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COPYRIGHT STATEMENT
‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'
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This study is the outcome of four years of research. It would not have been possible without the dedication and help from the colleagues I have worked along side with in the
David James Laboratory. Principally I would like to express my appreciation and gratitude to my PhD Supervisor, Prof. David James, who was a constant source of encouragement, guidance and support. I am a more passionate and curious scientist for having worked with him.
I was extremely fortunate to work in a laboratory with people who were wholly committed not just to the success of their own projects but also to the success of their colleagues. In this environment I found support at every turn and hope that I was able to reciprocate in kind. Particularly, I would like to mention Dr Jacqueline Stöckli for sharing her experience and providing continual guidance. Her support was a bulwark against the low moments.
Finally, I would like to express my deep gratitude to my family and friends. Thank you for putting up with me during my studies. You are the reason I made it. You are the reason to do it.
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Summary
1 Transport of cargo proteins through the eukaryotic vesicle trafficking system is a highly specific and regulated event. Vesicle trafficking is divided into five steps: (1) Formation and budding of the vesicle; (2) Movement of the vesicle along the actin and microtubule network; (3) Tethering of the vesicle to the target membrane; (4) Docking of the vesicle by formation of the SNARE (soluble N-ethylmaleimide-sensitive factor receptor protein) complex; (5) Fusion of the two membrane bilayers. Vesicle fusion results in the dispersal of SNARE proteins throughout the cell and mechanisms are required to maintain organelle identity in the face of membrane exchange. The regulation of SNARE complex formation is directed by Rab GTPase proteins at the tethering step.
Rab GTPases form the largest branch of the Ras family of small monomeric GTP-binding proteins with at least sixty members predicted in the Human genome. Rab proteins are localised to specific sub-cellular membranes where recruitment of effector proteins control tethering of the vesicle to membranes. Rabs cycle between the GDP-bound inactive and GTP-bound active state. This allows the Rabs to act as a switch whereby an active Rab will recruit it’s effectors. Two classes of proteins regulate the GDP/GTP state: Guanine nucleotide exchange factors (GEF) and the GTPase activating proteins
(GAP). RabGEFs promotes the loading of GTP and “activates” the Rab. RabGAPs enhance the intrinsic Rab GTP-hydrolysis activity, maintaining an inactive state. Recent investigation suggests that GAPs are the focus of regulatory pathways leading to activation of the cognate GTPase.
This study focuses on the function of the RabGAPs and how they are recruited to regulate vesicle trafficking. A major focus was to identify the substrate Rabs for members of the
RabGAP family. RabGAPs tested had a specific and unique Rab interaction profile
2 revealing a large diversity in Rab/RabGAP pairing. This study also focussed on cellular strategies that regulate RabGAP function. RabGAPs were found to be the targets for phosphorylation in signalling pathways and allow the cell to translate a signal into control of vesicle trafficking. Here, the RabGAPs present an avenue of highly specialised regulation of Rab GTPases and their associated trafficking pathways.
3
Chapter One
General Introduction
4
Membrane and Cargo trafficking
The eukaryotic cell is comprised of several membrane bound organelles that are structurally and biochemically distinct. These organelles are responsible for many of the cell’s vital functions including energy production, protein degradation and protein synthesis. Several of these functions are enhanced through the establishment of electrochemical gradients across internal membranes resulting in a distinct microenvironment. Partitioning these functions in membrane bound environments allows the modification of the lumen of these structures to establish the local conditions to carry out these functions that are required in a manner that does not influence the rest of the cell. One consequence of this membrane compartmentalisation is the need to be able to transport protein cargo and other solutes across the lipid bi-layer or from one compartment to another. This is achieved in part by the trafficking of small lipid vesicles to these organelles where the vesicle fuses with the target membrane to deliver the associated cargo molecules (Mellman and Warren, 2000).
Vesicle trafficking within the cell occurs through two major pathways the secretory pathway and the endocytic pathway (Fig. 1.1). The secretory pathway comprises three functionally discrete entities; the folding and assembly stage of vesicle transport, maturation of the cargo protein, and finally a sorting stage to the target destinations. This progression encompasses the functions of the ER, Golgi apparatus and Golgi associated cis and trans Golgi networks (ERGIC and TGN, respectively). Trafficking through the
5 Cilium
Secretory Macropinosome Recycling vesicles endosome
Early endosome
CCV TGN Golgi Late apparatus endosome
ER Lysosome
ERGIC
Phagosome
Nucleus
Caveosome
Early endosome
Fig. 1.1 Cellular membrane trafficking pathways. This is a representation of the major stations in the secretory and endocytic pathway. Vesicle trafficking of cargo molecules through the secretory pathway is initiated at the endoplasmic reticulum (ER) and passes through the endoplasmic reticulum intermedi- ate compartment (ERGIC) for delivery to the Golgi apparatus. Cargo molecule progress across the Golgi stack into the trans-Golgi network (TGN) and are sorted for delivery to the plasma membrane (PM) or endocytic system. The uptake and internalisation of cargo from the cell surface and exterior is initiated by budding of vesicles from the PM. Endocytic vesicles fuse to the early endosomes which sort cargo back to the PM or direct cargo to the recycling endosomes or late endosomes. Cargo molecules delivered to the recycling endosomes are sorted to the PM or the TGN. At the late endosome cargo is trafficked to the TGN or delivered to the lysosome. This diagram is a modified version of a figure presented by Stenmark, H. (2009) Nat Rev Mol Cell Biol. 10(8):513-525 6 secretory pathway is required for the delivery of trans-membrane proteins, proteins that undergo post-translational modifications in the Golgi apparatus, proteins that function in the lumen of organelles and proteins destined for cellular export. Nascent proteins destined for the secretory pathway are translated at the surface of the ER membrane and either fed through a pore into the lumenal space or in the case of a trans-membrane protein incorporated into the ER membrane. At this stage the protein undergoes folding and initial post-translational modifications (Sitia and Braakman, 2003). Once the protein passes through the quality control mechanisms in the endoplasmic reticulum pathway export from the ER can proceed. Budding of vesicles from the ER membrane is initiated through the recruitment of the COPII coat protein (Barlow, 1994). The COPII coat is a protein complex primarily comprised of the Sec23/Sec24 and Sec13/Sec31 hetero-dimers
(Barlow, 1994; Hicke et al., 1992). The Sec23/Sec24 hetero-dimer recognises sorting motifs in the membrane bound cargo destined for ER export and concentrates these proteins on the membrane (Kuehn et al., 1998). The ER lumenal proteins destined for transport bind trans-membrane cargo adaptors. These cargo adaptors are targets for
Sec23p/Sec24p sorting and allow the luminal cargo to be actively concentrated and packaged into budding COPII vesicles. The Sec13p/Sec31p hetero-dimer binds to the
Sec23p/Sec24p complex and forms the “structural cage” that shapes the membrane into a budding vesicle. COPII coat formation on the membrane is initiated by the Sar1p
GTPase which recruits the Sec23p/Sec24p to the ER (Aridor et al., 1998; Nakano and
Muramatsu, 1989). The formation of these large complexes between the COPII coat and the cargo and adaptor proteins distend the membrane and lead to the fission of a vesicle from the membrane. The next station in the early itinerary of the cargo proteins is a
7 tubulo-vesicular network also known as the endoplasmic reticulum Golgi apparatus intermediate compartment (ERGIC) (Bannykh et al., 1998). Here there is a sorting mechanism that ensures that proteins meant for further export and processing continue through the secretory system while ER specific proteins and proteins that have inadvertently escaped the ER are returned through retrograde vesicle trafficking.
It is an open question in the field whether the forward movement in the secretory pathway is mediated by vesicle trafficking out of a stationary ERGIC or whether parts of this network completely fuse with the cis-face of the Golgi apparatus. Regardless, the result is the transfer of cargo proteins to the next stage in the secretory pathway. The
Golgi apparatus is the site where extensive post-translational modification of the cargo proteins occurs (Emr et al., 2009). This involves a series of carbohydrate modifications and proteolyic cleavage steps that occur at the different faces across the Golgi (Dunphy et al., 1981; Dunphy and Rothman, 1985). Movement of cargo through the Golgi apparatus has been governed by two competing ideas for progression (Glick and Malhotra, 1998).
One describing the Golgi as an inert stack where cargo proteins moved through each cisternae by COPI coat mediated vesicle trafficking resulting in the final minting of proteins ready to be sorted to their functional compartments. Recently the weight of evidence suggests a Golgi model where each cisterna “matures” and progressively develops from the cis to the trans face (Bonfanti et al., 1998; Losev et al., 2006;
Matsuura-Tokita et al., 2006; Wooding and Pelham, 1998). While each cisternae move forward through the stack the native Golgi processing machinery undergoes retrograde trafficking to each incoming face maintaining the ordered sequence in post-translational processing. This results in the cargo molecules being carried in one stack through Golgi
8 processing. When the cisterna matures into the trans face it is disassembled by a series of vesicle fission events where each cargo molecule is sorted into the budding membranes and trafficked to the target sites within the secretory system (Griffiths and Simons, 1986).
After maturation in the Golgi apparatus the cargo proteins leaving the trans cisternae are trafficked through the TGN. This tubulo-vesicular network is distinct from the Golgi apparatus but is in part comprised of the vesicles and membrane compartments formed from the dismantling of the trans-Golgi cisternae. The TGN is a dynamic membrane platform where cargo proteins and lipids are sorted for transport into the exocytic and endocytic pathways (De Matteis and Luini, 2008). Cargo sorting and vesicle fission from the TGN is mediated by adaptor protein complexes and clathrin coats (Bonifacino and
Traub, 2003). The adaptor protein complex is a hetero-tetramer that binds to and concentrates specific membrane cargo molecules. This complex also recruits clathrin that shapes the budding membrane into a vesicle and leading to fission and progression of the cargo molecules to destinations beyond the Golgi (Ladinsky et al., 2002). This might be the plasma membrane, endosomes, late endosomes/lysosomes, secretory granules or other types of exocytic vesicles. Delivery to the PM results in the export of the vesicle lumenal cargo and the integration of membrane proteins into the cell surface.
Counterbalancing the secretory pathway with the uptake of external and cell surface membrane proteins is the endocytic pathway (Scita and Di Fiore, 2010). This is the mechanism where external proteins, solutes, and plasma membrane associated proteins such as receptors and channels are imported into the cell. Retrieval and uptake of cargo is the primary step in a sorting process where internalised cargo can be recycled back to the
PM, delivered to organelles such as the Golgi apparatus, or directed for degradation in the
9 lysosome . Cargo is internalised by vesicles that bud from the PM and enter the cytosol.
This process is initiated when membrane proteins and receptor bound external cargo are concentrated into endocytic sites on the plasma membrane. The co-ordination of endocytic cargo sorting is mediated by adaptor protein 2 (AP2) complexes and clathrin coats (Traub, 2009). In a manner similar to sorting in the secretory pathway these adaptors bind to the membrane cargo and recruit clathrin which forms the coat scaffold to initiate vesicle budding. These vesicles bud from the membrane and fuse with the early endosomes. Endocytosis can also be inititated through clathrin independent means
(Hansen and Nichols, 2009). These processes include caveolin (Henley et al., 1998; Oh et al., 1998) and flotillin mediated uptake (Glebov et al., 2006), macropinocytosis (Swanson and Watts, 1995)and uptake by scission of polymorphous tubule structures. These mechanisms of endocytosis are less-well understood but emerging studies suggest that different types of endocytosis in part determine the intra-cellular fate of the cargo.
The first platform for cargo-sorting in endocytosis occurs at the early endosomes . This provides a stage where cargo molecules can be sorted to various cell locations including delivery to the PM, recycling endosomes, or the late endosomal system. Recycling from the early endosome to the PM can ensure that inadvertently internalised cargo is quickly restored to the PM. Recycling of proteins between the early endosome and PM also plays an important role in signalling. Internalisation of receptor proteins through the early endosome can modulate signalling as the acidic pH of the organelle allows receptor- ligand complexes to dissociate and return the freed receptor back to the PM. From the early endosome cargo proteins can also be delivered to a “slower” recycling endosome and the TGN. The other outcome from trafficking through the early endosome is delivery
10 of the cargo into the late endosomal/lysosomal system. This system is used for degradation of cargo. In addition to the targeting of malfunctioning proteins, cellular processes such as phagocytosis and autophagy also utilise the late endocytic lysosomal system.
As outlined, the central component in the movement of cargo proteins between membrane compartments is vesicle trafficking. The trafficking of vesicles can be broadly divided into four observable steps; budding, vesicle movement, tethering and docking, and membrane fusion (Fig. 1.2). Budding involves the sorting and packaging of the intended cargo into forming vesicles (Kuehn et al., 1998; Traub, 2009). Here coat proteins are recruited to the membrane and with the aid of the associated budding machinery a vesicle is pinched from the donor membrane and undergoes fission releasing the vesicle into the cell. The vesicle is now translocated along a cytoskeletal network of microtubules or actin filaments that connect the membrane compartments (Hehnly and
Stamnes, 2007). This movement is carried out through the recruitment of motor proteins that attach the network and the vesicle. The motor proteins generate a locomotive force that drives the vesicle along the tubule or actin filaments to the target membrane. The next step involves the initial contacts between the vesicle and the target membrane. This tethering step is mediated over distances of 25 nm where long-range tether proteins bind to both membranes and draws the vesicle into close proximity with the target compartment (Cai et al., 2007). The tethering of the vesicle allows the interaction of the fusion machinery present on each membrane. These proteins, known as the SNAREs
(soluble N-ethylmaleimide-sensitive factor attachment protein receptor), form a tightly bound four-helix bundle that irreversibly attaches the incoming vesicle to the acceptor
11 Uncoating Rab GEF
Budding Rab Fusion Rab Movement
GDI Rab
GAP
Rab
Rab Rab
Donor membrane
Ligand Coat Protein Receptor Rab Rab GDP-bound Tethering Sorting adaptor inactive Rab PI kinase or phosphatase GTP-bound Rab active Rab Motor adaptor Q-SNAREs Tether R-SNAREs Cytoskeletal track Acceptor membrane Motor
Fig. 1.2 - Five stages of vesicle transport. Vesicle transport between a donor and acceptor mem- brane can be divided into five functionally distinct steps. The first is budding where cargo is sorted by interacting with coat proteins or adaptors. Accumulation of coat protein at the membrane surface distorts the lipid bi-layer and molds the membrane into a sphere where it scissions from the donor membrane and forms a vesicle. The vesicle sheds the protein coat in the uncoating step, allowing the coat complex to be recycled. The vesicle becomes associated with a motor protein attached to the cytoskeletal network that drives the vesicle towards it’s acceptor membrane. The interaction between the vesicle and the motor protein can be mediated by a Rab effector complex. The tethering step is mediated by the Rab and tether protein that binds to the Rab and the acceptor membrane. This is the first point of contact between the incoming vesicle and the acceptor membrane. The tethering step attaches the vesicle to the membrane and facilitates the interaction between the SNARE proteins. The SNARE proteins interact to form the SNARE complex that draws the vesicle and acceptor membrane into close proximity. In a mechanism that remains unclear, the two membranes fuse and the vesicle’s cargo content is delivered. This diagram is a modified version of a figure presented by Stenmark, H. (2009) Nat Rev Mol Cell Biol. 10(8):513-525
12 membrane (Sudhof and Rothman, 2009). The SNAREs are defined by the presence of one of the four different SNARE motifs that interact to form the hetero-oligermeric four-
helix bundle. The SNARE proteins provide at least one of the Qa, Qb, Qc, or R SNARE motifs, all of which are required to form the fusogenic helical bundle. For two membranes to undergo fusion, each of the membranes provides at least one member of the SNARE complex. Attachment through the SNARE complexes formed between the vesicle and acceptor membranes allow the repulsive forces of each bi-layer to be overcome leading to the fusion of the vesicle membrane with the acceptor compartment.
The trafficking of membrane results in the continuous and dynamic merging of lipid and protein between donor and acceptor compartments. Left unchecked this exchange and deposition compromises the identity of each compartment and increases the potential for mis-trafficking and break down in cellular function. To prevent disorder in the membrane systems the cell employs several mechanisms to preserve identity and exchange. One of these mechanisms involves the integration of membrane identity into the tethering and docking step of trafficking (Allan et al., 2000; Christoforidis et al., 1999). The tether molecules are recruited to one or both membranes by Rab GTPases . Tethers bind to specific Rab proteins thus requiring the presence of the cognate Rab on the membrane.
Indeed, the members of the Rab family populate specific membrane compartments within the cell (Chavrier et al., 1991; Zerial and McBride, 2001). This permits vesicle delivery to be regulated through the initial tether interaction between an incoming vesicle and it’s relevant target membrane. This places the Rab GTPase at the centre of determining compartment identity in vesicle trafficking.
13 The Rab GTPases
The Rab GTPases belong to the small Ras-like GTPase super family that includes the
Ras, Rho, Ran and Arf GTPases (Wennerberg et al., 2005). While members of this family share similar characteristics, each sub-group has a unique cellular function. This family is defined by several highly conserved sequence motifs that encode a common mechanism shared amongst all members (Fig. 1.3) (Valencia et al., 1991). These GTPases are guanine nucleotide binding proteins that bind either GDP or GTP. Binding to either nucleotide results in a conformation state of the GTPase specific to the associated nucleotide (Cherfils et al., 1997; Dumas et al., 1999; Hirshberg et al., 1997; Schlichting et al., 1990). These two conformations determine whether the GTPase is physiologically active or inactive. The GDP-induced conformation is termed “inactive” and the GTP bound conformation is termed “active”. The Ras-like proteins cycle between the GDP
“inactive” and GTP “active” states through an intrinsic nucleotide exchange and GTP hydrolysis activity. Although the GTPase itself binds both nucleotides with an equal affinity, the exchange of nucleotide generally promotes the “active” over the “inactive” conformation, as the cellular concentration of GTP is ten-fold higher than GDP. In the active conformation, the GTPase becomes an interaction partner and recruits proteins known as effectors. The effectors are a functionally diverse group of proteins with mechanisms that are highly specific to their site of recruitment. Thus, the Ras-like
GTPases function in their associated cellular process through recruitment of the cognate effectors. The cycle between the two conformations establishes a switch-like nature that affords the cell a spatial and temporal regulatory mechanism for effector recruitment and induced functional output.
14 Ypt32p M S ------NEDYGYDYD YLF K I V L I G D S G V G K S NLN L SRF TTDEFNIE Ypt1p ------MNSEYD YLF K L L L I G NSN S G V G K S CLL L R F SDD TYTND Sec4p M S GLRTV --SASSGNGKSYD SIMKI L L I G D S G V G K S CLL V R F V E D KFNPS Rab10 M A ------KKTYD LLF K L L L I G D S G V G K T CVL FRF SDD AFNTT Rab5a M A S RGATR--PNGPNTGNKICQFK L V L L G E S A V G K S SLV L R F V KGQFHEF Rab3a M A S ATDSRY GQKESSDQNFD YMFK I L I I G NSN S SVS V G K T SFLFRY A D D SFTPA Rab1a ------MSSMNPEYD YLF K L L L I G D S G V G K S CLL L R F A D D TYTES hRas ------MTEYK L V V V G AGGAGG G V G K S ALA TIQLIQN HFVDE
P - Loop
Ypt32p SKST I G V E F ATRT I E V E NKKIK A Q I W D T A G Q E R Y R AIT S AYY R G A VGA L I Ypt1p Y I S T I G V D F K I K T V E L D G K TVK L Q I W D T A G Q E R F R T I T S SYY R G SHGI I I Sec4p F I T T I G I D F K I K T V D I NGK KVK L Q L W D T A G Q E R F R T I T T AYY R G A MGI I L Rab10 F I S T I G I D F K I K T V E L QGK KIK L Q I W D T A G Q E R F H T I T T SYY R G A MGI ML Rab5a QEST I G A AFLTQTV CLD DTTVK FEIW D T A G Q E R Y H S L APMYY R G A QAA I V Rab3a F V S T V G I D F K V K T I YRNDKRIK L Q I W D T A G Q E R Y R T I T T AYY R G A MGFIL Rab1a Y I S T I G V D F K I R T I E L D G K TIK L Q I W D T A G Q E R F R T I T S SYY R G A HGI I V hRas Y DPTI E DSYR -KQVVID G ETCLLDILDD T A G Q E EYSAMRDQYMRTGEGFLC
Switch I Switch II
Ypt32p V Y D I S KSSSY EN CNHWL TELRENAD-DNVAV GL I G N K SDS D L AHLRA V PTDE Ypt1p V Y D V T DQESF NGVK MWL QEIDRYAT-STVLKLL V G N K C D L KDKR V V EYDV Sec4p V Y D V T DERTF TNIK QWFKTVNEHAN-DEAQLLL V G N K SDS D MET-RM V V TADQ Rab10 V Y D I T NGKSF ENISKWL RNIDEHAN-EDVERMLL G N K C D MDDKM R V V PKGK Rab5a V Y D I T NEESF ARAK NWV KELQRQAS-PNIVIAL SGG N K ADA D L ANKR A V DFQE Rab3a MYD I T NEESF NAVQDWSTQIKTYSW-DNAQV LL V G N K C D MEDERM V V SSER Rab1a V Y D V T DQESF NNVK QWL QEIDRYAS-ENVNKLL V G N K C D L TTKK V V DYTT hRas V F AINNTKSF EDIH QY REQIKRVKDSD DVPM VL V G N K C D L AA-RTVESRQ
Ypt32p A KNFAMENQMLFTET S A LNSDNV D KAF RELI VAIFQMVSKHQVDLSGSGT Ypt1p A KEFADANKMPFLET S A LDSTNV E DAF LTMARQIKESMSQQNLNETTQKK Sec4p G EALAKELGI PFIES S A K NDDNV NEIF FTL A KLIQEKI DSNKLVGVGN GK Rab10 G GQIAREHGI RFFET S A K ANINI E KAF LTL A EDILRKTP------VKEPN Rab5a A QSYADDNSLLFMET S A K TSMNV NEIF MAIA KKLPKNEPQN----PGANS Rab3a G RQLADHLGFEFFEASA S A K DNINV KQTFERLV DVICEKM SES----LDTAD Rab1a A KEFADSLGI PFLET S A K NATNV E QSFMTMAAEIKKRM GPG----ATAGG hRas A QDLARSY GI PYIET S A K TRQGVE DAF YTL V REIRQHKLRK----LN PPD
Ypt32p NNM GSNG A P K G P T I SLTPAPKEDKKKKSSN CC -- Ypt1p EDKGN------VNLKGQSLTN ---TGGGCC -- Sec4p EGNIS------INSGSGNS-----SKPNCC -- Rab10 SENVD------ISSGGGVTG----WKSKCC -- Rab5a ARGRG------VDLTEPTQP----TRNQCC SN Rab3a PAVTG------AKQGPQLSDQQVPPHQDCAC- Rab1a AEKSN ------VKIQSTPVKQ---SGGGCC -- hRas ESGPG------CMSCKCVLS
Variable Region Farnesyl/Geranylgeranyl site
Fig 1.3 Ras/Rab GTPase sequence comparison. - The protein sequences of hRas and several Rab/Ypt GTPases were aligned. The GTPase motifs are labelled and the Guanine interaction regions are high- lighted in yellow. The catalytic Glutamine residue is highlighted in red.
14 Rab GTPase Structure
The crystal structures for representatives each Ras GTPase sub-family have been solved and all share a broad structural similarity (Cherfils et al., 1997; Dumas et al., 1999;
Hirshberg et al., 1997; Schlichting et al., 1990). The GTPase secondary structure is comprised of a six-stranded β sheet core surrounded by five α helices (Fig. 1.4). Each sub-family has significant deviations that define the specific characteristics that are common within the members of the sub-families. These investigations have identified the underlying structural elements that determine the protein function. The small monomeric
GTPases contain a P-loop, guanine binding region, a Switch I and Switch II region, and a
C-terminal prenylation site. The P-loop is the major site of interaction with the nucleotide phosphate groups and a magnesium ion that is required for stabilisation of nucleotide binding. The guanine binding motifs provide the interaction with the guanine base and these residues are strictly conserved in structure and chemistry across the family. The
Switch I and Switch II regions are responsible for the shift in GTPase conformation
(Dumas et al., 1999; Esters et al., 2000; Huber and Scheidig, 2005). These regions each possess a phosphate interaction residue that binds to the GTP γ-phosphate. Binding to the phosphate brings these two Switch regions into close proximity that forms the effector domain in combination with α-3 helix and β-5 sheet. The glutamine residue critical for
GTP hydrolysis is conserved across all the sub-families of the GTPases and lies in the
Switch II region. This residue is utilised to co-ordinate the attacking water molecule in the hydrolysis of the GTP nucleotide.
Mutations of the residues in these regions can alter the functional properties of the
GTPases. This has resulted in the development of recombinant versions of the GTPases
16 Fig 1.4 Sec4p crystal structure. - The crystal structure for the Rab/Ypt GTPase Sec4p is presented as a 3D reconstruction highlighting the arrangement and orientation of α-helices (red) and β-sheets (yellow). A) Crystal structure of “inactive” GDP (black) bound Sec4p. Note the orientation of the catalytic Gln79 residue in this conformation. B) Crystal structure of “active” GTPγS (black) bound Sec4p. GTP conformational shift results in Gln79 orientation towards the GTP molecule and change in overall Sec4p structure. 17 that can be either “locked” in the active or inactive state as well as modification of effector recruitment and GTPase localisation. Alterations of the residues in the P-loop can destabilise nucleotide binding and indeed mutations of the residues that specifically interact with the γ-phosphate result in a binding preference for GDP over GTP, producing a constitutively “inactive” GTPase (Tisdale et al., 1992; Wagner et al., 1987). Mutation of the conserved catalytic glutamine residue completely abolishes the GTPase intrinsic hydrolytic activity and results in a constitutively “active” GTPase (Wagner et al., 1987).
Mutation and substitution of the effector and localisation sequences can change the associated effector partners and cellular localisation of the GTPase (Becker et al., 1991;
Chavrier et al., 1991). Due to the broad similarity between the Ras GTPases these mutational strategies produce generally analogous functional modifications across the members of the GTPase sub-families and have proven to be an invaluable tool in determining specific GTPase function.
The Rab GTPase Cycle
In vesicle trafficking the Rab GTPases traverse between an incoming and acceptor membrane. Once the vesicle has fused to the target compartment the Rab is extracted from this membrane and delivered as a cytosolic factor back to the donor compartment.
This is known as the Rab cycle, which allows the cell to maintain membrane identity, and is governed by the Rab activity states (Fig. 1.5).
When a Rab protein is synthesised it must undergo prenylation of the CCAAX motif with a geranylgeranyl-modification before it can be inserted into the membrane and become a functional part of the trafficking machinery (Khosravi-Far et al., 1992; Khosravi-Far et al., 1991).
18 GDI Rab Rab Effector Rab Rab dissociation insertion activation recruitment inactivation extraction
GDP RabGEF GDI Rab Rab effectors
GTP RabGAP GDF Rab
Fig. 1.5 - The Rab GTPase activation cycle. This diagram represents the each stage of the Rab cycle from delivery to the membrane to extraction by GDI. A Rab bound to GDI is recruited to the membrane by the Rab GDF. GDF triggers the release of the Rab from the GDI complex and the Rab inserts into the mem- brane via its geranylgeranyl modifications. The inactive GDP-bound Rab binds to its RabGEF and the GDP is displaced and replaced by GTP. The active GTP-bound Rab recruits its associated effectors to regulate membrane trafficking. The GTP-bound Rab binds to its RabGAP, which stimulates the Rab intrinsic GTP hydrolytic activity to convert the GTP to GDP and inactivate the Rab. The inactive Rab is now a target for GDI binding. GDI binds to the Rab and extracts it from the membrane where it is maintained as a cytosolic complex until dissociation by the GDF.
19 A nascent Rab is bound by the Rab GTPase escort protein (REP) (Alexandrov et al.,
1994), this complex now binds to the Rab Geranylgeranyl transferase (RGGT) protein which modifies the cysteine residues in the CCAAX motifs with the geranylgeranyl anchor (Andres et al., 1993). After prenylation the RGGT dissociates from the Rab-REP complex and the modified Rab is delivered to the membrane.
Rab GTPase function in cellular processes requires them to quickly switch between the active and inactive conformations. Although intrinsically capable of performing these reactions, the observed rates of hydrolysis and nucleotide exchange occur at a rate too slow for the associated functional events. To meet these limited time frames the cell engages two other classes of proteins that regulate GTPase function; the Guanine nucleotide Exchange Factor (GEF), and the GTPase activating protein (GAP) (Bos et al.,
2007). These proteins stimulate the GTPases’ intrinsic exchange and hydrolytic activity respectively. The GEF binds to the GTPase and destabilises it’s association with the nucleotide by modifying the nucleotide binding site and blocking the interactions between the phosphate groups and magnesium ion (Dong et al., 2007). This lowers the affinity for nucleotide binding and results in the release of the nucleotide and subsequent replacement with an available guanine nucleotide. Binding of an incoming nucleotide through the guanine interaction motifs disrupts the association of the GEF and Rab, releasing the GEF and allowing the binding of the nucleotide phosphate groups. In vivo this results in the favourable loading of GTP. Hence, the interaction with a GEF generally results in an “active” Ras-like GTPase. The GAP protein binds to the active
GTPase and inserts catalytic residues into the guanine nucleotide-binding pocket that
20 enhance the GTP hydrolysis (Pan et al., 2006). This establishes the regulated Ras-like
GTPase cycle between the inactive and active states.
After performing it’s required function, the Rab is inactivated by a cognate RabGAP allowing it to recycle back to it’s membrane of origin. This recycling is mediated by a protein known as the Guanine nucleotide Dissociation Inhibitor (GDI) (Soldati et al.,
1993; Ullrich et al., 1993). The GDI protein binds specifically to the GDP “inactive” Rab and extracts it from the membrane. This allows the Rab to be recycled back to the initial membrane location to facilitate the next round of vesicle transport. There are two GDI isoforms in the mammalian genome, GDIα and GDIβ. These isoforms are functionally redundant but are expressed differentially throughout the body. GDIα is expressed in the brain exclusively and GDIβ is expressed in all other tissues. Both isoforms display an indiscriminate Rab specificity and it remains as yet unclear whether the specific isoforms possess subtle functional differences (Yang et al., 1994).
