydrolysis and

thus predicted to be constitutively active (Polakis and McCormick, 1993). The

analogous mutants in Rabs have been used both in cells and in reconstituted

systems to probe requirements for nucleotide binding and hydrolysis in

different trafficking events.

Early cellular studies with Sec4Q71L and Rab5Q79L, mutants shown to be

deficient in GTP hydrolysis in vitro, gave somewhat conflicting results.

22

Secretion of invertase in Sec4 mutant cells was almost 3-fold slower at 13°C

compared to normal cells, and Sec4Q71L could not rescue other late acting

SEC mutants but instead generated synthetic lethal combinations (Walworth et

al., 1992). These mutant cells also accumulated secretory vesicles like the

original Sec4 temperature sensitive mutation (Novick and Schekman, 1980).

This suggested that the G3 motif Q to L mutant was a loss of function

mutation. In contrast, Rab5Q79L stimulated membrane fusion as shown by

the presence of enlarged early endosomes as well as increased fusion in a

cell-free endosome fusion assay (Stenmark et al., 1994). Compounding these

findings was the observation that GAP activity purified from lysates was able

to stimulate both wild-type and mutant Rab proteins. In addition, while GTPγS

was known to inhibit trafficking when added to cell free systems (Melançon et

al., 1987), it was unclear if this effect was due to Rab inhibition or other G

proteins involved in these events.

Zerial and co-workers took advantage of an analogous mutation in EF-Tu that

switches the nucleotide specificity of GTPases to xanthosine-5ʼ-triphosphate

(XTP) (Hwang and Miller, 1987). Using Rab5 mutated to bind XTP, Zerial and

co-workers discovered that Rab5 cycles between XDP and XTP on

membranes and that XTPγS does not inhibit in vitro endosome fusion (Rybin

et al., 1996). Studies of Ypt1Q67L corroborated these findings as this mutant

failed to block secretion (Richardson et al., 1998). This led to the hypothesis

23

that GTPase activity was needed simply for recycling of Rab proteins back to

the donor membranes, rather than playing an essential role in fusion, contrary

to the model of Bourne (Rybin et al., 1996; Richardson et al., 1998).

Termination of the active Rab signal would be key to keeping a target

membrane distinct from a donor membrane. Continual buildup of donor

microdomains at a target would otherwise lead to mis-sorting of receptors and

other cargo proteins within cells.

Deletion of RabGAPs in yeast revealed no obvious phenotypes, even when

putative GAPs for essential Rabs like Ypt1 and Sec4 were deleted. It is likely

that essential Rabs (such as Rab1/Ypt1) have multiple, dedicated GAPs. In

yeast, deletion of Gyp1, Gyp5 or Gyp8 alone (all GAPs with high in vitro

activity to Ypt1) did not yield any noticeable phenotype (De Antoni et al.,

2002). Synthetic, cold-sensitive growth phenotypes were observed in strains

harboring both the Ypt1Q67L mutant and various double deletions of Gyp1,

Gyp5 and Gyp8. The GAPs all have different cellular localizations, further

suggesting that GAP activity might be a way of preventing “stray” Rabs from

forming mis-localized microdomains at an incorrect membrane location.

In contrast with yeast, very little is known about RabGAP counterparts in

mammalian cells. The first mammalian RabGAP activity identified was

specific for Rab3A, an important regulator of exocytosis and synaptic vesicle

24

fusion (Burstein et al., 1991; Fukui et al., 1997). Interestingly, this protein

does not contain a TBC domain but does appear to catalyze hydrolysis by an

arginine finger mechanism, similar to Ras- and RhoGAPs (Clabecq et al.,

2000). GAPCenA was the first mammalian protein cloned containing a TBC

domain. This protein was originally discovered by a yeast two hybrid

interaction with Rab6Q72L via the GAPʼs C-terminus (a region with high

probability of coiled-coil structure). GAPCenA appeared to have GAP activity

on Rab6 and Rab4, the former requiring both prenylation of the Rab as well as

the coiled coil region C-terminal to the TBC domain (Cuif et al., 1999).

Approximately forty predicted GAPs for Rab GTPases in humans have been

identified by TBC domain sequence alignments (Bernards, 2003, Fuchs et al.,

2007; Table I). The key arginine in motif B is highly conserved but is not

absolute. For example, the arginine in Tre-2/USP6 is shifted one position

toward the N-terminus while TBC1D3 lacks this arginine. Interestingly,

TBC1D7 does not even have motif B, suggesting that it might completely lack

GAP activity altogether. Database searches also revealed that most

mammalian TBC domain proteins have multiple domains (Bernards, 2003).

These additional domains are very diverse and include lipid binding domains

such as the pleckstrin homology (PH) domain and glucosyltransferase/Rab-

like GTPase activators/myotubularins (GRAM) domains. Other domains

include phosphotyrosine-binding (PTB) domains and Src-homology-3 (SH3)

25

domains that are often involved in receptor tyrosine kinase signaling. Protein-

protein interaction domains such as coiled-coil domains and

RPIP8/Unc14/NESCA (RUN) domains are also sometimes found coupled with

TBC domains. Thus, RabGAPs may function as integrators of signals across

different trafficking events and perhaps even between different GTPase

families.

Recently, several efforts have been made to match mammalian RabGAPs with

their cognate Rabs partners. Initial attempts used yeast two hybrid screens

involving both mutant Rabs and GAPs to identify substrates with mixed

results. Through this approach, Barr and co-workers discovered a novel GAP

for Rab5 called RUTBC3/RabGAP-5. Overexpression of RUTBC3 blocked

uptake of both transferrin and epidermal growth factor (EGF), two known

Rab5-dependent processes (Haas et al., 2005). Fukuda and co-workers used

the same method to screen all Rabs against all RabGAPs and found many

GAPs that interacted with Rabs but most did not. Moreover, they found that

GAP activity did not seem to correlate with Rab binding in their screen (Itoh et

al., 2006).

Another method pioneered by the Barr laboratory, Rab inactivation screening,

has proven to be very successful at not only matching GAPs with their

substrate Rabs but also in identifying Rabs involved in fundamental cellular

26

processes. This method is based on the model that GAPs regulate the lifetime

of active Rabs. One prediction of this model is that overexpression of a GAP

in cells should lead to lower amounts of GTP-bound Rab. This in turn would

lead to a loss of microdomain integrity as evidenced by eventual loss of

effectors (and the Rabs themselves through GDI) from membranes. This loss

of microdomains would eventually lead to blocks in various transport steps.

These phenotypes would only be observed when overexpressing wild-type

GAPs and not their catalytically-inactive mutants, and would be predicted to be

yield phenotypes similar to that seen upon specific depletion of the substrate

Rabs.

The first example of this approach was used to probe which Rabs are required

for uptake of EGF and Shiga toxin (Stx; Fuchs et al., 2007). These ligands are

both internalized into small punctate structures within twenty minutes. The two

proteins then diverge in their trafficking pathways: STx is present in the Golgi

at sixty minutes post uptake, while EGF remains in endosomal structures.

Remarkably, Barr and co-workers discovered RabGAPs that differentially

regulate the uptake of these ligands (Fuchs et al., 2007). Overexpression of

RUTBC3/RabGAP-5 blocked EGF uptake but did not prevent STx from

trafficking to the Golgi. On the other hand, RN-tre, which previous work

suggested was involved in EGF signaling as a GAP for Rab5 (Lanzetti et al.,

2000), had no effect on EGF uptake, but did block STx trafficking to the Golgi.

27

These data suggested that these pathways are regulated by different Rab

GTPases. In vitro GAP assays confirmed that RUTBC3 was indeed a GAP for

Rab5, while RN-tre was a GAP for Rab43. Depletion of these Rabs produced

the same phenotypes as overexpression of their GAPs, confirming the

prediction of the model. Importantly, this work also highlighted possible

caveats of using so-called, constitutively active Rabs as probes in cells.

Rab5Q79L is known to block STx trafficking to the Golgi, although RUTBC3

overexpression does not block this process. Rab5Q79L causes enlarged

early endosomes that in the case of STx are less efficient in their transport

(Fuchs et al., 2007). Thus, constitutively active mutant phenotypes may not

reveal which Rabs are most important for a specific process but instead, might

shed light on the itinerary of a particular cargo. Stx passes through a Rab5

early endosome, but relies more heavily on Rab43 for its Golgi delivery.

Rab inactivation screening has also been used to probe Rab requirements in

maintaining Golgi structure, ciliagenesis, immunological synapse formation,

and melanosome aggregation, as well as for the identification of TBC domain

proteins that have Rab3 GAP activity. (Haas et al., 2007; Yoshimura et al.,

2007; Patino-Lopez et al., 2008; Itoh and Fukuda, 2006; Ishibashi et al., 2009).

In each of these cases, RabGAPs helped to define distinct requirements for

Rab GTPase function in a particular trafficking pathway. Perhaps most

striking was the discovery that only two Rab GAPs disrupted both Golgi

28

structure and protein secretion (Haas et al., 2007). Multiple GAPs caused

disruption of Golgi morphology in HeLa and hTERT-RPE1 cells, but only two

caused this phenotype in both cell types. These GAPs were TBC1D20, an

ER-localized GAP for Rab1 (Haas et al., 2007; Sklan et al., 2007) and RN-tre,

the above-mentioned GAP for Rab43. Overexpression of TBC1D20 caused

complete loss of Golgi structure, confirming the role of Rab1 in Golgi

biogenesis.

Do GAPs work alone in breaking down microdomains? Recent evidence

suggests that another layer of regulation exists above GAPs. In yeast, the

ability of Gyp7 to inactivate Ypt7 was shown to be commensurate with the

activity of Yck3, a kinase, (Brett et al., 2008). Yck3 helps complete

inactivation of Ypt7 by phosphorylating the HOPS tethering complex, which is

also a GEF for Ypt7; this phosphorylation is blocked by Ypt7-GTP. Perhaps

there are many different regulatory modes for GAPs in cells. Cells may have

evolved elaborate mechanisms to ensure tight regulation of highly conserved

Rabs. These results further highlight the complex integration of membrane

trafficking pathways in cells.

GAPs and GEFs: Defining Boundaries

Formation of Rab microdomains must be spatially and temporally regulated for

proper membrane transport. Coupling of different microdomains together on

29

the same organelle probably helps to maintain organelle identityand can

ensure vectorial flow of proteins and membranes. GAPs, as well as GEFs,

could provide many opportunities for regulation of these processes.

Current evidence supports a so-called Rab cascade model for microdomain

integration in which Rabs are activated and inactivated in sequential order to

coordinate vectorial flow of cargo. The first evidence in support of this model

came from the discovery that a late-Golgi localized Rab, Ypt32, recruits Sec2,

the GEF for the subsequent acting Sec4 onto secretory vesicles (Ortiz et al.,

2002).

The yeast secretory pathway has provided a large amount of evidence in

support of this model. The TRAPPI complex is a GEF for Ypt1, which functions

in the early secretory pathway between the ER and Golgi, and in intra-Golgi

transport (Wang et al., 2000). By binding Sec23, the TRAPP complex also

functions as a tether at the Golgi, for incoming COPII-coated vesicles (Cai et

al., 2007). This couples Ypt1 activation with cargo exit from the ER. With the

addition of two subunits, the specificity of the TRAPP complex (now TRAPPII)

may be switched to that of Ypt31/32, suggesting that there is a boundary at

the Golgi between active Ypt1 and active Ypt31/32 (Morozova et al., 2006 but

cf. Cai et al., 2008). This boundary would also require a way to inactivate Ypt1.

Satisfyingly, Novick and co-workers recently showed that Ypt32 is responsible

30

for recruiting Gyp1 to the Golgi, providing a mechanism for keeping early

Golgi, Ypt1 domains distinct from late Golgi, defined by Ypt31/32 (Rivera-

Molina and Novick, 2009). The cascade continues by recruitment of Sec2 onto

nascent secretory vesicles by Ypt32. This links Ypt32 to activation of Sec4,

the last Rab in the yeast secretory pathway. Activated Sec4 then binds Sec15,

a component of the large Exocyst tethering complex (Guo et al., 1999). Sec15

also associates with Sec2, leading to an intriguing mechanism by which a

Ypt32-regulated microdomain can be converted into a Sec4-regulated

microdomain (Medkova et al., 2006). Perhaps Sec15 displaces Ypt32 from

Sec2, thus incorporating a Sec4 GEF into a Sec4 microdomain to keep Sec4

active for tethering to the plasma membrane at the same time allowing access

for a Sec4-recruited Ypt32 specific GAP (Novick et al., 2006; Markgraf et al.,

2007).

There is also evidence for Rab cascades in mammalian cells. On endosomes,

Rab5 recruits the class C/VPS HOPS complex, which catalyzes nucleotide

exchange for the subsequently acting Rab7 (Rink et al., 2005). Using live cell

video microscopy, Zerial and co-workers showed that appearance of Rab7 is

followed by loss of Rab5, and that the ratio between the two Rabs controls the

identity of the compartment. This Rab conversion governs the maturation of

early endosomes into late endosomes (Rink et al., 2005). Presumably, a yet-

to-be identified GAP (perhaps recruited by Rab7) also inactivates Rab5 and

31

triggers its release from these membranes. It seems highly likely that similar

GEF/GAP recruitment will help other Rabs in all compartments of eukaryotic

cells to carry out their distinct functions. This mechanism provides for tight

control of the amount of active Rabs in cells.

