analysis of gtpase-activating proteins: rab1 and rab43 are ......2998 the sorting of secretory cargo...

2997Research Article

IntroductionVectorial protein transport between the ER and Golgi complexrequires the coordinated action of many different small GTP-binding proteins of the Ras superfamily, each controllingdistinct aspects of the cargo sorting, vesicle formation andvesicle fusion processes (Bonifacino and Glick, 2004; Zerialand McBride, 2001). These GTP-binding proteins share acommon mode of action involving the recruitment of cytosolicprotein complexes to localized domains on membrane surfaces(Munro, 2002; Short et al., 2005). Sar1 and ARF1 recruitCOPII and COPI coat complexes to ER and Golgi membranes,respectively, thus promoting vesicle formation (Bonifacino andGlick, 2004). Rabs recruit protein complexes important fordirected vesicle movement and target recognition (Pfeffer,1999; Waters and Hughson, 2000). To do this, these proteinscycle between a GDP bound inactive state, and a GTP boundmembrane-associated active state (Pfeffer and Aivazian, 2004;Vetter and Wittinghofer, 2001). It is this GTP bound state thatmakes specific interactions with so-called effector complexes,and thus promotes their recruitment to membranes. This cycleof activation and inactivation is under the control of GDP-GTPexchange factors (GEFs) and GAPs (Pfeffer and Aivazian,2004; Zerial and McBride, 2001).

In the case of ER to Golgi trafficking, Rab1 and Rab2 havebeen implicated in the tethering and fusion reactions, and arethought to act sequentially (Allan et al., 2000; Alvarez et al.,

1999; Segev, 1991; Tisdale and Balch, 1996; Tisdale et al.,1992). First, COPII vesicles, formed from specialized exit siteson the endoplasmic reticulum (ER exit sites: ERES), aretethered together by the action of Rab1 together with itseffector p115 to give rise to vesicular-tubular clusters (VTCs)adjacent to the Golgi (Allan et al., 2000; Alvarez et al., 1999;Bannykh et al., 1996; Cao et al., 1998). TRAPP, the GEF forRab1, promotes tethering of COPII vesicles by activating Rab1and also serves as a structural component of the tether(Barrowman et al., 2000; Jones et al., 2000; Sacher et al., 2001;Wang et al., 2000). Recent evidence shows that TRAPP isassembled onto forming COPII vesicles by direct interactionwith the coat, thus providing a mechanism to ensure all vesiclescan recruit active Rab1 (Cai et al., 2007). Second, Rab2 andits effectors promote the maturation of these VTC structuresand their incorporation into the Golgi (Short et al., 2001;Tisdale and Balch, 1996). Finally, in addition to its role inCOPII vesicle tethering, Rab1 may also have a function inregulating the exit of secretory cargo from the ER. This ideaarose from experiments showing that extraction of Rabs usingthe Rab chaperone GDI (guanine nucleotide dissociationinhibitor) prevented the exit of the vesicular stomatitus virusG-protein (VSV G) from the ER and that this could becomplemented by a complex of Rab1 and GDI (Peter et al.,1994). Later, Ypt1 and Uso1, the yeast homologues of Rab1and p115, respectively, were found to play a role in coupling

Rab GTPases control vesicle movement and tetheringmembrane events in membrane trafficking. We used the 38human Rab GTPase activating proteins (GAPs) to identifywhich of the 60 Rabs encoded in the human genomefunction at the Golgi complex. Surprisingly, this screenidentified only two GAPs, RN-tre and TBC1D20, disruptingboth Golgi organization and protein transport. RN-tre is theGAP for Rab43, and controls retrograde transport into theGolgi from the endocytic pathway. TBC1D20 is the ER-localized GAP for Rab1, and is the only GAP blocking thedelivery of secretory cargo from the ER to the cell surface.Strikingly, its expression causes the loss of the Golgicomplex, highlighting the importance of Rab1 for Golgi

biogenesis. These effects can be antagonized by reticulon, abinding partner for TBC1D20 in the ER. Together, thesefindings indicate that Rab1 and Rab43 are key Rabsrequired for the biogenesis and maintenance of a functionalGolgi structure, and suggest that other Rabs acting at theGolgi complex are likely to be functionally redundant.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/120/17/2997/DC1

Key words: Vesicle transport, Golgi complex, ER-exit site, RabGTPases, TBC-domain

Summary

Analysis of GTPase-activating proteins: Rab1 andRab43 are key Rabs required to maintain a functionalGolgi complex in human cellsAlexander K. Haas1,2, Shin-ichiro Yoshimura1,2, David J. Stephens3, Christian Preisinger2,*, Evelyn Fuchs1and Francis A. Barr1,2,‡1Cancer Research Centre, University of Liverpool, 200 London Road, Liverpool, L9 3AT, UK2Department of Cell Biology, Max-Planck-Institute of Biochemistry, Martinsried 82152, Germany3Department of Biochemistry, University of Bristol, Bristol, UK*Present address: Beatson Institute of Cancer Research, Glasgow, UK‡Author for correspondence (e-mail: [email protected])

Accepted 1 July 2007Journal of Cell Science 120, 2997-3010 Published by The Company of Biologists 2007doi:10.1242/jcs.014225

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the sorting of secretory cargo to vesicle formation (Morsommeand Riezman, 2002), providing further support for this idea.

To determine which specific Rabs are of general importancefor ER to Golgi trafficking and Golgi organization inmammalian cells, we have screened 38 human Rab GAPs fortheir ability to disrupt the organization of the Golgi complexand protein transport. Rab GAPs are characterized by aconserved catalytic domain, the TBC (Tre2/Bub2/Cdc16)domain (Albert and Gallwitz, 1999; Albert et al., 1999; Stromet al., 1993), that promotes GTP hydrolysis by a dual arginine-glutamine-finger catalytic mechanism related to the arginine-finger mechanism of Ras GAPs (Ahmadian et al., 1997; Albertet al., 1999; Pan et al., 2006; Rak et al., 2000). As we haveshown previously, in the case of Rab5 (Haas et al., 2005), RabGAPs can be used to specifically inactivate the endogenouspool of a Rab and thus interfere with the process that this Rabis involved in. By mutating the catalytic arginine residue of theTBC domain to alanine, the non-specific effects of GAPexpression can easily be discriminated from the specific effectsof Rab inactivation (Haas et al., 2005).