An aspect of the Rab cycle includes the delivery of the Rab to the functional membrane.
This requires dissociation of the Rab GTPase from GDI. This was hypothesised to be mediated by a theoretical guanine dissociation factor (GDF). The first evidence of the
GDF was observed in the delivery of Rab9 and other endosomal Rabs to the endosomal membrane. An unidentified factor was found to displace the GDI-Rab complex resulting in the insertion of the Rab into the membrane (Dirac-Svejstrup et al., 1997). This factor caused this displacement without nucleotide exchange and was specific to endocytic
Rabs. Yip3 was identified as the factor that facilitates the endocytic Rab-GDI complex disruption (Sivars et al., 2003). It was proposed that the members of the Yip family interact with different sub-sets of Rab-GDI complexes and deliver them to specific
21 membrane compartments. This closes the Rab GTPase cycle within the cell. Although the broad mechanics governing the cycle have been identified, the specific details as to how extraction and delivery are regulated and categorising a Rab with it’s cognate GDF, GEF and GAP remain the focus of investigations in the field.
Rab GTPase Function
The Rab GTPase family is recruited by the cell to regulate membrane trafficking and co- ordinate compartmental identity. Studies in yeast revealed that deletion of the Ypt family of proteins, the yeast homologs of mammalian Rabs, resulted in the missorting of cargo and promoted defects in organelle structure (Bacon et al., 1989; Goud et al., 1988;
Schmitt et al., 1986; Walworth et al., 1989; Wichmann et al., 1992). These studies provided the first evidence for the role of Rab GTPases in regulating vesicle transport and lead to the identification of the homologous family in mammalian cells (Segev et al.,
1988). The membranes of the secretory and recycling system are populated by at least one member of the Rab family and each Rab member is located at a specific membrane compartment (Fig. 1.6). This provides the basis for the Rab to be utilised as a membrane signpost and thereby facilitate the maintenance and function of an organelle through the recruitment of the functionally relevant Rab effectors. The Rab effectors are a diverse group of proteins that include sorting adaptors, vesicle tethers, kinases, phosphatases and motor proteins (Grosshans et al., 2006). This wide range of interaction partners provides the basis for Rab function at all points of membrane trafficking including vesicle budding, movement, tethering and fusion.
One of the outstanding questions in the field concerned the relationship between the Rab- mediated tethering step and the SNARE directed fusion step. The SNARE molecules are
22 Cilium RAB8 RAB17 RAB3 RAB23 RAB26 RAB34 RAB27 RAB17 RAB25 RAB5 RAB3 7 Apical GLUT4 vesicle Macropinosome Secretory recycling RAB8 vesicles and endosome granules RAB10 RAB27 RAB14 RAB15 RAB4 RAB5 Melanosome Early RAB5 RAB13 RAB38 RAB32 RAB22 endosome Tight RAB15 junction RAB8 RAB11 CCV Recyling RAB35 RAB5 TGN endosome RAB9 RAB22 Late endosome RAB6 RAB7 RAB3 3 RAB40 Early Golgi phagosome RAB2 Lysosome RAB5 RAB1 ER RAB7 RAB14 IC RAB22
RAB7 RAB18 Late phagosome Nucleus Lipid RAB3 3 droplet RAB24 Autophagosome RAB5 Caveosome
Early endosome RAB32 RAB21
Focal Mitochondria Integrin adhesions
Fig. 1.6 - The members of the Rab GTPase family localise to specific membrane compartments. Every membrane compartment in the cell is populated by at least one Rab GTPase. A functional pathway and location has been identified for most Rabs. The location of the Rabs on various organelles is illustrated and the arrows indicate direction of the trafficking pathways they regulate. This diagram is a figure presented by Stenmark, H. (2009) Nat Rev Mol Cell Biol. 10(8):513-525
23 indispensable for the fusion of the incoming membrane to the target bi-layer. A recent study by Ohya et al. (2009) demonstrates the necessity of Rab GTPases and their associated effector complexes in driving membrane fusion (Ohya et al., 2009). In this landmark investigation the authors sought to address the functional relationship between
Rabs and the SNARE proteins in membrane trafficking. Utilising an in vitro homotypic fusion assay the members of the endosomal tethering and fusion machinery were functionally interrogated by observing the fusion of purified endosomes and/or proteo- liposomes. Several important observations were made. Firstly, a core Rab5 effector machinery comprising of Rab5, Rabaptin-5, rabex-5, hVPS34, PIK3R4, EEA1,
Ranenosyn-5, and hVPS45 was necessary and sufficient to catalyse the same degree of fusion as observed for whole cell lysate. Secondly, stable membrane recruitment of Rab5 and it’s associated effectors was dependent on the presence of the cognate SNAREs.
Thirdly, the cognate SNAREs alone were unable to mediate fusion between membranes.
Rab5 and it’s effectors were required to produce fusion between membrane compartments. This study proposed that in addition to providing a layer of specificity on membrane fusion that the Rab GTPases and their associated effector complexes are in fact members of a core membrane fusion machinery which is more efficient than the
SNAREs alone. This study represented the culmination of a growing body of work linking the Rabs and their effectors to SNARE complex formation (Collins et al., 2005;
McBride et al., 1999; Simonsen et al., 1999; Tsuboi and Fukuda, 2005). Indeed several observed Rab effectors contain domains that interact with the membrane and in some cases influence membrane curvature. Such interactions could be utilised in a fusion reaction to distort the membranes to overcome the repulsive forces between the two bi-
24 layers. Furthermore, this suggests that correct identification of an incoming vesicle through the incorporation of tethers into a Rab effector complex is inextricably linked to fusion potential.
The Rab GTPase provides the cell with a versatile mechanism to temporally and spatially regulate membrane dynamics through a combination of effector recruitment, switchable activation state, and regulated localisation. As outlined above, it is becoming apparent that Rab function is an integral part of the membrane fusion apparatus of the cell. This highlights the need for understanding the regulatory mechanisms that govern Rab activity and localisation, as these will ultimately determine the spatial and temporal dynamics of membrane fusion. In combination with roles in vesicle budding and movement, this places the Rab GTPase family at the centre of cellular membrane trafficking.
The evolution of Rabs
The Rab GTPases family consists of 71 members making it the largest group in the Ras- like GTPase super family. The yeast S. cerevisiae genome encodes eleven equivalent Rab
GTPases, known as Ypt proteins. This expansion in the repertoire of Rabs at the mammalian cell’s disposal is thought to reflect the development and proliferation of specialised cell types and the requirement for regulation of the associated trafficking pathways. This can be observed in the restricted expression of particular Rabs in specific mammalian cell types. Indeed a study into the evolution of the Ras family found that Rab diversity was driven through positive selection (Jiang and Ramachandran, 2006).
The diversity in Rabs can be observed by the number of sub-branches in the family (Fig.
1.7). Broadly, each sub-branch can be assigned an equivalent yeast homologue from where each mammalian Rab has shared uni-cellular origins. Even with such broad
25 Rab36 Ypt6p Rab41 Rab6b Rab6a Ypt11p Rab15 Rab40c Rab40b Rab40a Rab37 Rab26 Rab44 Rab3d Rab3b Rab3a Rab3c Rab27b Rab27a Rab25 Rab11b Rab11a Ypt32p Ypt31p Rab2b Rab2a Rab14 Rab4b Rab4a Sec4p Rab13 Rab10 Rab8b Rab8a Rab35 Ypt1p Rab1b Rab1a Rab18 Rab12 Rab42 Rab39b Rab39a Rab33b Rab33a Rab30 Rab43 Rab19 Rab29 Rab38 Rab32 Rab7b Rab9b Rab9a Ypt7p Rab7a Rab20 Rab24 Rab17 Ypt10p Rab21 Rab31 Rab22 Rab5b Rab5c Rab5a Ypt52p Ypt53p Ypt51p Rab28 Rab23 Rab34 0.1
Fig 1.7 - Rab/Ypt GTPase phylogenetic tree. A phylogenetic tree comparing the H. sapiens Rab GTPases with the S. cerevisiae Ypt GTPases. The Rab and Ypt members are highlighted yellow and blue, respec- tively. The Rab-like and Ran GTPases have been excluded from this analysis.
26 evolutionary distance between the Ypt and Rab isoforms there remains a loose functional similarity. Ypt1p is most closely related to Rab1 and these GTPases regulate ER to Golgi as well as intra-Golgi trafficking. Indeed, mammalian Rab1 is able to compensate for
Ypt1p in yeast Ypt1 deletion strains (Haubruck et al., 1989). Furthermore, Ypt5p, the yeast Rab5 paralogue, can rescue endocytic Rab5 function in Rab5 mutational studies
(Singer-Kruger et al., 1995). Another example is illustrated by the Ypt7p and Rab7 as both these Rabs function in late endosomal and vacuole/lysosomal trafficking in yeast and mammalian cells respectively (Feng et al., 1995; Wichmann et al., 1992). Sec4p related Rabs. This demonstrates that a broad functional relationship exists for Rabs across these evolutionary distances. Together the studies of the Ypt proteins in S. cerevisiae can provide a guide into mammalian Rab function, however the proliferation of Rab GTPases and specialisation of mammalian cells types suggests the majority of the Rabs will function in innovative vesicle trafficking pathways.
Rab GTPase cross-talk.
Cellular cargo trafficking involves a transit route for cargo proteins that are directed by general and cargo specific trafficking pathways. These trafficking pathways converge through common membrane spaces and the cell must be able to sort the cargo from the common site into trafficking pathways to distinct cellular destinations. One of the clearest examples of this is endocytosis through the endosomal system (Mellman and Warren,
2000). Here, cargo from the PM is delivered to the early endosome where it is either returned to the PM in a “fast” exchange of membrane or travels via a “slower” pathway comprising the recycling endosomes (Mayor et al., 1993). This trafficking is governed by the three Rabs found at this stage, Rab4, Rab5 and Rab11 (Bucci et al., 1992; Ullrich et
27 al., 1996; van der Sluijs et al., 1992). These Rabs can inhabit the early endosome, Rab4 and Rab5, and recycling endosome, Rab4 and Rab11, to regulate the possible outcomes for cargo trafficked through the early stages of the endocytic system (Trischler et al.,
1999). Rab5 co-ordinates the fusion of the incoming vesicles from the PM and the homotypic fusion between the early endosomes (Bucci et al., 1992; Christoforidis et al.,
1999; Gorvel et al., 1991). Rab4 regulates the fast delivery of the internalised cargo back to the PM (van der Sluijs et al., 1992) and Rab11 governs the slower trafficking pathway through the recycling endosomes (Ullrich et al., 1996). In fact these Rabs were shown to form discrete sub-domains on a planar membrane. The study by Sönnichen et al. (2000) demonstrated that Rabs cluster in distinct domains on the endosome and that the relative amounts of these domains can distinguish an early endosomal from a recycling endosome
(Sonnichsen et al., 2000). The endocytosis of fluorescent transferrin in cells co- expressing fluorescently tagged Rab4, Rab5, and Rab11 was monitored by live cell and confocal microscopy. It was observed that transferrin quickly trafficked through endosomal compartments mainly comprised of Rab4/Rab5 and then progressed to a
Rab4/Rab11 positive compartment. Although when found on the same endosome each
Rab was observed to populate a distinct subsection of the membrane. Additionally it was observed that internalised transferrin first trafficked through the Rab5 compartment, then the Rab4 compartment and finally to the Rab11 compartment. This study demonstrated that Rabs do indeed segregate into separate domains. This observation of Rab sequestration and the movement of cargo between them demonstrated that endosomes are sub-divided into functional domains that direct the trafficking itinerary of internalised cargo. Implicit in these observations and model is a mechanism that somehow allows the
28 cell to establish exclusive Rab domains and regulate the movement of cargo between them.
A follow-up study performed by De Renzis et al. sought to identify whether there is cross-talk between Rab domains that govern domain organization and the flow of cargo through these sites (De Renzis et al., 2002). It had previously been reported that different
Rabs can recruit common effectors, and indeed Rab4 and Rab5 have been shown as
Rabaptin5 binding partners (Vitale et al., 1998). Hence the focus of this study was to determine whether the effectors common to both proteins orchestrated the Rab4/Rab5 domains. Rabenosyn5 was identified as an effector for Rab4 and Rab5. Rabenosyn5 was shown to bind each Rab simultaneously and analysis by light microscopy revealed that endosomes comprised of Rab4/Rab5 domains always contained Rabenosyn5.
Overexpression of Rabenosyn5 resulted in an increase in Rab4/5 domain endosomes with a decrease in the amount of Rab4/Rab11 endosomes and depended on the capacity for
Rabenosyn5 to bind both Rab4 and Rab5. This suggests that the common effectors promote the establishment of adjacent Rab domains. Rabenosyn5 overexpression also caused a diversion in trafficking through the endosomal system. Internalised transferrin was cycled through early endosomes and the PM and did not traffic through the Rab11 recycling endosomes. This study demonstrated that Rab domains are co-ordinated in part by effectors common to both Rabs and that this is required for regulating traffic of cargo through the domains.
Overall these studies suggest a model where specific regions on a planar membrane can be compartmentalised into exclusive Rab functional sites (De Renzis et al., 2002;
Sonnichsen et al., 2000). Effectors that are common to both Rabs establish a direct link
29 between the domains sharing an organelle. This allows for the co-ordination of several trafficking pathways that converge on a single organelle to ensure the sorting and progression of cargo molecules to their final stations.
Rab GTPase Cascades
Rab cross-talk is also employed to regulate the activity of each Rab. This regulation is mediated by recruitment of either the RabGEF or RabGAP by one Rab to stimulate the exchange or hydrolysis of the target Rab. This control mechanism has been observed in trafficking pathways in yeast and mammalian systems. In mammalian cells this cross-talk has been identified as the basis for the mechanism of early endosome to late endosome conversion. Two mechanisms of cargo progression between these endocytic compartments were possible; the first nominated vesicle trafficking as the major method of cargo delivery, the second hypothesised that the organelle converted from an early endosome to a late endosome through an exchange of molecules that define these compartments (Dunn and Maxfield, 1992; Thilo et al., 1995). Rink et al. established that organelle conversion provided this mechanism and it was intrinsically linked to the populating Rabs (Rink et al., 2005). In live cell microscopy experiments the uptake of exogenous cargo, low-density lipoprotein (LDL), was tracked through the endocytic system to the late endosome. Fluorescently tagged Rab5 and Rab7 were used as markers of early and late endosomes, respectively. Early endosomes are populated by Rab5, which regulates trafficking from the PM to these compartments in addition to homo- fusion between the endosomes (Bucci et al., 1992; Gorvel et al., 1991). The late endosome and lysosome compartments of the cell are populated by Rab7 (Bucci et al.,
2000; Meresse et al., 1995). After internalisation, LDL quickly accumulated in Rab5
30 positive early endosomes, which are located in the cell periphery and then traffics to the perinuclear region (Rink et al., 2005). This is accompanied by an increase in the size of the compartment and a loss in Rab5 from the membrane. The Rab5 loss is mediated by
GDI extraction, as no budding of Rab5 vesicles is evident. Concurrent with the observed
Rab5 loss was the population of the membrane by Rab7, demonstrating a switch of organelle identity from early to late endosomes. Cargo progression from early to late endosomes is mediated through a conversion of the organelle as distinguished by an exchange of the membrane associated proteins and occurred without vesicle trafficking between these membranes.
This co-ordinated exchange of membrane proteins suggests some level of cross-talk regulation between the molecules. It has been shown that a component of the class C
VPS/HOPS complex, Vps39, possesses Ypt7p GEF activity (Wurmser et al., 2000). The
VPS/HOPS complex is essential for late endosome/lysosome assembly and is recruited to endosomes by Rab5. Knockdown of Vps39 displayed defects consistent with a block in trafficking between the early and late ensosomes (Rink et al., 2005). This suggests a mechanism for the observed organelle conversion where the upstream Rab5 recruits the activating GEF for the downstream Rab7 and promotes the accumulation of active Rab7 on the membrane. This study not only illustrates that cargo trafficking can be mediated through organelle conversion but it also demonstrated that the mechanism for this conversion is in part mediated through the associated Rab GTPases and co-ordinated by cross-talk between these Rabs. This observed cross-talk between Rab5 and Rab7 suggested that Rabs in a transport pathway may be functionally linked through sequential regulation of each Rab’s activation state. This is known as a Rab cascade and was first
31 hypothesised in a study by Ortiz et al. The general model for a Rab cascade proposes that a Rab in a transport pathway will recruit the GEF for the next Rab downstream and lead to it’s activation, conversely this leads to the recruitment of the GAP to the upstream Rab by the downstream Rab resulting in upstream Rab inactivation (Ortiz et al., 2002).
Further evidence of Rab cascades have been found in yeast and mammalian systems. A study into the interplay between early endosome Rab GTPases identified a potential Rab cascade between Rab22 and Rab5 (Zhu et al., 2009). Rab22 localises to early endosomes and recruits the Rab5 GEF Rabex-5 (Mesa et al., 2001; Zhu et al., 2009). This association was shown to be important in the regulation of endosome integrity and delivery of internalised ligand to the degradation machinery. In S. cerevisiae a Rab cascade has been described between Ypt1p, Ypt31p/Ypt32p and Sec4p (Ortiz et al., 2002; Rivera-Molina and Novick, 2009; Wang and Ferro-Novick, 2002). Ypt1p was identified to recruit an as yet unidentified partner that stimulates the exchange of nucleotide on Ypt31p/Ypt32p
(Wang and Ferro-Novick, 2002). In the study by Ortiz et al. that first described the existence of Rab cascades Ypt31p/Ypt32p was found to recruit Sec2p and restored polarized trafficking in a mutant Sec2p strain (Ortiz et al., 2002). Sec2p is the nucleotide exchange factor for Sec4p. The interaction between Ypt31/32p and Sec2p demonstrates a direct link between the budding of secretory vesicles from the Golgi apparatus regulated by Ypt31p/Ypt32p to the Sec4p co-ordinated export of these vesicles to the exocytic sites.
According to the model of Rab cascades, regulation between each Rab can occur through
RabGEFs in a forward direction as well as through RabGAPs in the reverse. The initial studies identifying Rab cascades observed forward movement through the cascade by
32 GEF recruitment and Rab GTPase activation. Although RabGAP regulation through a cascade is hypothesised it was not observed until a recent study by Rivera-Molina and
Novick (2009). Here it was reported that Gyp1p, a Ypt1p GAP, was recruited by
Ypt31p/Ypt32p and regulated Ypt1p domains in specific compartments of the Golgi apparatus (Rivera-Molina and Novick, 2009). This was observed through the Gyp1p mediated conversion of Ypt1p membrane to Ypt31p/Ypt32p membranes in a pattern similar to the Rab5-Rab7 conversion of early to late endosomes (Rink et al., 2005). In the absence of Gyp1p the Ypt1p and Ypt32p domains were no longer distinct and resulted in an increased co-localisation of these yeast Rabs and their associated effectors. This study proposed a mechanism where Ypt32p targeted Gyp1p, inactivates Ypt1p resulting in GDI extraction from the membrane. This demonstrated how a Rab cascade can be established using a RabGAP to regulate the integrity of Rab domains and compartment identity.
Collectively, these yeast studies demonstrate how trafficking from the earliest secretory steps to delivery to the plasma membrane are co-ordinated across the entire length of the trafficking pathway by a Rab cascade. Ypt1p co-ordinates the delivery of cargo from the
ER and across the Golgi, a Ypt1p effector activates Ypt31/Ypt32p and these Rabs mediate the trafficking out of the Golgi. Ypt31/32p activation recruits Gyp1p, which inactivates Ypt1p and establishes a functional boundary between these Rabs. Ypt31/32p recruits the Sec4p GEF, Sec2p, and this regulates delivery of vesicles to the membrane of the budding daughter cell.
The Rab cross regulation that occurs in yeast and mammalian systems demonstrates the conserved nature of Rab cascades as a regulatory mechanism. This suggests that the trafficking pathways defined by the seventy-one Rab GTPases available in the human
33 genome will be incorporated into as yet unidentified Rab cascades. Co-ordination of the
Rab cascades across diverse trafficking pathways will require that the regulatory GEFs and GAPs reflect the necessity of specific temporal and spatial resolution. As there are approximately 52 RabGAPs encoded by the human genome this provides a cohort numerous enough to span the potential links between the Rab GTPases. Investigating the network in a Rab cascade by the connecting RabGAP partners can, in theory, map a trafficking pathway between seemingly unrelated Rabs and in conjunction with the activating GEFs possibly elucidate the entire Rab controlled itinerary of a cargo molecule. Although the majority of Rab GTPases have been functionally characterised to some extent (Stenmark, 2009), the RabGEFs and RabGAPs remain largely un- investigated. Discovering the functional Rab partners for these proteins and how they are regulated will be vital in detailing the regulated transport of cargo proteins and membrane in the cell.
The Rab GTPase activating proteins
The eukaryotic RabGAP family are defined by a tre-2/Bub2/Cdc16 (TBC) domain that confers the GTPase activating characteristic of these proteins (Neuwald, 1997). This domain was first identified in the S. cerevisiae Bub2 and S. pombe Cdc16 protein that are involved in the regulation of spindle assembly in mitosis (Fankhauser et al., 1993; Hoyt et al., 1991). The TBC domain was further identified in many other proteins across several eukaryotic species but the function of this domain was as yet unknown. A sequence analysis performed by Neuwald A.F. (1997) first expanded the presence of the
TBC domain to the Gyp proteins (GAP for Ypt proteins) and suggested that this domain may be responsible for the Ypt GAP activity (Neuwald, 1997).
34 Investigations in the primary sequences and crystal structures of the TBC domain have revealed the internal mechanics of the GAP activity (Albert et al., 1999; Pan et al., 2006;
Rak et al., 2000). The tertiary structure of the TBC domain represents a V-shaped molecule that is assembled from 16 α-helices and can spatially be resolved into two apparent sub-domains (Pan et al., 2006; Rak et al., 2000). The crystal structures of the
GAP domains for several other Ras-like GAP proteins have been determined and reveal that while several are composed entirely of α-helices these GAP domains cannot be superimposed (Bos et al., 2007). This suggests that the tertiary structure may be one of the factors that determine exclusivity of the GAPs on their monomeric GTPase partners.
The TBC domain is defined by six motifs (A-F) that are conserved across all RabGAPs
(Neuwald, 1997) (Fig. 1.8). Of these motifs A, B and C contain the most highly conserved sequences among the RabGAP family. The interactions between these motifs are thought to be critical in maintaining the overall structure and stability of the TBC domain (Pan et al., 2006). Motifs B and C exist in close proximity where conserved residues from each motif co-ordinate the hydrolytic activity on the GTP bound Rab.
Situated on the edge of the functional cleft both a highly conserved Arginine in motif B and Glutamine residue in motif C provide the catalytic machinery that destabilises GTP and co-ordinates the nucleophilic attack by the water molecule (Pan et al., 2006; Rak et al., 2000) (Fig 1.9). This is a unique feature of the RabGAP family as stimulated hydrolysis in other GAPs and Ras-like partners utilise a catalytic arginine provided by the
GAP and a catalytic glutamine provided by the GTPase (Bos et al., 2007). However these
35 TBC1D25 --GV EPSLR KVVW RYLL NVYPDGLTGRER MDYMKRKSREY E QLKSEWAQR TBC1D22a ---IP KPVR PM TWKLL SGY LPANV DR--RPATL QRKQKEY FAFIEHYYSS TBC1D16 --GI DVSIR G EVW PFLL RYYSHESTSEER EALRLQKRKEY SEIQQKRLSM TBC1D15 RGGL SHALR KQAW KFLL G YFPWDSTKEER TQLQKQKTDEY FRMKLQWKSI TBC1D14 --GNELNITHELF DICLA RAKERWRSLSTGGSEVENEDAGFSAADREASL TBC1D9a --GI P ESMRG ELW LLL SGAINEKAT---HPGYYEG---LVEKSM GKYNLA TBC1D8 ---IP ESLR G RLW LLFSDAVTDLAS---HPGYY GN---LVEQSLGRCCLV TBC1D6 --GV P LEHRA RVW MVL SGA QAQMDQN--PGYYHQLLQGERN PRLEDA--- TBC1D4 KEGV P KSRRG EIW QFLA L QYRLRHR---LPNKQQPPDISYKELLK QLTAQ TBC1D1 --GV P RHHRG EIW KFLA E QFHLKHQ---FPSKQQPKDV PYKELLK QLTSQ Gyp1p --GI P KIHRPVVW KLL I G YLPVNTKR--QEGFL QRKRKEY RDSLKHTFSD
TBC1D25 AN PEDLEFIRS -----TVLKDV LRR T DRAHPYYAGPEDGPHLRALH DLL TT TBC1D22a RN DEV H QDTY -----RQIHIDI PRR MSP--EALILQPK--VTEIFERIL FI TBC1D16 TPEEHRAFWRN--VQFTVDKDV VRR T DRNNQFFRGEDNP-NVESMRRIL LN TBC1D15 SQEQEKRNSRLRD Y RSLIEKDV NRR T DRTNKFYEGQDNP-GLILLH DIL MT TBC1D14 EL------IKLDI SRR T FPNLCIFQQGGPY--HDMLH SIL GA TBC1D9a TEE------IERDL HRR S LPEHPAFQNEM G---IAALR RVL TA TBC1D8 TEE------IERDL HRR S LPEHPAFQNETG---IAALR RVL TA TBC1D6 ------IRTDL NRR T FPDNVKFRKTTDPCLQRTLYNVL LA TBC1D4 QHA------ILVDL GRR T FPTHPYFSVQL GP-GQLSLFNLL KA TBC1D1 QHA------ILIDL GRR T FPTHPYFSAQL GA -GQLSLYNIL KA Gyp1p QHS-RDIPTW -----HQIEIDI PRR T NP--HIPLY QFKS-VQN SL QRIL YL
TBC1D25 Y A V THPQVSYC Q G M S DLA S PIL A VM DHEG------TBC1D22a W A I RHPASGY VQQ G INDLV T PFFVVFICEYTDREDVDKVDVSS----VPAE TBC1D16 Y A V YNPAVG Y SQQ G M S DLV APIL A EVLDE------TBC1D15 Y CM Y DF DL G Y VQQ G M S DLL S PLL YVMENE------TBC1D14 Y TCYRPDV G Y VQQ G M S FIA AVLI L NLDTAD------TBC1D9a Y A FRNPNIG Y C Q A M NIVTSVLL L YAKEE------TBC1D8 Y A HRNPKIG Y C Q SMNILTSVLL L YAKEE------TBC1D6 Y G HHNQGVG Y C Q G M NFIA GY LI L ITNNEE ------TBC1D4 Y SLLDKEVG Y C Q G ISFVA GV LL L HMSEE------TBC1D1 Y SLLDQEVG Y C Q G LSFVA GILL L HMSEE------Gyp1p W A I RHPASGY VQQ G INDLV T PFFETFLTEYLPPSQIDDVEIKDPS T Y MVDE
TBC1D25 ------HAFVCFCGIMKRLAANF H PDGRAM ATKFAHLKLLL R HADPDFY TBC1D22a VLRNIEA DTYW CMSKLLDGIQDNY TFAQPGI QM KV KM LEELV SRIDERVH TBC1D16 ------SDTFW CFVGLM QNTIFVSSPRDEDMEKQLLYLRELL R LTHV RFY TBC1D15 ------VDAFW CFASYMDQMHQNF EEQMQGMKTQLIQLSTLL R LLDSGFC TBC1D14 ------AF IAFSNLLNKPCQMAFFRV DH GLM LTYFAAFEVFFEENLPKLF TBC1D9a -----EA F WLLVALCERMLP-DYYNTRVVGA LVDQGVFEELA R DYV PQLY TBC1D8 -----EA F WLLVAVCERMLP-DYFNHRVIGA QVDQSVFEELI K EQLPELA TBC1D6 -----ESFWLLDALV GRILP-DYYSPAMLGL KTDQEVLGELV R AKLPAVG TBC1D4 -----QAF EM LKFLMYDLGFRKQY RPDMMSLQI QMY QLSRLL H DYHRDLY TBC1D1 -----EA F KM LKFLMFDMGLRKQY RPDMIIL QI QMY QLSRLL H DYHRDLY Gyp1p QITDLEA DTFW CLTKLLEQITDNY IHGQPGI LRQVKNLSQLV K RIDADLY
TBC1D25 QY L QEAG--ADDLFFCY RW LLLELKREFAFDDAL R MLEVTWSSL- TBC1D22a RHL DGHE--VRYL QFA FRW MNNLLM RELPL RCTIR L W D TY QSEPE TBC1D16 QHL VSLGED G L QM LF CHRW LLLCFKREFPEAEAL R I W E ACWAHY- TBC1D15 SYLESQD--SGYLYFCFRW LLI ------TBC1D14 AHFKKNN--LTPDIY L IDWIFTLY SKSLPL DLACRI W D VFCRDG E TBC1D9a D CM QDL G--VI STISLS-WFLTLF LSVM PFESAV VVVDCFFYEG - TBC1D8 EHMSDLS--AL ASISLS-WFLTLF LSIMPL ESAV H V VDCFFYDG I TBC1D6 ALMERLG--VL WTLLVSRW FICLF VDILPV ETVL R I W D CLFNEG - TBC1D4 NHL EENE--ISPSLY A APWFLTLF ASQFSLGFVA R V F D IIFL Q-- TBC1D1 NHL EEHE--IG PSLY A APWFLTM FASQFPL GFVA R V F D MIFLQG- Gyp1p NHFQNEH--VEFIQFA FRW MNCLLM REF QM GTVI R MWD TYLSET-
Fig 1.8 Comparison of RabGAP TBC domains. - The TBC domain amino acid sequences of a selection of RabGAPs were aligned. The six motifs that define the TBC domain are highlighted in red and the cata- lytic Arginine and Glutamine residues are highlighted in yellow. 36 Fig 1.9 - The crystal structure of a Rab/RabGAP interaction. The S. cerevisiae RabGAP Gyp1p TBC domain and the M. musculus Rab33 were co-purified and the crystal structure of this interaction was solved. The interaction was presevered with AlF treatment which stabilises the GTP to GDP transition state. A) Gyp1p TBC domain (yellow) binds to Rab33 (Blue), B) The interaction of the Rab and RabGAP catalytic amino acids with the guanine nucleotide. The Gyp1p Arg343 (Dark Blue) and Gln378 (Purple) form H-bonds with the GDP:AlF complex (GDP in Black and AlF in Orange), co-ordinating the nucleophilic attack of the water molecule (Red sphere). The Rab33 Gln92 (Red) does not interact with the GDP:AlF complex or the water molecule and faces away from the catalytic pocket forming an H-bond with the backbone of Gln378. 37 crystallisation studies reveal that the RabGAP provides both catalytic residues for GTP hydrolysis (Pan et al., 2006). Moreover mutating either of the RabGAP catalytic residues with an alanine resulted in the complete abolishment of RabGAP stimulated hydrolysis.