Given the essential nature of Ypt1/Rab1 in yeast, the observation that

depletion of the mammalian Rab1 GAP, TBC1D20, caused no apparent defect

in Golgi structure or secretion of VSV-G protein from the ER to the Golgi is

somewhat puzzling (Haas et al., 2007). It is possible that depletion was not

adequate or that another GAP may substitute for TBC1D20 function upon its

depletion. On the other hand, this result is consistent with studies in yeast that

GTP hydrolysis is not required for Ypt1 function (Richardson et al., 1998), but

it also highlights differences in how various GAPs themselves may function.

Depletion of RUTBC3 leads to enlarged early endosomes (like Rab5Q79L),

and also re-localized Rab5 to the Golgi, similar to the localization of dominant

negative Rab5 (Haas et al., 2005). Why is there an apparent difference

between TBC1D20 depletion and RUTBC3 depletion and as a corollary, why

do Rab1 and Rab5 then function differently? This might be due to difference

in the site of GAP action relative to GEF action. If a GEF and GAP for a Rab

are both present at the donor compartment, this might be a way of

32

proofreading a process like cargo collection. If they are segregated from each

other at donor and acceptor compartments, it is likelier that GAP depletion will

show a phenotype.

Also unclear is how GAPs “poach” Rabs from a microdomain in the first place.

Rab proteins on membranes exist in complexes with their cognate effectors,

which likely help to exclude other Rabs (and their effectors) from a given

region of membrane (Zerial and McBride, 2001). These effector networks

would be able to block access to the Rab from its GAP. Most characterized

Rab GAPs have relatively high KM values for their substrates, likely reflecting a

combination of low binding affinity and high catalytic rate (Pan et al., 2006). In

contrast, most Rab effectors bind with higher affinity and could compete with

GAPs for Rab binding. If a GAP is actively recruited to a microdomain, the

network of reversible Rab–effector interactions would still allow a GAP (even

with poor affinity) to eventually break down that microdomain in the absence of

Rab activation. Thus, competition between GEF and GAP activities is likely

pervasive throughout all membrane trafficking pathways and could regulate

the formation and breakdown of Rab microdomains.

In summary, the ordered delivery of proteins and lipids to their correct

compartments is important for a vast array of cellular functions. Initially the

goal of this thesis was to find nucleotide cycle regulators of the Rab9 GTPase.

33

Our laboratory is interested in the function of Rab9, which is required for the

recycling of mannose 6-phosphate receptors from late endosomes to the trans

Golgi network. We wanted to know the molecular requirements for Rab9

microdomain segregation and how the consequences of segregation is

integrated into other trafficking pathways in the cell. In this thesis, I present an

investigation into the role of GAPs in regulating Rab9-dependent trafficking

events. A novel GAP, RUN- and TBC domain-containing 1 (RUTBC1) was

identified as a Rab9 effector. Its role in classical, Rab9-mediated trafficking

and its in vitro substrate specificity were investigated. Surprisingly, we

discovered a novel link from Rab9, through RUTBC1, to the autophagic

pathway, a highly-conserved but specialized trafficking process. Another

novel GAP, RUTBC2 was also identified as a Rab9 effector. Though highly

related, these proteins appear to have different substrate specificities in vitro,

suggesting that these proteins may integrate Rab9-mediated trafficking into

the broader context of other trafficking pathways.

34

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Table I. Summary of mammalian Rab GTPase-activating proteins

TBC Protein

Aliases Reported substrates

Other domains

Function/Remarks References

EVI5 Rab11, -35

CC Regulates Shiga toxin uptake

Dabbeekeh et al., 2007; Fuchs et al., 2007

EVI5L Rab23, -10

CC Regulates primary cilia formation

Yoshimura et al., 2007; Itoh et al., 2006

RN-tre Rab5, -41 EGF receptor downregulation; Golgi biogenesis, regulates Shiga toxin uptake

Lanzetti et al., 2000; Haas et al., 2005; Fuchs et al., 2007

RUTBC1 SGSM2 Rab33b, -32

RUN Binds Rab9; interacts with Atg16L1

This study

RUTBC2 SGSM1 Rab34, -36

RUN Binds Nurr1 transcription factor

This study; Luo et al., 2008

RUTBC3 RabGAP-5, MAP, CIP85

Rab5 RUN; SH3

Regulation of endocytosis, binds tumor suppressor Merlin, gap junctions

Haas et al., 2005; Lee et al., 2004; Lan et al., 2005

TBC1D1 Rab8a, -10, -14

PTB Akt substrate; regulates insulin-stimulated GLUT4 receptor trafficking; genetically associated with obesity

Roach et al., 2004; Peck et al., 2009; Stone et al., 2006

TBC1D2A Armus Rab7 PH; CC Rac1 effector and regulator of E-cadherin degradation

Frasa et al., 2010

TBC1D2B KRAB; CC

Binds Rab22 Kanno et al., 2010

55

TBC Protein

Aliases Reported substrates

Other domains

Function/Remarks References

TBC1D3 PRC17 Hominoid specific; oncogene, activator of Ras?

Pei et al., 2002; Wainszelbaum et al., 2008

TBC1D4 AS160 Rab2a, -8a, -10, -14

PTB Akt substrate; regulates insulin-stimulated GLUT4 receptor trafficking

Kane et al., 2002; Mîlnea et al., 2005

TBC1D5 Rab7 Binds to retromer coat

Seaman et al., 2009

TBC1D6 GRTP1

TBC1D7 Rab17 Regulates primary cilia formation, oncogene

Yoshimura et al., 2007; Sato et al., 2010

TBC1D8A Vrp GRAM; CC

TBC1D8B GRAM

TBC1D9A GRAM; EF

TBC1D9B GRAM; EF

TBC1D10A EPI64 Rab27A, -35

Regulates microvillar structure; regulates Shiga toxin uptake

Itoh and Fukuda, 2006; Hanono et al., 2006; Fuchs et al., 2007

TBC1D10B Rab35, -3a

CC Regulates Shiga toxin uptake

Fuchs et al., 2007; Ishibashi et al., 2010

TBC1D10C Rab35 Regulates Shiga toxin uptake

Fuchs et al., 2007

56

TBC Protein

Aliases Reported substrates

Other domains

Function/Remarks References

TBC1D11 GAPCenA, RABGAP1

Rab4, -6 PTB; CC Controversial: has GAP activity on Rab6 but does not block Shiga toxin trafficking like Rab6 depletion; binds Rab36

Fuchs et al., 2007; Cuif et al., 1999; Kanno et al., 2010

TBC1D12

TBC1D13

TBC1D14 CC Crystal structure of TBC domain solved

Tempel et al., 2008

TBC1D15 Rab7 Binds Rab5A/B/C Zhang et al., 2005; Itoh et al., 2006

TBC1D16

TBC1D17 Rab21, -35

Regulates Shiga toxin uptake

Fuchs et al., 2007

TBC1D18 RabGAP1L Rab22 PTB Expression reduced in Alzheimer's patients

Itoh et al., 2006; de Yebra et al., 2004

TBC1D19

TBC1D20 Rab1 TM Regulates Golgi biogenesis; binds Hepatitis C NS5A protein

Haas et al., 2007; Sklan et al., 2007a; Sklan et al., 2007b

TBC1D21

TBC1D22A Crystal structure of TBC domain solved

Tempel et al., 2008

TBC1D22B

TBC1D23

57

TBC Protein

Aliases Reported substrates

Other domains

Function/Remarks References

TBC1D24 TLDc

TBC1D25 OATL1 Rab2a Itoh et al., 2006

TBC1D26

TBC1D27

TBC1D28

TBC1D29

TBC1D30 XM_037557 Rab8a Regulates primary cilia formation

Yoshimura et al., 2007

TBCKL PK; RHOD

USP6 Tre-2, TRE17

UCH Oncogene; founding member of TBC domain family, regulates plasma membrane to endomsome trafficking through Arf6 activation

Neuwald, 1997; Nakamura et al., 2002; Martinu et al., 2004

Domain Abbreviations:

CC - coiled-coli, RUN - RPIP8/Unc-14/NESCA, SH3 - Src homology domain 3,

PTB - Phosphotyrosine binding, PH - Pleckstrin homology, KRAB - Kruppel-

associated box, GRAM - Glucosyltransferases/Rab GTPase

Activators/Myotubularins, EF - EF hand, TM - transmembrane, TLDc - domain

in TBC and LysM containing proteins, PK - protein kinase, RHOD - rhodanese

homology domain, UCH - ubiquitin carboxy-terminal hydrolase

58

CHAPTER 2

RUTBC1: A NOVEL RAB9 EFFECTOR THAT

ACTIVATES GTP HYDROLYSIS BY RAB33B AND RAB32

(Manuscript in preparation)

Ryan M. Nottingham, Ian G. Ganley, Francis A. Barr,

David G. Lambright and Suzanne R. Pfeffer

Contributions: RMN contributed Fig. 1b, 2, 3a-c, 4 and 5. IGG contributed Fig.

3d. FAB contributed Fig. 1a and DGL contributed plasmids and reagents.

RMN and SRP conceived the project and wrote the paper.

59

ABSTRACT

Rab GTPases regulate all steps of membrane trafficking. In cells, their cycling

between active, GTP-bound states and inactive, GDP-bound states is

regulated by the action of antagonistic enzymatic activities called guanine

nucleotide exchange factors and GTPase-activating proteins (GAPs). The

substrates for most RabGAPs are unknown and the potential for cross talk

between different membrane trafficking pathways remains uncharted territory.

Rab9 and its effectors regulate recycling of mannose 6-phosphate receptors

from late endosomes to the trans Golgi network. We show here that RUTBC1

is a TBC domain-containing protein that binds to Rab9 specifically both in vitro

and in cultured cells but is not a GAP for Rab9. Biochemical screening of

RUTBC1ʼs Rab protein substrates revealed highest GAP activity toward

Rab33B and Rab32. These data support a model in which RUTBC1 is

recruited onto Rab33B positive microdomains adjacent to Rab9 microdomains.

This cross-talk between Rab domains suggests the existence of a Rab

cascade between endosomes and the Golgi.

60

INTRODUCTION

Spatial and temporal regulation are important for organizing and coordinating

movement of cargo throughout the secretory and endocytic pathways. Rab

proteins, small Ras-like GTPases, regulate all steps of intracellular trafficking

including cargo selection, vesicle motility along cytoskeletal elements,

tethering of vesicles near their targets and finally fusion of these vesicles with

target membranes (Stenmark, 2009). Rabs accomplish this regulation by

recruiting so-called effector proteins to create specific membrane

microdomains that help to identify different compartments (Zerial and McBride,

2001).

Rab proteins go through cycles of activation (i.e. nucleotide binding) that occur

against a cycle of membrane association. Active, GTP-bound Rabs bind so-

called effector proteins such as adaptors for coat or motor proteins, or

tethering factors that are the molecular machinery for each trafficking step.

Effectors display lower affinity for GDP-bound Rabs, thus favoring membrane

dissociation of effector proteins A GDP-bound Rab then becomes a substrate

for extraction from the membrane into the cytoplasm by a protein called GDI

(Pfeffer and Aivazian, 2004). GDI then recycles Rab proteins back to the

donor compartment for another round of membrane association.

61

In cells, the identity of the bound nucleotide is determined by the opposing

activities of two sets of enzymes: guanine nucleotide exchange factors

(GEFs), which catalyze the exchange of bound GDP for GTP, and GTPase-

activating proteins (GAPs), which catalytically accelerate a Rab proteinʼs slow,

intrinsic GTP hydrolysis rate.

Recently, the model of Rab cascades has been used to describe the linking of

one transport step to another to assure activation and inactivation of Rabs that

lie along the same transport pathway. In yeast, Ypt32p, a late Golgi Rab has

been found to be at the center of a far-ranging cascade in the secretory

pathway, not only recruiting Sec2p, the exchange factor for the next Rab in the

pathway (Sec4p; Ortiz et al. 2002) but also Gyp1p, the GAP for the previous

Rab in the pathway (Ypt1p; Rivera-Molina and Novick, 2009). Other examples

of Rab cascades have been found in mammalian cells including endosomal

maturation through Rab conversion from Rab5-positive early endosomes to

Rab7-positive late endosomes (Rink et al., 2005). It is reasonable to assume

that this satisfying model of Rab cascades will apply more generally

throughout trafficking pathways in each cell type.

Eukaryotes possess a family of proteins that contains the highly conserved

Tre2/Bub2/Cdc16 or TBC domain that has been shown to possess GAP

activity specifically on the Rab subfamily of GTPases (Neuwald, 1997, Strom

62

et al. 1993). TBC domains are often found in proteins that also contain

several other types of domains, suggesting a great amount of integration

between signaling pathways (Bernards, 2003). The human genome encodes

over 40 different TBC domain-containing proteins but only a few cognate pairs

of Rabs and GAPs have been functionally determined. Thus, much remains to

be learned about the functions of RabGAPs in cells: presumably, to

deconstruct established microdomains and form boundaries to focus Rab

microdomains, which would prevent mixing of different functional membrane

units.

The Rab9 GTPase is required for the recycling of mannose 6-phosphate

receptors from late endosomes to the trans Golgi network (Lombardi et al.,

1993; Barbero et al., 2002). It also plays a role in lysosome biogenesis

(Riederer et al., 1994) and late endosome morphology (Ganley et al, 2004). In

this study we analyze the role of a novel effector of Rab9 called RUTBC1. We

show here that RUTBC1 is a multi-domain protein that contains a TBC domain

and is not a GAP for Rab9 in cells but for other Rab GTPases.