ResultsA screen for TBC domain proteins that alter GolgistructureTBC-domain containing proteins, putative Rab GAPs, werescreened for their ability to alter Golgi structure whenoverexpressed in either HeLa or telomerase-immortalizedhuman retinal epithelial (hTERT-RPE1) cells (Fig. 1). Of the38 TBC domain proteins tested, only three showed anysignificant effects on Golgi organization in both HeLa andhTERT-RPE1 cells (Fig. 1A). One of these, TBC1D14 waseliminated since the catalytically inactive form showedequivalent or increased effects on the Golgi when compared tothe wild-type protein (Fig. 1A). This left two candidateregulators of Golgi-localized Rab GTPases, TBC1D20 andRN-tre, both capable of causing activity dependentfragmentation of both the cis-Golgi marker GM130 and thetrans-Golgi marker TGN46 (Fig. 1B,C).

To further define the site of action of the different GAPs, theeffects of their expression on the ER-Golgi intermediatecompartment (ERGIC) were examined (Fig. 2). In this screenTBC1D20 showed catalytic activity dependent ERGICfragmentation (Fig. 2A,B). Interestingly RN-tre caused onlysubtle changes to the ERGIC (Fig. 2A,B), suggesting that thisGAP is more important for regulating trafficking at other Golgicompartments. This is consistent with our previous findingsthat RN-tre, together with its target Rab43, is required forretrograde trafficking to the trans-Golgi, but not anterogradecargo transport through the Golgi (Fuchs et al., 2007) (seesupplementary material Figs S1 and S2). Interestingly,TBC1D22B, a potential GAP for Rab33 (Pan et al., 2006) wasfound to cause ERGIC disruption (Fig. 2A). Other GAPsshowed only weak or no effect on the ERGIC. BecauseTBC1D20 had a general effect on Golgi and ERGICorganization, and is a highly conserved protein (seesupplementary material Fig. S3), it was studied further.

TBC1D20 expression causes a loss of Golgi phenotypeTo establish what happens to the Golgi upon TBC1D20expression, markers for different Golgi sub-compartmentswere investigated (Fig. 3). TBC1D20 expression results in the

loss of recognizable Golgi or punctate vesicular staining forp115, golgin84 and golgin97, but has no effect on the stainingof the endosomal Rab5 effector EEA1 (Fig. 3A). TBC1D20,therefore, perturbs all compartments of the Golgi. None ofthese effects were seen when catalytically inactive forms ofTBC1D20 were used (Fig. 3B), indicating that these effects arelikely to be due to the catalytic inactivation of one or more Rabfamily GTPases.

These observations raised the question of where the Golgiproteins have redistributed to in TBC1D20-expressing cells.Peripheral membrane proteins such as GM130 and p115 canrelocate to the cytosol, but integral membrane proteins such asgolgin84 and Golgi enzymes must still be present in membranestructures. Alternatively, Golgi proteins could be degraded. Totest these possibilities a series of experiments was performed.First, the fate of a Golgi enzyme was followed. This revealedthat in TBC1D20-expressing cells, N-acetylglucosaminyl -transferase (NAGT-I) is redistributed to the ER, identified bythe marker calnexin (Fig. 3C). When a catalytically inactivemutant of TBC1D20 was used, NAGT-I remained in the Golgi(Fig. 3C). Second, to test if Golgi proteins were degraded,western blots were performed on extracts of cells expressingwild-type or catalytically inactive TBC1D20. Despite the hightransfection efficiency, which was over 50% as judged byfluorescence microscopy, there was no change in the levels ofeither GM130 or p115 in cells expressing either form ofTBC1D20 (Fig. 3D). Finally, the redistribution of peripheralGolgi membrane proteins to the cytosol was tested. This showedthat GM130 behaved like the integral membrane proteingolgin84 and was associated with the membrane pellet to thesame extent in control cells and in cells expressing wild-typeTBC1D20 (Fig. 3E). Interestingly, under the same conditions,the pool of membrane-associated p115 was lost in TBC1D20-expressing cells (Fig. 3E). This effect is not simply due toblocking ER-Golgi membrane trafficking or the disruption ofthe Golgi complex, since BFA, which specifically inactivatesCOPI, and the GTP-restricted form of Sar1, which blocks theCOPII pathway by preventing vesicle uncoating, did not causeloss of p115 from the membrane pellet or change thedistribution of GM130 (Fig. 3E). Together, these results suggestthat Golgi proteins are still associated with membrane structuresafter TBC1D20 expression, but their fates may be different.Similar to BFA treatment, Golgi enzymes are present in the ER,whereas Golgi matrix proteins such as GM130 are associatedwith a haze of small vesicular remnants. Significantly, the Rab1effector p115 was lost from membranes in TBC1D20-expressing cells, indicating that there may be defects in theRab1-dependent COPII vesicle-tethering pathway, andsuggesting that TBC1D20 may be a regulator of Rab1.

TBC1D20 is a GAP for Rab1 and 2 in vitroTo identify candidate target Rabs for TBC1D20, an unbiasedtwo-hybrid screen was performed, based on the use of mutantslocking the specific Rab-GAP interaction (Haas et al., 2005).This approach revealed that Rabs 1 and 2 are potentialcandidate targets of TBC1D20 (A.K.H. and F.A.B.,unpublished). To directly test this possibility, biochemicalGTP-hydrolysis assays were performed (Fig. 4). When testedagainst an extensive selection of Rabs, TBC1D20 stronglystimulated GTP hydrolysis by Rab1 and Rab2 (Fig. 4A).Although the majority of Rabs tested were not activated by

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TBC1D20, some increased activity of Rab8A, 13 18 and 35was observed (Fig. 4A). These Rabs are closely relatedmembers of the Rab1-Sec4 subfamily, and this probablyexplains why a Rab1 GAP can activate them in vitro.TBC1D20 activity towards Rab1 was dependent on the TBCdomain encoded by amino acids 1-317 but not other regions ofthe protein (Fig. 4B). This activity was abolished when eitherthe catalytic arginine 105 in the TBC domain was mutated toalanine, or the conserved glutamine 67 in the GTP-binding siteof Rab1 was mutated to leucine (Fig. 4B), in agreement with

the catalytic mechanism proposed for Rab GAPs (Pan et al.,2006). Furthermore, TBC1D20 did not display GAP activitytowards Sar1 and ARF1 (Fig. 4A), both non-Rab GTPasesknown to be required for protein trafficking and themaintenance of normal Golgi structure.

