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Correspondence to Francis A. Barr: f.a.barr{at}liverpool.ac.uk
Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A–C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.
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Once internalized, Shiga toxin is then transported directly from early and recycling endosomes to the trans-Golgi network (Mallard et al., 1998; Bonifacino and Rojas, 2006). This pathway is dependent on the function of clathrin and dynamin (Lauvrak et al., 2004), the small GTP-binding protein Rab6 and its effector proteins, the lipid phosphatase OCRL1, a defined set of SNAREs (Mallard et al., 2002; Monier et al., 2002; Tai et al., 2004; Choudhury et al., 2005; Hyvola et al., 2006), and a second GTP-binding protein Arl1 and its effector golgin-97 (Lu et al., 2004). From the TGN, the toxin is then transported by a retrograde pathway dependent on Rab6 through the Golgi apparatus to the ER (Sandvig et al., 1992; White et al., 1999), where the toxin A subunit enters the cytoplasm most likely by retrotranslocation (for review see Sandvig and van Deurs, 2002).
Despite the multistep nature of the Shiga toxin uptake pathway, only a single Rab GTP-binding protein, Rab6, has been shown to play any role in its transport (Girod et al., 1999; White et al., 1999; Mallard et al., 2002). Because each membrane trafficking step is thought to involve a discrete set of Rabs (Zerial and McBride, 2001), it seems likely that additional Rabs are involved in Shiga toxin uptake. To further define the pathway of Shiga toxin transport between the cell surface and the Golgi apparatus, we have focused on the identification of Rab GTPase-activating proteins (GAPs) that interfere with this step. Rab GAPs are characterized by a conserved catalytic domain, the TBC (Tre2/Bub2/Cdc16) domain (Strom et al., 1993; Albert and Gallwitz, 1999; Albert et al., 1999), 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, 1999; Rak et al., 2000; Pan et al., 2006). As we have shown previously in the case of Rab5 (Haas et al., 2005), Rab GAPs can be used to specifically inactivate the endogenous pool of a Rab and, thus, interfere with the process this Rab is involved in. Nonspecific effects of Rab GAP expression can easily be discriminated from the specific effects of Rab inactivation by use of an inactive point mutant in which the catalytic arginine residue is replaced by alanine (Haas et al., 2005).
Because the human genome encodes >60 Rabs and at least 39 TBC domain–containing proteins, the identification of specific Rab–Rab GAP pairs is not a trivial task (Zerial and McBride, 2001; Haas et al., 2005). In the present study, we have screened for human Rab GAPs that specifically inhibit the transport of Shiga toxin to the Golgi apparatus and do not have any effects of the uptake of EGF and its receptor. By combining this functional assay with biochemical analysis of GTP hydrolysis, we are able to identify discrete pairs of Rabs and Rab GAPs acting in the Shiga toxin and EGF transport pathways.
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Shiga toxin trafficking to the Golgi apparatus is independent of TBC1D11/GAPCenA
It has previously been reported that TBC1D11/GAPCenA is the GAP for Rab6 (Cuif et al., 1999), and, thus, this was tested for its ability to block the trafficking of Shiga toxin to the Golgi apparatus. Surprisingly, the expression of TBC1D11/GAPCenA had no obvious effect on the transport of Shiga toxin to the Golgi apparatus (Fig. 3 A) or on the ability of Rab6 or the Rab6-dependent effector Bicaudal-D1 (Matanis et al., 2002; Short et al., 2002) to target to Golgi membranes (Fig. 3 B).
Therefore, TBC1D11/GAPCenA was further investigated to find out whether it has the biochemical properties expected of a Rab6 GAP.
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A screen for Rab GAPs altering Shiga toxin trafficking
An unbiased approach was taken to identify which Rab GAPs act on the Shiga toxin transport pathway. Cells expressing the various predicted human Rab GAPs were tested blindly for their ability to transport Shiga toxin to the Golgi apparatus and EGF to early endosomes (unpublished data). Candidate positives from this first round of screening were then retested, comparing the effects of the wild-type GAP to that of a catalytically inactive point mutant. In this screen, 6 of 39 predicted GAPs showed catalytic activity–dependent effects on Shiga toxin trafficking to the Golgi apparatus (Table I and Fig. 4, A and B).
In contrast, in a parallel screen, only RabGAP-5 was able to block the uptake of EGF to early endosomes (Table I and Fig. 5, A and B).
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The ability of candidate positives to protect cells against the cytotoxic effects of complete Shiga-like toxin 1 was then tested (Fig. 4 C). This assay measures protein synthesis, which is inhibited if Shiga toxin can reach the ER and enter the cytoplasm and, therefore, is a more stringent measure of Shiga toxin trafficking than fluorescent assays (Spooner et al., 2004). This approach showed that EVI5, RN-tre, and TBC1D17 protect against Shiga-like toxin 1 between 1.87- and 3.35-fold, whereas RabGAP-5 with a fold protection of 1.07 could not (Fig. 4 C). The TBC1D10 family of GAPs showed variable extents of protection, and, therefore, it is unclear whether these represent specific positives with this assay. Interestingly, none of the positive GAPs were able to cause the release of the Rab6 effector Bicaudal-D1 from Golgi membranes (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200612068/DC1), even those that showed some effects on Golgi morphology (Fig. S2). This suggests that they do not act on Rab6. The trafficking pathway of Shiga toxin from the cell surface to the Golgi is therefore defined by the following Rab GAPs: EVI-5, RN-tre, the TBC1D10 family, and TBC1D17.