63

METHODS

Yeast Two-Hybrid Analysis

Yeast two-hybrid analysis was carried out as described previously (Fuchs et

al., 2007). Briefly, 56 mutant Rab proteins deficient for GTP hydrolysis (Q to

A) were cloned into pGBT9 bait vector (Clontech). RUTBC1 and RUTBC2

were amplified from cDNA libraries and were cloned into pACT2 prey vector

(Clontech); growth on selective media indicated an interaction between a Rab

and RUTBC1 or RUTBC2.

Plasmids

For mammalian expression, N-terminally 3xmyc-tagged RUTBC1 was

obtained by amplification from a cDNA library and ligation into a modified

version of pCDNA3.1(+) (Invitrogen) (Fuchs et al., 2007). This construct

encodes the shorter of two isoforms found in GenBank. GFP-RUTBC1 was

constructed by amplification of this isoform by PCR and ligated into pEGFP-C1

(Clontech). RUTBC1-N and RUTBC1-C truncation constructs were amplified

by PCR and ligated into 3xmyc-pCDNA3.1(+). RUTBC1-RUN was created by

addition of a stop codon directly after the RUN domain by site-directed

mutagenesis using QuikChange (Stratagene). Predicted RUTBC1 GAP-dead

mutant (R803A) was also created using QuikChange.

64

For bacterial expression, RUTBC1-C was ligated into pET28a (Novagen) in

frame with the N-terminal His-tag. His-RUTBC1-C R803A was created by site-

directed mutagenesis. Plasmid encoding GST-Rab9A was previously

described (Aivazian et al, 2006). GST-Rab9B was amplified by PCR from

pET14-Rab9B (Hayes et al., 2009) and ligated into pGEX-4T-1 (GE

Healthcare). GST-Rab6A was amplified by PCR from His-Rab6A (Burguete et

al., 2008) and ligated into pGEX-4T-1. His-Rab33Q72A was described by

Hayes et al., 2009; His-Rab33 wild-type was obtained by site-directed

mutagenesis of this construct.

Plasmids of Rab proteins for biochemical screening of GAP activity were

previously described (Pan et al., 2006). Phosphate Binding Protein (PBP)

from E. coli was amplifed by PCR from bacteria and cloned into modified

pET15. His-PBP A197C was constructed by site-directed mutagenesis.

Protein Expression and Purification

His-RUTBC1-C was transformed into Rosetta2 (DE3) cells (Novagen) and

grown at 37°C until OD600 = 0.5. The cells were induced with 0.4mM isopropyl

β-D-thiogalactoside (IPTG) and grown for an additional 4 hours at 22°C. Cell

pellets were resuspended in lysis buffer (25mM HEPES, pH 7.4, 300mM NaCl,

50mM imidazole) supplemented with 1mM PMSF and lysed by two passes at

20,000 psi through an EmulsiFlex-C5 apparatus (Avestin). Cleared lysates

65

(20,000 rpm for 45 min at 4°C in a JA-20 rotor; Beckman Coulter) were

incubated with Ni-NTA (Qiagen) for 1 hour at 4°C. The resin was then washed

with lysis buffer and eluted with 25mM HEPES, pH7.4, 300mM NaCl and

250mM imidazole. Fractions containing RUTBC1-C were pooled and

concentrated using an Amicon Ultra concentrator (Millipore). The sample was

dialyzed to remove imidazole and then brought to 10%(v/v) glycerol. The

sample was aliquoted, snap frozen in liquid nitrogen and stored at -80°C. His-

RUTBC1-C R803A was purified using the same procedure.

His-Rab33B wild-type was transformed into Rosetta2 (DE3) cells and grown at

37°C until OD600 = 0.6. The cells were induced with 0.4mM IPTG and grown

for an additional 3.5 hours at 37°C. Cell pellets were resuspended in lysis

buffer (50mM MES, pH 6.5, 8mM MgCl2, 2mM EDTA, 0.5mM DTT and 10µM

GDP) supplemented with 1mM PMSF and lysed by two passes at 20,000 psi

through an EmulsiFlex-C5 apparatus. Cleared lysates (20,000 rpm for 45 min

at 4°C in a JA-20 rotor) were loaded onto an 30mL SP-Sepharose column (GE

Healthcare) and then eluted with a 10CV gradient of 0-500mM NaCl.

Fractions containing Rab33B were pooled and brought to 50% ammounim

sulfate. Precipitated protein was collected by centrifugation and resuspended

in S100 buffer (64mM Tris-HCl, pH 8.0, 100mM NaCl, 8mM MgCl2, 2mM

EDTA, 0.2µM DTT, 10µM GDP) and gel filtered by FPLC on a 16/60 Superdex

75 column (GE Healthcare) into S100 buffer. Fractions containing Rab33B

66

were pooled and concentrated using an Amicon Ultra concentrator. The

sample then brought to 10%(v/v) glycerol, aliquoted and snap frozen in liquid

nitrogen and stored at -80°C. His-Rab33B Q72A was purified using the same

procedure.

GST-Rab9A expression and purification were previously described (Aivazian

et al., 2006) and GST-Rab9B and GST-Rab6A were purified by the same

procedure. Expression and purification of Rab proteins for the GAP screen

were previously described (Pan et al., 2006). His-PBP A197C was purified

and labeled according to the method in Shutes and Der (2005).

Antibodies

Mouse monoclonal anti-myc (9E10), mouse monoclonal anti-Rab9A, mouse

monoclonal anti-CI-MPR (2G11) and rabbit anti-CI-MPR, were all previously

described (Ganley et al., 2008). Rabbit anti-GFP antibody was from

Invitrogen; mouse anti-GFP antibody was from Roche. Rabbit anti-Rab2

antibodies were from Santa Cruz Biotechnology. HRP-conjugated goat anti-

mouse and goat anti-rabbit secondary antibodies as well as protein-A-HRP

were from Bio-Rad.

67

Binding assays

Constructs encoding 3xmyc-RUTBC1 or 3xmyc-RUTBC1 truncations were

translated in vitro using a TNT Quick Coupled Transcription/Translation

System (Promega) following the manufacturerʼs protocol. GST-tagged Rabs

were loaded with GTPγS or GDP as described (Aivazian et al., 2006) and

mixed with TNT lysate for 1.5 hours at 25ºC in binding buffer (25mM HEPES-

NaOH, pH7.4, 150mM NaCl, 5mM MgCl2, 1mM DTT, 0.1mM GTPγS).

RUTBC1 constructs bound to GST-Rabs were isolated using glutathione-

Sepharose, washed in binding buffer (with 400mM NaCl) and then eluted by

addition of 25mM glutathione and analyzed by immunoblot.

Cell Culture and Transfections

HeLa, COS-1 and HEK293T cells were obtained from American Type Culture

Collection and cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle's

medium supplemented with 7.5% fetal calf serum, 100U penicillin and

100µg/mL streptomycin. For overexpression studies, all cells were transfected

using Fugene 6 (Roche). Cells were harvested either 24 or 48 hours after

transfected as indicated.

Immunopreciptations, Protein Turnover and Lysosomal Enzyme Secretion

For immunoprecipitations, cells were transfected with indicated plasmids for

24 hours. Cells were then lysed in 50mM Tris-HCl, pH 7.4, 150mM NaCl, 1mM

68

MgCl2 and 1% Triton X-100 supplemented with Complete EDTA-free Protease

Inhibitor Cocktail (Roche) and spun for 15 minutes at full speed in a microfuge

at 4°C. Supernatants were pre-cleared using Protein-A agarose (Roche) for 15

minutes at room temperature and then immunoprecipitated with indicated

antibodies for 1.5 hours at room temperature. Immune complexes were

isolated on Protein-A agarose for 30 minutes at room temperature. The resin

was then washed 3 times with lysis buffer and once with PBS. The beads

were resuspended in 2X sample buffer and boiled and analyzed by

immunoblot. Protein turnover and lysosomal enzyme secretion assays were

performed as described previously (Ganley et al., 2008).

GAP Assays

For the biochemical screen of GAP substrates, the procedure followed by Pan

et al., 2006 was generally used with the exception that phosphate released

during the reaction was bound by modified PBP (A197C) labeled at position

197 with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide

(MDCC). Phosphate binding to MDCC-PBP causes a conformational change

in the phosphate binding cleft that results in an increase in MDCC

fluorescence (Brune et al., 1994). Reactions were started by adding a solution

containing GAP, MgCl2 and MDCC-PBP to desalted, GTP-exchanged Rabs by

a Precision 2000 liquid handling system (Biotek). Rab GTPases were at 2µM

for all reactions while the concentration of His-RUTBC1-C was varied.

69

Phosphate production was monitored by following fluorescence signal

continuously in a TECAN Saphire microplate reader using an excitation of

425nm and an emission cutoff filter of 455nm. Other assays were performed

as follows: purified Rab GTPases were exchanged with GTP-γ-32P as in

Aivazian et al. for 10 minutes at 25°C and desalted on PD-10 or PD-Mini

columns to remove free nucleotide. Loading efficiency was assayed by filter

binding and specific activity calculated from inputs. Various concentrations of

Rab-GTP were incubated with His-RUTBC1-C at 25°C. Aliquots of the

reaction were removed at various time points and quenched by addition of a

solution of 5% Norit-A in 50mM phosphoric acid. The quenched samples were

spun to pellet the charcoal and half of the supernatants were analyzed by

liquid scintillation counting in BioSafe-II scintillation fluid (Research Products

International) using an LS-6500 liquid scintillation counter (Beckman-Coulter).

70

RESULTS

We study the role of Rab9 GTPase in intracellular membrane traffic. As a

starting point to find regulators of Rab9, we used a two-hybrid screen

consisting of all TBC domain-containing proteins in the human genome as

prey against a comprehensive library of hydrolysis-deficient Rab GTPases as

bait (Haas et al., 2005, Fuchs, et al., 2007). This screen identified a TBC

domain-containing protein, RUTBC1, as a potential partner of both Rab9A and

Rab9B (Figure 1A). It also interacted to a lesser extent with Rab2 and Rab3

isoforms.

RUTBC1, and the closely related RUTBC2, are conserved proteins that

contain an N-terminal RPIP8/UNC-14/NESCA (RUN) domain and C-terminal

TBC domain (Figure 1B). RUN domains are entirely alpha-helical domains

that have been shown to interact with members of the small, Ras-like GTPase

superfamily including Rab6 and Rap1/2 (Callebaut et al., 2001; Recacha et al.,

2009; Janoueix-Lerosey et al., 1998). No enzymatic activity has been found to

be associated with RUN domains, suggesting that they likely mediate protein-

protein interactions. RUTBC1ʼs catalytic domain is unique in that there is a

large insertion between the first two “fingerprint” A and B motifs (Figure 1B,

sequence). In the structural model for Rab and RabGAP interaction, the

analagous region of Gyp1 is situated away from the Rab:GAP binding

interface (Pan et al., 2006). Most of the dissimilarity between RUTBC1 and

71

RUTBC2 TBC domains is found in this insertion. According to the NCBI

Homologene database, there is only one RUTBC1/2 protein in C. elegans (tbc-

8), Drosophila (CG1905) and zebrafish (LOC794373) while vertebrates have

both proteins. Drosophila actually has two RUTBC-like proteins but they are

thought to have diverged within flies, independent of the divergence that

occurred in vertebrates (Yang et al., 2007). Another protein, RabGAP-5, also

contains a RUN and TBC domain but the domain structure is reversed (Haas

et al., 2005; Yang et al., 2007).

RUTBC1 is a Rab9 effector

To confirm the results of the two hybrid screen and to test the nature of the

binding interactions, we tested if Rab9 could bind RUTBC1 in vitro. Full-length

RUTBC1 was difficult to express in E. coli so we utilized an in vitro

transcription/translation (IVT) system and assayed binding by GST-affinity

chromatography. As seen in Figure 2A, GST-Rab9A, but not GST-Rab9B or

GST-Rab6A, bound to in vitro translated full-length RUTBC1, confirming the

specificity seen in the screen. Rab9A and Rab9B are highly similar proteins

that localize to different organelles at steady-state: Rab9A on late endosomes

(Lombardi et al., 1993) and Rab9B at the Golgi (Yoshimura et al., 2007 and

our unpublished observations). Rab9A and 9B are most divergent in their C-

terminal, hypervariable domains. This suggests that RUTBC1 may recognize

part of the Rab9A hypervariable domain for binding. Next, we tested if

72

RUTBC1 preferred either the GTP or GDP-bound form of Rab9. Using the

GST-binding assay described above, in vitro translated RUTBC1 was bound

approximately 10-fold more efficiently by GST-Rab9 when loaded with GTPγS

than with GDP (Figures 1B and 1C). Thus, RUTBC1 is a bona fide effector of

Rab9A ( referred to as Rab9 below).