Rabs1 and Rab2 are important components of the ER-Golgitransport pathway in mammalian cells. Expression of a GAPfor either Rab1 or Rab2 should inactivate the cognate Rab, andtherefore block protein transport and disrupt Golgi structure.The screen described in Figs 1 and 2 revealed that only five

Fig. 1. A screen for TBC domainproteins that alter Golgi structure.(A-C) HeLa and hTERT-RPE1cells were transfected with theGFP-tagged wild-type andcatalytically inactive point mutantTBC-domain proteins listed in thefigure. After 24 hours they werefixed, and then stained withantibodies to GM130 or TGN46.(A) The number of cellsdisplaying a fragmented Golgiwas then counted (n>100 percondition), expressed as apercentage, and then plotted as abar graph; grey bars, wild-type(WT) GAPs; open bars, inactivemutants. (B) Cells triply stainedfor the expressed wild-type (WT)TBC1D20 and RN-tre, TGN46and GM130 are shown. (C) Cellstriply stained for the expressedcatalytically inactive mutant (RA)TBC1D20 R105A and RN-tre150A, TGN46, and GM130 areshown. Bars, 10 �m, in all panels.

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Fig. 2. A screen for TBC domain proteinsthat alter ERGIC structure. (A-C) HeLacells were transfected with the GFP-tagged wild-type and catalyticallyinactive point mutant TBC-domainproteins listed in the figure. After 24hours they were fixed, and then stainedwith antibodies to ERGIC53. (A) Thenumber of cells displaying a fragmentedERGIC was then counted (n>100 percondition), expressed as a percentage,and then plotted as a bar graph: grey bars,wild-type GAPs; open bars, inactivemutants. (B) Cells stained for theexpressed wild-type (WT) TBC1D20 andRN-tre, and ERGIC53 are shown.(C) Cells stained for the expressedcatalytically inactive mutant (RA)TBC1D20 R105A and RN-tre R150Aand ERGIC53 are shown. Bars, 10 �m,in all panels.

Fig. 3. TBC1D20 causes a‘loss of Golgi’ phenotype.HeLa cells transfected withGFP-tagged (A) wild-typeTBC1D20 (green), or (B) acatalytically inactive R105Apoint mutant (green) werefixed after 22 hours and thenstained with antibodies top115, golgin84, golgin97, andEEA1 (all in red) asindicated. (C) HeLa cellsexpressing GFP-taggedNAGT-I (green) weretransfected with Myc-taggedwild-type TBC1D20 or thecatalytically inactive R105Amutant (blue), fixed after 12hours, and then stained withantibodies to GM130 (red)and the Myc-epitope tag.(D) To determine the levels ofGolgi proteins, cell extractswere prepared from HeLacells transfected for 24 hourswith Myc-tagged wild-typeTBC1D20 (WT) or thecatalytically inactive R105Amutant (RA). Of theseextracts, 20 �g were westernblotted for GM130 and p115,the Myc-epitope to control forequal expression of theTBC1D20 constructs, and �-tubulin as marker for equalloading. The asteriskindicates a GM130 breakdown product. (E) HeLa cells left untreated,expressing Myc-tagged TBC1D20 or the dominant-negative Sar1 H79Gmutant for 24 hours, or treated with BFA for 30 minutes were fractionated.Equivalent amounts of the total lysate (T), membrane pellet (P), or solublecytosolic fraction (S) were western blotted for p115, GM130, golgin84 andthe Myc epitope. Bars, 10 �m, in all panels.

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TBC-domain proteins cause disruption of the Golgi complexin HeLa cells, and these were therefore tested against bothRab1 and Rab2 in biochemical GTP-hydrolysis assays (Fig.4C). This revealed that only TBC1D20 promotes GTPhydrolysis by Rab1 or Rab2 to a significant extent. Consistentwith these observations, target Golgi Rabs are already knownfor the TBC1D22 family, proposed to act on Rab33 (Pan et al.,2006), RN-tre, reported to act on Rab43 (Fuchs et al., 2007;Haas et al., 2005). TBC1D20 is therefore a specific GAP forRab1 and Rab2 in vitro.

TBC1D20 is a Rab1 GAP in vivoTo further narrow down the target of TBC1D20, the effects ofexpressing dominant-negative Rabs were compared (Fig. 5A).Dominant-negative Rab43 T32N was taken as a positivecontrol that caused Golgi fragmentation, but did not mimic theTBC1D20 phenotype (Fig. 5A,B). By contrast, dominant-negative Rab1 N121I, but not wild-type or activated Q67Lmutant, caused a loss of Golgi phenotype similar to that causedby TBC1D20 (Fig. 5A,B). Rab2 and the Rabs that gave weak

GTP-hydrolysis signals with TBC1D20 did not cause the lossof Golgi phenotype seen with dominant-negative Rab1 (Fig.5A,B). In addition, whereas Rab2 depletion caused Golgifragmentation, it did not cause the loss of Golgi phenotype seenwith TBC1D20 or in cells expressing dominant-negative Rab1(supplementary material Fig. S4A-C). Together, these findingssupport the idea that TBC1D20 acts as a Rab1 GAP in vivo.

To test if endogenous TBC1D20 is required for normalRab1 regulation, HeLa cells were depleted of TBC1D20 usingRNA interference (Fig. 5C,D). Western blot analysis showedthat TBC1D20 could be efficiently silenced using specificsiRNA duplexes (Fig. 5C). Endogenous TBC1D20 is a lowabundance protein spread throughout the ER and hencedifficult to detect (A.K.H. and F.A.B., unpublished). For thisreason a GFP-tagged, catalytically inactive form of the proteinwas used to monitor the efficiency of RNA interference.Consistent with the idea that TBC1D20 is a negative regulatorof Rab1 and hence its effectors, the intensity of p115 stainingwas increased in TBC1D20-depleted cells compared to thecontrol (Fig. 5D). The typical extended ribbon-like staining

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Fig. 4. TBC1D20 is a GAP for Rab1 and Rab2. Biochemical assays for GTP hydrolysis with the Rabs and GAPs indicated were performed asdescribed previously (Fuchs et al., 2007; Haas et al., 2005). All reactions were carried out for 60 minutes. (A) To determine the specific activitytowards a range of GTPases, 0.5 pmoles TBC1D20 were tested against 100 pmoles of the Rab GTPases indicated, as well as ARF1 and Sar1.The basal GTP hydrolysis seen with a buffer control was subtracted for each GTPase. (B) 5 pmoles of wild-type and R105A mutant TBC1D20full-length or the TBC-domain only (amino acids 1-317) were tested against 100 pmoles wild-type Rab1 or the Rab1 Q67L hydrolysis-defective mutant. Basal GTP hydrolysis seen with buffer is plotted as open bars, and GAP-stimulated GTP hydrolysis as grey bars. (C) Buffer(control) or 0.5 pmoles of the GAP indicated were tested against 100 pmoles Rabs 1 and 2. Basal GTP hydrolysis seen with buffer is plotted asopen bars, and GAP-stimulated GTP hydrolysis as grey bars.

