Rab Gtpase-Activating Proteins at the Golgi:Endosome Interface

Home , EVI5L, RABGAP1, Rab (G-protein), Ras GTPase, Small GTPase

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

This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/

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

ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in 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 adequate in 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 adequate in 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 in University Archives.

iii

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

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.

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

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.

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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

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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

LIST OF TABLES

Introduction

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

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

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.

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).

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

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

(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

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.

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

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

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

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

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

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

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

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

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).

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

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).

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

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.

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.

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.

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

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

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)

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

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.

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

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

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

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

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

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.

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.

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Table I. Summary of mammalian Rab GTPase-activating proteins

TBC Aliases Reported Other Function/Remarks References Protein substrates domains

EVI5 Rab11, CC Regulates Shiga Dabbeekeh -35 toxin uptake et al., 2007; Fuchs et al., 2007

EVI5L Rab23, CC Regulates primary Yoshimura et -10 cilia formation al., 2007; Itoh et al., 2006

RN-tre Rab5, -41 EGF receptor Lanzetti et downregulation; al., 2000; Golgi biogenesis, Haas et al., regulates Shiga toxin 2005; Fuchs uptake et al., 2007

RUTBC1 SGSM2 Rab33b, RUN Binds Rab9; This study -32 interacts with Atg16L1

RUTBC2 SGSM1 Rab34, RUN Binds Nurr1 This study; -36 transcription factor Luo et al., 2008

RUTBC3 RabGAP-5, Rab5 RUN; Regulation of Haas et al., MAP, CIP85 SH3 endocytosis, binds 2005; Lee et tumor suppressor al., 2004; Lan Merlin, gap junctions et al., 2005

TBC1D1 Rab8a, PTB Akt substrate; Roach et al., -10, -14 regulates insulin- 2004; Peck et stimulated GLUT4 al., 2009; receptor trafficking; Stone et al., genetically 2006 associated with obesity

TBC1D2A Armus Rab7 PH; CC Rac1 effector and Frasa et al., regulator of E- 2010 cadherin degradation

TBC1D2B KRAB; Binds Rab22 Kanno et al., CC 2010

TBC Aliases Reported Other Function/Remarks References Protein substrates domains

TBC1D3 PRC17 Hominoid specific; Pei et al., oncogene, activator 2002; of Ras? Wainszelbau m et al., 2008

TBC1D4 AS160 Rab2a, PTB Akt substrate; Kane et al., -8a, -10, regulates insulin- 2002; Mîlnea -14 stimulated GLUT4 et al., 2005 receptor trafficking

TBC1D5 Rab7 Binds to retromer Seaman et coat al., 2009

TBC1D6 GRTP1

TBC1D7 Rab17 Regulates primary Yoshimura et cilia formation, al., 2007; oncogene Sato et al., 2010

TBC1D8A Vrp GRAM; CC

TBC1D8B GRAM

TBC1D9A GRAM; EF

TBC1D9B GRAM; EF

TBC1D10A EPI64 Rab27A, Regulates microvillar Itoh and -35 structure; regulates Fukuda, Shiga toxin uptake 2006; Hanono et al., 2006; Fuchs et al., 2007

TBC1D10B Rab35, CC Regulates Shiga Fuchs et al., -3a toxin uptake 2007; Ishibashi et al., 2010

TBC1D10C Rab35 Regulates Shiga Fuchs et al., toxin uptake 2007

TBC Aliases Reported Other Function/Remarks References Protein substrates domains

TBC1D11 GAPCenA, Rab4, -6 PTB; CC Controversial: has Fuchs et al., RABGAP1 GAP activity on 2007; Cuif et Rab6 but does not al., 1999; block Shiga toxin Kanno et al., trafficking like Rab6 2010 depletion; binds Rab36

TBC1D12

TBC1D13

TBC1D14 CC Crystal structure of Tempel et al., TBC domain solved 2008

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

TBC1D16

TBC1D17 Rab21, Regulates Shiga Fuchs et al., -35 toxin uptake 2007

TBC1D18 RabGAP1L Rab22 PTB Expression reduced Itoh et al., in Alzheimer's 2006; de patients Yebra et al., 2004

TBC1D19

TBC1D20 Rab1 TM Regulates Golgi Haas et al., biogenesis; binds 2007; Sklan Hepatitis C NS5A et al., 2007a; protein Sklan et al., 2007b

TBC1D21

TBC1D22A Crystal structure of Tempel et al., TBC domain solved 2008

TBC1D22B

TBC1D23

TBC Aliases Reported Other Function/Remarks References Protein substrates domains

TBC1D24 TLDc

TBC1D25 OATL1 Rab2a Itoh et al., 2006

TBC1D26

TBC1D27

TBC1D28

TBC1D29

TBC1D30 XM_037557 Rab8a Regulates primary Yoshimura et cilia formation al., 2007

TBCKL PK; RHOD

USP6 Tre-2, UCH Oncogene; founding Neuwald, TRE17 member of TBC 1997; domain family, Nakamura et regulates plasma al., 2002; membrane to Martinu et al., endomsome 2004 trafficking through Arf6 activation

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

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.