We next investigated the location of the Rab9 binding site in RUTBC1. A

series of RUTBC1 truncations were generated (Figure 2C) and tested using

the GST-binding assay. GST-Rab9 bound to the N-terminal half of RUTBC1,

and not to the C-terminal half that contains the TBC domain (Figure 2D). This

suggests that Rab9 is likely not a substrate for the predicted GAP activity of

RUTBC1. Further truncation revealed that Rab9 did not seem to bind to a

construct composed of amino acids 1-185 that stopped right after the RUN

domain. A previous two-hybrid screen for GAP-Rab interactions failed to

detect Rab9 binding to either RUTBC1 or RUTBC2 (Itoh et al., 2006) using

constructs for both proteins containing only the TBC domain. Taken together,

these data suggest that Rab9 binds RUTBC1 in a region distinct from the RUN

or TBC domains in a guanine nucleotide dependent manner.

RUTBC1 interacts with Rab9 in cells

Rab9 regulates the recycling of mannose 6-phosphate receptors (MPRs) from

late endosomes to the trans Golgi network (TGN) (Lombardi et al., 1994,

73

Carroll et al., 2001). To explore whether Rab9 and RUTBC1 interact in cells,

HEK 293T cells were transfected with GFP-RUTBC1 or GFP as a control. As

shown in Figure 3A, endogenous Rab9 co-immunoprecipitated with GFP-

RUTBC1 and not GFP (Figure 3A). Rab2, which showed interaction with

RUTBC1 by two hybrid was not detected in the immunoprecipitates. This

experiment confirms that RUTBC1 can interact with Rab9 in living cells.

We characterized this interaction further by investigating the effects of

overexpressed, exogenous RUTBC1 on MPR recycling. When Rab9 function

is disturbed in cells by overexpression of a dominant negative mutant Rab9

(Riederer et al., 1994) or by depletion of its effectors (Reddy et al., 2006;

Espinosa et al., 2009), MPRs are mis-sorted to the lysosome. We reasoned

that if RUTBC1 were a GAP for Rab9, the phenotype of RUTBC1

overexpression should be similar to that of a GDP-preferring Rab9 mutant or

Rab9 depletion. When RUTBC1 was overexpressed in COS-1 cells, MPRs

levels at steady state were decreased by ~35% (Figure 3B). This observation

led us to investigate the turnover rate of MPR in HeLa cells overexpressing

RUTBC1; under these conditions, it was turned over more rapidly (Figure 3C),

consistent with lower levels of MPRs at steady state. This suggested that

some amount of MPR was being mis-sorted to lysosomes, consistent with a

block in Rab9 function. As a possible GAP for Rab9, the predicted GAP-

deficient mutant (R803A) of RUTBC1 should not have the same effect upon

74

exogenous expression. In a functional test of MPR trafficking, we measured

the amount of hexosaminidase activity secreted into the media by cells

transfected with RUTBC1. Hexosaminidase is usually sorted to the lysosome

but when MPR levels are deficient in the TGN due to mis-sorting, the

hydrolase is secreted. In 293T cells transfected with RUTBC1, slightly higher

hexosaminidase activity was detected in the media than in control cells (Figure

3D). Cells transfected with RUTBC1 R803A showed a similar effect. Taken

together, perturbation of MPR trafficking seen in our experiments is likely due

to titration of Rab9 by the overexpression of RUTBC1 rather than the GAP

activity encoded by RUTBC1ʼs catalytic domain.

RUTBC1 has narrow substrate specificity

RUTBC1 binds Rab9 in the region between the RUN and TBC domains. and

does not appear to function as a Rab9-GAP. This suggested that Rab9 might

be part of a Rab cascade, in which Rab9 may bind a GAP that inactivates a

prior acting Rab GTPase. In this case, discovering the substrates of RUTBC1

would provide insight into the significance of Rab9-RUTBC1 interaction and

the identity of a prior acting Rab candidate protein.

Thirty-two different mammalian Rab GTPases were screened in vitro, under

single turnover conditions, as substrates for RUTBC1 using purified His-

tagged RUTBC1-C (Figure 2C) containing the TBC domain. Figure 4

75

summarizes these results by comparing observed second order rate constants

for GAP-catalyzed hydrolysis to the rate constants for each Rab proteins

intrinsic hydrolysis rate. The TBC domain of RUTBC1 had the highest activity

against Rab33B and Rab32, while no activity was detected against Rab9,

Rab2 or Rab3 proteins.

Further characterization of the kinetic parameters of the TBC domain found

that RUTBC1 has similar activity on Rab33B and Rab32. These Rabs were

mixed with increasing concentrations of RUTBC1-C and the data obtained

under pseudo first order conditions were simultaneously fit to the integrated

pseudo first order Michaelis-Menten equation. Apparent second order rate

constants from this fit were 2980 M-1s-1 for Rab33B and 1930 M-1s-1 for Rab32

(Figure 5A). Classical analysis of Rab33B GTP hydrolysis stimulated by

RUTBC1-C yielded a kcat of 12 min-1, which is approximately 5000-fold higher

than Rab33Bʼs intrinsic rate (0.0019 min-1; Figure 5B). We also determined a

KM of ~150µM. This value for KM is likely a lower estimate because of difficulty

preparing Rab33B-GTP substrate at amounts high enough to show complete

saturation. Catalytic efficiency of RUTBC1-C for Rab33B was calculated to be

only 1.8-fold lower (1680 M-1s-1), showing good agreement between the two

methods.

76

In the co-crystal of Gyp1p and Rab33B, Pan et al. (2006) suggested that

RabGAPs catalyze GTP hydrolysis by a dual-finger mechanism where both a

catalytic arginine and glutamine are supplied by the GAP. This model predicts

that RabGAPs will still be able to stimulate so-called constitutively active Rabs

that harbor a glutamine to alanine mutation in their G3 motifs. As shown in

Figure 5C (left panel) RUTBC1-C can efficiently stimulate GTP hydrolysis of

Rab33BQ92A. The dual finger mechanism also predicts that mutation of the

conserved arginine in the B motif should abrogate GAP activity. RUTBC1-C

R803A does not stimulate Rab33B hydrolysis above the intrinsic rate (Figure

5C, right).

77

DISCUSSION

Rab9 plays an essential role in the recycling of mannose 6-phosphate

receptors from late endosomes to the TGN, and it plays a role in lysosome

biogenesis and late endosome morphology. Regulators of the Rab9

nucleotide binding cycle are currently unknown although it is likely that both a

GEF and GAP for Rab9 exist in cells. Here we have shown that a predicted

RabGAP protein, RUTBC1, is a novel Rab9 effector that seems to bind

specifically to Rab9A and not Rab9B. Rab9A binds at a site in the linker

region between the RUN and TBC domains, and the proteins can interact in

cells. Despite specific binding, RUTBC1 does not possess GAP activity on

Rab9 but instead displays GAP activity on Rab33B and Rab32. This implies

that Rab33B or Rab32 act in a pathway that feeds into Rab9, and Rab9

binding to RUTBC1 helps to clear these Rabs from a Rab9A membrane

microdomain

Currently there are no known direct links between Rab9-mediated trafficking

and either Rab33B- or Rab32-regulated events. Rab33B is a ubiquitously

expressed Rab localized to the medial Golgi (Zheng et al., 1998).

Overexpression of its GTPase-deficient form (Rab33BQ92L) re-localizes

resident Golgi enzymes, like N-acetylglucosamine transferase I to the

endoplasmic reticulum (ER) (Valsdottir et al., 2001). Overexpression of its

GDP-preferring form (Rab33BT47N) blocks the re-localization of Golgi resident

78

enzymes to the ER in cells induced by expression of a dominant negative

mutant of Sar1 (Valsdottir et al., 2001). Rab33B function is somehow related

to that of Rab6 in a COPI independent retrograde trafficking pathway from the

Golgi to ER (Jiang and Storrie, 2005). Depletion of Rab33B from cells using

siRNA impairs Shiga-like toxin B trafficking from the Golgi to the ER (Starr et

al., 2010). Interestingly, it also rescues the dispersed Golgi phenotype

observed upon depletion of ZW10 (human homologue of yeast Dsl1p) or Cog3

(human homologue of yeast Sec34), two distinct tethering complexes involved

in Golgi to ER retrograde traffic. Depletion of Rab33B alone has no effect on

Golgi morphology (Starr et al., 2010). These data suggest that Rab33B might

regulate flux of material through the medial Golgi cisterna.

Depletion of tethers often leads to transport vesicle accumulation. In Cog3

depleted cells, the vesiculated Golgi contains both matrix proteins like GM130

and resident proteins like glycosyl transferases (Zolov and Lupashin, 2005). If

these are indeed bona fide transport intermediates, Rab33B might be involved

in the formation of these structures, i.e. in vesicle formation. This is similar to

role of Rab9 may play on late endosomes by collecting MPRs into nascent

vesicle buds by establishment of a Rab9-TIP47-MPR microdomain (Carroll et

al., 2001).

79

Other TBC domain containing proteins are predicted to be GAPs for Rab33B.

TBC1D22A and TBC1D22B are the mammalian homologues of yeast Gyp1p.

Gyp1p is an extensively studied TBC domain protein that has GAP activity on

Ypt1p in vitro and in cells. Gyp1p is localized to the Golgi and can potently

stimulate Rab33B in vitro (Pan et al., 2006). Overexpression of TBC1D22 in

HeLa cells leads to disruption of the Golgi and this phenotype is dependent on

its GAP activity (Haas et al., 2007). This argues against TBC1D22 being a

GAP for Rab33B in cells, as depletion of Rab33B leads to no change in Golgi

morphology, at least at the level of light microscopy (Starr et al., 2010).

Little is known about specific Rab33B effectors. Affinity chromatography using

GST-Rab33BQ92L showed that GM130, Rabaptin-5, and Rabex-5 all

appeared to be binding partners for Rab33B (Valsdottir et al., 2001). GM130 is

a known Rab1 effector while Rababptin-5 and Rabex-5 are well characterized

interacting partners of Rab5 (Moyer et al., 2001; Horiuchi et al. 1997). A

recent study reported that Atg16L1, an protein essential for autophagy in yeast

and mammals, is a specific Rab33B effector (Itoh et al., 2008). Autophagy is

the process by which cells recycle cytoplasm and organelles by enclosing

them in a unique double membrane structure called an autophagosome. This

structure then fuses with the lysosome to begin degradation of the contents

(Glick et al., 2010).

80

Rab32 has been reported to have divergent roles in different cell types.

Originally characterized as an A-kinase anchoring protein, mutants of Rab32

were reported to alter mitochrondrial distribution (Alto et al., 2002), but this

finding does not appear to be common to all cell types (Hirota and Tanaka,

2009; our unpublished observations). In mouse melanocytes, Rab32 controls

post-Golgi trafficking of melanogenic enzymes to melanosomes, a lysosome-

related organelle. The protein seems to have a redundant role in this pathway

with the closely related Rab38 (Wasmeier et al., 2006). In the cht mouse that

is deficient in Rab38, pigment defects are relatively mild; however, cht

melanocytes depleted of Rab32 show severe pigmentation defects. Rab32

and Rab38 share a common effector, VARP (Vps9-domain and Ankryin

Repeat Protein), which is also necessary for proper melanogenic enzyme

trafficking and is a GEF for Rab21 (Tamura et al., 2009). Rab32 is also

thought to function as an AKAP in Xenopus melanophores, regulating

aggregation and dispersion of melanosomes in response to hormones (Park et

al., 2007). RUTBC1 specifically showed GAP activity to Rab32 and not

Rab38, suggesting that Rab32 and Rab38 are not entirely redundant and that

their functions might diverge in different cell types. Intriguingly, a recent report

also linked Rab32 to autophagy; overexpression of GDP-preferring mutants of

Rab32 blocked basal autophagy (Hirota and Tanaka, 2009).

81

Taken together, these data suggest the existence of a Rab cascade involving

cross-talk between the Golgi and late endosomes. According to the Rab

cascade model, Rab9 would recruit (or activate) a GAP for the Rab that acts

before it in a trafficking pathway. Rab33Bʼs role in retrograde trafficking to the

ER seems less likely to be regulated by Rab9 than its putative role in

autophagy, as key regulators of autophagy like Atg9 are known to cycle

between Golgi and late endosomes (Young et al., 2006). Indeed, Rab9 has

even been suggested to play a role in an alternative autophagy mechanism

that is independent of the Atg5/Atg12/Atg16L1 complex (Nishida et al., 2009).

Rab9ʼs role vis-à-vis Rab33B and Rab32 is an exciting avenue for additional

experimentation, and experiments to examine the role of RUTBC1 in

autophagy and other trafficking pathways are currently in progress.

82

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88

FIGURE LEGENDS

Figure 1: RUTBC1 interacts with Rab9. (A) RUTBC1 was screened for

binding against a comprehensive library of 56 human Rab GTPases. Growth

after streaking on selective media indicates an interaction between RUTBC1

and a Rab GTPase. (B) Diagram of RUTBC1 domain structure. The RUN

domain is shown in blue and the TBC domain is shown in red. The first three

conserved motifs of the TBC domain are shown. Conserved residues are in

bold and the predicted catalytic arginine and glutamine are shown in red. The

extended insert between motif A and motif B of RUTBC1 is compared to the

same regions of human RabGAP-5 (RUTBC3) and S. cerevisiae Gyp1p.