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pattern of GM130 became more compact, but did not increasein intensity (Fig. 5D). In addition, COPII-positive structuresdefined by Sec31 partially lost their perinuclear organizationadjacent to the Golgi, and become scattered throughout thecytosol (Fig. 5D). Despite these changes, the transport of VSVG from the ER to the plasma membrane was not altered(A.K.H. and F.A.B., unpublished). TBC1D20 thereforecontributes to the organization of COPII vesicles and theGolgi complex, but is not an essential component of thevesicle transport machinery.

ER exit is blocked by inactivation or depletion of Rab1If TBC1D20 is a GAP for Rab1, then it should be able to causea block in ER to Golgi transport. To test this, VSV G transportassays were performed using cells expressing wild-type andcatalytically inactive mutant forms of the TBC-domainproteins causing Golgi or ERGIC fragmentation (see Figs 1and 2). Strikingly, only TBC1D20 prevented the appearance ofthe VSV G at the cell surface (Fig. 6A,B). This effect wasdependent on the catalytic arginine, indicating that this is dueto specific inactivation of Rab1. Furthermore, depletion of


Fig. 5. Increased p115 staining at theGolgi in TBC1D20-depleted cells.(A) HeLa cells transfected withGFP-tagged dominant-negative Rabswere fixed after 24 hours, and thenstained with antibodies to GM130.The number of cells displaying afragmented Golgi (open bars) orscattered COPII staining (filled bars)was then counted (n>100 percondition), expressed as apercentage, and plotted as a bargraph. (B) HeLa cells transfectedwith GFP-tagged Rab1, dominantactive Rab1Q67L, and dominant-negative Rab1 N121I or Rab43T32N were fixed after 24 hours, andthen stained with antibodies toGM130 or COPII (red). Rabs werevisualized by GFP fluorescence(green), and DNA was stained withDAPI. (C) Cell lysates from HeLacells expressing GFP-taggedTBC1D20 treated with either controlor TBC1D20-specific siRNAduplexes were western blotted forGFP to detect TBC1D20, and�-tubulin as a loading control.(D) HeLa cells expressing the GFP-tagged R105A inactive form ofTBC1D20 (green) as a marker forthe efficiency of RNA interferencewere treated with either control orTBC1D20-specific siRNA duplexesfor 72 hours. The cells were thenfixed and stained with antibodies toCOPII, p115 or GM130 (all in red).DNA was stained with DAPI (blue).Bars, 10 �m.

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Rab2 did not block anterograde transport of VSV G from theER to the cell surface (supplementary material Fig. S4D).Therefore, the effect of TBC1D20 expression is mainly due tothe inactivation of Rab1.

Interestingly, in TBC1D20-expressing cells, VSV G wasapparently unable to exit the ER after 60 minutes of transport,and did not accumulate in punctate structures characteristic ofCOPII vesicles or ERES (Fig. 6C). The effects of depletingcomponents of ER-Golgi vesicle tethering complexes, Rab1,p115 and GM130, were then compared to those seen withTBC1D20 overexpression (Fig. 6D). Western blotting showedthat these proteins were specifically depleted (seesupplementary material Fig. S5A). Similar to TBC1D20,depletion of Rab1 or p115 prevented the appearance of VSV

G at the cell surface (Fig. 6D). In contrast to p115 depletion,where VSV G left the ER and accumulated in a collapsedpunctate structure reminiscent of GM130 staining in p115depleted cells (supplementary material Fig. S5B), VSV G wasunable to leave the ER in Rab1-depleted cells (Fig. 6D).Supporting this observation, similar results were obtained incell expressing dominant-negative but not dominant active orwild-type forms of Rab1 (Fig. 6E). GM130 although requiredfor normal Golgi-ribbon and COPII vesicle organization asexpected (Puthenveedu et al., 2006), was not required for VSVG transport (see supplementary material Fig. S5B and Fig. 6D).The similarity of the TBC1D20 overexpression, Rab1depletion, and Rab1 dominant-negative phenotypes indicatesthat TBC1D20 exerts its effects by inactivating Rab1. Although

Fig. 6. Rab1 is the only Rabessential for ER to cell surfacetransport. (A,B) VSV Gtransport assays wereperformed as described in theMaterials and Methods onHeLa cells expressing thewild-type and catalyticallyinactive arginine finger pointmutant TBC-domain proteinsindicated. The cell surfaceappearance of VSV G wasmeasured for all conditions(A). The bar graph (B) showsthe inhibition in transportobserved with the various wild-type (grey bars), andcatalytically inactive (openbars) TBC-domain proteins.(C) VSV G transport assayswere performed in HeLa cellsexpressing Myc-tagged wild-type (WT) or catalyticallyinactive arginine finger pointmutant (R105A) TBC1D20.After 60 minutes of transportcells were fixed and surfacestained for VSV G (red), thenpermeabilized and stained forthe Myc epitope (blue). TotalVSV G was observed by GFPfluorescence (green) at all timepoints. (D) VSV G transportassays were performed inHeLa cells treated with control,Rab1, p115, or GM130 siRNAduplexes for 72 hours. After 60minutes of transport cells werefixed and surface stained forVSV G (red), and total VSV Gwas observed by GFPfluorescence (green). (E) VSVG transport assays wereperformed in HeLa cellsexpressing Myc-tagged wild-type (WT), dominant-negative(N121I) or constitutive active(Q67L) point mutants of Rab1.After 60 minutes of transport the cells were fixed and surface stained for VSV G (red), then permeabilized and stained for the Myc epitope(blue). Total VSV G was observed by GFP fluorescence (green). Bars, 10 �m.

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Rab1 binding proteins, such as p115 and GM130, maycontribute to this phenotype, for example the loss of COPIIvesicle organization, they cannot explain why secretory cargoproteins are blocked in the ER. However, this would suggestthat TBC1D20 and Rab1 have some function at the level of theER.