RabGAP function
Although present across all eukaryotes, the size of the family of RabGAPs in each species family is diverse (Fig. 1.10). As with the Rab family this expansion in number is due to the proliferation of specialised cell types and their associated trafficking pathways in higher eukaryotes. Indeed the expression profiling of the Rabs and RabGAPs reveals that these members are expressed at varying levels between cell types. Where some Rabs and RabGAPs are expressed ubiquitously and likely function in general trafficking other
Rabs and RabGAPs are limited to a specific cell type and may function in discreet trafficking steps (Wu et al., 2009).
There are seven characterised RabGAPs expressed in S. cerevisiae. These proteins are collectively known as the Gyp proteins. These RabGAPs have been characterised in several organelle integrity and vesicle trafficking pathways (Albert and Gallwitz, 1999;
Brett et al., 2008; Chesneau et al., 2004; Du and Novick, 2001; Rivera-Molina and
Novick, 2009; Vollmer et al., 1999). Surprisingly there is a high level of promiscuity between the Gyp proteins and yeast Rab paralogues the Ypt proteins. Several of the Gyp proteins possess GAP activity towards Ypt proteins that are functionally unrelated
(Albert and Gallwitz, 1999; Du et al., 1998; Will and Gallwitz, 2001). This raises the question as to how these Gyp proteins are employed by a yeast cell to regulate Ypt activity. One possibility is that the Gyp proteins are used indiscriminately and stimulate the hydrolysis on any available active Ypt protein. This model is corroborated by Gyp
38 TBC1D27 Gyp6p TBC1D4 TBC1D1 TBC1D2b TBC1D2a Gyp5p Gyl1p TBC1D18 TBC1D11 TBC1D14 TBC1D12 Gyp7p RUTBC2 RUTBC1 TBC1D16 TBC1D21 TBC1D17 TBC1D15 TBC1D13 Gyp1p TBC1D22b TBC1D22a TBC1D19 TBC1D29 TBC1D23 TBC1D7 TBC1D30 RN-Tre TBC1D3 TBC1D28 TBC1D26 EVI5L EVI5 TBC1D10c TBC1D10b TBC1D10a RUTBC3 TBC1D9b TBC1D9a TBC1D8b TBC1D8 TBC1D6 Msb4p Msb3p TBC1D25 TBC1D20 Gyp8p TBC1D5 TBC1D24 0.05
Fig. 1.10 - RabGAP/Gyp phylogenetic tree. A phylogenetic tree comparing the H. sapiens RabGAPs with the S. cerevisiae Gyp proteins. The RabGAP and Gyp members are highlighted yellow and blue, respectively.
39 knock out studies where the yeast remain viable and there is no observable defect in membrane trafficking in contrast to the defects observed in Ypt knock outs (Brett et al.,
2008). This suggests that the function of one Gyp can be substituted for another and is likely due to the broad GAP specificities of these proteins. However, overexpression of the Gyp proteins can lead to vastly differing phenotypes. A study examining Ypt7p- mediated vacuole maintenance observed a diverse effect on the organelle structure when each Gyp protein was overexpressed (Brett et al., 2008). Despite sharing the ability to stimulate Ypt7p’s hydrolytic activity, Gyp1 overexpression had no effect on vacuole morphology whereas Gyp7 overexpression resulted in an enlarged vacuole compartment.
Here the application of ascribing the Gyp proteins to specific Ypt proteins based on in vitro analysis fails to accurately identify a functional Gyp/Ypt pairing. Instead, it has been shown that the recruitment of the yeast RabGAPs to the Ypt site in part determines the functional pairing. Indeed, the Gyp proteins that have been shown to possess Ypt1p
GAP activity, Gyp1p, Gyp5p and Gyp8p all localise to separate cell compartments.
Gyp1p localises to the Golgi apparatus where it regulates Ypt1p-mediated trafficking out of the Golgi (Du and Novick, 2001; Rivera-Molina and Novick, 2009). The role of
Gyp8p in trafficking remains largely unknown although it possesses robust Ypt1p activity and is a potential ER resident protein. However, the closest mammalian Gyp8 paralogue is TBC1D20, and this RabGAP activates the hydrolytic activity of Rab1, which is the mammalian Ypt1 paralogue (Haas et al., 2007; Sklan et al., 2007). TBC1D20 is one of the few RabGAPs to possess a transmembrane domain and is localised to the endoplasmic reticulum (ER) in mammalian cells where it regulates Rab1. Translating these observations back to the regulation of Ypt1p, this suggests that Gyp8p is recruited
40 to control Ypt1p function at the ER and Gyp1p is utilised to regulate Ypt1p mediated trafficking at the Golgi. This presents a model in yeast where RabGAP/Gyp proteins are recruited to regulate the cognate Rab/Ypt proteins at a specific membrane compartment.
Furthermore, a multi-substrate Gyp protein may be recruited in an effector like manner by a Ypt protein to regulate the activity of the Gyp’s substrate Ypt proteins. Recruitment at a specific membrane site would establish the functional dominance of only one Rab ensuring correct spatial and temporal regulation of vesicle trafficking.
In multi-cellular organisms, cell types have evolved more specialised roles and functions.
This has lead to the development of membrane trafficking pathways that are unique and intrinsic for the functions of these cells. The increase in the size of the Rab GTPase family from uni-cellular to multi-cellular organisms is an indication of this development.
The proliferation and specialisation of Rabs to regulate these trafficking pathways requires an equally attuned control mechanism for the Rabs. Although there are a limited number of studies dissecting the role of mammalian RabGAPs in trafficking, the investigations have so far revealed the specific roles for these RabGAPs.
The most extensive RabGAP investigations have been performed on the function of
TBC1D1 and TBC1D4. These RabGAPs have critical roles in the regulation of insulin and exercise stimulated GLUT4 translocation in muscle and adipose tissue (Kramer et al.,
2006; Taylor et al., 2008). TBC1D1 and TBC1D4 maintain GLUT4 in an intracellular storage compartment in the absence of insulin or exercise by negatively regulating a Rab that is required for GLUT4 translocation (Eguez et al., 2005; Larance et al., 2005; Roach et al., 2007; Sano et al., 2003). The insulin and exercise stimuli target these RabGAPs for phosphorylation and inhibits the GAP activity. This allows the active Rab to accumulate
41 and facilitate GLUT4 delivery to the PM. These RabGAPs demonstrate a cellular regulatory mechanism where an external signalling event is translated into membrane trafficking.
Trafficking pathways in mammalian cells can be co-ordinated by a series of RabGAPs.
Investigations into the uptake and delivery of Shiga toxin from the cell exterior to the ER demonstrated that several RabGAPs function to co-ordinate this endocytic process (Fuchs et al., 2007). TBC1D10a, TBC1D10b, TBC1D10c, TBC1D17, EVI5 and RN-Tre were shown to regulate this pathway and ability of these proteins to stimulate GTP hydrolysis was critical to this function. This study found no effect of RUTBC3, the Rab5 GAP, on this pathway. Rab5 regulates the fusion of endocytic vesicles with the early endosomes as well as establishing the early endosome compartment (Bucci et al., 1992; Gorvel et al.,
1991). Perturbation of RUTBC3 function by RNAi resulted in the enlargement of the early endocytic compartment, a phenotype of excessive Rab5 activation (Haas et al.,
2005). Overexpression of RUTBC3 resulted in the ablation of the Rab5 endocytic compartment and blocked the internalisation of EGF. Interestingly, no effect of RUTBC3 was observed on Shiga toxin trafficking and the RabGAPs that function in that pathway have no effect on EGF internalisation (Fuchs et al., 2007). These studies demonstrated that cargo molecules can have distinct endocytic trafficking itineraries regulated by
RabGAPs specific to each pathway.
Other RabGAP studies have identified the functional pairings between specific Rabs and
RabGAPs. TBC1D20 was identified as an ER resident protein that regulates Rab1 activity (Haas et al., 2007; Sklan et al., 2007). TBC1D20 overexpression resulted in fragmentation of the Golgi apparatus and a block in tsVSV-G trafficking, two effects also
42 observed in overexpression studies of constituently inactive Rab1 mutants. TBC1D10a, also known as EPI64, stimulates GTP hydrolysis on Rab27 and causes melanosome aggregation when overexpressed (Itoh and Fukuda, 2006). This is aggregation is also observed with expression of Rab27 active and inactive mutants. EVI5 has been identified as a Rab11 binding partner but there are conflicting reports as to whether this interaction stimulates Rab11 GTP hydrolysis (Dabbeekeh et al., 2006; Westlake et al., 2007).
TBC1D10c is a Rab35 GAP that is thought to regulate the trafficking from recycling endosomes to the PM (Patino-Lopez et al., 2008). These studies demonstrate a restricted action of mammalian RabGAPs to specific Rabs that is matched with a functional effect of the RabGAP on the trafficking pathway regulated by the Rab GTPase.
Hypothesis and Aims
Rab GTPases are central players in cellular membrane dynamics providing a temporal and spatial regulatory mechanism that orchestrates membrane trafficking and organelle identity. Each member of the Rab GTPase family has a distinct sub-cellular localisation and performs highly specialised functions in trafficking pathways at those membrane compartments. Co-ordination of Rab function at these locations requires a tight regulatory framework provided by two major classes of proteins the RabGEF and the
RabGAP. The members of these families are likely to be subject to their own specialised control mechanisms that ensure they are deployed at the correct time and place to regulate Rab function.
The RabGAP family possesses several characteristics that fulfil a role in Rab specific regulation. Across evolution the number of Rabs is closely matched by the number of
RabGAPs suggesting a tight coupling of the individual members of these families. The
43 proliferation in RabGAPs has seen an increase in the diversity of these members with additional functional domains and re-structuring in the design of the proteins. This indicates that these members have evolved to function in specific situations tailored to the role of each Rab GTPase. This affords the cell a unique regulatory strategy to control each Rab GTPase.
I propose the RabGAP family are co-ordinated in specialised regulatory mechanisms that actively control the function of the Rab GTPases in membrane trafficking. This involves a role not just in an on/off regulation of the Rab but also through co-ordination of the spatial and temporal layout of membrane trafficking pathways through mechanisms such as Rab cascades. Accordingly, the regulation of the RabGAP is likely to be highly specific to its role in regulating Rab GTPase function.
The specific aims of this study were;
1. To identify novel interaction profiles between the Rab GTPases and RabGAP
family.
2. To determine the functional relevance of the identified Rab/RabGAP pairings
3. To investigate the strategies used to regulate RabGAP activity on GTPase function.
44
Chapter Two
Materials and Methods
45
Reagents, Antibodies and Constructs
TBC1D5, TBC1D13 and TBC1D17 were amplified by PCR from a 3T3-L1 adipocyte cDNA library provided by Dr. Alan Saltiel (Ribon et al., 1998) and cloned into pGEMT-
Easy (Promega). These constructs were sequenced and identity of each confirmed as
NM_028162, NM_146252, NM_001042655, respectively. For RabGAPs TBC1D1,
TBC1D8, TBC1D10b, TBC1D11, TBC1D14, TBC1D15 TBC1D16 cDNA was obtained from OpenBiosytems corresponding to NM_015173, NM_018775, NM_015527,
NM_012197, NM_001113361, NM_022771.4, NM_172443, respectively and cloned into pGEMT-Easy (Promega) by standard TA cloning methodology. Gateway entry vectors were generated for Rab1, Rab4, Rab10, TBC1D13 and TBC1D15 using the Gateway
Technology (Invitrogen) cloning system by amplifying each cDNA with primers containing the relevant attB sites and performing BP reactions to recombine each PCR product in pDONR221 (Invitrogen).
The constitutively active Rab-pGBKT7 bait vectors were obtained from J. Junutula
(Genentech Inc, San Francisco, CA, USA). The list of Rabs and the mutated residues are summarised in Table 2.1. The catalytically inactive RabGAPs were sub-cloned from the pGEMT-Easy vectors described above into the vector pGADT7 by standard restriction enzyme and ligation techniques. The mutations generating inactive RabGAPs and the restriction enzymes used in sub-cloning are summarised in Table 3.2. The mutations outlined above were created using primer directed site-specific mutagenesis.
46 Gene Plasmid Mutation Gene Plasmid Mutation Gene Plasmid Mutation Rab1a pGBKT7 Q70L Rab15 pGBKT7 Q67L Rab34 pGBKT7 Q111L Rab2 pGBKT7 Q65L Rab17 pGBKT7 Q77L Rab35 pGBKT7 Q67L Rab3a pGBKT7 Q81L Rab18 pGBKT7 Q67L Rab37 pGBKT7 Q89L Rab3b pGBKT7 Q81L Rab20 pGBKT7 Q126L Rab39a pGBKT7 Q72L Rab3c pGBKT7 Q89L Rab21 pGBKT7 Q78L Rab40a pGBKT7 Q73L Rab3d pGBKT7 Q81L Rab22 pGBKT7 Q64L Rab40c pGBKT7 Q73L Rab4a pGBKT7 Q72L Rab23 pGBKT7 Q68L Rab43 pGBKT7 Q77L Rab4b pGBKT7 Q67L Rab24 pGBKT7 Wildtype TBC1D1 pGADT7 R854A Rab5a pGBKT7 Q79L Rab25 pGBKT7 Wildtype TBC1D4 pGADT7 R972A Rab6a pGBKT7 Q72L Rab26 pGBKT7 Q123L TBC1D5 pGADT7 R169A Rab6b pGBKT7 Q72L Rab27 pGBKT7 Q78L TBC1D10b pGADT7 R223A Rab7 pGBKT7 Q67L Rab28 pGBKT7 Q87L TBC1D11 pGADT7 R612A Rab8a pGBKT7 Q67L Rab29 pGBKT7 Q67L TBC1D13 pGADT7 R129A Rab9 pGBKT7 Q66L Rab30 pGBKT7 Q78L TBC1D14 pGADT7 R458A Rab10 pGBKT7 Q68L Rab31 pGBKT7 Q64L TBC1D15 pGADT7 R417A Rab11a pGBKT7 Q70L Rab32 pGBKT7 Q85L TBC1D16 pGADT7 R494A Rab13 pGBKT7 Q67L Rab33a pGBKT7 Q95L TBC1D17 pGADT7 R381A Rab14 pGBKT7 Q70L Rab33b pGBKT7 Q92L
Table 2.1 - Rab and RabGAP yeast two-hybrid constructs.
47 For all FLAG tagged constructs except TBC1D15 the corresponding cDNA was cloned into the p3xFLAG-CMV10 vector (Sigma) to form an in frame C-terminal fusion. The
FLAG-tagged TBC1D15 construct was generated with a Gateway LR reaction between the TBC1D15 entry vector and p3XFLAG-CMV10 containing the Gateway attL recombination cassette. The eGFP tagged constructs were generated by either sub- cloning the relevant cDNA into the pEGFP-C (Clontech) vectors or using Gateway technology to shuttle the cDNA from the entry vector into the pDEST53 (Invitrogen) vector. The mammalian GST tagged Rab GTPase constructs were constructed by
Gateway technology from the relevant entry vector into pDEST27 (Invitrogen).
Antibodies were purchased from Sigma (Mouse monoclonal anti-FLAG M2, Rabbit polyclonal anti-FLAG), Covance Research Products (HA antibody 16B12), Roche (GFP) and BD Biosciences (GM130). Antibodies were kindly provided by Dr. Wanjin Hong
(Syntaxin16, IMCB, Singapore) (Mallard et al., 2002). The antibody against GLUT4 has previously been described (James et al., 1988). TBC1D13 polyclonal rabbit antibody was raised against GST tagged full length TBC1D13 and affinity purified over a GST-
TBC1D13 column.
Rab GTPase effector pulldown
Wildtype Rab GTPase was cloned into pDEST27 (Invitrogen) by Gateway cloning to form a C-terminal fusion with a Glutathione S-Transferase (GST) tag. The construct was transfected into HEK293 FT (Invitrogen) cells by lipofection and recombinant Rab was isolated using Glutathione Sepharose 4B (GE Healthcare Biosciences). Briefly transfected HEK293 FT cells were incubated for 60 min in DMEM media with the addition of wortmannin 15 min before harvesting. Treated cells were washed twice with
48 cold PBS and lysed in Rab NP40 Buffer (1% NP40, 10% Glycerol, 25 mM Tris pH 7.5,
137 mM NaCl, 5 mM MgCl2, 0.1 mM GDP, EDTA-free Complete protease inhibitors
(Roche Applied Science), and phosphatase inhibitors (1 mM sodium pyrophosphate, 2 mM sodium vanadate, 10 mM sodium fluoride). The lysate was incubated on ice for 20 min then centrifuged at 18,000 x g for 20 min at 4˚C. Cleared lysate was incubated for 1-
2 h with glutathione sepharose on a rotating wheel at 4˚C. The beads were washed 3 times with Rab NP40 Buffer, followed by 2 times with PBS supplemented with 5 mM
MgCl2, 0.1 mM GDP (Sigma) and inhibitors.
The immobilised GST-Rab was loaded with either GDP or GTPγS (Sigma) before incubation with 3T3-L1 adipocyte lysate or HEK293 FT lysate (Christoforidis and Zerial,
2000). The Rab beads were washed once with Nucleotide Exchange (NE) Buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM EDTA, 5 mM MgCl2, 1 mM DTT) plus 10
μM GDP or 10 μM GTPγS, and then incubated for 20 min at 25˚C in NE Buffer plus 1 mM GDP or 1 mM GTPγS. This wash and incubation step was repeated twice. Rab beads were then washed once with Nucleotide Stabilisation (NS) buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT) plus 10 μM GDP or 10 μM GTPγS and incubated 20 min at 25˚C in NS buffer plus 1 mM GDP or 1 mM GTPγS.
3T3-L1 adipocytes were incubated for 2 h in DMEM (Invitrogen) and lysed in NP40
Buffer (1% NP40, 10% Glycerol, 25 mM Tris pH 7.5, 137 mM NaCl, EDTA-free
Complete protease inhibitors (Roche Applied Science), and phosphatase inhibitors (1 mM sodium pyrophosphate, 2 mM sodium vanadate, 10 mM sodium fluoride)).
Transfected HEK293 FT cells were incubated for 2 h in DMEM followed by incubation with wortmannin (100 nM) for 30 min and then lysed in NP40 Buffer. The lysate was
49 incubated on ice for 1 h and centrifuged at 18,000 x g for 20 min at 4˚C. Cleared lysate was supplemented with 5 mM MgCl2, and either 0.1 mM GDP or 0.1 mM GTPγS. The loaded Rab beads were incubated with the respective lysates for 1-2 h at 4˚C under rotation. Rab beads were washed twice with NS buffer, twice with NS buffer supplemented to 250 mM NaCl and washed once with Wash Buffer (20 mM HEPES pH
7.5, 250 mM NaCl, 1 mM DTT). Proteins bound to the GST-Rab were eluted by complexing Mg2+ in Elution Buffer (20 mM HEPES pH 7.5, 1.5 M NaCl, 20 mM EDTA,
1 mM DTT) plus 5 mM GDP or 1 mM GTPγS for 20 min at 25˚C.
Vamp2 Pulldowns
FLAG tagged Vamp2 constructs were previously described (Calakos et al., 1994). FLAG tagged Vamp2 was transfected alone or co-transfected with eGFP or eGFP tagged;
TBC1D5, TBC1D13, TBC1D15, TBC1D16, TBC1D17 into HEK293 FT cells with
Lipofectamine 2000 (Invitrogen) as described above. Transfected cells were allowed to recover for 48 h. Cells were lysed in NP40 Buffer (1% NP40, 20mM Tris-HCl pH 7.4,
137 mM NaCl, 10% v/v Glycerol, pH 7.4) supplemented with Complete protease
Inhibitors (Roche Applied Sciences) and phosphates inhibitors (2 mM sodium orthovanadate, 1 mM pyrophosphate, 10 mM sodium fluoride) by passing through a 25G syringe 8-10 times. Lysates were solubised on ice for 20 min and centrifuged at 18,000 x g for 20 min at 4°C to remove insoluble material. Each cleared lysate was incubated with
Protein G sepharose beads and 5 μg of monoclonal anti-FLAG M2 antibody (Sigma).
Incubations were performed at 4°C with rotation for 1-2 h. The sepharose beads were washed three times with NP40 Buffer and twice with PBS. FLAG-Vamp2 and interacting proteins were eluted in PBS with 1 μM FLAG peptide.
50 Yeast Two-Hybrid Screens and Interactions
Chemically competent Saccharomyces Cerevisiae yeast strain AH109 (BD Biosciences) were produced according to the LiAc method (Ref). The Rab bait and RabGAP prey vectors were co-transformed into the competent yeast by the DNA/PEG method (Ref).
Yeast co-transformants were plated on synthetic media lacking tryptophan and leucine and incubated at 30°C for 72 h. Yeast colonies that grew on the transformation selection plates were resuspended and diluted to form a dilution series10-1, 10-2, 10-3 which was spotted in parallel on media lacking leucine and tryptophan and media lacking leucine, tryptophan and histidine. The serial dilutions were incubated at 30°C for 72 h.
Immunoprecipitation
Cells were washed twice with ice-cold PBS and scraped down in NP40 Lysis Buffer (1%
NP40, 20 mM Tris-HCl pH 7.4, 137 mM NaCl, 10% v/v Glycerol, pH 7.4) supplemented with EDTA-free Complete protease inhibitors (Roche Applied Science), and phosphatase inhibitors (1 mM sodium pyrophosphate, 2 mM sodium vanadate, 10 mM sodium fluoride). Samples were passed through a 25G needle ten times and solubilized on ice for
20 min. Lysates were then cleared by centrifugation at 18,000 g for 20 min at 4°C. The cleared lysate was incubated with protein G speharose beads and either TBC1D13 antibody or a rabbit IgG antibody as the negative control. Immunoprecipitaions were incubated at 4°C for 2 h with rotation. After incubation the beads were washed five times by pelleting at 700 g centrifugation and resuspension with NP40 Lysis buffer for three washes then ice-cold PBS for two washes. The PBS was completely removed from the
51 beads which were boiled in Lamelli sample buffer at 95°C for 5 min. The supernatant was collected and resolved by SDS-PAGE.
Mass spectrometry analysis
Mass spectrometry was performed as previously described. Briefly protein samples were prepared in Lamelli sample buffer with 1 mM DTT as reducing agent. Samples were resolved by SDS-PAGE and proteins were visualised with Sypro-Ruby staining
(Molecular Probes) as per manufacturers instructions. Proteins were visualised under ultra-violet light and excised from the gel. Gel slices were diced and washed with 50% acetonitrile in 250 mM ammonium bicarbonate at room temperature for 30 min, then incubated in 100% acetonitrile at room temperature for 10 min. The gel slice was incubated with TPCK treated Trypsin (Promega) at 12.5 ng/μL in 100 mM ammonium bicarbonate overnight at 37°C. 5% formic acid was added to the gel and incubated for at least 1 h at 37°C. An equal volume of 100% acetonitrile was added to the slice and incubated at 37°C for at least an hour after which an additional two times volume of
100% acetonitrile was added and incubated at room temperature for 10 min. The solution was retained and evaporated until dry using a SpeediVac at 45°C. After sample was dried 20 μL of 5% formic acid was added and incubated for 10 min to solubilise the peptides. Samples were desalted and subjected to mass spectrometry and MS/MS data analysis.
For titanium dioxide enrichment of phosphopeptides the protocol used by Mann and colleagues was adhered to (Olsen et al., 2006). Briefly, 10 μL Titansphere-material (GL
Sciences) was layered on C8 STAGE Tips in 200 μL pipette-tips. The columns were washed once with 50 µL NH3 water in 40% MeCN pH~10.5 and re-equilibrated with 30
52 µL 0.1% TFA. Columns were preloaded with DHB by addition of 30 µL 3g/L DHB in
80% ACN/ 0.1% TFA. Peptides were loaded and incubated at RT for 1-2 h, the bound peptides were washed one time with 50 µL of 10% MeCN in 0.1% TFA and once with 50
µL of 80% ACN in a 0.1% TFA solution. The phosphopeptides were eluted from the columns by applying 30 µL NH3 water in 20% MeCN pH~10.5 followed by 50 µL NH3 water in 40% MeCN pH~10.5. The eluates were dried in speedi-vac and reconstituted in
10 µL 5% MeCN in 0.1% TFA. in vitro GAP assay
Recombinant GST-Rab was purified from HEK293 FT cells as described above.
RabGAPs were produced in HEK293 FT cells as a C-terminal FLAG tagged fusion and purified by immunoprecipitation with a FLAG antibody and subsequent elution in
Elution Buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 5 mM β-mercaptoethanol, 1 μM FLAG peptide). Purified GST-Rab on glutathione sepharose beads was washed twice with Wash Buffer (20 mM Tris-HCl pH 8.0, 100 mM
NaCl, 1 mM DTT) supplemented with 0.1 mM GDP and twice with Wash Buffer alone.
GST Rab (1 μM) was incubated with Loading Buffer (20 mM Tris-HCl pH 8.0, 100 mM
NaCl, 1 mM DTT) plus 0.1 μM [α-32P] GTP (MP Biomedicals) at 30˚C for 30 min. After loading, GST Rab was washed twice with Wash Buffer to remove unbound GTP. The loaded Rab was resuspended in Assay Buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT) and added to each GAP (final concentration 0.23 μM) or
Elution Buffer alone. The incubation was carried out at 30˚C and aliquots were taken at indicated time points. The reaction was terminated by mixing the aliquots with Stop
Buffer (0.2% SDS, 20 mM EDTA, 5 mM GDP, 5 mM GTP) and incubation at 65˚C for 5
53 min. Samples were spotted on a polyethyleneimine cellulose plate and thin layer chromatography (TLC) was performed in 0.75 M KH2PO4, pH 3.5.
Cell Culture and Transfections
3T3-L1 fibroblasts were cultured and passaged in DMEM (Invitrogen) supplemented with 10% v/v FCS () and penicillin/streptomycin/glutamine (Invitrogen) at 37˚C with
10% CO2. Fibroblasts were differentiated into adipocytes by supplementing standard media with IBMX, Biotin, Insulin and Dextran for 72 h and then replacing with standard media containing insulin for a further 72 h (Ramm et al., 2006). 3T3-L1 fibroblasts were infected with a pBabepuro-HA-GLUT4 retrovirus as previously described (Shewan et al.,
2003). After 24 h, infected cells were selected in standard media with 2 µg/mL puromycin. HA-GLUT4 fibroblasts were then grown and differentiated into adipocytes
(Shewan et al., 2003). Adipocytes at day 6-7 post differentiation were electroporated with indicated constructs as previously described (Larance et al., 2005) and used 48-72 h after electroporation.
HEK293 FT (Invitrogen) and HeLa cells were cultured in DMEM supplemented with
10% v/v FCS and penicillin/streptomycin/glutamine (Invitrogen) at 37˚C with 10% CO2.
For transient transfection a total of 10 μg of DNA was used per 10 cm dish with 30 μL of
Lipofectamine 2000 (Invitrogen) following standard protocols.
For SILAC labeling of 3T3-L1 adipocytes, arginine and lysine free Dulbecco’s minimal essential medium was used and supplemented with stable isotope labeled arginine and lysine in addition to dialysed FCS as described previously (Olsen et al., 2006).
54 Sub-cellular Fractionation of Adipocytes
Sub-cellular membrane fractions from 3T3-L1 adipocytes were prepared using a previously described differential centrifugation procedure (Marsh et al., 1995; Piper et al., 1991). Briefly, cells were homogenised in HES buffer (20 mM HEPES, 10 mM
EDTA, 250 mM sucrose pH 7.4) containing EDTA-free Complete protease inhibitors
(Roche Applied Science), and phosphatase inhibitors (1 mM sodium pyrophosphate, 2 mM sodium vanadate, 10 mM sodium fluoride) on ice. The lysates were centrifuged at
500 x g for 10 min to remove unbroken cells. The plasma membrane fraction was obtained after a 20 min centrifugation at 17,200 x g followed by centrifugation through sucrose. High-density microsomes (HDM) were obtained by centrifuging the 17,200 x g supernatant at 38,700 x g for 20 min and low-density microsomes (LDM) were obtained by spinning the 38,700 x g supernatant at 150,000 x g, 75 min.
Trafficking Assays and Immunofluorescence Microscopy
3T3-L1 adipocytes were electroporated at either day six or seven of differentiation and seeded on glass coverslips. Prior to incubation in the absence or presence of 100 nM insulin for 20 min, cells were serum depleted for 2 h at 37˚C. After washing in cold PBS, cells were fixed with 3% paraformaldehyde (ProSciTech) and quenched with 50 mM glycine in PBS and 2% bovine serum albumin. Cells were blocked in the same solution and either labelled for surface HA-GLUT4 with the monoclonal HA.11 antibody as previously described (Larance et al., 2005). The coverslips were then washed twice and incubated with the appropriate fluorophore conjugated anti-mouse antibody. Coverslips were washed twice and mounted on slides or for intracellular probing were permeablised
55 with 1% saponin (Sigma) in 50 mM glycine in PBS and 2% bovine serum albumin and labelled with the indicated antibodies.
Transferrin recycling was performed as previously described (Shewan et al., 2003).
Briefly; 3T3-L1 adipocytes retrovirally expressing the Transferrin receptor (Tfr) were electroporated with FLAG-TBC1D13 as described above. Cells were incubated for 1 h in serum free media and then in the presence of 50 μg/mL Tf-Alexa-488 (Invitrogen) for 2 h at 37˚C.