Figure 2: RUTBC1 is an effector of Rab9. (A) In vitro translated (IVT)

3xmyc-RUTBC1 (IVT-3xmyc-RUTBC1) proteins were incubated with various

bacterially purified GST-tagged Rabs pre-loaded with GTPγS. Bound material

was eluted with glutathione and half of the eluate was analyzed by immunoblot

using anti-myc antibodies. Rabs were detected by Ponceau S staining. (B)

IVT-3xmyc-RUTBC1 was incubated with GST-Rab9A pre-loaded with either

GTPγS or GDP and analyzed as in A. (C) Quantitation of RUTBC1 nucleotide

preference shown in B. Error bars represent SEM from two independent

experiments. (D) Diagram of 3xmyc-tagged RUTBC1 constructs used in

89

binding assays. (E) IVT-3xmyc-RUTBC1 truncation constructs were incubated

with GST-Rab9A and analyzed as in A. “Input” indicates 1% of the IVT

reaction used in the binding assay.

Figure 3: RUTBC1 binds to, but is not a GAP for Rab9 in cells. (A)

HEK293T cells were transfected with GFP-RUTBC1 or GFP for 24 hrs and

immunoprecipitated with anti-GFP antibodies followed by immunoblotting for

with specific antibodies to different Rab proteins. “Input” represents 2% of the

lysate subjected to immunoprecipitation. (B) Left panel - Quantitation of COS-

1 cells were transfected with 3xmyc-RUTBC1 for 48 hours and extracts were

immunoblotted with anti-CI-MPR anitbodies. Right panel - Quantitation of CI-

MPR half-life measured from pulse/chase analysis of HeLa cells transfected

with 3xmyc-RUTBC1 for 48 hours. Extracts were immunoprecipitated with

anti-CI-MPR and exposed to phosphor screen. (C) HEK293T cells transfected

with 3xmyc-RUTBC1 wild type or R803A for 24 hours were assayed for

secreted and intracellular hexosaminidase activity. Error bars in all panels

represent SEM from at least two independent experiments.

Figure 4: RUTBC1 TBC domain has GAP activity toward Rab33B and

Rab32 in vitro. Purified, mammalian Rab GTPases (32) were pre-loaded with

GTP for one hour at room temperature and then desalted to remove free

nucleotide. MDCC-PBP, MgCl2 and varying concentrations of purified His-

90

RUTBC1-C were added to start the reaction. Phosphate release was

monitored continuously by microplate fluorimeter (see Materials and Methods).

Catalytic efficiency (kcat/KM) relative to the intrinsic rate constant (kintr) for GTP

hydrolysis was determined. Plots represent the mean from duplicate wells.

Figure 5: RUTBC1 TBC domain stimulates GTP hydrolysis. (A) GTP

hydrolysis by Rab33B (left) and Rab32 (right) in the presence of increasing

concentrations of RUTBC1-C. Values indicate the concentration of RUTBC1-

C in µM. Values below graphs represent catalytic efficiency extracted from a

simultaneous fit of the data to the integrated pseudo first order Michaelis-

Menten equation for each Rab substrate. (B) Velocity of RUTBC1-C catalyzed

reaction in the presence of increasing concentrations of Rab33B. (C) Left

panel - GTP hydrolysis by Rab33BQ92A in the presence of wild-type

RUTBC1-C. Right panel - GTP hydrolysis by Rab33B in the presence of

RUTBC1-C or the R803A mutant.

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Figure 1

92

Figure 2

93

Figure 3

94

Figure 4

95

Figure 5

96

Figure 5, continued

97

CHAPTER 3

INTERACTION OF RUTBC1, A RAB33B GAP,

WITH THE RAB33B-EFFECTOR, ATG16L1

(Manuscript in preparation)

Ryan M. Nottingham and Suzanne R. Pfeffer

Contributions: RMN contributed Fig. 1-4.

RMN and SRP conceived the project and wrote the paper

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ABSTRACT

Macroautophagy is a highly-conserved pathway that degrades bulk

cytoplasmic constituents including proteins, organelles and microorganisms.

This material is sequestered in a double membrane autophagosome.

Autophagosomes fuse with both early and late endosomes and lysosomes

leading to the degradation of the enclosed material. Two ubiquitin-like

conjugation systems are required for formation and expansion of the isolation

membrane. Atg16L1 forms a complex with Atg5-Atg12, which is required for

autophagosome formation in vivo. Currently, Atg16L1 is the only identified

effector of Golgi-localized Rab33B. Here we show that RUTBC1, a GTPase-

activating protein (GAP) for Rab33B, also interacts with Atg16L1. Depletion of

RUTBC1 from cells causes a concomitant depletion of Atg16L1, consistent

with their presence in a complex in cells. These proteins interact in cells, and

overexpression of both proteins causes them to co-localize to large

cytoplasmic puncta under nutrient rich conditions. Taken together, these data

suggest a model whereby RUTBC1 binds to Atg16L1, recruiting RUTBC1 to a

Rab33B microdomain.

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INTRODUCTION

Macroautophagy is a highly-conserved pathway that degrades bulk

cytoplasmic constituents including proteins, organelles and microorganisms

and also plays a role in various disease states (Mizushima et al., 2008).

Macroautophagy involves building a membrane-bound structure called an

autophagosome around this material and then fusing this structure with

lysosomal compartments (Yorimitsu and Klionsky, 2005; Klionsky, 2007; Glick

et al., 2010). Macroautophagy is a non-selective process ; other kinds of

autophagy can be selective substrates, including mitochondria through

mitophagy, peroxisomes through pexophagy, chaperone-mediated autophagy,

and the cytoplasm-to-vacuole (Cvt) pathway).

Macroautophagy (simply called autophagy for the remainder of the chapter) is

unique among membrane trafficking pathways because the autophagosome is

bound by a double membrane whereas regular phagocytosis and conventional

trafficking involve only one bilayer. Autophagy occurs at basal levels in

vegetative cells but can be induced by different signaling pathways in

response to various stimuli such as starvation or inhibitors of master signaling

kinases such as mTOR. Autophagy can be suppressed by reagents that

induce lysosomal dysfunction, such as chloroquine, ammonium chloride or

protease inhibitors (Mizushima et al., 2010).

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The current model of autophagosome formation involves a unique structure

termed the pre-autophagosomal structure in yeast and the isolation membrane

or phagophore in mammalian cells. Initiation of autophagosome formation is

regulated by the Atg1 complex (Levine and Klionsky, 2004; Young et al.,

2006). Autophagosome formation also requires phosphatidylinositol-3-

phosphate, which is generated by a particular PI3K complex consisting of

Vps34, p150, Beclin1 and Atg14L (Itakura et al., 2008). Atg9, the only known

integral membrane protein required for autophagosome formation, cycles

between the Golgi and Rab7- and Rab9-positive late endosomes, suggesting

the involvement of endocytic transport machinery in regulating

autophagosome formation (Young et al., 2006). Indeed, autophagosomes

most likely merge with both early and late endosomes in addition to lysosomes

(Eskelinen, 2005).

Two ubiquitin-like conjugation systems are required for formation and

expansion of the phagophore in yeast and mammals (Yorimitsu and Klionsky,

2005). The first system catalyzes the conjugation of Atg12 to Atg5. The Atg5-

Atg12 conjugate is required for the proper function of the second conjugation

system, which covalently attaches Atg8/LC3 to phosphatidylethanolamine

(PE). The Atg5-Atg12 conjugate, a cysteine protease Atg4, the E1-like

enzyme Atg7, the E2-like enzyme Atg3 and PE-containing liposomes are

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sufficient for LC3 conjugation. In cells, another factor is required called Atg16

(Kuma et al., 2002).

Atg16 forms a complex with Atg5-Atg12 (called the Atg16 complex) and this

complex is thought to function as an E3-like enzyme, directing LC3-PE to the

correct membrane location (Fujita et al., 2008). This is consistent with the

observation that the Atg16 complex is observed on the isolation membranes

before LC3-PE. However, the Atg16 complex is not present on completed

autophagosomes, suggesting it is released before fusion of the expanding

double membrane or quickly thereafter. This has led some to propose that the

Atg5-Atg12:Atg16 complex might act as a type of coat complex similar to

those seen in conventional membrane trafficking.

Currently, Atg16L1 (mammalian Atg16) is the only identified effector of

Rab33B, a Golgi-localized small GTPase that has a role in retrograde

trafficking from the Golgi to the endoplasmic reticulum (Itoh et al., 2008;

Valsdottir et al., 2001). Overexpression of GTP-bound Rab33B leads to an

increase of the lipidated form of LC3, suggesting that Rab33B has a role in the

formation and expansion of autophagosomes through its interaction with

Atg16L1 (Itoh et al., 2008).

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Autophagy is a highly conserved process, but many mammalian autophagy

proteins have additional domains absent from their yeast counterparts. In

mammalian cells, Atg16L1 forms a complex with Atg5-Atg12 just as in yeast

(Mizushima et al., 2003). Yeast Atg16 has an N-terminal Atg5 binding domain

and a C-terminal coiled-coil domain that is required for dimerization of the

complex. In contrast, mammalian Atg16L1 also has an array of seven tandem

WD40 domain repeats at its C-terminus (Fig. 1; Mizushima et al., 2003).

These repeats are not required for proper canonical autophagy in mammalian

cells (Fujita et al., 2009).

Here we show that RUTBC1, a GTPase-activating protein (GAP) for Rab33B,

also interacts with Atg16L1. Depletion of RUTBC1 from cells causes a

concomitant depletion of Atg16L1, consistent with their presence in a complex

in cells. These proteins interact in cells, and overexpression of both proteins

causes them to co-localize to large cytoplasmic puncta under nutrient rich

conditions. Taken together, these data suggest a model whereby RUTBC1

binds to Atg16L1, recruiting RUTBC1 to a Rab33B microdomain.

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METHODS

Plasmids

For mammalian expression, N-terminally 3xmyc-tagged RUTBC1 was

obtained by amplification from a cDNA library and ligation into a modified

version of pCDNA3.1(+) (Invitrogen) (Fuchs et al., 2007). This construct

encodes the shorter of two isoforms found in GenBank. GFP-RUTBC1 was

constructed by amplification of this isoform by PCR and ligated into pEGFP-C1

(Clontech). The predicted RUTBC1 GAP-deficient mutant (R803A) was also

created using QuikChange (Stratagene). GFP-Rab33B was constructed by

amplification of Rab33B from His-Rab33BQ92A (Hayes et al., 2009) and

mutated back to wild-type using Quikchange. Myc-Atg16L1, encoding

Atg16L1 with an N-terminal, triple myc tag was a kind gift from Dr. Ramnik

Xavier.

Antibodies

Mouse monoclonal anti-myc (9E10) was previously described (Ganley et al.,

2008). Rabbit anti-GFP antibody was from Invitrogen; mouse anti-GFP

antibody was from Roche. Antibodies to RUTBC1 were produced in rabbits

(Josman, LLC) using the purified His-RUTBC1-C as antigen. Affinity

purification was carried out as described previously (Ganley et al., 2004).

Rabbit anti-Atg16L1 was from Abcam. HRP-conjugated goat anti-mouse and

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goat anti-rabbit secondary antibodies as well as protein-A-HRP were from Bio-

Rad. Alexa fluor-conjugated secondary antibodies were from Invitrogen.

Cell Culture and Transfections

HeLa and COS-1 cells were obtained from American Type Culture Collection

and cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium

supplemented with 7.5% fetal calf serum, 100U penicillin and 100µg/mL

streptomycin. For over-expression studies, all cells were transfected using

Fugene 6 (Roche). For siRNA transfections, HeLa cells were transfected

using DharmaFECT1 (Thermo Dharmacon). Cells were harvested for

immunblot analysis or processed for immunofluorescence at 24, 48 or 72

hours post transfection as indicated. The targeting sequence for RUTBC1

siRNA was 5ʼ-GGACGCAGTCAAAGAGAAA-3ʼ.

Immunopreciptations

For immunoprecipitations, cells were transfected with indicated plasmids for

24 hours. Cells were then lysed in 50mM Tris-HCl, pH 7.4, 150mM NaCl,

1mM MgCl2 and 1% Triton X-100 supplemented with Complete EDTA-free

Protease Inhibitor Cocktail (Roche) and spun for 15 minutes at full speed in a

microfuge at 4°C. Supernatants were pre-cleared using Protein-A agarose

(Roche) for 15 minutes at room temperature and then immunoprecipitated with

indicated antibodies for 1.5 hours at room temperature. Immune complexes

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were isolated on Protein-A agarose for 30 minutes at room temperature. The

resin was then washed 3 times with lysis buffer and once with PBS. The

beads were resuspended in 2X sample buffer and boiled and analyzed by

immunoblot.

Immunofluorescence Microscopy

Cells were transfected while attached to 22x22mm coverslips in a six-well

plate. After the indicated incubation time, cells were washed twice in

phosphate-buffered saline (PBS) and fixed for 20 min in 3.7% formaldehyde in

200 mM HEPES, pH 7.4. To quench fixation, cells were washed twice and

incubated for 15 min in DMEM and 10 mM HEPES, pH 7.4. Cells were

permeabilized and blocked for 5 min with 0.2% Triton X-100 in PBS, followed

by two washes and a 15-min incubation with 1% BSA in PBS. Cells were

incubated with primary antibody, diluted 1:500 (anti-Atg16L1) or undiluted

(anti-myc culture supernatant) in BSA/PBS for 30 min. This was followed by

three 5-min washes and a 30-min incubation in secondary antibody diluted

1:1000 in BSA/PBS. Micrographs were acquired using a microscope

(Axiophot2, Zeiss) fitted with a 63x/numerical aperture (NA) 1.25 Zeiss

NeoFluar objective lens and a charge-coupled device camera (Orca-R2;

Hamamatsu) and controlled by Zeiss Axiophot software. Pictures were

analyzed using Adobe Photoshop software.