COPII function in TBC1D20-expressing cellsThe block in cargo exit from the ER on TBC1D20overexpression, Rab1 depletion and Rab1 dominant-negativeN121I expression suggested a defect in the COPII vesicletransport pathway. Rab1 and its effector proteins are requiredfor COPII vesicle tethering, and this is an essential step in ERto Golgi transport (Allan et al., 2000). This is most easilyvisualized by analysis of COPII clustering adjacent to theGolgi complex in the perinuclear region. Low-level expressionof TBC1D20 (Fig. 7A) or expression of dominant-negativeRab1 (Fig. 5B) resulted in a loss of perinuclear COPII staining,leaving only a scattered peripheral pool of COPII. This effectwas not seen when the catalytically inactive R105A mutant ofTBC1D20 (Fig. 7A) or dominant active Rab1 were used,indicating that this effect is likely to be due to inactivation ofendogenous Rab1. TBC1D20, therefore, appears to interferewith tethering of COPII vesicles by the Rab1-p115 pathway.However, since depletion of p115 does not mimic the effectsof Rab1 inactivation, defective tethering cannot explain theobserved requirement for active Rab1 in cargo exit from theER (Fig. 6D).

One possibility is that Rab1 inactivation is interfering withthe assembly of the COPII vesicle coat, and thus trapping cargoin the ER. However, this is unlikely to be the case for tworeasons. First, TBC1D20 shows no activity towards Sar1 in

vitro. Second, fluorescence recovery after photobleaching(FRAP) studies, using previously published methods (Watsonet al., 2006), indicate that COPII coat turnover is the same inboth control and TBC1D20-expressing cells (Fig. 7B).

The other possibility is that cargo selection is defective inTBC1D20-expressing cells. To further investigate this, thebehaviour of ERGIC53 and p24, two proteins that cyclebetween the ER and the ERGIC, was investigated. In cellsexpressing wild-type TBC1D20 both endogenous ERGIC53and GFP-tagged p24 were present in large punctate structures(Fig. 8A). Significantly, p24 was absent from the punctatescattered COPII-positive structures seen in wild-typeTBC1D20-expressing cells (Fig. 8B, enlargement). This is incontrast to the distribution of p24 and ERGIC53 in cellsexpressing catalytically inactive TBC1D20, or in untransfectedcells, where they are present in the perinuclear ERGIC andsmall punctate COPII-positives structures in the cell periphery(Fig. 8B). Rab1 inactivation by TBC1D20 therefore causes adefect in COPII vesicle tethering, and a transport defect wherecargo molecules are no longer recruited into COPII positivestructures, despite normal COPII turnover.

TBC1D20 is an ER-associated Rab1 GAPThe results described so far indicate that TBC1D20 maycontrol the activity of Rab1, and that interfering with thisregulation prevents the exit of secretory cargo molecules fromthe ER. This raises the question of where TBC1D20 acts, andthis was therefore investigated further (Fig. 9). First thetopology of TBC1D20 was examined using a combination ofcarbonate extraction and proteinase K digestion experiments(Fig. 9A). These showed that TBC1D20 behaves as an integralmembrane protein in carbonate extraction, and is accessible to


Fig. 7. Loss of perinuclear ERES inTBC1D20-expressing cells. (A) HeLa cellstransfected with GFP-tagged wild-typeTBC1D20 or a catalytically inactive R105Apoint mutant (green) were fixed after 22 hoursand then stained with antibodies to the COPIIsubunit Sec31 (red). DNA was stained withDAPI (blue). The clustering of COPIIstructures in the perinuclear region, orTBC1D20-induced scattering was counted andthe results are show in the bar graph below(100 cells per experiments, n=2). Bar, 10 �m,in all panels. (B) FRAP was performed oncells expressing Myc-TBC1D20 and Venus-Sec16 using a Leica TCS SP3 AOBS scanningconfocal microscope with a pinhole size of 2Airy units, eightfold line averaging, at 1 frameper second, and region-of-interest bleachingwith 10 iterations of the 514 nm laser at 100%AOTF power. Cells used for photobleachingwere confirmed to express Myc-TBC1D20 bysubsequent immunofluorescence; 95% of allcell transfected with Venus-Sec16 were foundto be transfected with Myc-TBC1D20. Imagestaken from the time points indicated werequantified using ImageJ. Plot of the averageintensity of individual structures over time,error bars indicate the standard deviation (fivecells per experiment, n=3).

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proteinase K. This was similar to the type IItransmembrane golgin, golgin84. This suggests thatTBC1D20 is a type II membrane protein with theTBC domain oriented towards the cytosol. Asexpected, mapping experiments showed that the first317 amino acids of TBC1D20 containing the TBCdomain localize to the cytosol and cause disruption ofthe Golgi complex, and that deletions into this regionresult in a loss of this activity (Fig. 9B). These effectson the Golgi make it difficult to assess whereTBC1D20 is localized, and the catalytically inactiveform was therefore used for targeting experiments(Fig. 9C). This approach showed that full-lengthTBC1D20 was found to target to the ER and nuclearenvelope, and that this targeting information wascontained in amino acids 336-403 (Fig. 9C). Furtherdeletions of the C terminus, amino acids 364-403,resulted in a slightly altered distribution to the nuclearenvelope and Golgi complex, and a loss of thereticular ER staining pattern (Fig. 9C). Inspection ofthe TBC1D20 sequence revealed that it has ahydrophobic stretch from 364 to 387 contained withinthe putative ER-targeting region that could form amembrane-anchoring sequence or transmembranedomain (Fig. 9D). These observations fit with olderfindings that Ypt1 (yeast Rab1) GAP activity is foundin the particulate membrane fraction (Jena et al.,1992).

Reticulon interacts with and modulatesTBC1D20 functionTo identify partners for TBC1D20 that could explainits localization to the ER, or possibly act as regulatorsof its activity, a two-hybrid screen was performed using full-length TBC1D20 as a bait. This screen identified the C-terminal region of reticulon 1 variant 2 (reticulon) as aninteraction partner of TBC1D20 (Fig. 10A; A.K.H. and F.A.B.,unpublished). Mapping experiments revealed that the minimalregion of TBC1D20 able to interact with reticulon was the C-terminal fragment from amino acids 336-403 that shows thesame ER targeting as full-length TBC1D20 (Fig. 10A).However, the shorter fragment from amino acids 364-403showing nuclear envelope and Golgi targeting, could notinteract with reticulon. These results could be confirmed at theprotein level using immune precipitation, where full-lengthTBC1D20, irrespective of its catalytic activity, was found tointeract with reticulon (Fig. 10A). The first 337 amino acids ofTBC1D20, lacking the ER-targeting region did not show anysignificant binding to reticulon under the same conditions (Fig.10A). Finally, reticulon, like other family members, was foundto localize predominantly to the ER (Fig. 10B). TBC1D20therefore contains a specific ER-targeting motif that interactswith a known ER protein. Reticulon is particularly interestingin this regard, since reticulons have previously been shown tobe excluded from the nuclear envelope and to play a role in theorganization of the tubuloreticular ER (Voeltz et al., 2006).