VSV-G trafficking was performed as previously described. The construct encoding eGFP-tagged tsVSV-G was transfected into HeLa cells with standard lipofection by
Lipofectamine 2000 (Invitrogen). Transfected cells were allowed to recover for 24 h then were incubated at 40°C for 18 h. After this incubation the cells were either immediately washed twice with ice-cold PBS and fixed with 3% paraformaldehyde or incubated at
32°C for 30 min then washed and fixed.
In all immunoflourescence experiments the primary antibodies were detected with anti- rabbit Cy2, Cy3, Cy5; or anti-mouse Cy2, Cy3, Cy5; (Invitrogen) accordingly. Optical sections were obtained through separate scans for Cy2, Cy3 and Cy5 using the Leica TCP
SP confocal laser-scanning microscope. For quantification of HA, surface staining images were collected with the same confocal settings, and images were analysed using the Leica confocal software.
56
Chapter Three
Profiling Rab/RabGAP interactions by yeast two-hybrid
screening.
57
Introduction
The Rab proteins are small monomeric GTPases that comprise the largest branch of the
Ras-like GTPase super family (Chardin, 1988). The major role of these proteins is to regulate vesicle trafficking through linking membrane identity to the SNARE protein fusion machinery (McBride et al., 1999; Schmitt et al., 1986; Segev et al., 1988). This function is mediated through recruitment of binding partners known as Rab effectors by active Rabs to the membrane surface (Dunn et al., 1993; Plutner et al., 1990; Sapperstein et al., 1996). Intrinsic to Rab GTPase function is the oscillation between active and inactive states. The Rab can bind either to a GTP or GDP molecule and this results in a conformation specific to each nucleotide (Dumas et al., 1999). The activity state of the
Rab is regulated by the Rab guanine nucleotide exchange factor (RabGEF) (Wada et al.,
1997; Walch-Solimena et al., 1997) and the Rab GTPase activating protein (RabGAP)
(Cuif et al., 1999; Strom et al., 1993). The RabGEF binds to an inactive Rab and this association displaces the bound GDP molecule and, due to the high cellular concentration of GTP, results in GTP loading of the Rab. The RabGAP is recruited by the active Rab and stimulates the intrinsic GTPase hydrolytic activity to convert GTP to GDP. These regulatory proteins are crucial in directing vesicle trafficking as the intrinsic rates of exchange and hydrolysis occur at a rate far lower than the observed tempo of membrane trafficking.
A large number of RabGAPs have been identified in the human genome although very little is known about how they function in vivo (Bernards, 2003). The RabGAP family is
58 defined by the presence of the Tre/Bub2/Cdc16 (TBC) domain that confers the GTPase activating characteristic of these proteins (Neuwald, 1997). This lead to the identification of the human RabGAP family that is comprised of 52 members each containing a TBC domain. Although these proteins are predicted to be putative GAPs very few
Rab/RabGAP pairings have been established. The role of Rab proteins in specific trafficking pathways has been the focus of many investigations and almost every Rab has been ascribed to a specific trafficking pathway in the cell (Stenmark, 2009).
Consequently, determining the functional pairings between the Rab and RabGAPs should reveal which RabGAPs regulate the associated trafficking pathways and expose a further avenue for investigation into how those pathways are co-ordinated in the cell.
The catalytic function of the TBC domain was first investigated in the S. cerevisiae Gyp protein family and demonstrated a role in membrane trafficking (Strom et al., 1993; Tan et al., 1991). Although these studies revealed a specific role of the Gyp proteins in trafficking many were found to have a wide substrate specificity for the yeast Rab
GTPases (Albert and Gallwitz, 1999; Brett et al., 2008). In mammalian cells, there are sufficient RabGAPs such that the function of one RabGAP may be linked to one individual Rab GTPase. However, this has not been explored in detail and it will be critical to identify the functional Rab/RabGAP partnerships to understand how the
RabGAPs are employed overall to regulate vesicle trafficking.
Recently a yeast two-hybrid approach has been utilised to identify putative Rab-RabGAP pairings. This approach first identified an interaction between Rab6 and the coiled-coil domain of TBC1D11 (also known as RabGAP1/GAPCenA) (Cuif et al., 1999). In this report, TBC1D11 was also shown to display GAP activity against Rab6. This approach
59 was also used to characterise the functional relationship between Gyp6p and Ypt6p (Will and Gallwitz, 2001). Gyp6p stimulates the intrinsic activity of Ypt6p and it was observed by yeast two-hybrid that Gyp6p preferentially binds to the wild type Ypt6p and an active mutant over an inactive version of Ypt6p. This provided the first evidence that the Y2H approach could be employed as a strategy to identify Rab-RabGAP interactions. The first report where a Rab was screened against a library of RabGAPs showed that Rab5 binds to the RabGAP RUTBC3 (Haas et al., 2005). This screen relied on a strategy whereby specific mutant forms of the Rab and the GAP proteins were used to enhance or stabilise the interactions. GAP activity between RUTBC3 and Rab5 was observed in addition to functional effects of RUTBC3 on Rab5 regulated trafficking pathways. Based on these data it was proposed that RUTBC3 is the major Rab5 GAP. Another study by the same group identified the binding partners of TBC1D11 (Fuchs et al., 2007). In this study an interaction between Rab6 and the coiled-coil domain of TBC1D11 was observed although no interaction could be found with the full-length protein. Contradicting the study by Cuif et al., no GAP activity of TBC1D11was found against Rab6. Knockdown of Rab6 by siRNA demonstrated that the Rab6 GTPase was required for the trafficking of
Shiga toxin to the Golgi apparatus, however, there was no functional effect of TBC1D11 over-expression on this pathway. Full-length TBC1D11 interacted with Rab4 and it displayed a greater GAP activity than towards Rab6. Interestingly, the original report by
Goud and colleagues observed similar data but found that TBC1D11 had reduced activity on Rab4 when compared to Rab6.
A separate study by Fukuda and colleagues interrogated the complete Rab and RabGAP family for interactions by yeast two-hybrid (Itoh et al., 2006). This study, however,
60 yielded only a small number of interactions and failed to reproduce the results described by Barr and colleagues. An explanation for this disparity is that while Fukuda and colleagues had a larger library, the other studies utilised specific mutants to enhance the interactions. These studies indicate the usefulness of approaching Rab - RabGAP pairings using the yeast two-hybrid method while also highlighting the need for validation of these results through independent biochemical and functional analysis.
To determine the Rab GTPase interaction profile for each RabGAP the yeast two-hybrid approach as described by Haas et al. was adopted to screen for potential pairings. Each
RabGAP was screened against a yeast two-hybrid library comprising 43 of the mammalian Rabs. Although the Rab library does not include every Rab isoform in the
Human genome at least one representative from each subfamily was present. This should allow this screen to account for every possible Rab and RabGAP pairing as it has been demonstrated for several RabGAPs that they can act on all related Rab isoforms, e.g.
RUTBC3 with Rab5a/b/c (Haas et al., 2005). To increase the avidity of an interaction between these proteins the activity of the Rab and the RabGAP were disabled by mutating the respective catalytic residues. There are several lines of reasoning for this approach; 1) disabling the Rab intrinsic hydrolytic activity promotes an accumulation of the active over inactive Rab thus increasing the likelihood of obtaining an interaction. 2) disabling the catalytic activity of the RabGAP abolishes the stimulation of the hydrolytic activity. As GTP hydrolysis is likely coupled to dissociation of the Rab/RabGAP interaction this mutation should allow a stable interaction between the proteins.
These mutations have a defined action without affecting the broader ability of the protein to function in the cell and are used as a general tool in studies of Rab controlled
61 trafficking. Although these mutations have not been characterised for all Rab GTPases and RabGAPs the catalytic domains of both these proteins are generally well conserved among family members and where they have been employed they produce analogous results.
In the present study, I have used a yeast two-hybrid approach to screen a battery of
RabGAPs against a Rab library. Each RabGAP tested had a specific and unique Rab interaction profile and the RabGAPs tested generally displayed a binding profile of one to two Rab interactions. These results suggest that RabGAPs could be recruited to regulate several Rabs in a trafficking pathway. These interactions may be functionally important in regulating Rab hydrolytic activity or alternatively through the co-ordination of multiple Rabs into a Rab cascade.
Results
RabGAPs interact with specific Rab GTPases by yeast two-hybrid.
The Saccharomyces Cerevisiae AH109 strain were co-transformed with the catalytically inactive RabGAP and the hydrolytically inactive Rab GTPases cloned into the prey and bait vectors, respectively. The mutations used in this screen to generate the constitutively active Rab and catalytically inactive RabGAP are summarised in Table 2.1. Mutation of the catalytic Rab glutamine residue disables the intrinsic hydrolytic capability of the Rab thereby promoting the accumulation of GTP bound “active” Rab (Wagner et al., 1987).
Mutation of the catalytic arginine residue in the RabGAP abolishes the stimulatory effect on GTP hydrolysis and is predicted to stabilise the Rab-RabGAP interaction (Albert et al., 1999). A negative interaction control was provided by the empty pGADT7 and
62 pGBKT7 prey and bait vectors. All interactions were detected by spotting serial dilutions of co-transformed yeast onto –Leu/-Trp/-His agar. The RabGAPs tested and their respective Rab interactions are presented in Figures 3.1-3.10 and summarised in Table
3.1. Interactions were graded in strength based on whether growth was observed through the serial dilutions, the amount of growth on the interaction selection plate versus the transformation selection plate, and the time taken for growth to occur on the interaction plates. Based on these factors, interactions were defined as weak, medium and strong. A small number of co-transformations could not be defined as positive interactions despite repetition in multiple screens and so have been marked as potential binders. These interactions are not due to auto-activation by the RabGAP fusion protein as no growth was observed with the empty bait vector and each interaction was repeated successive times.
Interactions with Rab3a were observed for several RabGAPs. However, repeated analysis demonstrated these interactions were variable. The intermittent growth with Rab3a leads to the conclusion that this is a non-specific interaction in the system.
Rabs bind to discrete regions in the cognate RabGAPs
The structural organisation of RabGAPs is quite heterogeneous. Some RabGAPs, such as
TBC1D13, are comprised of little more than the TBC domain while others, such as
TBC1D4, are much larger containing several discrete domains (Bernards, 2003). It is possible that the Rab/RabGAP interactions are mediated either by binding to the TBC domain or an interaction may occur at a distal site and this could have significant implications on how these pairings function in vivo. Binding mediated through the TBC domain would be expected to indicate a substrate Rab, whereas binding outside this
63 TBC1D 1 4 5 10b 11 13 14 15 16 17 Rab1a ----++++---- Rab2 ------+++- Rab3a + - - + - + ++ + + - Rab3b - - - Rab3c - - - Rab3d - - - Rab4a - +++ - - +++ - + - - - Rab4b - +++ Rab5a ------Rab6a ----++----- Rab6b - - ++ Rab7 ------Rab8a ----+----- Rab8b - - Rab9 ------Rab10 -----+++---- Rab11 - - - +/- - - +++ - +/- - Rab13 ------Rab14 ----++----- Rab15 ------Rab17 ------Rab18 ------Rab20 ------Rab21 ------Rab22 ------+/---- Rab23 ------Rab24 ------+++--- Rab25 ------+++--- Rab26 ------++--- Rab27 - - + - - - +++ - - - Rab28 ------+++--- Rab29 - - - +/- - - +++ ++ +/- +/- Rab30 ------+++--- Rab31 ------Rab32 ------++--- Rab33 ------+--- Rab34 ----+----- Rab35 ------Rab37 ------Rab38 ------+++--- Rab39a ------Rab39b ----+-+--- Rab40a ------Rab40c ------Rab43 ++ ++ ------
Table 3.1 - Rab GTPase and RabGAP interactions. This table summarises the yeast two-hybrid interactions detected between the listed Rab GTPases and RabGAPs. Interactions were graded as none observed (-), weak (+), medium (++) or strong (+++). Growth that could not be distinguished from background but could not be excluded are indicated as potential interactions (+/-).
64 Transformation Interaction Transformation Interaction Selection Selection Selection Selection
Rab 1a Rab 25 Rab 26 Rab 2a Rab 3a Rab 27
Rab 3b Rab 28 Rab 3c Rab 29
Rab 3d Rab 30
Rab 4a Rab 31
Rab 32 Rab 4b Rab 33a Rab 5a Rab 33b Rab 6a Rab 6b Rab 34 Rab 7 Rab 35
Rab 8a Rab 37
Rab 8b Rab 38
Rab 9 Rab 39a
Rab 10 Rab 39b
Rab 11a Rab 40a
Rab 13 Rab 40c Rab 14 Rab 43 Rab 15
Figure 3.1 - Yeast two-hybrid interactions between Rab 17 TBC1D1 and Rab GTPases. Yeast two-hyrbid assay; S. cerevisiae Rab 18 AH109 cells were co-transformed with the TBC1D1 pGADT7 and each Rab GTPase pGBKT7 Rab 20 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for transformants and Rab 21 onto -Leu, -Trp, -His media to select for interaction. Growth was observed for TBC1D1 co-transformed with Rab 22a Rab3a and Rab43. Rab 23 Rab 24
65 Transformation Interaction Transformation Interaction Selection Selection Selection Selection
Rab1a Rab25 Rab2 Rab26 Rab3a Rab27a Rab3b Rab28 Rab3c Rab29 Rab3d Rab30 Rab31 Rab4a
Rab4b Rab32 Rab5a Rab33a Rab6a Rab33b Rab6b Rab34 Rab35 Rab7 Rab37 Rab8a Rab38 Rab8b
Rab9 Rab39a Rab10 Rab39b Rab11a Rab40a Rab13 Rab40c Rab14 Rab43 Rab15 Rab17
Rab18 Figure 3.2 - Yeast two-hybrid interactions between TBC1D4 and Rab GTPases. Yeast two-hyrbid assay; S. Rab20 cerevisiae AH109 cells were co-transformed with the TBC1D4 pGADT7 and each Rab GTPase pGBKT7 Rab21 vector. Co-transformed yeast were serially diluted on Rab22 synthetic media -Leu, -Trp, to select for transformants and onto -Leu, -Trp, -His media to select for interaction. Rab23 Growth was observed for TBC1D4 co-transformed with Rab4a, Rab4b, and Rab43. Rab24
66 Transformation Interaction Transformation Interaction Selection Selection Selection Selection
Rab1 Rab28 Rab2 Rab29 Rab3a Rab30
Rab4a Rab31
Rab5a Rab32 Rab6a Rab33
Rab7 Rab34
Rab8a Rab35
Rab9 Rab37
Rab10 Rab38 Rab11 Rab39a Rab13 Rab39b
Rab14 Rab40
Rab15 Rab43 Rab17 -ve Rab18 Rab20 Rab21
Rab22
Rab23
Rab24
Rab25
Rab26
Rab27
Figure 3.3 - Yeast two-hybrid interactions between TBC1D5 and Rab GTPases. Yeast two-hyrbid assay; S. cerevisiae AH109 cells were co-transformed with the TBC1D5 pGADT7 and each Rab GTPase pGBKT7 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for transformants and onto -Leu, -Trp, -His media to select for interaction. Growth was observed for TBC1D10b co-transformed with Rab27.
67 Transformation Interaction Transformation Interaction Selection Selection Selection Selection
Rab1a Rab22 Rab2 Rab23 Rab3a Rab24 Rab4a Rab25 Rab5a Rab26 Rab6a Rab27
Rab7 Rab28 Rab8a Rab29 Rab9 Rab30 Rab10 Rab31 Rab11 Rab32 Rab13 Rab33
Rab14 Rab34 Rab15 Rab35 Rab17 Rab37 Rab18 Rab38 Rab20 Rab39a Rab21 Rab39b
Rab40a Rab43 -ve
Figure 3.4 - Yeast two-hybrid interactions between TBC1D10b and Rab GTPases. Yeast two-hyrbid assay; S. cerevisiae AH109 cells were co-transformed with the TBC1D10b pGADT7 and each Rab GTPase pGBKT7 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for transformants and onto -Leu, -Trp, -His media to select for interaction. Growth was observed for TBC1D10b co-transformed with Rab3a, Rab11, and Rab29.
68 Transformation Interaction Transformation Interaction Selection Selection Selection Selection
Rab1a Rab25 Rab2a Rab26 Rab3a Rab27 Rab4a Rab28 Rab5a Rab29 Rab6a Rab30 Rab7a Rab31
Rab32 Rab8a Rab33 Rab9 Rab34 Rab10 Rab35 Rab11a Rab37 Rab13 Rab38 Rab14 Rab39a Rab15
Rab39b Rab17 Rab40a
Rab20 Rab43
Rab21 Rab22 Rab23 Rab24
Figure 3.5 - Yeast two-hybrid interactions between TBC1D11 and Rab GTPases. Yeast two-hyrbid assay; S. cerevisiae AH109 cells were co-transformed with the TBC1D11 pGADT7 and each Rab GTPase pGBKT7 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for transformants and onto -Leu, -Trp, -His media to select for interaction. Growth was observed for TBC1D17 co-transformed with Rab1a, Rab4a, Rab6a, Rab8a, Rab11, Rab14, Rab26, Rab34, and Rab39b.
69 Transformation Interaction Transformation Interaction Selection Selection Selection Selection
Rab1 Rab 25 Rab2 Rab 26 Rab3a Rab 27a Rab3b Rab 28 Rab3c Rab 29 Rab3d Rab 30 Rab4a Rab 31
Rab4b Rab 32 Rab5a Rab 33a Rab 33b Rab6a Rab 34 Rab6b Rab 35 Rab7 Rab 37 Rab8a Rab 38 Rab8b Rab 39a
Rab 9 Rab 39b Rab 10 Rab 40a Rab 11a Rab 40c Rab 13 Rab 43 Rab 14 Rab 15 Rab 17
Rab 18 Rab 20 Rab 21 Rab 22a Rab 23 Rab 24
Figure 3.6 - Yeast two-hybrid interactions between TBC1D13 and Rab GTPases. Yeast two-hyrbid assay; S. cerevisiae AH109 cells were co-transformed with the TBC1D13 pGADT7 and each Rab GTPase pGBKT7 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for transformants and onto -Leu, -Trp, -His media to select for interaction. Growth was observed for TBC1D13 co-transformed with Rab1, Rab3a and Rab10.
70 Transformation Interaction Transformation Interaction Selection Selection Selection Selection
Rab1a Rab25 Rab2 Rab26 Rab3a Rab27 Rab4a Rab28 Rab5a Rab29 Rab6a Rab30 Rab7 Rab31
Rab8a Rab32 Rab9 Rab33 Rab10 Rab34 Rab11 Rab35 Rab13 Rab37 Rab14 Rab38 Rab15 Rab39a Rab17 Rab18 Rab39b Rab20 Rab40a Rab21 Rab43 Rab22 -ve Rab23 Rab24
Figure 3.7 - Yeast two-hybrid interactions between TBC1D14 and Rab GTPases. Yeast two-hyrbid assay; S. cerevisiae AH109 cells were co-transformed with the TBC1D14 pGADT7 and each Rab GTPase pGBKT7 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for transformants and onto -Leu, -Trp, -His media to select for interaction. Growth was observed for TBC1D14 co-transformed with Rab3a, Rab4a, Rab11, Rab22, Rab24, Rab25, Rab26, Rab27, Rab28, Rab29, Rab30, Rab32, Rab33, Rab38, and Rab39b.
71 Transformation Interaction Transformation Interaction Selection Selection Selection Selection
Rab 1a Rab 27 Rab 28 Rab 2 Rab 29 Rab 3a Rab 30 Rab 4a Rab 31 Rab 5a Rab 32 Rab 6a Rab 33 Rab 7a Rab 34
Rab 8a Rab 35 Rab 9 Rab 36 Rab 10 Rab 37 Rab 11 Rab 38 Rab 39 Rab 13 Rab 40 Rab 14 Rab 43 Rab 15 Rab 17
Rab 18 Rab 20 Rab 21 Rab 22 Rab 23 Rab 24 Rab 25 Rab 26
Figure 3.8 - Yeast two-hybrid interactions between TBC1D15 and Rab GTPases. Yeast two-hyrbid assay; S. cerevisiae AH109 cells were co-transformed with the TBC1D15 pGADT7 and each Rab GTPase pGBKT7 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for transformants and onto -Leu, -Trp, -His media to select for interaction. Growth was observed for TBC1D15 co-transformed with Rab3a, Rab36, and Rab29.
72 Transformation Interaction Transformation Interaction Selection Selection Selection Selection
Rab1 Rab22 Rab2 Rab23 Rab3a Rab24 Rab4a Rab25 Rab5a Rab26 Rab6a Rab27
Rab7 Rab28 Rab8a Rab29 Rab9 Rab30 Rab10 Rab31 Rab11 Rab32 Rab13 Rab33
Rab14 Rab34 Rab15 Rab35 Rab17 Rab37 Rab18 Rab38 Rab20 Rab39a Rab21 Rab39b
Rab40a Rab43 -ve
Figure 3.9 - Yeast two-hybrid interactions between TBC1D16 and Rab GTPases. Yeast two-hyrbid assay; S. cerevisiae AH109 cells were co-transformed with the TBC1D16 pGADT7 and each Rab GTPase pGBKT7 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for transformants and onto -Leu, -Trp, -His media to select for interaction. Growth was observed for TBC1D16 co-transformed with Rab2, Rab11, and Rab29.
73 Transformation Interaction Transformation Interaction Selection Selection Selection Selection
Rab1a Rab25
Rab2a Rab26
Rab3a Rab27
Rab4a Rab28
Rab5a Rab29
Rab6a Rab30
Rab7 Rab31
Rab8a Rab32 Rab9 Rab33 Rab10 Rab11 Rab34 Rab13 Rab35 Rab14 Rab37 Rab15 Rab38
Rab39a
Rab17 Rab39b Rab18 Rab20 Rab40a
Rab21 Rab43 Rab22 Rab23 Rab24
Figure 3.10 - Yeast two-hybrid interactions between TBC1D17 and Rab GTPases. Yeast two-hyrbid assay; S. cerevisiae AH109 cells were co-transformed with the TBC1D17 pGADT7 and each Rab GTPase pGBKT7 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for trans- formants and onto -Leu, -Trp, -His media to select for interaction. Growth was observed for TBC1D17 co-transformed with Rab3a and Rab29.
74 domain would suggest the RabGAP is a Rab effector. We sought to determine whether the Rabs bound to the TBC domains of the respective RabGAPs or whether binding occurred at another site in the protein. To this end we screened the TBC domain of
TBC1D4 against the Rab binding partners in Figure 3.2, Rab4a and Rab43. The N- terminal regions of TBC1D4 were screened to determine if the site of Rab interaction lay outside the TBC domain. The truncation mutants for TBC1D4 are summarised in Table
2.1.
Constructs encoding the full-length TBC1D4 (amino acids 1-1299), an N-terminal region of the protein (amino acids 1-518), and the TBC domain (amino acids 865-1299) were screened by yeast two-hybrid against Rab4a and Rab43 (Fig. 3.11). Again, the constitutively “active” Rab GTPase mutants were used and where applicable the catalytically inactive version of TBC1D4 was employed. As observed earlier, full-length
TBC1D4 interacted with Rab4 and Rab43. When comparing the truncation mutants I found that Rab4 only displayed an interaction with the TBC1D4 1-518 construct and no growth was observed when co-transformed with the TBC1D4 865-1299 construct. The interaction with the N-terminal construct was comparable to that observed with the full- length protein. The interaction with Rab43 was recapitulated with the TBC domain alone and no interaction was observed with the TBC1D4 1-518 mutant. The interaction between the TBC domain of TBC1D4 and Rab43 was much stronger than the interaction with the full-length protein. These results indicate that the Rab GTPases bind to discrete regions within the RabGAP and that the sequence encoded in the region is necessary and sufficient for this interaction.
75 Transformation Interaction Selection Selection
TBC1D4 1-1299
TBC1D4 1-518 Rab4a
TBC1D4 865-1299
TBC1D4 1-1299
TBC1D4 1-518 Rab43
TBC1D4 865-1299
Figure 3.11 - Yeast two-hybrid interactions between TBC1D4 truncation mutants and Rabs 4 and 43. Yeast two-hybrid assay; S. cerevisiae AH109 cells were co-transformed with the TBC1D4 pGADT7 encod- ing an N-terminal mutant (1-518), a TBC domain mutant (865-1299), or full-length TBC1D4 (1-1299) and each indicated Rab GTPase pGBKT7 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for transformants and onto -Leu, -Trp, -His media to select for interaction. Rab4 was found to interact with N-terminal mutant and not the TBC domain of TBC1D4. Rab43 displayed an interac- tion with the TBC domain of TBC1D4 and not the N-terminal domain.
76 Discussion
Until recently, the identification of Rab/RabGAP pairings was mainly described through the outcome of in vitro assays. The development of a yeast two-hybrid approach allows a fast method of navigating through all possible Rab/RabGAP combinations to reveal each cognate partner. The RabGAPs RUTBC3, known as RabGAP5, and TBC1D11 have been the subject of investigations using this approach (Fuchs et al., 2007; Haas et al., 2005).
Here, these investigations have been expanded to identify novel Rab/RabGAP interactions. This yeast two-hybrid approach has yielded a spectrum of Rab/RabGAP interactions and allowed dissection of these binding properties. The effectiveness of this approach as a tool to reveal the Rab/RabGAP partnerships are evinced in the following;
1) There is an extraordinary degree of specificity in the interaction between different
RabGAPs and different Rabs. 2) The binding profile of each Rab and RabGAP was unique.
One of the possible drawbacks to this approach is the chance of false positives due to overexpression of functionally linked classes of proteins. The most important observations illustrating the authenticity of these results are the diversity in the detected interactions, the conserved Rab binding of highly similar TBC domains, and the maintenance of the original full-length interactions across the truncation profiles. This can be observed in the profiling of two of the screened RabGAPs, TBC1D1 and
TBC1D4. TBC1D1/TBC1D4 are involved in insulin-stimulated GLUT4 translocation sharing 61 and 91% similarity across the full-length and TBC domains, respectively. The highly conserved nature of the TBC domains suggests that these regions would bind the same Rab and, indeed, it was found that both domains bound to Rab43. This
77 demonstrates that there is a shared specificity encoded in the TBC domains of these proteins that facilitate the Rab43 interaction. Furthermore, highlighting the sequence divergence between these RabGAPs outside the TBC domains, the TBC1D4 N-terminus, but not that of TBC1D1, interacted with Rab4. TBC1D1/TBC1D4 illustrate that the specificity of the Rab/RabGAP interactions is due to the specific sequence of the proteins rather than artefacts introduced by the yeast two-hybrid system. Further dissection of the binding between TBC1D4 and Rab4/Rab43 revealed the Rab4 interaction occurs in the
N-terminus while the Rab43 interaction occurs in the TBC domain. Screening of the truncation constructs did not result in the loss or generation of Rab interactions indicating a RabGAP can possess modularity in Rab binding while presenting a fidelity to the full- length RabGAP/Rab binding profile.
Importantly, we were able to recapitulate the results of other reports of Rab-RabGAP interactions in the field. The RabGTPase interaction profile by yeast two-hybrid screening has previously been characterised for TBC1D11 (Fuchs et al., 2007). The results published in that study largely agree with the interactions we report here but there are some notable differences. Haas et al screened a full-length version and a truncation encoding the coil-coiled domain of TBC1D11. The reported full-length interactions occurred with Rab4, Rab11 and an extremely weak interaction with Rab6. When the
TBC1D11 coiled-coil domain was used as prey, interactions were observed with Rab1
Rab4, Rab6, Rab8, Rab11, Rab14, Rab25, Rab30, and Rab37. In my studies (Table 3.1 and Figure 3.5) interactions were observed between full length TBC1D11 and Rab1,
Rab4a, Rab6a, Rab6b, Rab8a, Rab14, Rab34, and Rab39b. The major differences between these results and the study by Haas et al are an improved Rab6 binding, Rab1
78 binding and binding to Rab39b. We were unable to detect binding with Rab25, Rab30 or
Rab37. These observations provide a level of confidence that these results are functionally relevant, and reproducible.
The screens between our Rab library and the RabGAPs TBC1D10b and TBC1D17 did not return any reproducible interactions. This is an interesting result considering the majority of the other RabGAPs bound several Rabs. There are several possibilities that could account for this observation. 1) The target Rab for these proteins may not have been present in the screen. The library was missing Rab12, Rab36, Rab41, Rab42,
Rab44, and did not include isoforms that could compensate for these. 2) Fusion to the
GAL4 domains may in some cases interfere with the binding ability of either the Rab or the RabGAP. 3) Interaction may be mediated or require an additional factor not available in the yeast two-hybrid system. 4) It is also possible that despite possessing a putative
TBC domain these proteins may not bind Rab GTPases. Further strategies would have to be developed to identify the Rab interaction partners for these RabGAPs.
One of the more striking features of my results is the diversity of binding between the
Rabs and RabGAPs, ranging from single interactions observed for TBC1D1 and
TBC1D16 through to TBC1D11 and TBC1D14, which displayed multiple interaction partners. This challenges the original notion that each Rab has a single specific RabGAP showing that it may be possible for some Rabs to be the target of multiple activators. This may reflect the expression profiles of these proteins across different tissue types. A Rab
GTPase that functions in a cell type where the cognate RabGAP is not expressed would require an alternate member of this family for regulation. The differential expression of
TBC1D1 and TBC1D4 across adipose and muscle tissue is thought to be an example of
79 this (Roach et al., 2007; Taylor et al., 2008). These RabGAPs regulate trafficking of the
GLUT4 molecule in these tissues through activity on the same Rab (Miinea et al., 2005;
Roach et al., 2007; Sano et al., 2003). By expressing at least one of these RabGAPs in these tissues ensures the correct regulation of the Rab and hence the trafficking pathway.
TBC1D1 and TBC1D4 have been shown to be responsive to distinct signalling pathways and the differential expression is hypothesised to be a part of a mechanism that allows the cell to activate this trafficking pathway in response to metabolic demands (Chen et al.,
2008; Kane et al., 2002). This arrangement may provide a template for other Rab
GTPases that are targeted by multiple RabGAPs.