106

RESULTS

We have shown previously that RUTBC1 is a Rab9 effector that displays GAP

activity on Rab33B and Rab32 in vitro. In order to characterize the functional

significance of RUTBC1's GAP activity, we investigated the ability of RUTBC1

to act as a GAP for these Rabs in cells. GAPs regulate the lifetime of active,

GTP-bound Rabs. Overexpression of a GAP in cells should decrease the

amount of GTP-bound Rab and thus lead to a shift in the equilibrium between

a Rab and its effectors. Effector proteins bind preferentially to GTP-bound

Rabs; this allows effectors to act as biosensors of the amount of GTP-bound

Rabs in cells. One predicted consequence of GAP overexpression would be

solubilization of membrane-localized effectors due to conversion of their

cognate Rab-GTP substrate(s) to RabGDP. Presumably, this would also lead

to solubilization of the Rab itself by subsequent GDI-mediated extraction.

Overexpression of GFP-Rab33B in HeLa cells recruited endogenous Atg16L1

to the Golgi apparatus (Fig. 2A) consistent with previous findings in NIH3T3

cells (Itoh et al., 2008). Since Atg16L1 is a direct effector of Rab33B,

overexpression of RUTBC1 might disrupt the Golgi-associated Atg16L1. As

shown in Figure 2B, overexpression of myc-tagged RUTBC1 dissociated

Atg16L1 from the Golgi. This suggests that RUTBC1 does indeed function as

a GAP for Rab33B in cells.

107

We next investigated the consequences of depleting RUTBC1 in cultured cells.

According to the GenBank database, the RUTBC1 gene encodes two isoforms

that differ by the inclusion of one exon in the linker region between the RUN

and TBC domains (Fig. 1). Our siRNA was designed to target the 5ʼ end of

the RUTBC1 ORF to deplete both isoforms. Amplification of RUTBC1 from a

HeLa cDNA library indicated that only isoform 2 (the shorter isoform) appeared

to be expressed in these cells (data not shown). HeLa cells were mock

transfected or transfected with RUTBC1 siRNA and further incubated for 72

hours. As shown in Fig. 3A and B, RUTBC1 was efficiently silenced upon

siRNA treatment (~95%). Levels of Cog3, a subunit of the COG tethering

complex, were unchanged and used as a loading control. To our surprise,

Atg16L1 levels also decreased (Fig. 3A). The Atg16L1 gene encodes three

splice variants. The alpha and beta isoforms are both expressed in HeLa cells

(REF). The larger beta isoform was depleted approximately 40%, while the

alpha isoform was depleted by almost 70% (Fig. 3B). All three isoforms can

form the oligomeric Atg16L1 complex, thus the functional significance of the

different isoforms remains unclear.

Concomitant depletion of Atg16L1 with RUTBC1 strongly suggests that these

two proteins interact in cells. We tested this directly by co-

immunoprecipitation analysis. Myc-Atg16L1 co-immunoprecipitated with GFP-

RUTBC1 and not with GFP (Fig. 3C). Furthermore, myc-Atg16L1 was also

108

immunoprecipitated with GFP-RUTBC1 R803A, a GAP-deficient mutant. In

the reverse experiment, GFP-RUTBC1 was co-immunoprecipitated with myc-

Atg16L1 (data not shown). Thus, RUTBC1 interacts with Atg16L1, and this

interaction is independent of its GAP activity.

The immunoprecipitation results led us to investigate whether RUTBC1 and

Atg16L1 colocalize in cells. Atg16L1 is usually cytosolic, but upon stimulation

of autophagy, it is recruited to the phagophore. Overexpression of Atg16L1

caused the formation of large puncta throughout the cytoplasm (Fig. 4A,

middle cell). GFP-RUTBC1 also co-localizes to these puncta, the size of

which seemed to correlate with RUTBC1 expression levels (Fig4A, compare

upper and lower panel). Interestingly, GFP-Rab33B also co-localized to

puncta labeled by Atg16L1 (Fig. 4B). These puncta were somewhat larger in

size than the RUTBC1-positive structures. It is currently unclear if RUTBC1-

positive structures are the equivalent to those labeled by Rab33B.

Alternatively, both kinds of structures could represent intermediates in

maturation of a compartment.

109

DISCUSSION

Here we show that RUTBC1 and a Rab33B-effector, Atg16L1, can interact in

cells. RUTBC1 displays GAP activity on Rab33B in vitro. It also appears to

have GAP activity in cells as shown by its ability to solubilize Rab33B-recruited

Atg16L1 from the Golgi. In co-immunoprecipitation experiments using

Atg16L1 as a biosensor for the level of Rab33B-GTP in cells, we showed that

both wild-type and the GAP-deficient mutant of RUTBC1 decreased the

amount of Rab33B co-immunoprecipitating with Atg16L1 (data not shown).

RUTBC1 may be recruited to a Rab33B microdomain containing Atg16L1,

displacing Atg16L1 from Rab33B in a sort of GAP “invasion” of a microdomain,

perhaps by indirectly competing for the Rab33B binding site on Atg16L1. This

interaction would also lead to inactivation of Rab33B by stimulation of GTP

hydrolysis. This model could be tested in vitro by adding wild-type and GAP-

deficient RUTBC1 to Rab33B-loaded liposomes bearing Atg16L1. Both

proteins would release Atg16L1 from membranes, but only addition of wild-

type RUTBC1 would lead to extraction of Rab33B from membranes by

addition of GDI. Alternatively, we could use Golgi enriched fractions from cells

expressing tagged Atg16L1 and Rab33B proteins.

The observation that depletion of RUTBC1 also leads to partial co-depletion of

Atg16L1 suggests that RUTBC1 is required for the stability of Atg16L1.

Indeed, the two proteins interact in cells as shown by co-immunoprecipitation

110

experiments. This could be through formation of a stable complex or by

RUTBC1-mediated regulation of Atg16L1 degradation.

Most Atg16L1 is found in a high molecular weight complex composed of the

Atg5-Atg12 conjugate and Atg16L1 (~800kDa; Mizushima et al., 2003). That

work also showed that a protein of 144kDa was also present in purified

Atg16L1 complex from mammalian cells but its identity was not reported.

RUTBC1 has a predicted molecular weight of ~118kDa and apparent

molecular weight of ~125kDa by SDS-PAGE. Gel filtration of bovine brain

cytosol revealed that RUTBC1 is in a large complex (~450kDa; data not

shown). Future experiments will characterize the composition of this complex.

Finally, Atg16L1 appears to be degraded by the ubiquitin proteasome system,

at least when overexpressed (Fujita et al., 2009). How RUTBC1 might

influence Atg16L1 stability will be another area for future work.

111

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114

FIGURE LEGENDS

Figure 1: Domain architecture of RUTBC1 and Atg16L1. RUTBC1

contains an N-terminal RUN domain (RPIP8/Unc-14/NESCA) and a C-terminal

TBC domain (Tre2/Bub2/Cdc16). Atg16L1 contains an N-terminal Atg5-

binding domain (BD) and a middle coiled-coil domain (CC). Mammalian

Atg16L1 also has a C-terminal WD40 repeat domain.

Figure 2: RUTBC1 solubilizes Atg16L1 from the Golgi. HeLa cells were

transfected with GFP-Rab33B and myc-RUTBC1 for 48 hours. Cells were

stained with rabbit anti-Atg16L1 antibodies and mouse anti-myc antibodies

followed by goat anti-rabbit-Alexa568 and goat anti-mouse-Alexa647

secondary antibodies. Scale bars represents 5µm.

Figure 3: RUTBC1 and Atg16L1 interact in cells. (A) HeLa cells were either

mock transfected or transfected with siRNA targeted to RUTBC1 and

incubated for 72 hours. Extracts were blotted with affinity purified RUTBC1

antibodies and anti-Atg16L1 antibodies. Cog3 was used as a loading control.

Asterisks indicate non-specific bands. (B) Quantitation of data presented in A.

Error bars represent standard deviation. (C) COS-1 cells transfected with

GFP-RUTBC1, GFP-RUTBC1 R803A and myc-Atg16L1 as indicated for 24

hours. Extracts were immunoprecipitated with anti-GFP antibodies and

115

immune complexes were analyzed by immunoblot. Immunoprecipitated GFP-

RUTBC1 and GFP were detected by Ponceau S stain.

Figure 4: RUTBC1 localizes to Atg16L1-positive puncta. (A) HeLa cells

were transfected with GFP-Rab33B and myc-Atg16L1 for 24 hours. Cells

were stained with mouse anti-myc antibodies and Alexa 594-conjugated goat

anti-mouse secondary antibodies. (B) HeLa cells were transfected with myc-

Atg16L1 and GFP-RUTBC1 for 24 hours. Cells were stained as in A. Scale

bars represents 5µm.

116

Figure 1

117

Figure 2

118

Figure 3

119

Figure 3 continued

120

Figure 4

121

Figure 4 continued

122

CHAPTER 4

CHARACTERIZATION OF RAB SUBSTRATES

AND BINDING PARTNERS OF RUTBC2

(Manuscript in preparation)

Ryan M. Nottingham, Ian G. Ganley, Francis A. Barr,

David G. Lambright and Suzanne R. Pfeffer

Contributions: RMN contributed Fig. 1b, 2, 3a-c, 4 and 5. IGG contributed

Fig.3d. FAB contributed Fig. 1a and plasmids. DGL contributed plasmids and

reagents. RMN and SRP conceived the project and wrote the paper

123

ABSTRACT

Rab GTPases regulate vesicle budding, motility, docking and fusion. In cells,

their cycling between active, GTP-bound states and inactive, GDP-bound

states is regulated by the action of opposing enzymes called guanine

nucleotide exchange factors and GTPase-activating proteins (GAPs). The

substrates for most RabGAPs are unknown and the potential for cross talk

between different membrane trafficking pathways remains uncharted territory.

Rab9 and its effectors regulate recycling of mannose 6-phosphate receptors

from late endosomes to the trans Golgi network. We show here that RUTBC2

is a TBC domain-containing protein that binds to Rab9 specifically both in vitro

and in cultured cells but is not a GAP for Rab9. Biochemical screening of

RUTBC1ʼs Rab protein substrates revealed highest GAP activity toward

Rab34 and Rab36, both Golgi-localized Rabs. These data suggest a model in

which RUTBC2 somehow links Rab9 function to Rab34/36 function at the

Golgi. This cross-talk between Rab domains suggests the existence of a Rab

cascade between endosomes and the Golgi.

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INTRODUCTION

Rab GTPases are master regulators of membrane trafficking. They catalyze

the formation of functional membrane microdomains by recruiting effectors to

membranes while in their GTP-bound states. These effectors help Rabs

regulate every step of a trafficking event.

RUTBC2 is a putative Rab GTPase-activating protein (GAP) conserved in C.

elegans, Drosophila and vertebrates. The primary sequence reveals the

presence of two domains, an N-terminal RPIP8/Unc-14/NESCA (RUN) domain

and a C-terminal Tre-2/Bub2/Cdc16 (TBC) domain. RUN domains are thought

to function as protein-protein interaction domains. The TBC domain

possesses RabGAP activity and is conserved from yeast to man. There are

approximately 70 Rabs encoded by the human genome and likely ~40

expressed in any given cell. There are at least 40 human RabGAPs,

suggesting that RabGAPs are highly specific for their Rab protein substrates.

Little is known about either the biochemistry or cell biology of RUTBC2. It was

first detected in purified lysosomal membranes (Ausseil et al., 2006). The

original goal of that work was to find the causative agent of

mucopolysaccharidosis IIIC, a disease with defective lysosomal enzyme

activity. Later, Shimizu and co-workers analyzed the tissue distribution of both

the human and mouse transcripts by Northern blot. They found that RUTBC2

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has a more restricted expression pattern than the highly related RUTBC1 or

RUTBC3 and that RUTBC2 was highly enriched in mouse brain (Yang et al.,

2007). Furthermore, RUTBC2 localized at steady-state to the trans Golgi

network in mouse neuroblastoma cells (Yang et al., 2007). RUTBC2 co-

immunoprecipitates with Rap1a/b and Rap2a/b via a region just C-terminal to

the RUN domain and with multiple Rab GTPases (Yang et al., 2007). This

suggests a link between Rap- and Rab-mediated signaling in cells but the

current Rab substrates of the RUTBC2 TBC domain are unknown.

Only one interacting partner of RUTBC2 has been identified. Nurr1 is an

orphan nuclear receptor required for the development of dopaminergic

neurons in the mouse brain. Co-expression of RUTBC2 and Nurr1 increased

transcription of endogenous tyrosine hydroxylase, part of the dopamine

synthesis pathway (Luo et al., 2008). Moreover, suppression of RUTBC2 by

siRNA led to decreased cell division and decreased expression of the

dopamine transporter, a Nurr1 target gene (Luo et al., 2008). This has led to

speculation that RUTBC2 may be important in the pathogenesis of Parkinsonʼs

disease, which is known to be influenced by Nurr1 (Federoff, 2009).

Rab9 GTPase is required for the recycling of mannose 6-phosphate receptors

from late endosomes to the trans Golgi network (Lombardi et al., 1993;

Barbero et al., 2002). It also plays a role in lysosome biogenesis (Riederer et

126

al., 1994) and late endosome morphology (Ganley et al, 2004). In this study

we report that RUTBC2 is a specific effector of Rab9. Rab9 binds RUTBC2 in

the linker region between the RUN and TBC domains. The RUTBC2 TBC

domain does not possess GAP activity on Rab9 but instead shows GAP

activity toward highly-related Rab34 and Rab36 proteins.