These results suggested that reticulon might be important forcontrolling TBC1D20 function. As shown previously, wild-type TBC1D20 causes the apparent loss of GM130 whenexpressed alone (Fig. 10C,D). This effect can be antagonizedby the expression of reticulon, judged by the recovery of

GM130 staining into a fragmented perinuclear ribbon structurein cells expressing both TBC1D20 and reticulon (Fig. 10C,D).When co-expressed with a catalytically inactive form ofTBC1D20, reticulon and TBC1D20 were both found in the ER,and GM130 showed a normal Golgi staining (Fig. 10C). If thiseffect of reticulon is specific, it should require the ER-targetingproperties of TBC1D20. As predicted, amino acids 1-337, aTBC-domain containing fragment of TBC1D20 capable ofcausing Golgi fragmentation but lacking the ER-targetingdomain, was resistant to the effects of reticulon (Fig. 10C,D).Importantly, the effect of reticulon was not mediated throughalterations in the expression levels of the various TBC1D20constructs tested, since these were equal under all conditions(Fig. 10E). Together these finding support the idea thatreticulons help to functionally compartmentalize the ER, in thiscase by controlling the activity of TBC1D20, the GAP forRab1.

DiscussionWe have identified functions at the Golgi complex for a subsetof the large family of TBC domain proteins encoded in thehuman genome. One of the most interesting of these isTBC1D20, which is unique amongst the TBC-domain proteinsin that it possesses a predicted transmembrane anchor thattargets it to the ER and when overexpressed causes a completedisruption of the Golgi complex. TBC1D20 shows specificGAP activity towards Rab1 and Rab2, unlike the other GAPstested. This agrees with previous studies on budding yeast

Fig. 8. Loss of recycling ERGIC markers from ERES in TBC1D20-expressing cells. HeLa cells expressing GFP-tagged p24 (green) weretransfected with wild-type (WT) or catalytically inactive (RA) Myc-taggedTBC1D20 (blue). After 18 hours cells were fixed and then stained withantibodies to either (A) the ERGIC marker ERGIC53 or (B) the Sec31subunit of the COPII vesicle coat (red), the Myc epitope (blue). GFP wasvisualized directly (green). The enlargements correspond to 10�10 �mregions of the merged images. Bar indicates 10 �m in all panels.

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Fig. 9. TBC1D20 is an ER membrane protein. (A) A schematic showing the proposed topology of TBC1D20. Carbonate extractionexperiments were performed as described in the Materials and Methods to investigate the membrane association of TBC1D20. Golgin84 andGM130 were taken as representative integral and peripheral membrane proteins, respectively. The topology of TBC1D20 was investigatedusing proteinase K (PK) digestion experiments in the presence or absence of Triton X-100 (TX). TGN46 and Golgin84 were taken as controlsfor proteins with large lumenal or cytosolic domains, respectively. (B) HeLa cells transfected with Myc-tagged TBC1D20 and a series of C-terminal deletions were fixed after 24 hours, and then stained with antibodies to GM130 (green) and the Myc epitope (red). The reticular orcytosolic nature of TBC1D20 constructs is more clearly visible in the enlargements. (C) HeLa cells expressing the GFP-tagged R105A mutantof TBC1D20, or a series of N-terminal deletions were fixed after 24 hours, and then stained with antibodies to GM130 (red). TBC1D20constructs were visualized by GFP fluorescence (green). DNA was stained with DAPI (blue). Bars, 10 �m in all panels. (D) A summary ofthe TBC1D20 constructs used and results obtained in B and C. Activity refers to the ability to cause the ‘loss of Golgi’ phenotype;localization of the different constructs is scored as ER, including nuclear envelope, and Golgi. The TBC domain is marked in red, and theputative transmembrane domain in green.

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Gyp8, the homologue of human TBC1D20, which waspreviously reported to have Ypt1 GAP activity, and to suppressthe cold-sensitive defect in strains expressing the Ypt1 Q67L

activated mutant (De Antoni et al., 2002). Further investigationrevealed that TBC1D20 blocks protein transport at the level ofER exit, by inactivating Rab1. As expected, this phenotype was

Fig. 10. Reticulon interacts with and modulates TBC1D20 function at the ER. (A) A schematic of reticulon 1 variant 2 (reticulon 1v2) showingthe two hydrophobic regions (HR), which by analogy with other reticulons may form transmembrane or membrane anchoring sequences(Voeltz et al., 2006). The minimal binding fragment identified by yeast two-hybrid (Y2H) screening is shown in blue. Directed Y2H analysiswas performed using reticulon 1v2 as the prey and full-length TBC1D20 and the various deletion constructs indicated. All combinations growon non-selective media (–LW), whereas only a subset can grow on the selective media (QDO). GFP-immunoprecipitations were performedfrom HeLa S3 cells cotransfected with Myc-tagged reticulon, and the GFP-tagged TBC1D20 constructs indicated, using a published method(Voeltz et al., 2006). The immunoprecipitates were blotted with GFP and Myc antibodies to detect TBC1D20 and reticulon, respectively.(B) HeLa cells transfected with reticulon 1v2 (reticulon; green) were fixed after 24 hours, and then stained with antibodies to calnexin orGM130 (red). (C,D) HeLa cells expressing Myc-tagged wild-type, R105A catalytically inactive mutant TBC1D20, or the minimal TBC domain(amino acids 1-317) in the absence or presence of GFP-tagged reticulon 1v2 (reticulon) were fixed, and then stained for GM130 and antibodiesto the Myc epitope. In some panels DNA was stained with DAPI. Bars, 10 �m. (D) Quantification of the cells showing intact Golgi, the ‘loss ofGolgi’ phenotype, or Golgi fragmentation in cells expressing TBC1D20 constructs and reticulon as indicated. (E) Western blots were performedto control for the expression levels of TBC1D20 and reticulon, with tubulin as a loading control.