There is growing evidence in the field that Rab controlled checkpoints in a trafficking pathway can be directly regulated by GEF and GAPs bound to the preceding or subsequent stations (Ortiz et al., 2002; Rivera-Molina and Novick, 2009; Wang and
Ferro-Novick, 2002). In the maturation of early endosomes to the late endocytic compartment it has been shown that Rab5, an early endosome marker, recruits the
RabGEF for the late endosome Rab GTPase Rab7 (Rink et al., 2005). This work demonstrates how an upstream Rab can recruit effectors that are required for activation of a downstream Rab. More recently, a study has been reported where in the exocytic pathway in S. cerevisiae the downstream Rab Ypt32 recruits the RabGAP Gyp1p to inactivate the upstream Rab Ypt1p (Rivera-Molina and Novick, 2009). This illustrates how Rabs can establish cascades by recruiting GEFs and GAPs to spatially and temporally regulate Rab trafficking checkpoints. This role for RabGAPs could explain the results generated from these yeast two-hybrid Screens. TBC1D4 and TBC1D13 bind two Rabs each suggesting that these RabGAPs could co-ordinate a linear trafficking
80 pathway between Rab4 – Rab43 and Rab1 – Rab10, respectively. TBC1D11 and
TBC1D14 bind many Rabs and indicate that these RabGAPs may be involved in regulating independent pathways or that these Rabs regulate several checkpoints that converge into one pathway. This would suggest that the Rabs that bind to the same
RabGAP could be functionally related. Indeed, within the RabGAP interaction profiles there are sub-groups of Rabs that have been functionally characterised in the same trafficking pathway. TBC1D14 binds Rab24, Rab32 and Rab33a/b, all of which have been implicated in autophagy (Hirota and Tanaka, 2009; Itoh et al., 2008; Munafo and
Colombo, 2002). Rab32 and Rab38 are required for the transport of melanosome enzymes in melanogenesis (Lopes et al., 2007; Wasmeier et al., 2006), a process involving Rab27 (Strom et al., 2002), which we also observed as a TBC1D14 binding partner. TBC1D11 interacted with Rab4, Rab6, Rab8 and Rab14, which are all, involved in trafficking through endosomal-TGN recycling pathways (Henry and Sheff, 2008;
Junutula et al., 2004; Nery et al., 2006; van der Sluijs et al., 1992).
This idea is further supported by our observations that the Rab proteins bound either the
TBC domain or another discrete region of the protein. This observation suggests that the
Rabs that bind the TBC domain will act as RabGAP substrates while the other Rabs will recruit the RabGAPs in an effector-like manner. In the context of Rab cascades these binding profiles can be used to link Rabs together in a trafficking pathway through the common RabGAP. The Rabs that recruits the RabGAP in an effector-like manner could either be upstream or downstream of the next Rab that is the target of the TBC domain. In the case of TBC1D4, Rab4 may recruit the RabGAP to a specific location within the cell to regulate the activity of Rab43.
81 In addition to revealing the intricacies of Rab-RabGAP interactions these results provide an initial starting point to investigate RabGAP action in trafficking. It will be important to determine whether these interactions can be recapitulated in a different experimental scenario and investigations would include GAP activity assays, affinity binding and functional assay analysis.
82
Chapter Four
TBC1D13 is a Rab10-interacting protein involved in
GLUT4 trafficking
83
Introduction
In the screens for Rab/RabGAP pairings the RabGAP TBC1D13 yielded an interaction with Rab1 and Rab10 (Chapter 3). This was of interest as Rab10 is a major regulator of insulin-stimulated GLUT4 trafficking in adipocytes and polarised trafficking in epithelial cells (Babbey et al., 2006; Roland et al., 2009; Sano et al., 2007; Sebastian et al., 2007).
Rab1, on the other hand, regulates the trafficking of vesicles from the ER to the Golgi as well as maintaining overall Golgi morphology (Allan et al., 2000; Plutner et al., 1991;
Tisdale et al., 1992).
In muscle and adipose tissue, the glucose transporter GLUT4 is required to facilitate glucose uptake in response to insulin stimulation (James et al., 1988). Insulin-stimulated
Akt signalling in adipocytes results in the translocation of GLUT4 from an intracellular storage compartment to the plasma membrane (Bryant et al., 2002; Huang and Czech,
2007). In the basal state, GLUT4 is excluded from the plasma membrane and actively traffics between endosomes, the trans-Golgi network (TGN) and GLUT4 storage vesicles
(GSV) (Martin et al., 2000; Shewan et al., 2003; Slot et al., 1991). Although several of the players in GLUT4 vesicle trafficking are known, the complete pathway remains elusive. One of the major goals has been to establish the identity of the Rab GTPases that regulate GLUT4 trafficking. Recent progress toward this goal was made with the identification of the RabGAP TBC1D4 as a negative regulator of insulin-stimulated
GLUT4 translocation. TBC1D4 was first identified as an Akt substrate in adipocytes
(Kane et al., 2002) that is localised to GLUT4 containing vesicles (Larance et al., 2005).
84 An insulin resistant phosphorylation mutant of TBC1D4 inhibited GLUT4 translocation and this was shown to be dependant on TBC1D4 GAP activity (Sano et al., 2003).
Insulin-stimulated phosphorylation of TBC1D4 through Akt was shown to play a crucial role in it’s GAP activity at least in part due to regulated 14-3-3 binding (Ramm et al.,
2006; Stockli et al., 2008). These studies indicated that insulin stimulation controlled the activity of a Rab GTPase critical to GLUT4 translocation through TBC1D4. The GTPase activity of Rab10 was shown to be stimulated by TBC1D4 (Miinea et al., 2005). and several studies have now implicated Rab10 in insulin dependent movement of GLUT4 to the PM (Sano et al., 2007; Sano et al., 2008). However, the exact step in the GLUT4 trafficking pathway controlled by Rab10 has not been identified.
Rab10 localises to a tubulo-vesicular network and endosomes where it has been shown to play a role in endosomal traffic (Babbey et al., 2006; Roland et al., 2009; Sebastian et al.,
2007). A proteomic survey of constituent members of GLUT4 vesicles identified a number of resident Rab GTPases including Rab10 (Larance et al., 2005). This provided the first evidence that Rab10 may play a role in GLUT4 trafficking. Overexpression of constitutively active Rab10 in 3T3-L1 adipocytes led to increased surface GLUT4 in the absence of insulin and RNAi mediated knock down of Rab10 in adipocytes inhibited insulin-stimulated GLUT4 translocation (Sano et al., 2007; Sano et al., 2008). Hence, these studies indicated that Rab10 is a key component of the regulatory machinery that catalyses the docking and fusion of GLUT4 vesicles with the PM. Based on these findings a model has been put forward whereby TBC1D4 controls the GTP loading of
Rab10 in an insulin-dependent manner.
85 It was perhaps surprising then to identify a new member of the RabGAP family
TBC1D13, as a binding partner of Rab10 by Yeast Two Hybrid. Since TBC1D4 did not bind to Rab10 in the Yeast Two Hybrid screen this gave rise to the possibility that
TBC1D13 may be an alternate in vivo GAP for Rab10. Although TBC1D4 plays a crucial role in GLUT4 translocation the evidence that this is mediated through Rab10 is less compelling. The most convincing argument for TBC1D4 Rab10 GAP activity is based on the output of an in vitro GAP assay (Miinea et al., 2005). However, TBC1D4 also displayed GAP activity toward a number of other Rabs in this assay. This assay used a truncation mutant of TBC1D4 that encoded the GAP domain alone and no activity was found with the full-length protein. This is an important distinction to make as it is reported in the RabGAP field that specificity of GAP activity is reduced when only the
TBC domain is employed in these assays (Albert and Gallwitz, 1999; Albert et al., 1999;
Brett et al., 2008). This is reflected in the broad GAP activity observed with the TBC1D4
GAP domain.
Yeast Two Hybrid analysis also revealed the TBC1D13 binds to the Rab1 GTPase. Rab 1 plays a critical role in the earliest trafficking steps of the secretory pathway (Plutner et al., 1991; Tisdale et al., 1992). Rab1 is localised to the ER, ERGIC, and cis-face of the
Golgi where it regulates the anterograde movement of the early secretory pathway.
Studies involving the overexpression of dominant negative Rab1 mutants observed a complete dispersal of these organelles that is caused by the block in the early secretory pathway (Tisdale et al., 1992; Wilson et al., 1994). Using a temperature sensitive mutant of the VSV-G protein as a general marker of vesicle trafficking from the ER it was observed that this trafficking block occurs at ER exit sites. A similar block in tsVSV-G
86 trafficking has been observed in cells overexpressing the Rab1 GAP TBC1D20 (Haas et al., 2007; Sklan et al., 2007). It is thought that active Rab1 co-ordinates budding from the
ER in an as yet unknown manner. In addition to a functional role on the ER Rab1 also mediates the tethering step in vesicle fusion at the cis Golgi. Rab1 recruits the effector proteins p115 and GM130 (Allan et al., 2000; Moyer et al., 2001). p115 is a tether on the incoming vesicle and interacts with GM130 a Golgi resident protein. This interaction draws in the incoming vesicle to promote the subsequent formation of the SNARE complex and eventual fusion. Rab1 is also thought to play a role in organization of Golgi superstructure. Rab1 recruits several effectors such as GRASP65, and golgin84 that regulate trafficking between Golgi cisternae, and stabilise the Golgi laterally across each ribbon (Moyer et al., 2001; Puthenveedu et al., 2006; Satoh et al., 2003).
I have found TBC1D13 to interact with both Rab1 and Rab10. This is an intriguing result as both these Rabs have been shown to have RabGAPs that stimulate GTP hydrolysis and play a role in associated trafficking functions. Perhaps TBC1D13 can act as a GAP on these Rabs or it may play an additional role as a Rab effector to co-ordinate trafficking.
The following investigations will focus on recapitulating the Yeast Two-Hybrid interactions, ascertaining a direct role on each Rab, and determining if TBC1D13 has functional relevance to the specific trafficking pathways.
87 Results
TBC1D13 is a single domain putative RabGAP expressed in all tissues and adipocyte cell lines
TBC1D13 possesses the six conserved motifs shared by all members of this family
(Albert and Gallwitz, 1999) including the two catalytic residues that accelerate Rab GTP hydrolysis (Fig. 4.1A) (Pan et al., 2006). An alignment between TBC1D13 and its closest yeast and mammalian homologues revealed a unique sixty-five amino acid sequence insertion in TBC1D13 between the two catalytic motifs that is predicted to encode for an
α-helix. The ‘extended’ TBC domain comprises the majority of the TBC1D13 400 amino acid protein, with no other identifiable domains.
To probe the TBC1D13 expression profile and interaction partners an antibody was raised against a GST-TBC1D13 fusion protein. The TBC1D13 antibody immuno-labelled a single band in a 3T3-L1 adipocyte cell lysate of the predicted molecular weight at ~ 45 kDa (Fig. 4.1B) and specifically recognised purified TBC1D13 (Fig. 4.1C). This antibody was used to probe for TBC1D13 from 3T3-L1 cells throughout the course of adipocyte differentiation (Fig. 4.1D) where I observed the up-regulation of TBC1D13 protein levels from day 4 of differentiation. I surveyed several mouse tissue types for endogenous
TBC1D13 expression (Fig. 4.1E) and found that TBC1D13 was present at varying levels in all tissues tested.
TBC1D13-interacting proteins by immunoprecipitation
To identify TBC1D13-interacting proteins the TBC1D13 antibody was used to immunoprecipitate TBC1D13 and interaction partners from 3T3-L1 adipocytes. As
88 )C D) C) B) across severaltissuetypes. Figure 4.1-TBC1D13containsasingleTBCdomainandisexpressedin3T3-L1adipocytes amino acidpositionsarenoted.B) TBC1D13 antibodyrecognises TBC1D13 ina3T3-L1adipocytewholecell with TBC1D13 antibody.with TBC1D13 time course. Tubulin ispresentedasacontrol.E)Lysates fromindicatedmousetissueswereimmunoblotted FLAG and TBC1D13 antibody. D) TBC1D13 and Tubulin expressionduringa3T3-L1adipocytedifferentiation lysate. C)PurifiedFLAGtagged TBC1D4 GAP domain, TBC1D13 and TBC1D15 wereimmunoblottedwith kDa 172 251 24 34 49 64 86 4 A) 172 251 kDa 86 34 49 64
TBC1D4 GAP 1 LGTBC1D13 FLAG 35 TBC1D13 A) A schematicoffull-length TBC1D13 showingthe TBC domain (green). The
TBC1D15
TBC1D4 GAP
TBC1D13 TBC Domain
TBC1D15 TBC1D13 kDa E) kDa 45 55 45 3T3-L1 Adipocyte Differentiation (Day) Brain 1 2345678
Lung 345
Brown Fat 400 White Fat
Kidney Spleen
Heart TBC1D13
Muscle Tubulin
Liver 87 shown (Fig. 4.2), a number of bands were specifically enriched with the TBC1D13 antibody. These bands were excised, digested with trypsin and identified with LC-
MS/MS analysis. The identified proteins that were isolated with TBC1D13 antibody and not detected in the IgG control are shown (Fig. 4.2 and Table 4.1). TBC1D13 and ten other co-immunoprecipitated proteins were identified. Six of these interactions with
TBC1D13 were insulin responsive; two from the basal state and four from the insulin- stimulated adipocytes. Of particular note is the interaction of TBC1D13 with Rab1b and
Rab10. This interaction was observed in both the basal and insulin-stimulated fractions.
Two other notable interacting partners were Alix, a late endosomal multi vesicular body
(MVB) constituent, and Sac3 a PIP2 phosphatase that has recently been implicated in
GLUT4 translocation.
TBC1D13 binds Rabs in a nucleotide dependent manner
To validate the TBC1D13 interaction with Rab1 and Rab10 the Rab activity states necessary for binding were examined. The Yeast Two Hybrid approach (Chapter 3) was employed to screen constitutively inactive or active mutants of Rab1 and Rab10 to determine how this might regulate the interaction of these Rabs with wild-type and catalytically inactive TBC1D13. I observed a significant interaction between constitutively active Rab1 Q70L mutant and the constitutively active Rab10 Q68L mutant with the inactive TBC1D13 R129A (Fig. 4.3). No interaction was observed using constitutively inactive Rabs (Rab1 SN; Rab10 TN). No detectable interaction was observed between Rabs 1 or 10 and wild-type TBC1D13 bait.
90 Protein Identified Unique Peptides kDa Basal Insulin Acetyl-CoA Carboxlyase 2 (ACC2) 12 276 + + Acetyl-CoA Carboxylase 1 (ACC1) 57 265 + + Sac3 5 103 - + Matrin 3 794-+ Programmed cell death 6 interacting protein (Alix) 3 92 + + Calcium-binding and coiled-coil domain-containing protein 1 3 77 + - Synaptotagmin binding, cytoplasmic RNA interacting protein (SYNCRIP) 5 69 + - TBC1D13 30 45 + + Sideroflexin-1 9 35 - + Prohibitin-2 333-+ Ras-related protein Rab-10 1 25 + - Ras-related protein Rab-1B 13 22 + +
Table 4.1 - TBC1D13 immuno-precipitation isolates interaction partners from 3T3-L1 adipocytes. The proteins isolated with the TBC1D13 antibody were identified by LC-MS/MS analysis. The proteins and number of peptides identified are listed. kDa Basal Insulin ACC1 251 172 ACC2 Matrin
86 Sac3 Alix 64 SYNCRIP
49 TBC1D13
Prohibitin 2 34 Sideroflexin 1
Rab1 Rab10 17
IgG TBC1D13
Figure 4.2 - TBC1D13 interacting proteins isolated by immunoprecipitaion. 3T3-L1 adipocyte lysate was either incubated with a Rabbit IgG control antibody or with a TBC1D13 antibody. Proteins immunopre-cipitated are resolved by SDS-PAGE and visualised with Sypro Ruby staining. 3T3-L1 adipocytes were either basaled or treated with 100 nm insulin for 30 min as indicated.
91 Transformation Interaction Selection Selection
TBC1D13 RA + Rab1 SN
TBC1D13 RA + Rab1 QL
TBC1D13 RA + Rab10 TN
TBC1D13 RA + Rab10 WT
TBC1D13 RA + Rab10 QL
Figure 4.3 - TBC1D13 interacts with Rab1 and Rab10 in a nucleotide specific manner by Yeast Two Hybrid. Yeast were co-transformed with pGADT7 TBC1D13-RA vector and pGBKT7 Rab1-SN, pGBKT7 Rab1-QL, pGBKT7 Rab10- TN, Rab10-WT, or Rab10-QL respectively. Co-transformants were serially diluted onto –HLW nutrient plates and interaction was determined by growth.
92 Specificity of the TBC1D13 and Rab10 Interaction
In the Yeast Two Hybrid screens an interaction between Rab1 and Rab10 with TBC1D13 was established. The interaction with Rab1 and Rab10 was recapitulated in the TBC1D13 immunoprecipitation where I was able to purify both Rabs. Peptides were identified that spanned the entire length of Rab1. I was only able to identify a single unique peptide for
Rab10. To confirm the Rab10-TBC1D13 interaction, an affinity purification technique was employed, using recombinant GST-Rab10, chemically loaded with either GDP or the non hydrolysable GTP analogue GTPγS. The efficiency of nucleotide association was increased by performing three consecutive loading cycles as described (Christoforidis and
Zerial, 2000). The nucleotide loaded Rabs were incubated with HEK293 FT lysate in which FLAG-TBC1D13 was expressed and proteins that interacted with the Rab in a nucleotide-dependent way were selectively eluted by complexing Mg2+ (Christoforidis and Zerial, 2000). The eluted proteins were subjected to SDS-PAGE and western blotting analysis. To further investigate the interaction between TBC1D13 and Rab10 the binding properties of TBC1D13 and an unrelated endosomal Rab GTPase, Rab4a was tested as a negative control. Nucleotide-specific eluates were immunoblotted with a FLAG specific antibody (Fig. 4.4). A significant increase in FLAG-TBC1D13 binding to GST-Rab10 following pre-loading with GTPγS was observed. A small amount of FLAG-TBC1D13 was found associated with Rab4a but this was not nucleotide dependent and likely represents non-specific binding. These data suggest that the interaction between Rab10 and TBC1D13 is specific and occurs in a GTP-dependent manner.
94 SM GST Pulldown
αFLAG
Rab4 GDP + - - - Rab4 GTPγS - + - - Rab10 GDP - - + - Rab10 GTPγS - - - + FLAG-TBC1D13 WT
SM GST Pulldown
αFLAG
Rab4 GDP + - - - Rab4 GTPγS - + - - Rab10 GDP - - + - Rab10 GTPγS - - - + FLAG-TBC1D13 RA
Figure 4.4 - TBC1D13 binds to Rab10. The ability of Rab4 and Rab10 binding to TBC1D13 was determined using GST bead binding assay. GST-Rabs loaded with GDP or GTPγS were incubated with HEK293 FT lysate expressing either FLAG-TBC1D13WT or FLAG-TBC1D13RA. Rab interacting proteins were eluted by complexing free Mg2+. Starting material and eluates were immunoblotted with FLAG antibody. Rep-resentative image of three independent experi- ments is shown.
95 TBC1D13 co-localises with Rab1 and Rab10
TBC1D13, like many members of this family, does not possess a trans-membrane domain. However, several members of this family have been shown to localise to discrete membranes. As shown in Figure 4.5, this was also the case for TBC1D13 as there was a concentration of labelling in the peri-nuclear region as well as peripheral vesicles in adipocytes (Fig. 4.5A). Notably, eGFP Rab1, eGFP Rab10 and GLUT4 partially co- localised with TBC1D13 in the peri nuclear compartment in adipocytes (Fig. 4.5A-B).
However, for the most part TBC1D13 and GLUT4 were found in distinct structures.
Comparison of the localisation of TBC1D13 to the Rab1 effector GM130 also demonstrated a partial overlap in the perinuclear region. These results indicate that
TBC1D13 is localised to internal membranes in adipocytes where there is significant overlap with both its interacting Rabs. To further explore the sub-cellular location of
TBC1D13, sub-cellular fractions were obtained from 3T3-L1 adipocytes by differential centrifugation (Marsh et al., 1995; Piper et al., 1991). Four separate fractions were obtained: the PM, which was enriched in markers of the plasma membrane, LDM, containing vesicles and large protein complexes, and organelles such as the Golgi, and the HDM which contained endosomes, endoplasmic reticulum, and lysosomes (HDM) and cytosol. Immunoblotting using the TBC1D13 specific antibody revealed that
TBC1D13 was concentrated in the HDM, LDM, and cytosol. (Fig. 4.5D). This localisation was not significantly affected by insulin.
TBC1D13 does not display GAP activity toward Rab1 or Rab10
As TBC1D13 binds to Rab1 and Rab10 in a nucleotide-specific manner I next set out to determine if TBC1D13 has GAP activity towards these Rab GTPases in vitro. The GAP
96 blots stained for TBC1D13 andIRAP areshown. PM, HDMand LDMfractionswereobtained bydifferential cellfractionation. Representative Western then incubatedintheabsence orpresenceof100nMinsulinfor20min.Cellswere lysedandcytosol, nuclear regionofthiscell.Scale bar10μm.(D)3T3-L1adipocyteswereincubated inDMEMfor2hand antibody. The outlineboxindicatesthearea ofthepanelimmediatelybelowthatmagnified peri- 3T3-L1 adipocyteswere electroporated witheGFP-Rab1andstained GM130 and TBC1D13 indicates theareaofpanel immediatelybelowthatmagnifiedtheperi-nuclear regionofthiscell.(C) electroporated witheGFP-Rab10QL andstainedwithGLUT4 TBC1D13 antibody. The outlinebox eGFP-Rab10WT andstainedwithGLUT4 TBC1D13 antibody. (B)3T3-L1adipocytes were Figure 4.5-IntracellularlocalisationofTBC1D13. TBC1D13 D) C) B) A) IRAPIRAPIRAP Rab10QL Rab10WT Rab1 B I
HDM GM130 GLUT4 GLUT4 LDM (A)3T3-L1adipocyteswereelectroporatedwith
PM TBC1D13 TBC1D13 TBC1D13
CYT 97 domain of TBC1D4 accelerates Rab10 GTP hydrolysis and this was used as a positive control (Miinea et al., 2005). The Rab10 intrinsic GTP hydrolysis rate was very low
(0.004 min-1) (Fig. 4.6A). The catalytic efficiency for Rab10 GTP hydrolysis in the
-1 -1 presence of the TBC1D4 GAP domain (865-1299) was 0.259 min μM (kcat/Km). This value is similar to the catalytic efficiency of TBC1D4 for Rab14 (0.2 min-1μM-1) reported by Lienhard and colleagues (Miinea et al., 2005). In contrast to that seen with TBC1D4,
TBC1D13 had a minimal effect on Rab10 GTP hydrolysis beyond its intrinsic rate
-1 -1 (kcat/Km = 0.0113 min μM ). Similarly, there was little if any effect of TBC1D13 on
Rab1 GTP hydrolysis (Fig. 4.6B). The Rab1 intrinsic GTP hydrolysis rate was low
(0.0059 min-1) and this increased slightly with the addition of TBC1D13 (0.0325 min-
1μM-1) but this was not reversed with catalytically inactive TBC1D13 (0.0372 min-1μM-
1). Based on these studies it seems likely that TBC1D13 is neither a Rab1 nor a Rab10
GAP.
TBC1D13 overexpression does not affect Rab1 regulated trafficking
Rab1 regulates vesicle transport between the ER and the Golgi (Plutner et al., 1991;
Tisdale et al., 1992), whereas Rab10 appears to function in endosomal vesicle transport.
To dissect the in vivo specificity of TBC1D13 action the effect of TBC1D13 overexpression on ER to Golgi transport was examined using a temperature-sensitive mutant of the vesicular stomatitis virus glycoprotein (tsVSV-G) (Presley et al., 1997).
This protein is a well-characterised marker of trafficking in the early secretory pathway.
When cells are incubated at 40°C the mutation causes the mis-folding and aggregation of the G protein in the ER. When the temperature is reduced to 32°C the VSV-G is optimally folded and can now be trafficked to the Golgi. GFP-tagged tsVSV-G and
98 A)
GDP
GTP
0 5 10 30 0 5 10 30 0 5 10 30 Time (min) Rab10 Rab10 Rab10 TBC1D4 (865-1299) TBC1D13
B)
GDP
GTP
Time 0 5 10 30 0 5 10 30 0 5 10 30 (min) Rab1 Rab1 Rab1 TBC1D13 WT TBC1D13 RA C) D)
% GDP % GDP 100 100
80 80
60 60
40 40
20 20
0 10 20 30 0 10 20 30 Time (min) Time (min)
Figure 4.6 - Rab GAP assay with TBC1D4 GAP domain, TBC1D13, and Rab10. (A) TLC seperation of α32P GDP and GTP in assays with GST-Rab10 alone, GST-Rab10 plus FLAG tagged TBC1D4 GAP domain, and Rab10 plus FLAG-TBC1D13. (B) Quantification of the data in (A) is presented as the percentage GDP versus time for Rab10 ( ), Rab10 plus TBC1D4 GAP ( ), and Rab10 plus TBC1D13 ( ).
99 FLAG-tagged TBC1D13 were co-transfected into HeLa cells and tsVSV-G trafficking was monitored by immunofluorescence. Overexpression of FLAG-TBC1D13 had no detectable effect on ER to Golgi transport of the tsVSV-G protein (Fig. 4.7). This is consistent with other published findings (Haas et al., 2007).
Insulin-stimulated GLUT4 translocation is blocked by TBC1D13 overexpression
TBC1D13 binds to Rab10 (Fig. 4.2, 4.3, and 4.4) and it partially co-localises with Rab10 and GLUT4. However, it has no demonstrable in vitro Rab10 GAP activity (Fig. 4.6), indicating that TBC1D13 might act as an effector for Rab10. To explore this hypothesis I set out to analyse whether TBC1D13 regulates a known Rab10 regulated trafficking process. Rab10 localises to an endosomal tubulo-vesicular network and it has been reported to control the delivery of GLUT4-containing vesicles, as well as other carriers, to the plasma membrane (Babbey et al., 2006; Sano et al., 2007; Sebastian et al., 2007).
Insulin-stimulated translocation of GLUT4 to the plasma membrane was analysed in
3T3-L1 adipocytes with and without overexpression of TBC1D13. 3T3-L1 adipocytes expressing HA-GLUT4 were electroporated with FLAG-TBC1D13 and insulin- stimulated HA-GLUT4 translocation was quantified by immunofluorescence microscopy using surface HA labelling to detect translocated HA-GLUT4 (Fig. 4.8) (Ng et al., 2008;
Ramm et al., 2006; Stockli et al., 2008). Overexpression of TBC1D13 completely inhibited insulin-stimulated HA-GLUT4 translocation to the PM. To determine whether this effect was due to the potential TBC1D13 GAP activity the catalytically inactive mutant was electroporated into the HA-GLUT4 adipocytes. Disabling the catalytic activity of TBC1D13 did not reverse this inhibitory effect (Fig. 4.8A-B). The catalytic
100 tsVSV-G GM130
40ºC tsVSV-G
40ºC ↓ 32°C
tsVSV-G FLAG GM130
40ºC tsVSV-G + TBC1D13WT + tsVSV-G 40ºC ↓ 32°C
Figure 4.7 - TBC1D13 does not effect ER to Golgi tsVSV-G trafficking. HeLa cells were transfected to express eGFP tagged tsVSV-G with or without FLAG tagged TBC1D13. The cells were incubated at either 40°C for 12hrs or 40°C for 12hrs then shifted to 32°C for 15min. GM130 staining is representative of cis-Golgi localization. Scale Bar 10 μM.
101 Figure 4.8 - TBC1D13 overexpression (A) blocks insulin stimulated GLUT4 trans- location. (A) 3T3-L1 adipocytes were HA FLAG electroporated with FLAG tagged full-length TBC1D13WT or TBC1D13RA as indicated. Transfected cells were detected with the FLAG antibody and indicated by arrowheads. HA-GLUT4 expressing 3T3-L1 adipocytes were incubated in DMEM without serum and
13WT then incubated with or without 100nM in-sulin for 20 min and GLUT4 transloca- tion to the PM was determined by surface HA staining. (B) Quantification of surface HA staining in (A) for independent experi- ments (n=3) and detectable surface GLUT4 is expressed as a percentage of control untransfected adipocytes ( ), TBC1D13WT ( ), and TBC1D13RA ( ). Scale bar 10μm.
13RA
100 Control TBC1D13 WT TBC1D13 RA 75
50
25 Surface GLUT4 (%)
0 Basal Insulin
102 Arg in TBC1D13 is adjacent to a second Arg and it is possible this second residue may be able to compensate for the loss of the catalytic Arg. To test this, a double Arg mutant construct, FLAG-TBC1D13-RRAA, was electroporated into adipocytes but this also blocked GLUT4 translocation similar to the wild type protein (data not shown). If this observed block in trafficking is mediated through one of TBC1D13’s interacting Rabs it may be possible to reverse the effect by co-overexpressing each Rab. To this effect either
GFP tagged Rab1 or Rab10 were co-electroporated with FLAG-TBC1D13 into 3T3-L1 adipocytes (Fig. 4.9). Adipocytes co-transfected with both Rabs and TBC1D13 still displayed a block in insulin-stimulated HA-GLUT4 translocation.
Overexpression of TBC1D13 does not disrupt general trafficking or insulin signalling
Overexpression of RabGAPs can disrupt vesicle trafficking and organelle structure in cells (Haas et al., 2005; Haas et al., 2007; Itoh and Fukuda, 2006; Patino-Lopez et al.,
2008; Roach et al., 2007; Sano et al., 2003). Therefore, it was important to test whether the block in GLUT4 trafficking was a consequence of a general trafficking defect, rather than a specific effect on the GLUT4 pathway. I first chose to investigate whether the distribution of GLUT4 had been altered in cells overexpressing TBC1D13 (Figure
4.10A). 3T3-L1 adipocytes overexpressing TBC1D13 displayed a typical perinuclear staining for GLUT4 with no aberrant localisation or reduction in overall protein levels.