127

METHODS

Yeast Two-Hybrid Analysis

Yeast two-hybrid analysis was carried out as described previously (Fuchs et

al., 2007). Briefly, 56 mutant Rab proteins deficient for GTP hydrolysis (Q to

A) were cloned into pGBT9 bait vector (Clontech). RUTBC2 was amplified

from cDNA libraries and was cloned into pACT2 prey vector (Clontech); growth

on selective media indicated an interaction between a Rab and RUTBC2.

Plasmids

For mammalian expression, N-terminally 3Xmyc-tagged RUTBC2 was

obtained by amplification from a cDNA library and ligation into a modified

version of pCDNA3.1(+) (Invitrogen) (Fuchs et al., 2007). This construct

encodes the shortest of four isoforms found in GenBank and is missing the

first 25 amino acids at the N-terminus. GFP-RUTBC2 was constructed by

amplification of this isoform with the addition of the missing N-terminus by

PCR and ligated into pEGFP-C3 (Clontech). The predicted RUTBC2 GAP-

inactive mutant (R829A) was created using QuikChange.

For bacterial expression, RUTBC2-C was ligated into pET28a (Novagen) in

frame with the N-terminal His-tag. A plasmid encoding GST-RUTBC2 was

constructed by ligation of RUTBC2 into pGEX-6P-1 (GE Healthcare).

Untagged Rab9CLLL and GST-Rab9A were previously described (Aivazian et

128

al, 2006). GST-Rab9B was amplified by PCR from pET14-Rab9B (Hayes et

al., 2009) and ligated into pGEX-4T-1. GST-Rab6A was amplified by PCR

from His-Rab6A (Burguete et al., 2008) and ligated into pGEX-4T-1. GST-

C110 and GST-C123 were previously described (Reddy et al., 2006). His-p40

was previously described (Diaz et al., 1997). HisGFP was made by amplifying

GFP from pEGFP-C1 (Clontech) and ligation into pET14b (Novagen).

Rab proteins tested for GAP activity were previously described (Pan et al.,

2006). Phosphate Binding Protein (PBP) from E. coli was amplified by PCR

from bacteria and cloned into modified pET15. His-PBP A197C was

constructed by site-directed mutagenesis.

Protein Expression and Purification

His-RUTBC2-C was transformed into Rosetta2 (DE3) cells (Novagen) and

grown at 37°C until OD600 = 0.5. The cells were induced with 0.4mM isopropyl

β-D-thiogalactoside (IPTG) and grown for an additional 4 hours at 22°C. Cell

pellets were resuspended in lysis buffer (25mM HEPES, pH 7.4, 300mM NaCl,

50mM imidazole) supplemented with 1mM PMSF and lysed by two passes at

20,000 psi through an EmulsiFlex-C5 apparatus (Avestin). Cleared lysates

(20,000 rpm for 45 min at 4°C in a JA-20 rotor; Beckman Coulter) were

incubated with Ni-NTA (Qiagen) for 1 hour at 4°C. The resin was then washed

with lysis buffer and eluted with 25mM HEPES, pH7.4, 300mM NaCl and

129

250mM imidazole. Fractions containing RUTBC2-C were pooled and

concentrated using an Amicon Ultra concentrator (Millipore). The sample was

dialyzed to remove imidazole and then brought to 10%(v/v) glycerol. The

sample was aliquoted, snap frozen in liquid nitrogen and stored at -80°C.

GST-RUTBC2 was transformed into Rosetta2 (DE3) cells and grown at 37°C

until OD600 = 0.6. The cells were induced with 100µM IPTG and grown for an

additional 4.5 hours at 30°C. Cell pellets were resuspended in lysis buffer

(50mM HEPES, pH 7.4, 250mM NaCl, 1mM DTT, 1mM EDTA) supplemented

with 1mM PMSF and lysed by two passes at 20,000 psi through an

EmulsiFlex-C5. Cleared lysates (19,000 rpm for 30 min at 4°C in a JA-20

rotor; Beckman Coulter) were incubated with Glutathione-Sepharose FastFlow

(GE Healthcare) for 1.5 hours at 4°C. The resin was then washed with 25

column volumes lysis buffer and eluted with 50mM Tris-HCl, pH8.0, 250mM

NaCl and 20mM glutathione. Fractions containing RUTBC2-C were pooled

and concentrated using an Amicon Ultra concentrator (Millipore). The sample

was dialyzed to remove glutathione and brought to 10%(v/v) glycerol, snap

frozen in liquid nitrogen and stored at -80°C.

Untagged Rab9CLLL and GST-Rab9A expression and purification were

previously described (Aivazian et al., 2006) and GST-Rab9B and GST-Rab6A

were purified by the same procedure. His-p40 purification was previously

described (Diaz et al., 1997). His-GFP was purified using the Qiaexpressionist

130

Kit (Qiagen) according to the manufacturer. Expression and purification of

GST-C110 and GST-C123 were previously described (Reddy et al., 2006).

Expression and purification of Rab proteins for the GAP screen were

previously described (Pan et al., 2006). His-PBP A197C was purified and

labeled according to the method described (Shutes and Der, 2005).

Antibodies

Mouse monoclonal anti-myc (9E10), mouse monoclonal anti-Rab9A, mouse

monoclonal anti-CI-MPR (2G11) and rabbit anti-CI-MPR, were all previously

described (Ganley et al., 2008). Rabbit anti-GFP antibody was from

Invitrogen; mouse anti-GFP antibody was from Roche. Rabbit anti-RUTBC2

was purchased from Sigma. HRP-conjugated goat anti-mouse and goat anti-

rabbit secondary antibodies as well as protein-A-HRP were from Bio-Rad.

Binding assays

Constructs encoding 3xmyc-RUTBC2 were translated in vitro using a TNT

Quick Coupled Transcription/Translation System (Promega) following the

manufacturerʼs protocol. GST-tagged Rabs were loaded with GTPγS or GDP

as described (Aivazian et al., 2006) and mixed with TNT lysate for 1.5 hours at

25ºC in binding buffer (25mM HEPES-NaOH, pH7.4, 150mM NaCl, 5mM

MgCl2, 1mM DTT, 0.1mM GTPγS). RUTBC1 constructs bound to GST-Rabs

were isolated using glutathione-Sepharose, washed in binding buffer (with

131

400mM NaCl) and then eluted by addition of 25mM glutathione and analyzed

by immunoblot. Binding assays using purified proteins were as described

(Hayes et al., 2009).

Cell Culture and Transfections

HeLa, HEK293T, SK-N-SH and Vero cells were obtained from American Type

Culture Collection and cultured at 37°C and 5% CO2 in Dulbecco's modified

Eagle's medium supplemented with 7.5% fetal calf serum, 100U penicillin and

100µg/mL streptomycin. For overexpression studies, all cells were transfected

using Fugene 6 (Roche). Cells were harvested either 24 or 48 hours after

transfected as indicated. For siRNA treatment, HEK293T cells were

transfected using Lipodectamine 2000 (Invitrogen).

Immunopreciptation, Protein Turnover, Lysosomal Enzyme Secretion and

Fractionation

For immunoprecipitation, cells were transfected with indicated plasmids for 24

hours. Cells were then lysed in 50mM Tris-HCl, pH 7.4, 150mM NaCl, 1mM

MgCl2 and 1% Triton X-100 supplemented with Complete EDTA-free Protease

Inhibitor Cocktail (Roche) and spun for 15 minutes at full speed in a microfuge

at 4°C. Supernatants were pre-cleared using Protein-A agarose (Roche) for 15

minutes at room temperature and then immunoprecipitated with indicated

antibodies for 1.5 hours at room temperature. Immune complexes were

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isolated on Protein-A agarose for 30 minutes at room temperature. The resin

was then washed 3 times with lysis buffer and once with PBS. Beads were

resuspended in 2X sample buffer boiled and analyzed by immunoblot. Protein

turnover and lysosomal enzyme secretion assays were performed as

described (Ganley et al., 2008). Cell fractionation was as previously described

(Ganley et al., 2004) except SK-N-SH cells were swollen in 10mM HEPES

pH7.4 for 5 minutes.

GAP Assays

For the biochemical screen of GAP substrates, the procedure followed by Pan

et al. (2006) was generally used with the exception that phosphate released

during the reaction was bound by modified PBP (A197C) labeled at position

197 with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide

(MDCC). Phosphate binding to MDCC-PBP causes a conformational change

in the phosphate binding cleft that results in an increase in MDCC

fluorescence (Brune et al., 1994). Reactions were started by adding a solution

containing GAP, MgCl2 and MDCC-PBP to desalted, GTP-exchanged Rabs by

a Precision 2000 liquid handling system (Biotek). Rab GTPases were at 2µM

for all reactions while the concentration of His-RUTBC2-C was varied.

Phosphate production was monitored by following fluorescence signal

continuously in a TECAN Saphire microplate reader using an excitation of

425nm and an emission cutoff filter of 455nm.

133

RESULTS

We are interested in the function and localization of the Rab9 GTPase. As a

starting point to find regulators of Rab9, we used a two-hybrid screen

consisting of all TBC domain-containing proteins in the human genome as

prey against a comprehensive library of hydrolysis-deficient Rab GTPases as

bait (Haas et al., 2005; Fuchs, et al., 2007). This screen revealed that a TBC

domain-containing protein, RUTBC2, interacted with both Rab9A and 9B

(Figure 1A). RUTBC2 also interacted to a lesser extent with Rab3 isoforms.

Like the closely-related RUTBC1, the TBC domain of RUTBC2 has a large

insertion between the first two “fingerprint” A and B motifs (Figure 1B,

sequence). In the structural model for Rab and RabGAP interaction, the

analogous region of Gyp1 is situated away from the Rab:GAP binding

interface (Pan et al., 2006). Most of the dissimilarity between RUTBC1 and

RUTBC2 in the TBC domain is found in this insertion. According to the NCBI

Homologene database, there is only one RUTBC1/2 protein in C. elegans (tbc-

8), Drosophila (CG1905) and zebrafish (LOC794373) while vertebrates have

both proteins.

RUTBC2 is a Rab9 effector

To confirm the results of the screen and to test the nature of the interactions,

we tested if Rab9 could bind RUTBC2 in vitro. Full-length RUTBC2 was

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produced both in an in vitro transcription/translation system as well as in E.

coli, and assayed for binding to immobilized GST-Rabs. As shown in Figure

2A, GST-Rab9A but not GST-Rab9B or GST-Rab6A could bind in vitro

translated, full-length RUTBC2. These results seem contradictory to the

screen results but stronger Rab9B binding in two hybrid assays has been

observed before (Hayes et al., 2009). Next, we asked if RUTBC2 preferred

either the GTP or GDP-bound form of Rab9. Using the GST-binding assay

described above, in vitro translated RUTBC2 was bound much more efficiently

by GST-Rab9 when loaded with GTPγS than with GDP (Fig. 2B). Thus,

RUTBC2 is a bona fide effector of Rab9A (called Rab9 from now on).

We next investigated the location of the Rab9 binding site in RUTBC2. Using

purified proteins, we found that full-length GST-RUTBC2 bound as well to

Rab9 as the positive control, the GST-tagged, C-terminal 110 amino acids of

GCC185 (Reddy et al., 2006) (Fig. 2D, left panel). There was little Rab9

binding to the negative control, the GST-tagged, C-terminal 123 amino acids of

Golgin245. Rab9 bound poorly to the TBC domain alone (His-tagged

RUTBC2-C) but very well to a known Rab9 effector, His-p40 (Fig. 2D, right

panel). This suggests that the Rab9 binding site is likely located in the N-

terminus. An earlier two-hybrid screen for GAP-Rab interactions failed to

detect Rab9 binding to RUTBC2 (Itoh et al., 2006). This was likely due to the

135

use of constructs for both proteins that contained only the TBC domain.

Taken together, these data suggest that Rab9 binds directly to RUTBC2 in its

N-terminal region.

RUTBC1 interacts with Rab9 in cells

Rab9 regulates the recycling of mannose 6-phosphate receptors (MPRs) from

late endosomes to the trans Golgi network (TGN) (Lombardi et al., 1993;

Riederer et al., 1994). To ask whether Rab9 and RUTBC2 interact in cells,

HEK 293T cells were transfected with GFP-RUTBC2 or GFP as a control.

Endogenous Rab9 was immunoprecipitated with GFP-RUTBC2 and not GFP

(Figure 3A). Thus, RUTBC2 can interact with Rab9 in living cells.

We further characterized this interaction by investigating the effects of

exogenously expressed RUTBC2 on MPR recycling. When Rab9 function is

disturbed in cells by overexpression of a dominant negative mutant Rab9

(Riederer et al., 1994) or by depletion of its effectors (Reddy et al., 2006;

Espinosa et al., 2009), MPRs are mis-sorted to the lysosome. We reasoned

that if RUTBC2 had GAP activity on Rab9, the phenotype of RUTBC2

overexpression should be similar to that of a GDP-preferring Rab9 mutant or

Rab9 depletion. When RUTBC2 was overexpressed in HeLa cells, MPR

levels at steady state were decreased by ~40% (Figure 3B). This observation

led us to investigate the turnover rate of MPR in HeLa cells overexpressing

136

RUTBC1 and found it to be essentially unchanged (Figure 3C). This

suggested that though some amount of MPR was being mis-sorted to

lysosomes, there was likely an increase in MPR translation. As a possible

GAP for Rab9, the predicted GAP-deficient mutant (R829A) of RUTBC2

should not have the same effect when overexpressed.