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also observed in cells depleted of Rab1. This is consistent witholder observations that extraction of Rab1 using the Rabchaperone GDI prevents the ER exit of VSV G (Peter et al.,1994). The common factor in these situations is that theactivated GTP form of Rab1 is absent. Depletion of the Rab1effector p115 did not cause the same effect, and as expectedsecretory cargo could exit the ER, but accumulated in apunctate cluster adjacent to the nucleus and did not reach theplasma membrane. Importantly, p115 depletion is not expectedto alter the amount of activated Rab1, suggesting this is thedifference between TBC1D20 expression and Rab1 depletion.These findings suggested that TBC1D20 and Rab1 have p115-independent functions in controlling the exit of secretory cargoand Golgi enzymes from the ER. Interestingly, a similar rolehas already been proposed for Ypt1 in budding yeast, whichhas been shown to play a role in the sorting of some cargomolecules exiting the ER (Morsomme and Riezman, 2002).Since neither TBC1D20 in human cells nor Gyp8 in yeast isessential for secretion, regulation of GTP hydrolysis by GAPsappears not to be critical for Rab1 or Ypt1 function underlaboratory conditions (De Antoni et al., 2002; Richardson etal., 1998). However, this may be a simplification of what isrequired to maintain secretory pathway function under morephysiological conditions.

Limiting Rab1 activity under stress conditionsA recent study on the mechanism of �-synuclein toxicityshows that this creates a stress situation resulting in a block ofER to Golgi trafficking (Cooper et al., 2006). This genomicscreen in yeast revealed Ypt1 but not other Rabs can suppress�-synuclein toxicity and recover ER to Golgi trafficking, andthat the TBC1D20 homologue Gyp8 significantly enhanced �-synuclein toxicity (Cooper et al., 2006). Interestingly, COPIIvesicle components and the Ypt1 effector Uso1 were notrecovered, suggesting that general enhancement of COPIIvesicle formation and tethering is not the mechanism by whichYpt1 is acting. The explanation that we favour is that theactivity of Ypt1, and Rab1, becomes limiting for the exit ofsecretory proteins from the ER under stress conditions, and thismay be mediated through the action of the Gyp8/TBC1D20family of Rab GAPs.

Does reticulon restrict TBC1D20 to subdomains of theER?Recent work has uncovered a role for the reticulon family oftransmembrane proteins in the organization and shaping of theER, and the regulation of ER to Golgi transport (De Craene etal., 2006; Voeltz et al., 2006; Wakana et al., 2005). It istherefore interesting that the ER targeting region of TBC1D20interacts with at least one member of the reticulon family, andthat this interaction exerts a negative effect on the ability ofTBC1D20 to disrupt the Golgi complex. These findings raisethe question of why is ER exit defective in cells where Rab1has been inactivated? One possible reason is that it may beimportant to couple the sorting of cargo and Rab1 into COPIIvesicles to ensure that they will recruit the correct tetheringfactors and therefore dock with the correct destination. HowRab1 could exert such effects is less clear, and will requirefurther investigation of its interaction partners, for example theTRAPP Rab1 GEF complex (Barrowman et al., 2000; Jones etal., 2000; Wang et al., 2000), and in mammalian cells the

MICAL family of proteins (Weide et al., 2003). Interestingly,TRAPP has recently been shown to bind the COPII coat andmay therefore be recruited to ER exit sites as COPII vesiclesform (Cai et al., 2007; Yu et al., 2006). Thus, TRAPP mightprovide a means to ensure loading of Rab1 into the vesicle asit forms. A second explanation may relate to the proposedfunction of reticulons in the compartmentalization of the ER(De Craene et al., 2006; Voeltz et al., 2006), and the fact thatvesicle formation from the ER is a highly controlled processrestricted to specific ER exit sites. It is therefore conceivablethat reticulon together with TBC1D20 may be important fordefining these sites, or perhaps restricting them to subdomainsof the ER.

Rab1 and Golgi biogenesisOne striking aspect of TBC1D20 expression is that unlike anyof the other 38 Rab GAPs tested, it causes the apparent loss ofcis-, medial and trans-Golgi markers. This effect is not simplydue to blocking vesicle trafficking, since treatments that do this,such as the GTP-restricted form of Sar1, BFA or depletion ofthe vesicle tether p115, block ER to Golgi traffic, yet only causepartial fragmentation of the Golgi complex. As discussed, thismay be due to the fact that these treatments will not directlyalter the levels of activated Rab1, whereas TBC1D20expression will. Why then, should blocking Rab1 function atthe ER cause such extreme changes in Golgi structure?

Although debated for many years, most recent findingssupport the view that the Golgi complex is not formed of staticcisternae but rather that it is made up of highly dynamic andcontinuously maturing cisternae that receive and exchangematerial through vesicle trafficking pathways (Bonfanti et al.,1998; Losev et al., 2006; Matsuura-Tokita et al., 2006;Mironov et al., 1997). Exactly how new Golgi cisternae formis equally hotly debated, and under some conditions Golgi mayform by division of pre-existing structures (Pelletier et al.,2002; Shima et al., 1997). However, there is also good evidencethat the Golgi can arise directly from the ER (Bevis et al., 2002;Glick, 2002; Mironov et al., 2003), and that availability ofcargo such as Golgi enzymes is a key factor in determining thesize of the resulting Golgi cisternae (Guo and Linstedt, 2006).Furthermore, the organization of ERES and the COPII vesicletrafficking pathway is of key importance for normal Golgibiogenesis and function (Connerly et al., 2005). Our findingsthat ER exit of secretory cargo, ERGIC markers such as p24and Golgi enzymes is blocked in cells where Rab1 isinactivated may be significant in this context. Interestingly,these cargo molecules fail to accumulate at ERES even thoughCOPII turnover, as measured by FRAP experiments appearsnormal. By interfering with the sorting of Golgi enzymes anddirectly preventing the formation of Golgi precursors, Rab1inactivation may therefore cause a much more serious defectin Golgi biogenesis than depleting individual tethering factorsor Golgi matrix proteins.

Yeast and mammalian Golgi – not so different after allThe results presented here suggest that despite its greater sizeand morphological complexity the mammalian Golgi complexis more similar to its budding yeast counterpart in terms of Rabfunction than previously thought. In budding yeast, althoughadditional Rabs are needed for transport from the Golgi to thecell surface, Ypt1 appears to be the sole Rab required for ER


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to Golgi transport, and transport though the Golgi (Brennwaldand Novick, 1993). Surprisingly, the results presented herereveal a similar picture in mammalian cells. Rab1 depletion,inactivation by expression of TBC1D20, or expression ofdominant-negative Rab1 all cause a block in ER to Golgiprotein transport, and the collapse of the Golgi. Inactivation ofother Rabs by expression of other TBC-domain proteins hadno effect on secretion, despite the fact that a number of thesecaused Golgi fragmentation. This might indicate that otherRabs functioning at the Golgi are redundant, or not requiredfor general anterograde cargo transport. These ideas and othersdiscussed here, provide many avenues for further investigation.