No morphological change was detected in the Golgi and TGN system in cells overexpressing TBC1D13 as determined by labelling with the markers, GM130 and
Syntaxin16, respectively (Fig 4.10B-C). Moreover, the general protein recycling system was unperturbed as determined by uptake of Alexa 488 labelled transferrin in adipocytes
103 eGFP FLAG TBC1D13 HA-GLUT4
Rab10
Rab1
Figure 4.9 - Rab1 or Rab10 overexpression does not reverse TBC1D13 inhibited GLUT4 translo- cation. (A) 3T3-L1 adipocytes were electroporated with FLAG tagged full-length TBC1D13WT and either eGFP tagged Rab1 or eGFP tagged Rab10. Co-transfected cells were detected with the FLAG antibody and eGFP fluorescence. HA-GLUT4 expressing 3T3-L1 adipocytes were incubated in DMEM without serum and then incubated with or without 100nM in-sulin for 20 min and GLUT4 translocation to the PM was determined by surface HA staining. Scale bar 10μm.
104 GLUT4 FLAG TBC1D13 A)A)
STX16 FLAG TBC1D13 B)
GM130 FLAG TBC1D13
C)
Transferrin FLAG TBC1D13
D)
Figure 4.10 - TBC1D13 overexpression does not disrupt general membrane trafficking. FLAG- TBC1D13WT was electroporated into 3T3-L1 adipocytes and the labelled with FLAG antibody and GLUT4 (A) or Syntaxin16 (B) or GM130 (C). (D) 3T3-L1 adipocytes expressing transferrin receptor were electropo- rated with FLAG-TBC1D13 and incubated for 2hr in the presence of Transferrin-Alexa488. Scale bar 10μm.
105 expressing Transferrin receptor (Fig 4.10D). Finally, overexpression of TBC1D13 did not impair the insulin-dependent phosphorylation of Akt (Fig 4.11). This suggests that overexpression of TBC1D13 does not simply disrupt the overall integrity of the cell and its inhibitory effect is likely due to a specific block in GLUT4 vesicle transport, possibly through modulating Rab10 function.
Discussion
In this study I have used several independent approaches to show that the RabGAP
TBC1D13 binds specifically to Rab1 and Rab10 and likely acts as a novel Rab10 effector. An impairment in GLUT4 trafficking was observed with the overexpression of
TBC1D13 in adipocytes and was quite specific to this pathway as I failed to observe any disruption in ER to Golgi trafficking or transferrin recycling. In view of previous findings implicating Rab10 in GLUT4 trafficking this implicates TBC1D13 as an important effector of Rab10 involved in this insulin regulated pathway.
Despite an emergence of studies on TBC proteins there are no reports concerning the function of TBC1D13. TBC1D13 is an unusual member of the TBC family because whereas most TBC proteins are multi-domain proteins, TBC1D13 is comprised of a GAP domain alone. In addition, the GAP domain of TBC1D13 is much larger than that found in other TBC proteins due to the presence of several inserts, with one notable insert between the catalytic motifs. Here several pieces of evidence are provided indicating that
TBC1D13 is a specific Rab10 binding protein (Fig. 4.2, 4.3, 3.6). It should be noted that
TBC1D13 was also shown to interact with the ER to Golgi Rab1. I was unable to obtain any functional evidence for a role of TBC1D13 in ER/Golgi trafficking or on Rab1 GTP hydrolytic activity leaving the role of the TBC1D13/Rab1 interaction unclear. Notably,
106 TBC1D13 AKT p473 Basal Insulin
Figure 4.11 - TBC1D13 does not inhibit insulin stimulated phosphorylation of Akt. 3T3-L1 adipocytes electroporated with FLAG-TBC1D13 were basaled for 2 hr in DMEM and were either stimulated with 100 nm insulin for 20 min or received no dose (Basal). Cells were fixed and probed for TBC1D13 overexpression with a mouse monoclonal FLAG antibody and Akt activation was monitored with a rabbit polyclonal Akt pSer473 antibody. Phosphorylation of Akt Ser473 was imaged at the bottom and middle of the cell. Scale Bar 10 μM.
107 overexpression of TBC1D20, a RabGAP that stimulates the Rab1 hydrolytic activity, has been shown to disrupt ER to Golgi trafficking and Golgi morphology, both of which were reversed when TBC1D20 was rendered catalytically inactive (Haas et al., 2007; Sklan et al., 2007). Nevertheless, I did observe some co-localisation between TBC1D13 and
GM130, and as TBC1D20 is described as an exclusive ER membrane protein we cannot exclude a role for TBC1D13 on Rab1 at the Golgi.
The interaction between Rab10 and TBC1D13 combined with the inhibitory effect of
TBC1D13 overexpression on insulin-stimulated GLUT4 translocation is of interest due to
Rab10’s GSV localisation and its role in GLUT4 trafficking (Larance et al., 2005; Miinea et al., 2005; Sano et al., 2007). It should be noted that of all the Rabs residing on GSVs,
TBC1D13 bound specifically to Rab10. This interaction between TBC1D13 and Rab10 was GTP-dependent, with Rab10:GTPγS binding TBC1D13 >3-fold more compared to
Rab10:GDP. This indicates at least two possible functions for TBC1D13: as a
Rab10GAP or a Rab10 effector protein. Three pieces of evidence suggest that TBC1D13 is not a Rab10GAP: 1) TBC1D13 has an insert in a highly homologous region within the
RabGAP family of proteins that possibly disrupts any potential GAP activity; 2) There was no detectable in vitro GAP activity of TBC1D13 for Rab10; 3) The block in GLUT4 translocation seen with TBC1D13 overexpression could not be overcome by mutating the catalytic Arginine in the putative GAP domain. Hence, based on these data I suggest
TBC1D13 is a Rab10 effector. It is becoming apparent that RabGAPs have Rab effector type roles beyond their GTPase activating function. For example, The RabGAP EVI5 has been described as a binding partner for Rab11 but there is controversy in the literature as to whether it can also accelerate Rab11 GTP hydrolysis (Dabbeekeh et al., 2006;
108 Westlake et al., 2007). Overexpression of TBC1D14 disrupts Golgi morphology and this effect was independent of its catalytic ability (Haas et al., 2007). A recent study reported the RabGAPs in yeast are recruited to co-ordinate Rab cascades. Ypt32p recruited Gyp1p as an effector to regulate Ypt1p activity (Rivera-Molina and Novick, 2009). It is also well described that other GAPs, like IQGAPs, interact significantly with Rac and Cdc42 but display no GAP activity for these proteins, but rather function as an important scaffolding protein (Briggs and Sacks, 2003).
As a Rab10 effector, it is of interest that overexpression of TBC1D13 has a relatively specific effect to inhibit GLUT4 trafficking to the PM in adipocytes. This block in insulin-stimulated GLUT4 translocation likely involves a block in vesicle transport as no defect in insulin signalling was detected, at least to Akt. (Fig 4.11). I have been unable to reverse this effect either by mutating the GAP domain in TBC1D13 nor by co- overexpressing wild type Rab1 or Rab10. The mechanism for this inhibitory effect is not yet clear. I propose two possible mechanisms; overexpression of TBC1D13 sequesters active Rab10 and prevents its accessibility to other key effectors; alternatively, TBC1D13 acts as a negative regulator of Rab10 playing an important role in the basal intracellular sequestration of GLUT4 where the overexpression of TBC1D13 leads to an insulin insensitive dominant negative effect. Notably, TBC1D13 also binds to a number of proteins that may be linked to GLUT4 trafficking. In particular this includes the Bro1 domain containing protein Alix. Alix has been implicated in a range of functions including endosomal sorting and cytoskeletal rearrangement in the cell cortex (Cabezas et al., 2005; Morita et al., 2007).
109 I conclude that TBC1D13 is a highly specific Rab10 effector protein that plays an important role in GLUT4 trafficking in adipocytes. Several questions remain to be addressed from these studies. What is the functional outcome of the Rab10/TBC1D13 interaction and how does this interaction relate to some of the other TBC1D13 interactions observed here such as with Alix? Is there cross talk between TBC1D13 and
TBC1D4 regulated Rab10 activity on the one hand and between Rab10 and Rab1 regulation of traffic on the other? Although Rab1 and Rab10 govern trafficking events that are apparently remote a convergence in certain situations cannot be ruled out. In these circumstances TBC1D13 could be recruited to regulate the interplay. This latter point is of interest in view of recent findings that link the function of multiple Rabs and
RabGAPs in a coupled manner to ensure both vectoriality and specificity in protein sorting.
110
Chapter Five
Cellular strategies in regulating RabGAP function.
111
Introduction
Rab GTPases regulate eukaryotic vesicle trafficking and organelle identity (Stenmark,
2009). One of the features of this system is the characteristic Rab cycle. This cycle is composed of two major components; Rab membrane association, and Rab activity. The
Rab GTPases can bind to GDP and GTP resulting in a nucleotide-specific structural conformation (Dumas et al., 1999). These conformations recruit specific interaction partners. The GDP bound Rab becomes a target for the Rab recycling machinery such as the Guanine nucleotide Dissociation Inhibitor (GDI) which extracts the Rab from the membrane (Soldati et al., 1993; Ullrich et al., 1993), while the GTP associated Rab recruits Rab effector proteins that function in vesicle trafficking and organelle identity pathways (Allan et al., 2000; Christoforidis et al., 1999; Fukuda et al., 2002). Hence, when bound to GDP the Rab is “inactive” and binding to GTP results in an “active” conformation.
The Rab cycles between the inactive and active state through it’s intrinsic nucleotide exchange and GTP hydrolytic activities. These mechanisms underpin the switch-like nature of the Rab where it can alternate between the GDP and GTP conformations.
However, these intrinsic rates of exchange and hydrolysis are slow and the cellular action governed by these proteins requires more rapid regulation. To overcome this limitation the cell recruits two types of proteins to regulate activity by enhancing the intrinsic actions. The Rab GTPase exchange factors (RabGEFs) stimulate the exchange of nucleotides bound to the Rab. In vivo this results in the loading of a GTP molecule onto
112 the Rab, as the cellular concentration of GTP is ten-fold greater than GDP. The hydrolytic activity of the Rab is activated by the Rab GTPase activating protein
(RabGAP).
One of the consequences of this system is as soon as the Rab is switched into it’s active conformation it becomes a target for inactivation by the associated RabGAP. To ensure that functional complexes can form between the Rab and it’s effector molecules the cell must possess a strategy to regulate the function of the RabGAPs. This is required not only to facilitate fast processes mediated by Rabs such as vesicle tethering and fusion but also to enable Rab effector complexes to persist and define organelle identity through resident
Rab domains (Sonnichsen et al., 2000). The functional regulation of the RabGAPs is mediated via upstream signalling pathways and via protein-protein interactions.
The RabGAP TBC1D4, also known as AS160, was identified as a target of Akt phosphorylation in response to insulin stimulation in 3T3-L1 adipocytes (Kane et al.,
2002; Sano et al., 2003). This study identified two putative Akt sites that were phosphorylated in response to insulin. More recent studies indicate that TBC1D4 is phosphorylated at six sites in response to insulin stimulation (Chen et al., 2008; Geraghty et al., 2007). In insulin stimulation the binding of the insulin receptor to it’s ligand results in auto-phosphorylation that initiates a signal cascade to Akt, among other kinases, and regulates metabolic and biosynthetic processes (Manning and Cantley, 2007).
Overexpression studies revealed that phosphorylation of TBC1D4 at these residues is required for normal insulin-stimulated GLUT4 translocation (Sano et al., 2003).
Overexpression of a TBC1D4 mutant in which four of these sites were mutated to alanine inhibited insulin-stimulated GLUT4 translocation in adipocytes. Intriguingly, replacing
113 the catalytic arginine residue in the TBC domain of the phosphorylation mutant restored insulin-stimulated translocation of GLUT4 to normal levels. This demonstrated that insulin-directed phosphorylation of TBC1D4 is employed to regulate GLUT4 translocation by controlling an unidentified Rab GTPase. Reinforcing these observations were RNAi studies revealing that TBC1D4 silencing results in an increase of GLUT4 at the PM in unstimulated adipocytes (Eguez et al., 2005; Larance et al., 2005). Although the precise mechanism behind the alleviation in TBC1D4 regulation is unknown two important observations provide insight into this process. TBC1D4 was identified as a
GLUT4 vesicle resident protein that redistributes to the cytosol after insulin stimulation, and phosphorylation of TBC1D4 leads to 14-3-3 binding at the Akt phosphorylation sites
(Larance et al., 2005; Ramm et al., 2006). Although redistribution of TBC1D4 provides a simple explanation for its effects in GLUT4 trafficking it has been demonstrated that dissociation from the GLUT4 vesicle is not necessary for insulin stimulated GLUT4 translocation (Stockli et al., 2008). In fact, it was found that 14-3-3 association with
TBC1D4 was the crucial factor in controlling TBC1D4 regulation.
These observations provided the first model of how the insulin signal is translated into a
GLUT4 trafficking event. It is proposed that in the non-stimulated state TBC1D4 regulates the activity of a Rab in the GLUT4 trafficking pathway and maintains it in the
GDP inactive form. Insulin signalling results in the phosphorylation of TBC1D4 and 14-
3-3 association. 14-3-3 binding inhibits the ability of TBC1D4 to stimulate GTP hydrolysis on the relevant GLUT4 vesicle Rab possibly by altering the conformation of
TBC1D4, redistributing the RabGAP to the cytosol, or both. This results in the
114 accumulation of the active Rab, which facilitates translocation of GLUT4 to the cell surface.
These studies demonstrated how the cell could use phosphorylation to regulate the function of RabGAPs. Further examples of phosphorylation-directed control of a
RabGAP were observed for TBC1D1 (Chen et al., 2008; Peck et al., 2009; Roach et al.,
2007). TBC1D1 is closely related to TBC1D4 and shares 47% and 79% identity and a
61% and 91% similarity across the full-length protein and TBC domains, respectively.
The level of homology shared between the proteins and specifically across the TBC domains suggested the possibility that these proteins regulate the same Rab GTPases and vesicle pathways in the cell. Indeed the two most critical insulin responsive phosphorylation sites for GLUT4 translocation found in TBC1D4 are conserved in
TBC1D1 and are phosphorylated in response to insulin (Chen et al., 2008). Measurement of GAP activity using the TBC domains of each protein, revealed that they share a similar
Rab specificity. Both RabGAPs displayed activity toward Rab2, Rab8, Rab10 and Rab14 but not to any other of the Rabs found on GLUT4 vesicles (Miinea et al., 2005; Roach et al., 2007). In 3T3-L1 adipocytes, the overexpression of TBC1D1 resulted in an almost complete inhibition of insulin-stimulated GLUT4 translocation. In contrast to TBC1D1,
TBC1D4 can only block GLUT4 translocation when a phosphorylation resistant mutant is employed. Similarly to the phosphorylation resistant TBC1D4 mutant the TBC1D1 inhibition was reversed when the putative catalytic Arg in TBC1D1 was mutated. This demonstrates that, although TBC1D1 and TBC1D4 share the same Rab GTPase substrates and are phosphorylated in response to insulin on conserved sites, there is an additional layer of regulation in TBC1D1 function. Furthermore, reduction in the levels
115 of ectopically expressed TBC1D1 was able to partially relieve the block in GLUT4 translocation but not restore normal cell surface levels (Peck et al., 2009). Using reduced amounts of expression it was shown that mutation of the insulin responsive sites in
TBC1D1 resulted in a total block in GLUT4 translocation, a result that mirrors the use of the TBC1D4 phospho-resistant mutants. This reveals that while insulin signalling is a component in regulating TBC1D1 function, this stimulus alone cannot disable the negative regulation of this RabGAP on GLUT4 translocation. Studies into TBC1D1/14-
3-3 association revealed that, in contrast to TBC1D4, insulin stimulation does not promote TBC1D1/14-3-3 binding (Chen et al., 2008). 14-3-3/TBC1D4 association was shown to be critical in regulating insulin-stimulated translocation of GLUT4 and hypothesised to alter TBC1D4 conformation and/or redistribute the RabGAP away from its target Rab GTPase (Ramm et al., 2006; Stockli et al., 2008). Here, insulin-stimulation fails to initiate an inhibitory 14-3-3/TBC1D1 association and this accounts for the continued TBC1D1 suppression of GLUT4 translocation. These studies demonstrated that although TBC1D1 and TBC1D4 are both targets for insulin-stimulated phosphorylation and that both proteins regulate GLUT4 translocation; there is a distinct regulatory strategy that governs the function of each protein.
Phosphorylation profiling of TBC1D1 revealed several sites that were phosphorylated in response to different agonists. The most important observations in these studies were the identification of potential AMP-activated protein kinase (AMPK) sites (Chen et al., 2008;
Taylor et al., 2008). AMPK is a target in a signalling cascade that is responsive to the energy state of the cell and is activated when AMP levels rise and ATP levels fall (Kahn et al., 2005). This activation indicates the cell is facing an energy crisis. To overcome this
116 AMPK inhibits ATP consuming functions and switches on pathways that generate ATP.
Exercise of skeletal muscle is a physiological condition that causes such an energy crisis and is relieved through the activation of fatty-acid oxidation and stimulating glucose transport. AMPK activators stimulated the phosphorylation on TBC1D1 and resulted in
14-3-3 binding (Chen et al., 2008). These observations were applied to GLUT4 translocation studies in adipocytes where pre-treatment with an AMPK activator before insulin stimulation was able to partially reverse the inhibitory effects of TBC1D1 overexpression (Chavez et al., 2008). This suggests a model where signalling regulates
14-3-3 association with these RabGAPs to control their function in GLUT4 translocation.
Regulation of GAP function by phosphorylation is not restricted to TBC1D1/TBC1D4 and indeed one of the most studied phosphorylation regulated GAP functions is the regulation of the TSC1-TSC2 complex (Dan et al., 2002). This complex is a component of the insulin stimulated mTOR-Raptor complex (mTORC1) activation pathway. TSC-2 is the GAP for the Rheb small GTPase and regulates activation of the mTOR pathway
(Tee et al., 2003b). TSC-1 is a binding partner for TSC-2 and stabilises TSC-2 by preventing ubiquitination and targeted proteasomal degradation. Akt phosphorylates the
TSC1-TSC2 complex and this is proposed to change the sub-cellular localisation of the complex to relieve the negative regulation of the Rheb GTPase. This leads to the activation of mTORC1 and protein synthesis. This complex only functions as a hetero- dimer and mutation in either of these proteins can interfere with the overall function of the complex. The disease Tuberous-Sclerosis is caused by mutations in either TSC-1 or
TSC-2 highlighting the importance of each component of this dimer.
117 The investigations into the TSC1-TSC2 complex reveal that, in addition to phosphorylation-dependant regulation, protein complex formation can be critical to GAP function. In S. cerevisiae the RabGAPs Gyl1 and Gyp5 have been shown to form a functional hetero-dimer (Chesneau et al., 2004). These proteins were shown to interact directly and form a complex with the yeast Rab Sec4p (Chesneau et al., 2008). This interaction was found to be functionally significant as both are required for the correct formation of budding sites. Although it is not understood how or why these proteins function as a dimer it demonstrates that RabGAP function depends on more than availability of a Rab substrate.
This study seeks to further elaborate the regulation strategies used to control RabGAP function. Interrogating the action of insulin-stimulated phosphorylation has revealed an expanded cohort of responsive RabGAP members. In combination with the identification and profiling of RabGAP dimerisation these results demonstrate the broad strategies employed by the cell to regulate RabGAP function.
Results
TBC1D1 and TBC1D4 do not posses GAP activity to Rab43
TBC1D1 and TBC1D4 were both found to bind to Rab43 in Yeast Two Hybrid Screens of the Rab GTPase family (Chapter 3). Furthermore it was found that Rab43 bound specifically to the TBC domain of these proteins indicating that these RabGAPs could potentially stimulate the Rab hydrolytic activity. The ability of TBC1D1 and TBC1D4 to activate Rab43 GTP hydrolysis was determined in vitro. The TBC domain of TBC1D4, amino acids 865-1299, was employed in these assays as this construct has been observed
118 to stimulate hydrolytic activity whereas the full-length protein was inactive (Miinea et al.,
2005). Full-length TBC1D1 was used in these studies and the RabGAP TBC1D13 was included as a negative control (Fig. 5.1). The Rab43 intrinsic GTP hydrolysis rate was low, 0.007 min-1. Each RabGAP tested had a minimal effect on the hydrolytic rate and was found to be similar across all pairings including the catalytically inactive TBC1D4
TBC domain. This indicates that the small effects on the hydrolytic activity were unlikely to be due to GTPase activating potential of these proteins. These studies indicate that neither TBC1D1, TBC1D4 nor TBC1D13 are Rab43 GAPs.
TBC1D4 but not TBC1D1 homo-oligomerises
GAP proteins have been found in functional hetero-dimers that can regulate the activity of the GAP (Chesneau et al., 2004; Tee et al., 2003b). This oligomerisation may also be required for the normal function of the GAP as well as be. TBC1D4 has been observed to exist in a large complex of a molecular weight that suggests a possible hetero/homo- dimer arrangement (Ramm et al., 2006). To investigate the capability of TBC1D1 and
TBC1D4 to dimerise we utilised a Yeast Two Hybrid strategy to detect interactions between these proteins. No interaction was observed between TBC1D1 proteins or
TBC1D4 (Fig. 5.2). In contrast, TBC1D4 was observed to bind to itself in this screen. To investigate where the homo-dimerisation sites occur with TBC1D4, truncation mutants were screened. A construct that encodes the first 924 residues of TBC1D4 and a construct encoding the TBC domain, residues 812-1299, were employed in the Yeast Two Hybrid screen. Full-length TBC1D4 interacted with the TBC domain but not with the N-terminal
119 GDP
GTP
Time (min) 0 5 10 30 0 5 10 30 0 5 10 30
Rab43 Rab43 Rab43 TBC1D4 (865-1299) TBC1D4 (865-1299) RA
100
80 GDP
60
% GDP 40
20 GTP
0 0 10 20 30 Time (min) Time (min) 0 5 10 30 0 5 10 30
Rab43 Rab43 TBC1D1 TBC1D13
Figure 5.1 - Rab GAP assay with TBC1D1, TBC1D4 and Rab43. (A) TLC seperation of α-32P GDP and GTP in assays with GST-Rab43 alone, GST-Rab43 plus FLAG-TBC1D4 GAP domain, Rab43 plus FLAG-TBC1D4 GAP domain RA, Rab43 plus FLAG-TBC1D1, and Rab43 plus FLAG-TBC1D13. (B) Quantification of the data in (A) is presented as the percentage GDP versus time for Rab43 (♦), Rab43 plus TBC1D4 GAP (■), Rab43 plus TBC1D4 GAP RA (▲), Rab43 plus FLAG-TBC1D1 (+), and Rab43 plus FLAG-TBC1D13 (●).
120 Transformation Interaction Selection Selection
TBC1D1 1-1168 TBC1D1 1-1168 TBC1D4 1-1299
TBC1D4 1-1299
TBC1D4 812-1299 TBC1D4 1-1299
TBC1D4 1-924
TBC1D4 1-1299
TBC1D4 1-924 TBC1D4 812-1299
TBC1D4 812-1299
TBC1D4 1-1299 TBC1D4 1-924 TBC1D4 1-924
Fig. 5.2 - TBC1D4 forms a homodimer and a heterodimer with TBC1D1. Yeast two-hybrid assay; S. cerevisiae AH109 cells were co-transformed with the full-length TBC1D1 (1-1168) pGADT7 construct and full-length TBC1D1 or full length TBC1D4 (1-1299) in pGBKT7. TBC1D4 pGADT7 encoding an N-terminal mutant (1-924), a TBC1D4 TBC domain mutant (812-1299), or full-length TBC1D4 were co-transformed with each of the indicated TBC1D4 truncation in the pGBKT7 vector. Co-transformed yeast were serially diluted on synthetic media -Leu, -Trp, to select for transformants and onto -Leu, -Trp, -His media to select for interaction. No interaction was observed with TBC1D1. Full-length TBC1D4 was found to interact with full-length TBC1D4 and the TBC1D4 TBC domain. The TBC1D4 TBC domain mutant interacted with the full-length TBC1D4, the N-terminal mutant and the TBC1D4 TBC domain. The N-terminal mutant interacted with the TBC1D4 TBC domain only.
121 truncation mutant. The TBC domain of TBC1D4 was found to self-associate and to interact with the N-terminus of TBC1D4. The N-terminal TBC1D4 truncation mutant bound only to the TBC domain. This suggests that TBC1D4 can form homo-dimers with two possible configurations, either a head to head or head to tail arrangement (Figure
5.2). These results show that TBC1D4 possesses the ability to form homo-dimers, which may have implications for it’s cellular function.
TBC1D1 inhibits insulin-stimulated GLUT4 translocation
Due to the similarity between TBC1D1 and TBC1D4 in sequence and Rab specificity I undertook studies to determine whether TBC1D1 played a role in GLUT4 translocation.
While these studies were being performed the papers by Lienhard and colleagues on
TBC1D1 and GLUT4 translocation were published (Chavez et al., 2008; Peck et al.,
2009; Roach et al., 2007). FLAG-tagged TBC1D1 was electroporated into 3T3-L1 adipocytes that were expressing HA-tagged GLUT4 (Fig. 5.3). Overexpression of
TBC1D1 resulted in a block in insulin-stimulated GLUT4 translocation to the plasma membrane. The phosphorylation profiling of TBC1D1 revealed potential AMPK phosphorylation sites that were required for full insulin stimulation of the RabGAP (Chen et al., 2008). Therefore, I next tested the effects of activating AMPK on the TBC1D1 block in 3T3-L1 adipocytes. HA-GLUT4 expressing 3T3-L1 adipocytes were electroporated with FLAG-TBC1D1. These cells were then incubated with the AMPK activator AICAR before insulin stimulation. Pre-activation of AMPK resulted in a small reversal of the block in GLUT4 translocation but did not restore normal GLUT4 surface levels. These data are consistent with other published data (Chavez et al., 2008).
122 Insulin AICAR + Insulin
FLAG-TBC1D1HA-GLUT4 FLAG-TBC1D1 HA-GLUT4
FLAG-TBC1D4 HA-GLUT4FLAG-TBC1D4 HA-GLUT4
100
80
60 Insulin AICAR + Insulin
40
20 Cells with HA-GLUT4 rims (%)
0
TBC1D1 TBC1D4
Figure 5.3 - TBC1D1 overexpression inhibits insulin stimulated GLUT4 translocation. 3T3-L1 adipo- cytes were electroporated with FLAG tagged full-length TBC1D1 or TBC1D4 as indicated. Transfected cells were detected with the FLAG antibody. HA-GLUT4 expressing 3T3-L1 adipocytes were incubated in DMEM without serum and then incubated with 100nM insulin for 20 min or were treated with 2 mM AICAR for 30 mins before addtion of 100 nm insulin. GLUT4 translocation to the PM was determined by surface HA stain- ing. Quantification of HA-GLUT4 rims for independent experiments (n=2) is presented in the graph. Scale bar 10μm.
123 TBC1D1 overexpression does not interfere with organelle integrity
Due to the almost total inhibition in insulin-stimulated GLUT4 trafficking in 3T3-L1 adipocytes overexpressing TBC1D1 it was important to clarify whether this was a
GLUT4 specific effect or due to a broader defect in cellular trafficking. Disruption of
Rab GTPase and RabGAP function has, in some cases, resulted in a significant disruption of elements of the secretory pathway, such as the Golgi apparatus structure and endosomal maintenance (Bacon et al., 1989; Bucci et al., 1992; Haas et al., 2005; Segev et al., 1988). To determine that the basic organelle structures important for GLUT4 trafficking are intact in cells overexpressing TBC1D1 the Golgi apparatus and TGN were visualised using the markers GM130 and Syntaxin16, respectively. 3T3-L1 adipocytes were electroporated with FLAG-TBC1D1 and the Golgi apparatus and the TGN were visualised (Fig. 5.4) Neither GM130 nor Syntaxin16 localisation was affected by
TBC1D1 overexpression.
Insulin-stimulated phosphorylation of RabGAPs
Vesicle trafficking pathways have been observed as multi-station pathways that can involve several Rab GTPase and RabGAP regulated points (Fuchs et al., 2007). In
GLUT4 trafficking it is unknown whether insulin stimulation results in direct delivery from the GLUT4 storage compartment to the PM or whether a more circuitous route is taken through the TGN/endosomal systems. In the case of the latter this would require the participation of several Rab checkpoints and hence regulation by the associated
RabGAPs. This may require a regulated insulin response at these checkpoints akin to the proposed actions on TBC1D1 and TBC1D4. Therefore, changes in the phosphorylation
124 Syntaxin 16 FLAG-TBC1D1
A)
GM130 FLAG-TBC1D1
B)
Figure 5.4 - Overexpression of TBC1D1 does not disrupt the integrity of the Golgi apparatus or TGN. 3T3-L1 adipocytes were electroporated with FLAG tagged TBC1D1 and organelle integrity was examine by confocal microscopy. A) The TGN is unaffected by TBC1D1 overexpression as represented by Syntaxin16 labelling. B) The Golgi apparatus is unaffected by TBC1D1 overexpression as represented by GM130 label- ling. Scale bar 10 μm.
125 profiles of cellular RabGAPs in response to insulin would potentially indicate which members of this family regulate insulin responsive GLUT4 translocation and responsive membrane trafficking in general.
There are 52 RabGAPs in the human genome and each is postulated to regulate discrete vesicle transport steps (Bernards, 2003). To identify which if any of these proteins might be insulin regulated I took a global approach using phospho-proteomic analysis. To obtain quantitative information about phosphorylation I used stable isotope labelling with amino acids in cell culture (SILAC) combined with mass spectrometry. Here 3T3-L1 adipocytes were cultured in media containing “Light”, “Medium”, or “Heavy” isotopes of
Lysine and Arginine. Pre-adipocytes were passaged at least 5 times before differentiation was initiated. This resulted in 3T3-L1 adipocytes with at least 80% incorporation of the
Light, Medium or Heavy isotopes in the cellular protein component. To determine the insulin responsiveness of the RabGAPs three conditions were tested. All cells were serum depleted for two hours; the serum depleted “Light” adipocytes were the basal sample, the
“Heavy” adipocytes were insulin stimulated after serum depletion, and the “Medium” adipocytes were pre-treated with the PI3K inhibitor wortmannin before insulin stimulation. Adipocytes were lysed and the cleared whole cell lysate was passed over a strong cation exchange column and each fraction was digested with trypsin.
Phosphorylated peptides were isolated using Titanium oxide and then eluted for identification by LC-MS/MS analysis.