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.

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.

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

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.

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.

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

(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

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.

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

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.

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).

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

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

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,

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

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

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

-1 RUTBC1-C yielded a kcat of 12 min , 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.

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).

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

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).

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).

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).

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.

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Zolov and Lupashin. Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells. J Cell Biol (2005) vol. 168 (5) pp. 747-59.

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

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-

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.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 5, continued

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

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.

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).

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

(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.

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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.

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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

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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.

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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

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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.

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REFERENCES

Eskelinen. Maturation of autophagic vacuoles in Mammalian cells. Autophagy (2005) vol. 1 (1) pp. 1-10.

Fuchs et al. Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways. J Cell Biol (2007) vol. 177 (6) pp. 1133-43.

Fujita et al. Differential involvement of Atg16L1 in Crohn disease and canonical autophagy: analysis of the organization of the Atg16L1 complex in fibroblasts. J Biol Chem (2009) vol. 284 (47) pp. 32602-9.

Fujita et al. The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol Biol Cell (2008) vol. 19 (5) pp. 2092- 100.

Ganley et al. A syntaxin 10-SNARE complex distinguishes two distinct transport routes from endosomes to the trans-Golgi in human cells. J Cell Biol (2008) vol. 180 (1) pp. 159-72.

Ganley et al. Rab9 GTPase regulates late endosome size and requires effector interaction for its stability. Mol Biol Cell (2004) vol. 15 (12) pp. 5420- 30.

Glick et al. Autophagy: cellular and molecular mechanisms. The Journal of pathology (2010) pp.

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Hayes et al. Multiple Rab GTPase binding sites in GCC185 suggest a model for vesicle tethering at the trans-Golgi. Mol Biol Cell (2009) vol. 20 (1) pp. 209- 17.

Itakura et al. Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Mol Biol Cell (2008) vol. 19 (12) pp. 5360-72.

Itoh et al. Golgi-resident small GTPase Rab33B interacts with Atg16L and modulates autophagosome formation. Mol Biol Cell (2008) vol. 19 (7) pp. 2916-25.

Klionsky. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol (2007) vol. 8 (11) pp. 931-7.

Kuma et al. Formation of the approximately 350-kDa Apg12-Apg5.Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast. J Biol Chem (2002) vol. 277 (21) pp. 18619-25.

Levine and Klionsky. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell (2004) vol. 6 (4) pp. 463-77.

Mizushima et al. Methods in Mammalian Autophagy Research. Cell (2010) vol. 140 (3) pp. 313-326.

Mizushima et al. Autophagy fights disease through cellular self-digestion. Nature (2008) vol. 451 (7182) pp. 1069-75.

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Mizushima et al. Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate. J Cell Sci (2003) vol. 116 (Pt 9) pp. 1679-88.

Valsdottir et al. Identification of rabaptin-5, rabex-5, and GM130 as putative effectors of rab33b, a regulator of retrograde traffic between the Golgi apparatus and ER. FEBS Lett (2001) vol. 508 (2) pp. 201-9.

Yorimitsu and Klionsky. Autophagy: molecular machinery for self-eating. Cell Death Differ (2005) vol. 12 Suppl 2 pp. 1542-52.

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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

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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.

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Figure 1

116

Figure 2

117

Figure 3

118

Figure 3 continued

119

Figure 4

120

Figure 4 continued

121

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

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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

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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.

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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

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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

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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

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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

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

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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

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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

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.

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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

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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

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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

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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.

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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

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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

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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.

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Aivazian et al. TIP47 is a key effector for Rab9 localization. J Cell Biol (2006) vol. 173 (6) pp. 917-26.

Ausseil et al. An acetylated 120-kDa lysosomal transmembrane protein is absent from mucopolysaccharidosis IIIC fibroblasts: a candidate molecule for MPS IIIC. Mol Genet Metab (2006) vol. 87 (1) pp. 22-31.