Materials and MethodsAntibody reagentsAntibodies reagents were as follows: mouse anti-GM130, Sec31 and EEA1 (BectonDickinson, Heidelberg, Germany); sheep anti-TGN46 (Serotec, Düsseldorf,Germany); sheep anti-GM130, p115 and GRASP55 (Barr et al., 2001; Shorter etal., 1999). Donkey secondary antibodies conjugated to HRP, AMCA, CY2 and CY3were obtained from Jackson ImmunoResearch Labs (West Grove, PA).

Molecular biology and protein expressionHuman Rab GAPs were identified by searching the GenBank database using theTBC-domain signature motifs defined by Goody and co-workers (Rak et al., 2000).The 38 human Rab GAPs identified by this method and expression constructs havebeen described previously (Fuchs et al., 2007). Yeast two-hybrid assays, proteinexpression and purification were performed as described previously (Haas et al.,2005).

Cell culture and RNA interferencehTERT-RPE1 cells (Clontech Laboratories) were grown at 37°C and 5% CO2 in a1:1 mixture of DMEM and HAMS F12 containing 10% calf serum, 2.5 mM L-glutamine, and 1.2 g/l sodium bicarbonate. HeLa cells were cultured at 37°C and5% CO2 in DME containing 10% FCS. HeLa or hTERT-RPE1 cells plated on glasscoverslips at a density of 50,000 cells/well of a 6-well plate were used for plasmidtransfection and RNA interference. All siRNA duplexes were obtained fromDharmacon Inc, Lafayette, CO and are described in Table SI in supplementarymaterial. Where multiple siRNA duplexes are indicated the same results wereobtained with all. For western blotting, cells from three wells of the six-well platewere washed in 2 ml PBS, then lysed in 70-80 �l 50 mM Tris-HCl pH 7.4, 150mM NaCl, 0.1% (wt/vol) Triton X-100. For each lane of a minigel 10 �g of theprotein lysate was loaded.

Cell fractionation and membrane topologyFor fractionation experiments HeLa cells were grown on 15 cm dishes to 70%confluence. Cells were either transfected using 15 �g of each plasmid DNA andleft for 24 hours to express the protein of interest, or incubated for 30 minutes ingrowth medium containing 5 �g/ml BFA. The cells were then harvested and washedtwice in ice cold PBS. The cell pellet was resuspended in 1 ml of 25 mM Tris-HClpH 7.4, 130 mM KCl, 5 mM MgCl2 containing a protease inhibitor cocktail (RocheDiagnostics). Cells were then broken open by passing them 40 times through a 27Gneedle using a 1 ml syringe. Nuclei and cell debris were removed by centrifugationfor 5 minutes at 1000 g and 4°C. This post-nuclear supernatant was split into twoaliquots, one was kept on ice and represented to the total material. The other halfwas centrifuged for 30 minutes at 100,000 g and 4°C. The supernatant, which wasthe soluble cytosolic fraction, was transferred to a fresh tube. The pellet, which wasthe membrane fraction, was resuspended in 100 �l sample buffer. Aliquots of thesupernatant and total of each sample were precipitated by adding 0.5 �l of 10%(wt/vol) sodium deoxycholate and 30 �l of 100% (wt/vol) TCA. After 30 minutesincubation on ice, precipitated protein was collected by centrifugation at 20,000 gand 4°C. Pellets were washed with ice cold acetone, and then resuspended in 100�l of sample buffer.

For proteinase K digestion experiments, HeLa S3 cells from two 70% confluent15 cm dishes and one 10 cm dish transfected with GFP-tagged TBC1D20 constructsfor 24 hours were washed twice in cold PBS, then scraped off the dishes, and pooled.The cell pellets were resuspended using six passes through a 21G needle in 50 mMHepes pH 7.5, 200 mM sucrose, 1 mM MgCl2, 1 mM EDTA, to give a final volumeof 1 ml. This cell suspension was passed though an EMBL cell cracker (EuropeanMolecular Biology Laboratories, Heidelberg, Germany) fitted with an 8.002 mmdiameter ball. The broken cell suspension was centrifuged twice at 1000 g and 4°Cfor 5 minutes to remove cell debris and leave a post nuclear supernatant. ProteinaseK (New England Biolabs) was incubated at 37°C for 30 minutes prior to use toinactivate any contaminating lipase activity. The post nuclear supernatant was thenadjusted to 10 mM CaCl2, and 100 �l aliquots treated with 2 �g proteinase K, 0.5%

(vol/vol) Triton X-100, or both proteinase K and Triton X-100 for 30 minutes onice. To stop the reaction, a half volume of 100% (wt/vol) TCA was added, sampleswere then vortexed and incubated on ice for 30 minutes. Proteins were recoveredby centrifugation at 20,000 g for 15 minutes, the pellets washed in 1 ml of –20°Cacetone, resuspended in 100 �l of sample buffer, and 20 �l analysed by westernblotting.

For carbonate extraction experiments, 100 �l aliquots of the post nuclearsupernatant was centrifuged at 100,000 g for 30 minutes at 4°C to generate amembrane pellet. The pellet was resuspended in 100 mM Na2CO3 and incubatedon ice for 30 minutes. To recover the membrane the sample was centrifuged at100,000 g for 30 minutes at 4°C. The supernatant was precipitated using 25%(wt/vol) TCA, and equal amounts of the total, carbonate-extracted supernatant andmembrane pellet fractions analysed by western blotting.

Protein transport assays and microscopyVSV G ts045 protein transport assays were carried out using an adaptation of apublished protocol. HeLa cells plated on glass coverslips were treated with specificRNA duplexes for 56 hours at 37°C, then transfected with a plasmid encoding greenfluorescent protein (GFP)-tagged VSV G protein for 2 hours at 37°C, then for 12hours at 39.5°C. The cells were then incubated at 4°C to promote VSV G proteinfolding, and afterwards the growth medium was replaced with pre-warmed mediumat 31.5°C. After the required chase period cells were fixed with 3% (wt/vol)paraformaldehyde in PBS. Cell surface VSV G was detected with a monoclonalantibody to the VSV G lumenal domain and a donkey anti-mouse secondary coupledto CY3, and total VSV G by GFP fluorescence. The ratio of surface to totalmeasured fluorescence was used to calculate the extent of VSV G transport. Shigatoxin transport assays were performed using a published method (Fuchs et al.,2007). Cells were processed for immunofluorescence and images collected asdescribed previously (Fuchs et al., 2007).

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analysis of gtpase-activating proteins: rab1 and rab43 are ......2998 the sorting of secretory cargo...

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