In addition to TBC1D4 nine other RabGAPs were identified as phosphorylated proteins
(Table 5.1). Comparing the abundance of a specific peptide across the three possible isotope labels allows for a quantification expressed as a ratio of the non-stimulated basal
126 Protein Sites identified Position Residue Insulin/Basal Wortmannin/Basal Insulin/Wortmannin 2D1CBT 1 S419 - - - B2D1CBT 6 S764 - - - 468 S 4.47 1.05 4.61 474 S 4.47 1.05 4.61 S574 - - - 959 T 0.99 1.02 0.88 965 S 0.95 1.08 0.94 TBC1D4 4 348 S 4.48 1.58 2.36 S153 - - - 577 S 7.22 1.48 4.78 595 S 7.81 1.95 3.68 B9D1CBT 3 S5801 - - - 1090 S 0.43 0.44 0.99 1092 T 0.80 0.98 0.81 TBC1D10B 3 643 S 0.57 0.9 0.64 644 S 0.57 0.9 0.64 664 S 0.62 0.89 0.72 TBC1D11 1 42 S 1.01 0.99 1 51D1CBT 2 S102 - - - 205 S 1.22 1.13 1.06 TBC1D16 3 98 T 2.81 1.27 2.44 101 S 2.61 1.28 2.26 102 S 2.81 1.19 2.44 TBC1D22A 1 169 S 0.89 0.81 0.94 TBC1D24 2 482 S 1.38 13.71 0.22 494 S 1.38 13.71 0.22
Table 5.1 - RabGAP phosphorylation is responsive to insulin stimulation. Phosphorylated peptides were identified from the listed RabGAPs. The number of phosphorylation sites discovered, position, and phosphorylated residue are detailed. The abundance of phosphorylated peptides are expressed as a ratio between the indicated treatments.
127 (Light) state. In this analysis I identified twenty-six phosphorylation sites across ten
RabGAPs. Among these sites seven were increased with insulin and these phosphorylation sites were found in TBC1D2b, TBC1D4 and TBC1D16. All of the sites that increased with insulin were sensitive to wortmannin to some degree. Three sites were found to decrease with insulin and these were found in TBC1D9b and TBC1D10b. Only the sites in TBC1D10b were wortmannin sensitive. TBC1D11, TBC1D15 and
TBC1D22a showed little or no change across the treatments. Wortmannin treatment increased phosphorylation on TBC1D24, while insulin stimulation only had a slight effect at this site. Although phosphorylation was detected on TBC1D2 there was not enough detected signal to calculate ratios across the treatments. These results demonstrate that insulin-stimulated phosphorylation occurs on RabGAPs other than TBC1D1 and
TBC1D4 suggesting a role for these proteins in regulating insulin responsive membrane trafficking.
Discussion
Understanding the regulation of RabGAPs will provide a vital clue as to how the cell orchestrates the dynamics of membrane trafficking. The emerging details of the integration of the signalling and trafficking of the GLUT4 molecule through the TBC1D1 and TBC1D4 RabGAPs serve as an example of how critical these roles can be (Chavez et al., 2008; Peck et al., 2009; Sano et al., 2003). In these studies it has been demonstrated that the observed regulation strategies through signalling and complex formation are not unique to specific RabGAP members but are likely to provide the basis of regulatory models for the entire family.
128 Dimerisation has been observed as a regulatory mechanism for other GAP proteins such as the regulatory protein-GAP interaction of TSC1-TSC2 (Tee et al., 2003a), and the
GAP-GAP architecture of the Gyp5p-Gylp complex (Chesneau et al., 2008). TBC1D4 appears to be able to form a homo-dimer. Dimerisation was found to specifically occur between TBC1D4 molecules and excluded the closely related TBC1D1, which was unable to dimerise with TBC1D4 or itself. The Yeast Two Hybrid profiling of the
TBC1D4 interaction provides two alternative arrangements of the homo-dimer. One possibility is a parallel configuration between the proteins with the alternate possibility a head to tail arrangement. It is unknown whether this dimerisation is required for functional regulation of the TBC1D4 Rab. It has been observed that the Rab4 GTPase binds to the N-terminus region of TBC1D4 (Chapter 3). This region can bind to the TBC domain in TBC1D4 in the head to tail configuration and suggests a possible mechanism where the N-terminus co-ordinates the delivery of a Rab to the GAP domain. While this provides an attractive mechanism it should be noted that the large TBC1D4 complex is observed in cytosolic fractions of the cell and it is unknown whether this complex exists on the membrane. As TBC1D4 translocates to the cytosol after stimulation it is consistent that dimerisation is a regulatory mechanism to negatively regulate the RabGAP function.
One possibility is that 14-3-3 facilitates TBC1D4 dimerisation (Ramm et al., 2006). 14-3-
3 forms homo-dimers while allowing each member of the dimer to retain binding to the phosphorylated 14-3-3 recognition motif. This is unlikely however because we have observed large oligomeric TBC1D4 structures in the cytosol of non-insulin stimulated cells. Although, one of the 14-3-3 binding sites in TBC1D4 appears to be constitutively phosphorylated (Sano et al., 2003) and so in this case it is conceivable that 14-3-3 bridges
129 two molecules of AS160 via interaction with this phosphorylation site in each molecule.
In response to insulin Akt phosphorylates TBC1D4 creating an additional 14-3-3 binding site. This would mean that under these circumstances 14-3-3 may switch from mediating
TBC1D4 dimerisation to binding both sites on TBC1D4 forming a monomer.
Alternatively, two 14-3-3 dimers may bind to two TBC1D4 molecules at these two different sites. However, this model would require that TBC1D4 is phosphorylated at the
14-3-3 constitutive site in yeast as we observed dimers in this system. This needs to be further explored in the yeast two-hybrid setting. To reveal the role dimerisation plays in
TBC1D4 function it will be crucial to determine whether TBC1D4 is present as homo- dimers on the membrane and also whether dimerisation inhibits or enhances GAP activity.
Previous studies have revealed a conundrum in the GAP activity profiling of TBC1D1 and TBC1D4. While full-length versions of these proteins have failed to stimulate Rab
GTPase hydrolysis, the TBC domain of each protein activates hydrolysis on a significant cohort of Rabs (Miinea et al., 2005; Roach et al., 2007). Activation of GTP hydrolysis has not been tested for every Rab but so far the TBC domain stimulates activity on Rab2,
Rab8, Rab10 and Rab14. These Rabs have been shown to directly function in post Golgi trafficking events and all could possibly be responsible for insulin-regulated GLUT4 trafficking. Investigating the Rab binding profiles of TBC1D1 and TBC1D4 revealed that both of these RabGAPs bind to Rab43 (Chapter 3). This suggested that TBC1D4 and
TBC1D1 would stimulate Rab43 hydrolytic activity. In vitro hydrolysis assays showed a very small effect of these RabGAPs on Rab43 hydrolytic activity and this activity was not reversed when the catalytic Arg was disabled. This suggests that neither TBC1D1 nor
130 TBC1D4 are Rab43 GAPs. This is an intriguing result as the binding profile between the
Rab and the RabGAP show an interaction in the TBC domain. The crystal structure of a
Rab/TBC domain demonstrates that interactions between the TBC domain and the Rab result in a configuration that promotes GTP hydrolysis (Pan et al., 2006). This would argue for a catalytic relationship between these two proteins. However experimentally hydrolytic activity is absent in these pairings. An alternative explanation could be that
Rab43 recruits TBC1D1/4 to regulate another Rab in a cascade-like manner. However, as
Rab43 recruitment occupies the TBC domain it is difficult to envisage how such an arrangement would be orchestrated. Dimerisation of TBC1D4 could fit this model.
Recruitment through Rab43 of a TBC1D4 homo-dimer in theory could be utilised to stimulate hydrolysis by the partner TBC1D4 on the true substrate Rab.
Phosphorylation-dependant regulation of TBC1D4 is critical for insulin stimulation of
GLUT4 translocstion. TBC1D1 and TBC1D4 are highly homologous; both RabGAPs are phosphorylated at conserved sites in response to insulin and share the same Rab substrates suggesting a highly related function. However, studies into the roles of these proteins in insulin-stimulated GLUT4 translocation reveal markedly different regulatory function. In contrast to TBC1D4, overexpression of wildtype TBC1D1 in 3T3-L1 adipocytes resulted in a complete block in GLUT4 delivery to the PM (Fig. 5.3). This is due to the failure of insulin to stimulate 14-3-3 binding which is proposed to inactivate the RabGAP. Activation of AMPK was able to partially relieve this block in GLUT4 translocation consistent with the observation that AMPK phosphorylation of TBC1D1 regulates 14-3-3 association (Fig. 5.3). My studies agree with the reports that were published by Lienhard and colleagues during these investigations. These studies observed
131 that pre-activation of AMPK by AICAR doubled the insulin stimulated surface level of
GLUT4 in adipocytes overexpressing TBC1D1, although this was still much lower than the response of control cells (Chavez et al., 2008; Peck et al., 2009).
Despite the similarity between these proteins, divergent strategies are employed in their regulation. In muscle cells it has been shown that AMPK activation can lead to GLUT4 translocation to the PM with no effect observed on translocation in adipocytes
(Fazakerley et al., 2010). This difference is thought to reflect the mechanism of exercise- stimulated GLUT4 translocation in muscle tissue, a pathway not required in adipose tissue. This has lead to a model where TBC1D1 and TBC1D4 are employed by a cell to regulate GLUT4 translocation in response to exercise or insulin, respectively (Taylor et al., 2008). Indeed expression profiling of these RabGAPs has revealed that TBC1D1 expression in adipocytes is almost negligible reflecting the requirement of insulin regulation of the GLUT4 translocation pathway only (Roach et al., 2007). Intriguingly profiling of different muscle types revealed a mixed expression profile for TBC1D1 and
TBC1D4 (Taylor et al., 2008). This indicates that in some muscle types GLUT4 translocation may be exclusively regulated by one of these RabGAPs and in cells co- expressing TBC1D1 and TBC1D4 both RabGAPs regulate GLUT4 translocation. This may indicate that in vivo some muscle types are required to be more sensitive to exercise induced glucose uptake, where TBC1D1 would dominate, while other cell types are more sensitive to insulin-stimulated uptake where TBC1D4 dominates. In muscle types with mixed expression it would be important that the signal from one stimulus is not inhibited by the alternate RabGAP and this may be overcome by distinct recruitable pools. Indeed, distinct GLUT4 pools were observed in detailed electron microscopy of muscle fibres
132 (Ploug et al., 1998). These studies also showed that stimulation with insulin or exercise exclusively recruited separate GLUT4 pools. It is possible that recruitment of the separate pools does not involve TBC1D1 or TBC1D4. GLUT4 vesicles may contain a mix of the regulatory RabGAPs and inactivation of TBC1D1 by exercise or TBC1D4 by insulin allows a critical threshold of active Rab to accumulate on the vesicle membrane. Recent studies show that TBC1D1 function is somewhat responsive to insulin and that TBC1D4 possesses AMPK phosphorylation sites (Peck et al., 2009; Treebak et al., 2010). This may partially inactivate the alternate RabGAP and further promote accumulation of the active Rab.
Quantitative phospho-proteomic profiling have revealed several other RabGAPs in adipocytes that display changes in phosphorylation in response to insulin (Table5.1).
Analogous to TBC1D4 the RabGAPs TBC1D2b and TBC1D16 were phosphorylated in response to insulin-stimulation and this was found to be wortmannin sensitive. This indicates that these proteins are the targets of a kinase that is downstream of PI3K. As the function of these two RabGAPs is largely unknown, the implications of insulin- stimulated phosphorylation of these proteins are unclear. TBC1D2b has been reported to bind Rab22 without stimulating GTP hydrolysis (Kanno et al., 2010) and TBC1D16 interacts with Rab2 by yeast two-hybrid (Fig. 3.9). Phosphorylation may facilitate the
Rab/RabGAP interaction, enhance the GAP activity or regulate protein complex formation/dissociation of these RabGAPs. This provides a mechanism for regulation of these proteins whether it is activation or inhibition of the RabGAP and allows the cell to integrate cellular signalling to regulation of membrane trafficking.
133 These studies indicate that signalling to the RabGAPs is likely to be a common strategy for regulation of membrane trafficking. These results already point towards further insulin-regulated RabGAPs and identifying their substrate Rab GTPases and associated trafficking pathways will further elaborate the cellular outcomes of insulin signalling.
Proteomic surveys in other signalling pathways have also identified RabGAPs as targets for phosphorylation. This represents a broad strategy of regulation where the cellular action of the RabGAPs is actively controlled to determine Rab GTPase function and provides a key avenue for further exploration into the mechanisms behind cargo trafficking and organelle identity.
134
Chapter Six
Rab10 recruits the RabGAP TBC1D15 in an effector-like
manner.
135
Introduction
In muscle and adipose tissue the insulin-stimulated trafficking of the GLUT4 transporter from an intra-cellular compartment to the PM is a vital mechanism in the maintenance of whole body blood glucose homeostasis (Huang and Czech, 2007). The GLUT4 trafficking itinerary is a complex multi-station pathway located between the plasma membrane (PM), recycling endosomes, the trans-Golgi network (TGN) and tubular- vesicular GLUT4 storage compartments (Martin et al., 2000; Shewan et al., 2003; Slot et al., 1991). During this itinerary GLUT4 will travel through many trafficking stations each mediated and regulated by a host of molecules. Although several key molecules have been revealed in this pathway many remain unknown. One of the features of this pathway is the significant overlap between this pathway and other more general pathways such as the Transferrin receptor (TfR) recycling pathway (Livingstone et al., 1996). These observations suggest that GLUT4 trafficking is governed by regulatory steps common to both general trafficking as well as the more specific pathways. Identifying and establishing the function of these players will reveal the intricacies of this pathway and further our understanding of how the body regulates glucose disposal.
Recent investigations into GLUT4 trafficking have revealed that the RabGAP members
TBC1D1, TBC1D4 and TBC1D13 have a critical role in insulin-stimulated translocation of GLUT4 (Eguez et al., 2005; Larance et al., 2005; Roach et al., 2007). RabGAPs are regulatory proteins for the Rab GTPase family that bind a specific Rab to stimulate GTP hydrolysis and switch the Rab to an inactive state (Stenmark, 2009). As the trafficking pathway of GLUT4 is likely to be mediated by several Rabs there will also be several
136 RabGAPs that regulate these steps. However, the RabGAPs identified thus far as functioning in insulin-stimulated GLUT4 translocation have all been associated with one
Rab GTPase, Rab10 (Miinea et al., 2005; Roach et al., 2007; Sano et al., 2007). Rab10 localises to a tubulovesicular network and endosomes where it has been shown to play a role in endosomal traffic and basolateral sorting (Babbey et al., 2006; Roland et al., 2009;
Sebastian et al., 2007). A proteomic survey of GLUT4 vesicles derived from 3T3-L1 adipocytes identified several Rabs, including Rab10, as a GLUT4 vesicle constituent
(Larance et al., 2005; Miinea et al., 2005). Moreover, overexpression of constitutively active Rab10 in 3T3-L1 adipocytes led to increased surface GLUT4 in the absence of insulin and RNAi mediated knock down of Rab10 levels in adipocytes inhibited insulin- stimulated GLUT4 translocation (Sano et al., 2007; Sano et al., 2008). Hence, these studies indicate that Rab10 is a key component of the regulatory machinery that catalyses the docking and fusion of GLUT4 vesicles with the PM. This observation suggests that the effects on GLUT4 translocation of TBC1D1, TBC1D4 and TBC1D13 may all be mediated through Rab10. Notably, the GTPase activity of Rab10 is stimulated by
TBC1D1 and TBC1D4 (Miinea et al., 2005). This was intriguing since TBC1D4 was first identified as an Akt substrate in adipocytes (Kane et al., 2002) that is localised to GLUT4 containing vesicles (Larance et al., 2005). Phosphorylation was shown to play a crucial role in TBC1D4 GAP activity at least in part due to regulated 14-3-3 binding (Ramm et al., 2006). Based on these findings a model has been put forward whereby TBC1D4 controls the GTP loading of Rab10 in an insulin-dependent manner.
Considering the role of Rab10 in GLUT4 trafficking we sought to isolate and identify
Rab10 effectors. Research into Rab10 function has so far been limited to defining the
137 trafficking pathways regulated by this Rab and the Rab10 effectors have yet to be established. An isoform of Rim1 and the Class V Myosin motors have been demonstrated to be Rab10 interaction partners (Fukuda, 2003; Roland et al., 2009). However, no functional mechanism has been characterised for these interactions. Identification of
Rab10 effectors will help elucidate the specific GLUT4 trafficking step that is governed by Rab10 and may help to define how this is regulated as opposed to Rab10’s broader functions.
Surprisingly, we have identified a new member of the RabGAP family, TBC1D15, as a binding partner of Rab10 that conforms with the profile of a Rab effector. Intriguingly we also confirm that TBC1D15 is a potential VAMP2 binding partner. Functional studies in adipocytes indicate that TBC1D15 has no detectable effect on insulin-dependent delivery of GLUT4 to the PM but unlike TBC1D4, it does not display significant GAP activity on
Rab10.
Results
Rab10 GTPase isolates two RabGAPs from 3T3-L1 adipocytes
We sought to determine the effectors that bound one of the GSV Rab GTPases, Rab10.
GST Rab10 was purified from HEK293-FT cells and loaded with either GDP or GTPγS.
The nucleotide-loaded Rab was incubated with 3T3-L1 adipocyte lysate from insulin- stimulated or basal adipocytes. After washing, the Rab10 interacting proteins were eluted by complexing the Mg2+ and incubating with the opposite nucleotide. The eluted proteins were resolved by SDS-PAGE and visualised with SyproRuby staining. As shown (Fig.
6.1A), a number of bands were specifically isolated with the active and inactive GST-
138 Basal Insulin A) GDPGTPγS GDP GTPγS kDa
205
116
97
80
66
55
45
30
21
14
B) Basal Insulin GDP GTPγS kDa GDP GTPγS 251
172 IB: TBC1D4
86 IB: TBC1D15 64
Figure 6.1 Rab10 isoates TBC1D15 in a nucleotide specific manner. GST-Rab10 loaded with GDP or GTPγS was incubated with lysate prepared from 3T3-L1 adipocytes. Adipocytes were pre-treated with serum starvation for 2 hrs followed by 100 nm insulin for 30 mins (Insulin) or recieved no insulin stimulation (Basal). A) Eluted Rab10 binding proteins were resolved by SDS-PAGE and visualised with Sypro Ruby staining. Arrows indicate protein bands that are either enriched or only present in the GTP γS conditions. B) Eluted Rab10 binding proteins were probed with TBC1D4 and TBC1D15 antibodies.
139 Rab10. Gel slices were excised, digested with trypsin,peptides were eluted from the gel and subjected to LC-MS/MS analysis. One of the proteins identified was the RabGAP
TBC1D15 and this interaction was found in the insulin-stimulated fraction.
I sought to confirm this result using a TBC1D15 antibody for immuno-detection. GST
Rab10 was purified from HEK293-FT cells and loaded with either GDP or GTPγS. The nucleotide loaded Rab was incubated with 3T3-L1 adipocyte lysate from insulin- stimulated or basal adipocytes. Interacting proteins were eluted by complexing the Mg2 and association with TBC1D15 was determined by immuno-blotting (Fig. 6.1B).
TBC1D15 interacted with GTPγS but not GDP-loaded GST-Rab10 in an insulin- independent manner. TBC1D4 was not detected in these affinity purifications.
TBC1D15 binds to VAMP2
TBC1D15 has been reported to bind to VAMP2 by Yeast Two-Hybrid and in a pull-down approach (Zhang et al., 2005). In addition to Rab10, VAMP2 is one of the major constituents of GSVs and these interactions may link TBC1D15 to GLUT4 trafficking
(Malide et al., 1997; Volchuk et al., 1995). Here we sought to recapitulate these binding data and expand the analysis to several other members of the RabGAP family. FLAG tagged VAMP2 and GFP tagged RabGAPs were co-transfected into HEK293-FT cells.
FLAG-VAMP2 was immunoprecipitated with the monoclonal FLAG antibody and the
RabGAP interaction was determined by probing for GFP (Figure 6.2). Immunopurified
VAMP2 associated with eGFP tagged TBC1D15 and with TBC1D17. However, no interaction was observed with eGFP alone, TBC1D5 or TBC1D13. Interestingly,
TBC1D15 and TBC1D17 display 65% amino acid sequence identity. Although binding
140 eGFP eGFP-TBC1D5 eGFP-TBC1D13 eGFP-TBC1D15 eGFP-TBC1D17 eGFP eGFP-TBC1D5 eGFP-TBC1D13 eGFP-TBC1D15 eGFP-TBC1D17
IB: GFP
Start Material FLAG-VAMP2 IP
Figure 6.2 - Vamp2 co-purifies the RabGAPs TBC1D15 and TBC1D17. HEK 293-FT cells were co-transformed with FLAG-tagged VAMP2 and the indicated eGFP tagged RabGAPs. VAMP2 was isolated by purification with FLAG antibody. FLAG-VAMP2 was eluted with the FLAG peptide and co-purified RabGAPs were detected with GFP antibody.
141 between TBC1D17 and VAMP2 is weaker than with TBC1D15 it suggests a related or analogous function for these two RabGAPs.
TBC1D15 localises to the cytosol and perinuclear region in adipocytes
Several members of the RabGAP family are localised to internal membranes despite the lack of readily identifiable membrane targeting domains. TBC1D15 does not possess such a domain and has been previously observed as a cytosolic protein in CHO cells
(Zhang et al., 2005). Sub-cellular fractionation analysis of 3T3-L1 adipocytes revealed that TBC1D15 is only found in the cytosol fraction and this was unaltered with insulin
(Fig. 6.3B). This localisation agrees with the observations by Zhang et al. However, these data are not consistent with TBC1D15 as a stable VAMP2 or Rab10 binding protein, since VAMP2 is a membrane protein concentrated in the LDM and PM fraction, and
Rab10 resides in the HDM, LDM and PM fractions (Sano et al., 2008). We sought to further determine the localisation of this protein in 3T3-L1 adipocytes. I was unable to detect specific labelling using the TBC1D15 antibody so eGFP tagged TBC1D15 was electroporated into 3T3-L1 adipocytes. eGFP-TBC1D15 was found both in the cytosol and associated with internal membrane structures localised to the peri-nuclear region
(Fig. 6.3A).
TBC1D15 does not possess GAP activity to Rab10
TBC1D15 binds to Rab10 in a nucleotide specific manner and localises to a similar region in 3T3-L1 adipocytes. We sought to investigate whether TBC1D15 accelerates
Rab10 GTP hydrolysis in vitro. The GAP domain of TBC1D4 was used as a positive
142 eGFP-TBC1D15 PM CYT HDM LDM
B I B I B I B I kDa
165 IB: IRAP
72 IB: TBC1D15
Fig 6.3 - TBC1D15 localises to the peri-nuclear region and the cytosol. A) 3T3-L1 adipocytes were electroporated with eGFP-tagged TBC1D15. TBC1D15 was visualised by GFP fluorescence. Scale bar 10 μm. B) 3T3-L1 adipocytes were serum starved for 2 hr (B) and stimulated with 100 nM insulin for 20 min where indicated (I). Treated adipocytes were seperated in high density membranes (HDM), low density membranes (LDM), plasma membrane (PM), and cytosol (CYT) fractions by differential centrifugation.
143 control (Miinea et al., 2005) and full-length TBC1D13 was used as a negative control
(Fig 6.4A). As observed in the experiments in Figure 4.6, Rab10 possesses a low intrinsic
GTPase activity (0.0038 min-1.) I observed a catalytic efficiency for Rab10 GTP hydrolysis in the presence of the TBC1D4 GAP domain (865-1299) of 0.165 min-1μM-1
(kcat/Km).
-1 -1 TBC1D13 had minimal catalytic efficiency for Rab10 –0.0075 min μM (kcat/Km) and I
-1 -1 found no activity of full-length TBC1D15 towards Rab10 –0.0185 min μM (kcat/Km). It should be noted that no full-length RabGAP has been found to stimulate Rab10 hydrolytic activity and only truncations encoding the TBC domain of TBC1D1 and
TBC1D4 have reported activity. Hence, I decided to test the ability of the TBC domain of
TBC1D15 to stimulate Rab10 GTP hydrolysis (Fig 6.4B). A FLAG-tagged TBC1D15
GAP fusion was generated comprising amino acids 294-640. As above, the low intrinsic activity of Rab10 was observed, 0.0106 min-1, and the TBC1D4 GAP domain increased
-1 -1 this activity, 0.135 min μM (kcat/Km). Consistent with the data using full-length
TBC1D15, no stimulatory effect of the TBC1D15 GAP domain on Rab10 GTP
-1 -1 hydrolysis was observed, -0.0225 min μM (kcat/Km). A catalytic inactive version of the
TBC1D15 GAP domain, generated by mutating the putative catalytic arginine residue to
-1 -1 an alanine had a catalytic efficiency of –0.0385 min μM (kcat/Km). These studies indicate that TBC1D15 does not posses Rab10 GAP activity.
Overexpression of TBC1D15 does not effect GLUT4 translocation
Rab10 and VAMP2 are both GSV constituents and play important roles in insulin- stimulated GLUT4 translocation. Several RabGAPs linked to Rab10 have been shown to modulate GLUT4 trafficking (Roach et al., 2007; Sano et al., 2003), Chapter 4, Chapter
144 A)
GDP
GTP
Time 0 5 10 30 0 5 10 30 0 5 10 30 0 5 10 30 (min) Rab10 Rab10 Rab10 Rab10 TBC1D4 (865-1299) TBC1D13 TBC1D15
Time (min) 0 5 10 30 0 5 10 30 0 5 10 30 0 5 10 30 Rab10 Rab10 Rab10 Rab10 TBC1D4 (865-1299) TBC1D15 (294-600) TBC1D15 (294-600) RA B) D) 100 100
80 80
60 60 % GDP 40 % GDP 40
20 20
0 0 0 10 20 30 0 10 20 30 Time (min) Time (min) Figure 6.4 - Rab GAP assay with TBC1D15 GAP and Rab10. (A) TLC seperation of α-32P GDP and GTP in assays with GST-Rab10 alone, GST-Rab10 plus FLAG-TBC1D4 GAP domain, Rab10 plus FLAG-TBC1D13, and Rab10 plus FLAG-TBC1D15. (B) Quantification of the data in (A) is presented as the percentage GDP versus time for Rab10 (♦), Rab10 plus TBC1D4 GAP (■), Rab10 plus TBC1D13 (▲), and Rab10 plus TBC1D15 (●). (C) TLC seperation of α-32P GDP and GTP in assays with GST-Rab10 alone, GST-Rab10 plus FLAG- TBC1D4 GAP domain, Rab10 plus FLAG-TBC1D15 GAP domain, and Rab10 plus FLAG-TBC1D15 GAP RA domain. (D) Quantification of the data in (C) is presented as the percentage GDP versus time for Rab10 (♦), Rab10 plus TBC1D4 GAP (■), Rab10 plus TBC1D15 GAP (▲), and Rab10 plus TBC1D15 GAP RA(●).
145 5). TBC1D15 may be recruited by VAMP2 and Rab10 to regulate a substrate Rab and co- ordinate GLUT4 translocation. To determine this, overexpression studies were performed investigating the effect of TBC1D15 on GLUT4 translocation. 3T3-L1 adipocytes expressing HA-GLUT4 were electroporated with FLAG-TBC1D15 and insulin- stimulated HA-GLUT4 translocation was quantified by immunofluorescence microscopy using surface HA labelling to detect HA-GLUT4 (Fig. 6.5). Overexpression of TBC1D15 resulted in no defect in GLUT4 translocation with surface HA detection being similar to the control eGFP electroporated cells.
Discussion
The investigation into identifying Rab10 effector molecules (Figure 6.1) found a strong
GTP-dependent association between Rab10 and TBC1D15. Although the initial identification was from insulin-stimulated adipocyte lysate, subsequent analysis found this association from both basal and insulin treated cells. This increases the number of
RabGAPs associated with Rab10 and suggests a complex regulatory role for Rab10.
TBC1D15 has been identified as a Rab7 and Rab11 GAP (Zhang et al., 2005). This assignment was determined by an in vitro GAP assay against a limited number of Rab
GTPases that did not include Rab10. We found no GAP activity of TBC1D15 towards
Rab10 while the GAP domain of TBC1D4 was able to stimulate hydrolysis. This suggests that TBC1D15 is recruited by Rab10 in an effector-like manner. TBC1D15 could act as a bona fide member of a Rab10 effector complex that mediates membrane trafficking or it could be recruited by Rab10 to establish a Rab cascade with a sequential
Rab. Due to Rab10’s activity in endosomal trafficking and the possibility of TBC1D15 possessing Rab7 and Rab11 GAP activity this could place Rab10 in a trafficking pathway
146 HA-GLUT4 FLAG-TBC1D15 A)
B) 100
80
60
40
20 Cells with HA-GLUT4 rims (%) 0 Control TBC1D15
Fig. 6.5 - TBC1D15 overexpression does not affect insulin-stimulated GLUT4 translocation. FLAG tagged TBC1D15 was electroporated into retrovirally infected HA-GLUT4 3T3-L1 adipocytes. A) Trans- fected adipocytes were serum starved for 2 hr then stimulated with 100nm insulin for 20 min. Plasma mem- brane GLUT4 levels were determined by surface HA antibody staining. Scale bar 10 μm. B) Quantification of cells with HA-GLUT4 rims after insulin stimulation. Control cells were electroporated with an eGFP expressing construct. n=2
147 involving Rab7 and/or Rab11. Rab7 and Rab11 have been characterised to function in the late endosomal trafficking pathway where Rab7 regulates lysosomal trafficking and
Rab11 regulates trafficking through recycling endosomes (Casanova et al., 1999; Feng et al., 1995; Meresse et al., 1995; Ullrich et al., 1996). Rab10 has been implicated in trafficking through a specific endosomal compartment that was distinct from early endosomal machinery as well as classical recycling endosome markers (Babbey et al.,
2006; Sebastian et al., 2007). Hence, an association between Rab10 and TBC1D15 suggests a trafficking pathway to late endosomal compartments through a Rab10 positive endosome.