Barbero et al. Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells. J Cell Biol (2002) vol. 156 (3) pp. 511-8.

Burguete et al. Rab and Arl GTPase family members cooperate in the localization of the golgin GCC185. Cell (2008) vol. 132 (2) pp. 286-98.

Chen et al. Rab36 regulates the spatial distribution of late endosomes and lysosomes through a similar mechanism to Rab34. Mol Membr Biol (2010) vol. 27 (1) pp. 24-31.

Díaz et al. A novel Rab9 effector required for endosome-to-TGN transport. J Cell Biol (1997) vol. 138 (2) pp. 283-90.

Espinosa et al. RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. Cell (2009) vol. 137 (5) pp. 938-48.

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Federoff. Nur(R1)turing a notion on the etiopathogenesis of Parkinson's disease. Neurotoxicity research (2009) vol. 16 (3) pp. 261-70.

Fuchs et al. Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways. J Cell Biol (2007) vol. 177 (6) pp. 1133-43.

Ganley et al. A syntaxin 10-SNARE complex distinguishes two distinct transport routes from endosomes to the trans-Golgi in human cells. J Cell Biol (2008) vol. 180 (1) pp. 159-72.

Ganley et al. Rab9 GTPase regulates late endosome size and requires effector interaction for its stability. Mol Biol Cell (2004) vol. 15 (12) pp. 5420- 30.

Goldenberg and Silverman. Rab34 and its effector munc13-2 constitute a new pathway modulating protein secretion in the cellular response to hyperglycemia. Am J Physiol, Cell Physiol (2009) vol. 297 (4) pp. C1053-8.

Goldenberg et al. Golgi-bound Rab34 is a novel member of the secretory pathway. Mol Biol Cell (2007) vol. 18 (12) pp. 4762-71.

Haas et al. Analysis of GTPase-activating proteins: Rab1 and Rab43 are key Rabs required to maintain a functional Golgi complex in human cells. J Cell Sci (2007) vol. 120 (Pt 17) pp. 2997-3010.

Haas et al. A GTPase-activating protein controls Rab5 function in endocytic trafficking. Nat Cell Biol (2005) vol. 7 (9) pp. 887-93.

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Hayes et al. Multiple Rab GTPase binding sites in GCC185 suggest a model for vesicle tethering at the trans-Golgi. Mol Biol Cell (2009) vol. 20 (1) pp. 209- 17.

Hynes et al. Induction of midbrain dopaminergic neurons by Sonic hedgehog. Neuron (1995) vol. 15 (1) pp. 35-44.

Itoh et al. Screening for target Rabs of TBC (Tre-2/Bub2/Cdc16) domain- containing proteins based on their Rab-binding activity. Genes Cells (2006) vol. 11 (9) pp. 1023-37.

Lombardi et al. Rab9 functions in transport between late endosomes and the trans Golgi network. EMBO J (1993) vol. 12 (2) pp. 677-82.

Luo et al. Identification of a novel nurr1-interacting protein. J Neurosci (2008) vol. 28 (37) pp. 9277-86.

Mori et al. Cloning and characterization of a novel Rab-family gene, Rab36, within the region at 22q11.2 that is homozygously deleted in malignant rhabdoid tumors. Biochem Biophys Res Commun (1999) vol. 254 (3) pp. 594- 600.

Pan et al. TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature (2006) vol. 442 (7100) pp. 303-6.

Reddy et al. A functional role for the GCC185 golgin in mannose 6-phosphate receptor recycling. Mol Biol Cell (2006) vol. 17 (10) pp. 4353-63.

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Riederer et al. Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network. J Cell Biol (1994) vol. 125 (3) pp. 573-82.

Shutes and Der. Real-time in vitro measurement of GTP hydrolysis. Methods (2005) vol. 37 (2) pp. 183-89.

Speight and Silverman. Diacylglycerol-activated Hmunc13 serves as an effector of the GTPase Rab34. Traffic (2005) vol. 6 (10) pp. 858-65.

Vokes et al. Genomic characterization of Gli-activator targets in sonic hedgehog-mediated neural patterning. Development (2007) vol. 134 (10) pp. 1977-89.

Wang and Hong. Interorganellar regulation of lysosome positioning by the Golgi apparatus through Rab34 interaction with Rab-interacting lysosomal protein. Mol Biol Cell (2002) vol. 13 (12) pp. 4317-32.

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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

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Figure 3

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Figure 4

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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

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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.

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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

Espinosa et al. RhoBTB3: a Rho GTPase-family ATPase required for 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.

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