In a functional test of MPR trafficking, we measured the amount of

hexosaminidase activity secreted into the media by cells transfected with

RUTBC2. Hexosaminidase is usually sorted to the lysosome but when MPR

levels are deficient in the TGN due to missorting events, the hydrolase is

secreted. In 293T cells transfected with RUTBC2, slightly higher

hexosaminidase activity was detected in the media than in control cells (Figure

3D). Cells transfected with RUTBC2 R829A showed a similar effect. Taken

together, perturbation of MPR trafficking is likely due to titration of Rab9 by the

overexpression of RUTBC2 rather than the GAP activity of this protein.

RUTBC2 has narrow substrate specificity

Discovering the Rab substrates of RUTBC2 should provide insight into the

significance of Rab9-RUTBC2 interaction. Thirty-two different mammalian

Rab GTPases were screened under single turnover conditions as substrates

for RUTBC2 using purified, His-tagged RUTBC2-C (Figure 2C) that contains

the catalytic TBC domain. Figure 4 summarizes these results by comparing

observed second order rate constants for GAP-catalyzed hydrolysis to the rate

137

constants for intrinsic hydrolysis by each Rab protein. The TBC domain of

RUTBC2 had the highest activity against Rab36 and Rab34 while no activity

was detected against Rab9 or Rab3. Both Rab36 and Rab34 are localized to

the Golgi (Wang and Hong, 2002; Chen et al., 2009).

RUTBC2 is enriched in neural cells

Analysis of different cell lines revealed that RUTBC2 is present in different

amounts in HEK293T, Vero and SK-N-SH cells (Fig 5A). SK-N-SH cells are a

human neuroblastoma cell line that might be similar to mouse Neuro2a

neuroblastoma cells used by Shimizu and co-workers in earlier RUTBC2

studies (Yang et al., 2007). RUTBC2 seems to be highly expressed in these

cells compared to kidney cells, consistent with that report. RUTBC2 could be

efficiently depleted by siRNA treatment of 293T cells (Fig. 5B). Quantitation

revealed an approximate ~90% level of depletion. RUTBC2 also stably

associated with membranes (Fig. 5C). Fractionation of SK-N-SH cells showed

that most of RUTBC2 was cytosolic; nevertheless, a small pool of RUTBC2

was stably associated with membranes. This is also consistent with the

steady-state localization observed previously (Yang et al., 2007).

Unfortunately, our antibody could not be used for immunofluorescence

microscopy to determine the specific membranes with which RUTBC2

associates. Together, these data suggest a model where RUTBC2 in some

way links late endosome and Golgi trafficking pathways.

138

DISCUSSION

Here we have shown that a predicted RabGAP protein, RUTBC2, is a novel

Rab9 effector. RUTBC2 does not display GAP activity on Rab9 but instead

has GAP activity for Rab36 and Rab34. RUTBC2 is present in many cell

types but is enriched in cells of neural origin. A small pool of RUTBC2 also

stably associates with membranes. Although Rab9B appeared to interact with

RUTBC2 in a two hybrid screen, only purified Rab9A bound directly to

RUTBC2. Binding to either Rab would be consistent with the observation that

murine RUTBC2 localizes to the trans Golgi network (Yang et al., 2007).

Rab9B is known to be on the Golgi while Rab9A regulates the transport of

MPRs from late endosomes to the TGN. It is not yet clear precisely where

Rab9A and RUTBC2 interact.

Currently there are no known direct links between Rab9-mediated trafficking

and either Rab36- or Rab34-regulated events. However, both Rab36 and

Rab34 are thought to mediate the localization of lysosomes (Wang and Hong,

2002; Chen et al., 2009), despite the fact that both Rabs localize to the Golgi.

GTP-restricted Rab34 or Rab36 cause LAMP1-positive lysosomes (and not

MPR-positive structures) to cluster in the perinuclear area (Wang and Hong,

2002; Chen et al., 2009; Goldenberg et al., 2007). This phenomenon is

mediated by RILP, an effector of both Rab34 and Rab36 as well as Rab7, a

Rab known to play a role in lysosomal biogenesis. A mutation in the switch I

139

region of Rab34 (K82Q) that blocks association with RILP fails to redistribute

lysosomes (Wang and Hong, 2002). Little else is known about Rab36 other

than it is deleted in many cases of malignant, rhabdoid tumors (Mori et al.,

1999).

Rab34 has also been suggested to play additional role in constitutive

secretion. It binds to hmunc-13, a diacylglycerol binding protein (Speight and

Silverman, 2005). Depletion of Rab34 and hmunc-13 from HeLa cells blocks

temperature sensitive Vesicular stomatitis virus glycoprotein (VSV-G) arrival at

the plasma membrane (Goldenberg et al., 2007; Goldenberg and Silverman,

2009). Brefeldin A treatment revealed that blocked VSV-G returned to the

endoplasmic reticulum, suggesting that Rab34 depletion causes a block in

intra-Golgi transport rather than exit from the trans Golgi network (Goldenberg

et al., 2007). A previous screen for the ability of overexpressed RabGAPs to

block VSV-G secretion in multiple cell types showed that RUTBC2 had no

effect on this process (Haas et al., 2007). This suggests that RUTBC2 may

only have activity on Rab36 in living cells.

There is also no known connection between Nurr1 and either Rab34 or Rab36.

One attractive, but highly speculative connection might be that either of these

Rabs are required for proper trafficking of the dopamine transporter. RUTBC2

might be able to integrate signals from the Golgi apparatus in regard to the

140

status of secretion to the Nurr1 transcriptional response. Interestingly, Rab34

has been shown to be a direct target of Hedgehog signaling (Vokes et al.,

2007). Hedgehog signaling helps mediate the specification of distinct cell

identities in the ventral neural tube through a Gli-mediated transcriptional

network, including dopaminergic neurons (Hynes et al., 2005). Experiments

elucidating the connections, if any, between these diverse signaling and

trafficking pathways could have potential impact on the pathogenesis and

treatment of Parkinson’s disease.

141

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FIGURE LEGENDS

Figure 1: RUTBC2 interacts with Rab9. (A) RUTBC2 was screened against

a library of 56 human Rab GTPases. Growth after streaking on selective

media indicates an interaction between RUTBC2 and a Rab GTPase. (B)

Diagram of RUTBC2 domain structure. The RUN domain is shown in blue; the

TBC domain is shown in red. The first three conserved motifs of the TBC

domain are indicated. Conserved residues are in bold and the predicted

catalytic arginine and glutamine are shown in red, respectively. The extended

insert between motif A and motif B of RUTBC2 is compared to the same

regions of human RUTBC1 and RabGAP-5 (RUTBC3) and S. cerevisiae

Gyp1p.

Figure 2: RUTBC2 is an effector of Rab9. (A) In vitro translated (IVT)

3xmyc-RUTBC2 proteins were incubated with various bacterially purified,

GST-tagged Rabs pre-loaded with GTPγS. Bound material was eluted by

glutathione and half of the eluate was analyzed by immunoblot using anti-myc

antibodies. Rabs were detected by Ponceau S staining. (B) IVT-3xmyc-

RUTBC2 was incubated with GST-Rab9A pre-loaded with either GTPγS or

GDP and analyzed as in A. (C) Diagram of 3xmyc-tagged RUTBC1 constructs

used in binding assays. (D) Left panel: purified GST-RUTBC2 and control

proteins were incubated with Rab9A loaded with GTPγ[35S] and immobilized

using glutathione-Sepharose. Bound Rab was detected by scintillation

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counting. Right panel: His-RUTBC2-C and control proteins were incubated

with Rab9A loaded with GTPγ[35S] and isolated using Ni-NTA. “Input”

indicates 1% of the IVT reaction used in the binding assay.

Figure 3: RUTBC2 binds to, but is not a GAP for Rab9A in cells. (A)

HEK293T cells were transfected with GFP-RUTBC2 or GFP for 24 hrs and

RUTBC2 was immunoprecipitated with rabbit anti-GFP antibodies followed by

immunoblotting with specific antibodies to Rab9A and GFP. “Input” represents

2% of the lysate subjected to immunoprecipitation. (B) Left panel - COS-1 cells

were transfected with 3xmyc-RUTBC2 for 48 hours and extracts were

immunoblotted with anti-CI-MPR antibodies. Right panel - Quantitation of CI-

MPR half-life measured from pulse/chase analysis of HeLa cells transfected

with 3xmyc-RUTBC2 for 48 hours. Extracts were immunoprecipitated with

anti-CI-MPR antibodies and exposed to a phosphor screen. (C) HEK293T

cells transfected with 3xmyc-RUTBC2 wild type or R829A for 24 hours were

assayed for secreted and intracellular hexosaminidase activity. Error bars in

all panels represent SEM from at least two independent experiments.

Figure 4: RUTBC2 TBC domain has GAP activity toward Rab36 and

Rab34 in vitro. Purified, mammalian Rab GTPases (32) were pre-loaded with

GTP for one hour at room temperature and then desalted to remove free

nucleotide. MDCC-PBP, MgCl2 and varying concentrations of purified His-

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RUTBC2-C were added to start the reaction. Phosphate release was

monitored continuously by microplate fluorimeter (see Materials and Methods).

Catalytic efficiency (kcat/KM) relative to the intrinsic rate constant (kintr) for GTP

hydrolysis was determined. Plots represent the mean from duplicate wells.

Figure 5: RUTBC2 is stably associated with membranes in SK-N-SH

cells. (A) Detergent extracts (100µg) of indicated cell lines were resolved by

SDS-PAGE and immunoblotted using anti-RUTBC2 antibodies. (B) HEK293T

cells were either mock transfected or transfected with siRNA targeting

RUTBC2 for 72 hours. Shown is quantitation of the band corresponding to

RUTBC2. (C) SK-N-SH cells were fractionated into crude membranes and

cytosol. Increasing volumes of each fraction were analyzed by immunoblot

using RUTBC2 specific antibodies.

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Figure 1

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Figure 2

150

Figure 3

151

Figure 4

152

Figure 5

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SUMMARY AND FUTURE PERSPECTIVES

Rab9 plays an essential role in the recycling of mannose 6-phosphate

receptors from late endosomes to the TGN, and it is also important for

lysosome biogenesis and late endosome morphology. Regulators of the Rab9

nucleotide binding cycle remain unknown, although it is likely that both a GEF

and GAP for Rab9 exist in cells. This thesis has added to our understanding

of Rab9 function in cells by providing new clues to links between different

trafficking pathways.

We have shown that two Rab GAP proteins, RUTBC1 and RUTBC2, are novel

Rab9 effectors that show preference for different Rab protein substrates for

their GAP activities. This implies that the substrate Rabs act in pathways that

feed into a Rab9-regulated pathway or organelle. Rab9 binding to RUTBC1 or

RUTBC2 might help to clear these Rabs from a Rab9 microdomain. It is now

imperative to investigate the significance of Rab9 interaction with these GAP

proteins. Does Rab9 simply localize these proteins transiently to late

endosomal membranes? Does Rab9 affect the catalytic activity of RUTBC1

and/or RUTBC2? Recent evidence suggests that binding of Rabs can

influence nucleotide hydrolysis activity directly (Rab9 and RhoBTB3: Espinosa

et al., 2009) or GEF activity (Rab11 and Rabin8: Knödler et al., 2010). The

latter case is especially germane to the proteins in this study because it

supports the Rab cascade model in a process (ciliagenesis) distinct from the

154

secretory pathway or the early-to-late endosome transition. It will thus be

important to test the ability of Rab9 to alter the catalytic activity of RUTBC1

and RUTBC2.

One of the more surprising findings in this thesis is the connection between

RUTBC1 and Atg16L1. Atg16L1 is required for conventional autophagy and

RUTBC1 depletion leads to concomitant depletion of Atg16L1. The most

important next goal must be to determine the effects of RUTBC1 depletion of

autophagosome formation and maturation. Rab9 does not have a

characterized role in autophagy but it would be reasonable to speculate that

mannose 6-phosphate receptor trafficking is important for autophagy, both for

maturation of the autophagosome into a late endosome-like structure and in its

canonical role delivering hydrolases to lysosomes.

Also of interest is our characterization of RUTBC2. Its enrichment in brain

tissue and connections to dopaminergic neuron biogenesis are important

justifications for further study. There is also no known connection between

Nurr1 (a reported binding partner for RUTBC2) and either Rab34 or Rab36.

One attractive, but highly speculative connection might be that either of these

Rabs are required for proper trafficking of the dopamine transporter. RUTBC2

might be able to integrate signals from the Golgi apparatus in regard to the

status of secretion to influence the Nurr1 transcriptional response.

155

Experiments elucidating the possible connections between these diverse

signaling and trafficking pathways could have potential impact on our

understanding of the fundamental pathogenesis of Parkinson’s disease.

References

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endosome to Golgi transport. Cell (2009) vol. 137 (5) pp. 938-48.

Knödler et al. Coordination of Rab8 and Rab11 in primary ciliogenesis.

Proceedings of the National Academy of Sciences (2010) vol. 107 (14) pp.

6346-51.