Our initial Yeast Two-Hybrid screens identified an interaction between TBC1D15 and
Rab29 (Chapter 3). No interaction was found with Rab10 in this system in contrast to the association observed in the Rab10 pulldowns. This suggests that the interaction between
Rab10 and TBC1D15 may be indirect and possibly mediated through an intermediary protein, such as VAMP2. Alternatively, the interaction between TBC1D15 and Rab10 may require an additional post-translational modification not observed in yeast.
TBC1D15 has been found to possess several phosphorylation sites (Chapter 5) and so this deserves further investigation.
TBC1D15 was reported to interact with one of the major GSV constituent proteins,
VAMP2 (Zhang et al., 2005), by Yeast Two Hybrid and co-immunoprecipitation. In these studies, this observation was recapitulated and expanded. It was found that TBC1D15 interacts with VAMP2 although association was also detected between VAMP2 and
TBC1D17. As noted above, TBC1D15 and TBC1D17 are highly homologous. This suggests that these proteins may have a similar function in vesicle trafficking. An
147 example of this is the role of the highly related TBC1D1 and TBC1D4 in GLUT4 trafficking. It is hypothesised that TBC1D1 and TBC1D4 act on the same Rab GTPase but respond to differing regulatory signalling. This model could also apply for TBC1D15 and TBC1D17. Determining if both function in the same trafficking pathways and how each RabGAP is regulated will be critical in testing this theory.
The association of a RabGAP with the membrane fusion machinery adds a further element to the co-ordination between Rab GTPases and SNARE proteins. The interaction between VAMP2 and TBC1D15/TBC1D17 suggest that these RabGAPs may be involved in regulating some aspect of VAMP2 vesicle fusion. VAMP2 is a v-SNARE that interacts with t-SNARE proteins, such as Syntaxin4 and SNAP23, to form SNARE complexes that mediate membrane fusion (Kawanishi et al., 2000). It has been shown that Rab effector complexes associate with SNARE machinery and play an essential role in this fusion
(McBride et al., 1999; Ohya et al., 2009). Hence, RabGAP binding to VAMP2 presents a possible mode of regulation for assembly of the Rab effector complexes with the SNARE proteins. If these RabGAPs bind to VAMP2 specifically this would present a mechanism to link trafficking regulation to membrane identity. A similar version of this model is demonstrated by the interaction between TBC1D1/TBC1D4 and IRAP, another major
GSV constituent. These RabGAPs have been shown to bind to the cytosolic tail of IRAP and it is proposed that this targets TBC1D1/4 to GSVs to control those GSV resident
Rabs (Peck et al., 2006; Ramm et al., 2006). This suggests a common mechanism where
RabGAPs could be targeted to their functional sites through interactions with cargo proteins or the membrane fusion machinery. This would allow a mechanism of linking specific cargo trafficking directly to its regulatory machinery.
148 As Rab10 and VAMP2 are important participants in GLUT4 trafficking, the association with TBC1D15 suggests this RabGAP could be recruited to regulate a Rab in the GLUT4 trafficking pathway. In contrast to other Rab10-associated RabGAPs we were unable to find a functional effect of this interaction on insulin-stimulated GLUT4 translocation.
This observation suggests that if TBC1D15 does function in GLUT4 translocation that it’s role may be more layered and complex; i.e. investigations into TBC1D4 only yielded effects on GLUT4 trafficking or Rab10 hydrolysis when phosphorylation and truncation mutants were used in the analysis (Miinea et al., 2005; Sano et al., 2003). Future investigation of TBC1D15 function in GLUT4 trafficking would focus on testing a series of phosphorylation mimetic and resistant TBC1D15 mutants as well as determining the exact nature of Rab10 binding and identifying whether the association is direct or mediated through another Rab10 effector. Alternatively the Rab10/TBC1D15 interaction may have no functional consequence in GLUT4 translocation and instead may be utilised in the Rab cascade regulation of a trafficking pathway defined in part by Rab7, Rab10, and Rab11.
Rab10 appears to be the target of RabGAPs TBC1D1, TBC1D4, TBC1D13, and
TBC1D15. The relationship to each RabGAP is specific and robust yet the functional relationship and whether these associations intersect remain elusive. There are several possible explanations for the multiple RabGAP association of this Rab. Rab10 could be a substrate for the GAP activities of all these RabGAPs. This interpretation is complicated by the fact that no activity has been reported here or in the literature for these proteins against Rab10. Indeed, only utilising fragments of the protein encoding the TBC domain yield appreciable hydrolysis and this observation is limited to TBC1D1/4 (Miinea et al.,
149 2005; Roach et al., 2007). Another interpretation posits that these RabGAPs are Rab10 effectors that are recruited to co-ordinate the regulation of Rab GTPases upstream or downstream in the trafficking pathway, a parallel pathway or possibly establish membrane identity. In fact, all these RabGAPs have been demonstrated to interact with at least one other Rab apart from Rab10 (Chapter 3, Chapter 4) providing a mechanism for this cross-talk between the noted Rabs and Rab10. This could place Rab10 at the convergence of several trafficking pathways where it is involved in a sorting step that resolves cargo delivery to the late endosome, TGN, or PM.
This study has discovered an additional Rab10 effector that indicates a high level of complexity at Rab10 regulated trafficking stations. It will be critical to resolve whether
Rab10 is a member of a Rab cascade as this could explain why it has numerous RabGAP binding partners. It will also be important to identify further Rab10 effectors as well as define the precise compartment where Rab10 and the GLUT4 trafficking pathway intersect.
150
Chapter Seven
General Discussion
151
The regulation of Rab GTPase inactivation by the RabGAPs is a critical arm of the Rab cycle. Spatial and temporal regulation of Rab activity is a necessary component in the cellular orchestration of membrane identity and transport. The members of the Rab
GTPase family are targeted to specific functional locations within the cell. Here the Rab recruits Rab effectors to mediate its regulatory role in membrane dynamics. A tight regulatory environment for these processes is required to ensure the precision accuracy of vesicle trafficking and organelle identity is preserved.
The yeast and mammalian genomes reveal that there has been a proportional expansion in both Rab and RabGAP families during the course of evolution. This suggests a tight coupling between individual members of each family. The sequential expansion of these pairings affords cells a greater fidelity for regulation presumably as a feature of the development from uni-cellular to multi-cellularity. Intriguingly this expansion has been accompanied by a somewhat restricted diversification in Rab structure presumably because its function is relatively fixed or conserved between different vesicle transport steps (Dumas et al., 1999; Esters et al., 2000; Huber and Scheidig, 2005). Conversely, there has been a dramatic rearrangement in the shape and appearance of the RabGAPs indicating that the major capacity for functional diversity is likely embedded in these proteins. For example, aside from the TBC domain, the conserved region that stimulates
Rab GTP hydrolysis, members of the RabGAP family display an assortment of functional domains arranged in a variety of architectures (Bernards, 2003).
The major experimental approach to identify the Rab/RabGAP pairings has been through in vitro assays measuring GTP hydrolysis. Investigations into the yeast RabGAPs, the
152 Gyp proteins, revealed a broad specificity with one individual Gyp displaying GTPase activity towards as many as five Ypt proteins (Albert and Gallwitz, 1999; Brett et al.,
2008; Will and Gallwitz, 2001). Investigation into mammalian RabGAP activity has been more limited due to the sheer number of possible combinations between the sixty Rabs and forty RabGAPs. So far the mammalian RabGAPs demonstrate a more restricted profile in activating Rab hydrolysis (Fuchs et al., 2007; Haas et al., 2005; Haas et al.,
2007; Miinea et al., 2005), however the majority of RabGAPs remain untested.
The specialised nature of Rab function will require that its regulatory mechanisms be equally as focussed. To this end my studies concentrated on identifying the functional
Rab/RabGAP interactions, determine the role of these pairings in membrane and cargo trafficking, and explore the cellular strategies employed to regulate the RabGAPs.
Rab-RabGAP association
A major focus of my investigations was to determine the Rab GTPase interaction profile for each RabGAP. This would potentially identify the RabGAPs that regulate the tested
Rabs and provide a starting point for investigation of RabGAP function on Rab mediated vesicle trafficking. To this end a yeast two-hybrid approach described by Haas et al. was adopted to screen for potential pairings. Each RabGAP was screened against a yeast two- hybrid library comprising 43 of the mammalian Rabs. This methodology provided an efficient means to navigate and test the vast combinatorial possibilities between these families.
The RabGAPs studied were found to bind to multiple Rab GTPases in the yeast two- hybrid screens (Chapter 3.0). These studies revealed a high degree of specificity such that each RabGAP displayed a unique profile of Rab interaction. These interactions
153 potentially identify the substrate Rabs for these RabGAPs. The yeast two-hybrid screens provide a rapid and systematic approach to reveal the pairings for further investigation. It was important to validate these results by independent experimental means. Although the yeast two-hybrid screens resulted in the generation of specific Rab/RabGAP interactions this approach can be criticised as being highly artificial as, the detected interactions occur between overexpressed fusion proteins that are forced into the same cellular location and the assay utilises catalytic mutants for both the Rab and the RabGAP.
The TBC1D13 yeast two-hybrid interaction with Rab1 and Rab10 was confirmed by two independent experimental approaches. The first approach employed a co- immunoprecipitation strategy using a TBC1D13 antibody. Endogenous TBC1D13 was purified from 3T3-L1 adipocytes and mass spectrometry analysis of the co-purified binding partners identified Rab1 and Rab10 (Fig 4.2 and Table 4.1). The second approach employed an affinity purification method. Recombinant GST-tagged Rab10 was produced and used to purify FLAG tagged TBC1D13 from HEK293-FT lysate in a GTP dependent manner (Fig 4.4). These TBC1D13 binding studies demonstrate the validity of the TBC1D13 interactions with Rab1 and Rab10 detected by the yeast two-hybrid screens. TBC1D13 was also found to have an effect on insulin-stimulated GLUT4 translocation, which is a Rab10-regulated trafficking pathway (Larance et al., 2005; Sano et al., 2007; Sano et al., 2008). This suggests that the interactions generated by the yeast two-hybrid screens represent functional pairings that exist within mammalian cells to regulate membrane trafficking.
The Rabs detected by yeast two-hybrid are potential substrates for their associated
RabGAPs. I hypothesised that interactions mediated by the TBC domain in the RabGAP
154 would reflect a pairing between the RabGAP and a substrate Rab. To this end, the interaction sites between TBC1D4 and it’s partner Rabs, Rab4 and Rab43 were mapped.
Rab binding was mediated by discrete locations in TBC1D4. The interaction between
Rab4 occurred in the N-terminal region of TBC1D4 while the interaction with Rab43 was mediated through the TBC domain (Fig 3.11). This suggests that Rab43 may be a substrate for TBC1D4 GAP activity. It is possible that binding outside the TBC domain helps recruit the RabGAP in a way that co-ordinates activity on Rab4. However, this seems unlikely as it has been reported that the TBC domain of the Rab4 GAP, TBC1D11, is sufficient and necessary to bind Rab4 indicating that there is no impediment for stimulation of Rab4 GTP hydrolysis by it’s associated TBC domains (Fuchs et al., 2007).
Additionally it has been reported that TBC1D4 has no Rab4 GAP activity (Miinea et al.,
2005).
The absence of GTP hydrolytic activity in the interaction between TBC1D4 and Rab4 suggests the recruitment of the RabGAP by the Rab in an effector like manner. This presents two possibilities; that TBC1D4 is a bona fide member of a Rab4 effector complex that mediates a membrane trafficking event, or that TBC1D4 is recruited by
Rab4 to regulate the activity of a substrate Rab. Although the first possibility cannot be ruled out, the second is observed in Rab cascades and provides an attractive mechanism to co-ordinate cross-talk and trafficking between Rabs (Rivera-Molina and Novick,
2009). This suggests that the Rabs that bind to the same RabGAP are functionally linked and regulate the same membrane trafficking pathway. Indeed, recruitment by one Rab may be required for a RabGAP to function on its substrate Rab. Mapping the RabGAP interaction sites for the Rab/RabGAP pairings will reveal which Rabs are potential GAP
155 substrates and which Rabs recruit the RabGAP in an effector like manner. Investigations into membrane trafficking pathways could then be performed using RabGAPs with selective mutations that abolish binding to its partner Rabs. This could identify which of these Rabs co-ordinate trafficking pathways through Rab cascades and reveal the sequential layout of Rabs in that pathway.
RabGAPs can be recruited by Rab GTPases in an effector-like manner
One of the major aims of my studies was to identify and functionally evaluate novel
Rab/RabGAP interactions. The yeast two-hybrid screens yielded several novel interactions and validation experiments demonstrated these pairings extend beyond the two-hybrid system. Profiling the interaction domains in the yeast two-hybrid screens and publication of studies during my investigations demonstrate that Rabs may recruit
RabGAPs in an effector-like manner that does not result in stimulation of GTP hydrolysis. Consequently, the results garnered from the yeast two-hybrid studies could represent RabGAP binding to substrate Rabs in addition to effector-like interactions. This potentially links the Rabs that bind to same RabGAP in a Rab cascade where one Rab recruits the RabGAP to inactivate the substrate Rab.
Whether a Rab recruits a RabGAP in an effector-like manner or is a substrate for hydrolytic activation can be distinguished through GTP hydrolysis assays. Somewhat surprisingly, none of the tested pairings from the yeast two-hybrid screens generated a functional interaction in the hydrolysis assays (Fig 4.6 and 5.1), despite the observation that these Rabs bound to the TBC domain of the associated RabGAPs. As outlined above,
156 the interactions observed by yeast two-hybrid could be recapitulated by other experimental means demonstrating that these pairings are not an artefact of that binding assay. This leads to the conclusion that the interactions generated through the yeast two- hybrid screen identify the effector-like pairings between the Rab and RabGAPs rather than identifying the catalytic pairings. This suggests that the Rabs identified in the yeast two-hybrid screen will recruit the RabGAP and co-ordinate the activity on the substrate
Rab. This arrangement may regulate the convergence of several trafficking pathways through a single Rab checkpoint, or may reflect a mechanism to ensure that an active Rab does not accumulate in a specific cellular location, or may have developed to overcome variations in tissue specific expression of the Rab GTPases.
The results provided by the GTP hydrolysis assays are unambiguous. However, the robustness of these assays can be questioned. This is an in vitro analysis that overlooks the potential involvement of other binding partners, membrane composition and localisation, or phosphorylation profiles. This could account for the incongruous results that have been observed when profiling RabGAP function. TBC1D1 and TBC1D4 have been shown to exhibit Rab hydrolysis in vitro only when expressed as a C-terminal truncation encoding the GAP domain alone (Miinea et al., 2005; Roach et al., 2007).
Full-length versions of these RabGAPs yielded no activity under the same conditions.
Assuming the Rabs activated by the TBC domain of TBC1D1/4 are true substrates, there must be some critical factor absent from the assay required for full-length RabGAP hydrolysis. The RabGAP EVI5 has been described as a Rab11 GAP, however this result is also subject to controversy as it was found to stimulate Rab11 hydrolysis in one study but showed no activity in another (Dabbeekeh et al., 2006; Westlake et al., 2007). This
157 indicates that in the cases where a co-factor is required, profiling RabGAPs in this manner may not reveal their substrate Rab. However, it remains the only method available to distinguish between an effector-like or substrate interaction between the Rab and RabGAP and should be included in the experimental approach to functionally dissect
Rab/RabGAP associations.
These studies have revealed that RabGAPs associate with specific Rabs and suggests the cell will utilise these proteins as part of a tailored mechanism to regulate the Rab co- ordinated trafficking pathways. It was observed that Rab interaction can be mediated through regions of the RabGAP that lie outside the TBC domain. This suggests that these
Rabs can recruit the associated RabGAP in a manner that does not result in stimulation of
GTP hydrolysis. This is an important characteristic for the co-ordination of a Rab cascade by a RabGAP. The yeast two-hybrid screen potentially identifies functional relationships between different Rabs that are linked together by their common RabGAP. These associations provide a starting point for functionally dissecting Rab/RabGAP-regulated vesicle trafficking and provide insight into how these pathways may be co-ordinated through cascades between the Rabs.
The role of TBC1D13 in GLUT4 trafficking
One of the interactions identified in the yeast two-hybrid screens was between TBC1D13 and Rab10 (Fig 3.6). Rab10 is an important component of the insulin-stimulated GLUT4 trafficking pathway (Sano et al., 2007; Sano et al., 2008). Investigations into the functional effect of the TBC1D13/Rab10 interaction on Rab10 mediated trafficking revealed the overexpression of TBC1D13 had an inhibitory effect on GLUT4 translocation (Fig 4.9). In accordance with the observation that TBC1D13 is not a Rab10
158 GAP (Fig 4.6) expression of a catalytically inactive version of TBC1D13 did not restore
GLUT4 translocation. This suggests that the defect in GLUT4 trafficking may be due to the disruption of a Rab cascade between Rab10 and an as yet unidentified TBC1D13 substrate Rab. It will be critical to identify the substrate Rab and an approach expanding the GTP hydrolysis assay to survey the entire Rab family should be employed. As outlined above the interaction with Rab1 and Rab10 nominates potential TBC1D13 Rabs, and the first members that should be investigated are the Golgi resident Rabs such as
Rab2, Rab6, Rab33 and trans-Golgi network Rabs such as Rab8 and Rab13. Identifying the TBCD13 substrate Rab will allow experiments to be designed to test whether this Rab and Rab10 function in a cascade through TBC1D13. This will be important to distinguish whether TBC1D13 acts in GLUT4 translocation through it’s associated Rabs or by another mechanism potentially indicated by it’s non-Rab interaction partners (Fig 4.2 and
Table 4.1).
To further investigate the role of TBC1D13 in insulin-stimulated GLUT4 translocation it will be important to perturb TBC1D13 function in several ways. As the overexpression of
TBC1D13 blocks delivery of GLUT4 to the plasma membrane, investigations to determine the effect of reduced TBC1D13 through RNAi will be critical. It is tempting to predict that knockdown should result in an increase in basal or insulin-stimulated
GLUT4 levels at the plasma membrane caused by the unregulated activity of the substrate Rab. However, as there appears to be a GAP-independent component to the
TBC1D13 inhibition of GLUT4 translocation the outcome of knocking down TBC1D13 may not yield such a clear outcome.
159 It will be important to identify how TBC1D13 is regulated in controlling Rab GTPase function and cargo trafficking. Studies into TBC1D1 and TBC1D4 function have revealed that phosphorylation of RabGAPs is a key method for regulating their function
(Kane et al., 2002; Peck et al., 2009; Roach et al., 2007; Sano et al., 2003). My investigations have also revealed that a number of RabGAPs undergo changes in their phosphorylation profile in response to insulin signalling (Table 5.1). TBC1D13 was not identified in those studies, this approach was not designed to specifically isolate any particular protein and TBC1D13 phosphorylated peptides may not have been abundant.
This could be overcome with the use of the TBC1D13 antibody to purify endogenous
TBC1D13 for the analysis of its phosphorylation profile. Discovering if insulin- responsive phosphorylation sites exist on TBC1D13 will help reveal whether TBC1D13 functions at an actively regulated station in the GLUT4 translocation pathway, or alternatively, that TBC1D13 controls a more constitutive stage of GLUT4 trafficking.
RabGAPs integrate signalling into membrane trafficking
The regulation of the Rab GTPase cycle is a critical component of the overall control of membrane trafficking by the Rab family (Stenmark, 2009). RabGEF and RabGAP activity not only switch the activation state of the Rab but also determine where and how long a Rab state is maintained. One of the aims of these investigations was to identify the cellular regulatory strategies employed to control RabGAP function.
GLUT4 trafficking to the PM is stimulated by insulin in adipocytes, and by insulin and exercise in muscle tissue (Huang and Czech, 2007). The distinct regulation strategies of
TBC1D1 and TBC1D4 integrate the insulin and exercise signals to release GLUT4 to the
PM (Kane et al., 2002; Peck et al., 2009; Roach et al., 2007; Sano et al., 2003). A model
160 is evolving where TBC1D4 comprises the insulin responsive arm in GLUT4 translocation and TBC1D1 governs the exercise response (Taylor et al., 2008). TBC1D4 is expressed at much higher levels than TBC1D1 in adipocytes. In fact, the level of TBC1D1 expression in adipocytes is negligible (Roach et al., 2007). This may explain the observation that AMPK activation has little to no effect on GLUT4 translocation in adipocytes. Muscle tissue appears to express both of these RabGAPs (Taylor et al.,
2008). This presents an interesting problem as to how muscle cells co-ordinate GLUT4 trafficking in response to exercise versus insulin. TBC1D1 and TBC1D4 are hypothesised to regulate GLUT4 trafficking by maintaining the same Rab in the GDP state. In the absence of insulin or exercise, GLUT4 is stored in GSVs away from the PM.
Stimulation by insulin or exercise likely relieves the inhibitory effect of TBC1D4 or
TBC1D1, respectively. However, under this situation one of the RabGAPs will still be active and thus capable of regulating Rab activity in a manner that may oppose the actions of the other. This suggests that muscle cells have developed a strategy that allows
GLUT4 translocation to proceed while one arm of the basal GLUT4 regulatory mechanism is still active.
One possibility is that in muscle, TBC1D1 and TBC1D4 regulate distinct pools of GSVs that can be mobilised to the PM. Electron microscopy studies of muscle fibres has revealed two distinct GLUT4 pools (Ploug et al., 1998). These pools were distinguished through the level of co-localisation of GLUT4 with the Transferrin receptor (TfR) and were shown to be differentially recruited to the PM in response to insulin or exercise.
Insulin resulted in the mobilisation of vesicles that contained GLUT4 and very little TfR while exercise recruited vesicles that contained both GLUT4 and TfR. The differential
161 regulation of these pools could be mediated through TBC1D1 and TBC1D4. In this model, TBC1D1 would regulate the TfR-positive GLUT4 vesicles while TBC1D4 would regulate the vesicles that only contain GLUT4. One outstanding question in this model is what is the mechanism that directs the specific RabGAP to each pool? It is thought that
TBC1D1/4 are recruited to GSVs through interaction with the GSV co-resident protein
IRAP (Peck et al., 2006; Ramm et al., 2006). As there is no evidence to suggest that the
IRAP interaction can be controlled to favour a specific RabGAP, it is unclear how such a model could convey distinct targeting of these RabGAPs to different populations of vesicles. Interestingly, the yeast two-hybrid studies revealed crucial differences in Rab interactions for TBC1D1 and TBC1D4 (Fig 3.1 and 3.2). TBC1D4 bound Rab4 and this interaction was not observed for TBC1D1. Rab4 has been found on GLUT4 vesicles and could participate in recruiting TBC1D4 to GSVs (Hashiramoto and James, 2000; Kessler et al., 2000). Indeed, studies in adipocytes revealed that Rab4 is enriched in the insulin responsive vesicles while it was present at reduced levels on the non-responsive vesicles containing GLUT4 and TfR (Hashiramoto and James, 2000). This would allow the cell to establish a population of GLUT4 vesicles that favour the presence of TBC1D4 over
TBC1D1. Alternatively, the association between Rab4 and TBC1D4 may influence the formation of the insulin responsive GLUT4 pool. The formation of GLUT4 vesicles that translocate to the cell surface are thought to be derived from the endosomal system
(Lampson et al., 2001; Xiong et al., 2010). Rab4 is a key regulator of trafficking through the sorting and recycling endosomes. The TBC1D4/Rab4 interaction may somehow co- ordinate the sorting of GLUT4 into the insulin responsive vesicles through a Rab cascade-like mechanism. Formation of vesicles in this way could intrinsically link the
162 establishment of this pool to its ability to be recruited by insulin stimulation through
TBC1D4.
However, if formation of these pools is independent of TBC1D1/4 and the association to the GSV is mediated only through IRAP then another model must be developed. A single pool of GSVs regulated by TBC1D1 and TBC1D4 would be comprised of vesicles that possess both RabGAPs on the membrane. If the majority of vesicles have equal amounts of each RabGAP it may be feasible that inactivation of either TBC1D1 or TBC1D4 allows the accumulation of a critical threshold of active Rab to facilitate GLUT4 trafficking. Recent studies have shown TBC1D1 to be somewhat responsive to insulin- stimulation (Peck et al., 2009). In the same vein, TBC1D4 possesses AMPK phosphorylation sites (Treebak et al., 2010). This suggests that both RabGAPs may be partially inactivated by both stimuli allowing for the accumulation of sufficient active
Rab. Furthermore, studies in muscle cells have shown that insulin or exercise stimulated
GLUT4 translocation result in similar levels of GLUT4 at the cell surface and co- stimulation results in a greater response (Wallberg-Henriksson et al., 1988; Zorzano et al., 1986). The co-stimulation produces an additive effect that is much greater than stimulation with a single agonist. These data fits the two-pool model of regulation, as in the single pool model only a minority of the GLUT4 vesicles would have been retained in response to a single agonist and fail to account for the level of increase in response to insulin and exercise stimulation.
There are several further avenues for investigation into the dual TBC1D1/4 control of
GLUT4 translocation. In addition to identifying the true substrate Rab for these
RabGAPs it will be important to functionally evaluate the interaction between Rab4 and
163 TBC1D4. Rab4 recruitment could establish a bias for TBC1D4 association to GLUT4 vesicles or it could be involved in forming the distinct GLUT4 pools in muscle. This could be examined by mapping the IRAP and Rab4 binding sites on TBC1D4 and developing binding mutants for these and examining their role in GLUT4 trafficking.
Generating a TBC1D1 chimera that binds Rab4 and investigating its effects in GLUT4 translocation could examine functional evaluation of Rab4 binding. As recent studies have implicated a role for insulin signalling in TBC1D1 function it will be important to investigate the insulin directed mechanism for TBC1D1 regulation.
The investigations into TBC1D1 and TBC1D4 have shown how the cell employs distinct signalling strategies to regulate RabGAP function and that this is critical in co-ordinating trafficking of GLUT4. I sought to determine whether signalling is employed as a broad strategy to regulate RabGAP function. Using a quantitative mass spectrometry analysis of phosphorylated proteins (Mann, 2006) I was able to identify several RabGAPs that are phosphorylated (Table 5.1). As previously outlined two RabGAPs, TBC1D2b and
TBC1D16, were insulin responsive as phosphorylation at their sites increased in response to insulin and were sensitive to wortmannin treatment. This is similar to the insulin- response of TBC1D1 and TBC1D4. Although insulin-stimulated phosphorylation of
TBC1D4 relieves its inhibitory action on a substrate Rab, the effect on TBC1D2b and
TBC1D16 cannot be assumed to be similar. Little is known about either of these
RabGAPs. A very recent study has identified TBC1D2b as a Rab22 interaction partner that does not stimulate GTP hydrolysis (Kanno et al., 2010). Rab22 plays an important role in trafficking through the early endosomal compartments and may recruit TBC1D2b to regulate an endocytic Rab in a cascade arrangement (Mesa et al., 2001; Zhu et al.,
164 2009). This suggests that insulin may potentially have a role in regulating aspects of endocytic trafficking. This is an intriguing result as recent models have emerged describing endosomes as platforms that help organise the spatial and temporal circuitry of cellular signalling pathways (Scita and Di Fiore, 2010). This suggests a model where insulin signalling to TBC1D2b could potentially modulate signalling through changes to in endocytic compartments and would establish a cross-talk mechanism between signalling and trafficking. To investigate the model several pieces of information will have to be established; 1) TBC1D2b will have to be confirmed as a bona fide Rab22 effector. 2) The substrate Rab for TBC1D2b will need to be identified. 3) The effect of
Rab22 and the TBC1D2b substrate Rab on signalling from the endosomes will need to be determined. 4) The insulin-stimulated phosphorylation of TBC1D2b will need to be assessed for a functional role in regulating its associated Rabs. 5) TBC1D2b co- ordination of signalling endosomes will need to be established.
TBC1D16 was identified as a Rab2 interacting protein. This potentially places TBC1D16 function at either the ERGIC, the medial-Golgi, or at the trans-Golgi network as Rab2 has been characterised as functioning at all of these locations (Chun et al., 2008; Short et al., 2001; Tisdale et al., 1992). It is often overlooked that Rab2 is a GLUT4 vesicle resident (Larance et al., 2005) and that was found to be a substrate Rab for TBC1D1 and
TBC1D4 hydrolytic activity (Miinea et al., 2005). It is possible that Rab2 has a more important role in regulation of insulin-stimulated GLUT4 trafficking than previously anticipated. It will be important to identify the substrate Rab for TBC1D16 and establish the function of insulin-stimulated phosphorylation on this RabGAP. The regulation of
165 insulin-stimulated GLUT4 trafficking may be co-ordinated by a Rab cascade defined by
Rab2, TBC1D4, TBC1D16 and the TBC1D16 substrate Rab.
These studies have illustrated that signalling to the RabGAPs is a critical mechanism for regulating their function on Rab GTPases and the associated membrane trafficking pathways. Cellular signalling pathways can be stimulated with a myriad of agonists.
Although my studies focussed on phosphorylation in response to insulin it is almost certain that RabGAPs will be signalling targets in several pathways. Quantitative mass spectrometry analysis of phosphorylated proteins will provide an excellent means to identify RabGAPs targeted in response to different stimuli and presents a powerful means to reveal the cellular regulation of RabGAPs.
Mapping the network
The Rab GTPases are central players in cellular membrane dynamics providing a temporal and spatial regulatory mechanism that orchestrates membrane trafficking and organelle identity. The role of the RabGAP in regulating Rab function is developing from a passive member of the Rab cycle to an active component in co-ordination of vesicle trafficking. This is best illustrated by the emerging roles of members of this family in co- ordinating trafficking between Rabs through cascades and integration of cellular signalling with membrane trafficking. The human RabGAP family is comprised of 52 members and only a minority have been investigated. This study has made novel discoveries about the shared characteristics of members of this family related to the interactions between Rabs and RabGAPs and highlighted the role signalling plays in
RabGAP regulation. Furthermore, investigations into TBC1D13 have identified a novel role for this protein in GLUT4 trafficking, highlighting the complexity of this trafficking
166 pathway. As outlined above and in preceding chapters there are many questions and avenues of investigation to explore. These will help unlock the intricacies of the cargo and membrane trafficking networks within the cell and identify the strategies employed to regulate their function.
167
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168
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