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The role of Rab6 in the Shiga toxin trafficking pathway
None of the GAPs capable of blocking Shiga toxin transport was able to cause the release of Bicaudal-D1 from the Golgi to the cytoplasm (Fig. S3), and this indicates that they do not act on Rab6. Why the Rab6 GAP was not found in the screen is unclear, but because the depletion of Rab6 reduces Shiga toxin transport to the Golgi (Fig. 2; Del Nery et al., 2006), this has to be viewed as a false negative. There is evidence that some TBC domain proteins form heterodimeric complexes (Pereira et al., 2001) and that this is necessary for their activity. If this is the case for the Rab6 GAP, it could have been missed in the current screen.
What is clear is that TBC1D11/GAPCenA is unlikely to be the GAP for Rab6. It displays robust biochemical activity toward Rab4 but not Rab6 and has none of the cell biological properties expected for a Rab6 GAP. It cannot block Shiga toxin trafficking or cause release of the Rab6 effector Bicaudal-D1 from the Golgi. Therefore, at this time, the identity of the Rab6 GAP remains mysterious and will require further investigation.
Some cautions on the use of dominant-active Rabs
Some of the results presented here suggest that there are problems with the use of dominant-active GTP-locked Rabs. Although the expression of dominant-active Rab5Q79A results in the trapping of Shiga toxin in an early endocytic compartment, indicating its transport is Rab5 dependent, this interpretation is not supported by the use of specific Rab GAPs. Although RabGAP-5 does block EGF and transferrin trafficking (Haas et al., 2005), it has no effect on the transport of Shiga toxin to the Golgi (Fig. 6). This suggests that although Shiga toxin trafficking is normally Rab5 independent, when dominant-active Rab5 is expressed, it becomes trapped in the enlarged early endocytic compartment created under these conditions (Stenmark et al., 1994). One explanation for this could be that Shiga toxin, although normally trafficking in a Rab5-independent fashion, does pass through a Rab5-positive endocytic compartment, and this can also be perturbed by the overexpression of active mutant forms of Rab5. In contrast, EGF trafficking is blocked by both RabGAP-5 and Rab5Q79A (Figs. 2 and 6) and, therefore, does follow a Rab5-dependent pathway through early endosomes. Thus, dominant-active Rab5 appears to create a situation in which the function of the early endocytic pathway is perturbed such that molecules that normally traffic in a Rab5-independent manner are also affected. Dominant-active Rabs should therefore be used with some caution, and, as shown here, specific Rab GAPs provide valuable and specific tools to manipulate the activity of endogenous Rabs.
Are Rab GAPs specific for particular Rab GTPases?
It has been suggested that Rab GAPs are not particularly specific toward their target Rab (Albert and Gallwitz, 1999; Pan et al., 2006). However, this is somewhat counterintuitive because Rabs are argued to be important components acting at specific membrane trafficking steps and for the specification of organelle identity (Pfeffer, 2001; Munro, 2002), and one would therefore expect their regulators to be equally specific. We believe that Rab GAPs are likely to be specific toward particular target Rabs or Rab subfamilies and have several arguments to support this statement. We show that GAPs such as TBC1D11/GAPCenA, RabGAP-5, and RN-tre are highly active toward specific Rabs in biochemical assays. In addition, we can demonstrate that Rab GAPs have highly specific effects on discrete membrane trafficking pathways, which is consistent with the idea that they act on specific Rabs in vivo.
A possible explanation for this discrepancy comes from our investigation of TBC1D11/GAPCenA, which was previously suggested to act on Rab6 (Cuif et al., 1999). In our hands, TBC1D11/GAPCenA acts on Rab4 but not Rab6 and does not have the effects on Rab6 effectors or Shiga toxin transport that is expected for a Rab6 GAP (Fig. 3). Investigation of the interaction between TBC1D11/GAPCenA and Rabs shows that regions outside of the minimal predicted catalytic domain are required for specific Rab binding (Figs. 3 and S1). Strikingly, deletion of these regions relaxes the interaction specificity of the protein. Previous studies have used truncated GAP domains rather than full-length proteins (Albert and Gallwitz, 1999; Pan et al., 2006), and, in light of our findings, this may be problematic because these proteins may have a relaxed specificity and, thus, may be able to accelerate GTP hydrolysis on a broader spectrum of Rabs. An analogous situation has been described for the GAP1 family of bifunctional Ras and Rap GAPs (Kupzig et al., 2006). In this case, regions outside of the core catalytic domain are required for the recognition of Rap (Kupzig et al., 2006). Further investigation of Rab GAP specificity is needed before any general conclusions can be made, but it is clearly important to study the full-length proteins and not only the truncated fragments corresponding to predicted domains.
Understanding Rab GAP function
There are >60 Rab family GTPases encoded by the human genome and at least 39 TBC domain–containing Rab GAPs. However, little is known about the function of the majority of these proteins. This is partly the result of the enormous complexity of membrane trafficking systems and the lack of suitable cell biological models for Rabs with functions in specific tissues. As a first step in unravelling this complex network, we have screened for Rab GAPs that can influence the trafficking of Shiga toxin to the Golgi or of EGF to early endosomes. Using biochemical assays, we have then identified target Rabs for several of these GAPs. This approach should be useful for the study of other trafficking events, for trafficking between the ER and Golgi apparatus, or for a wide variety of different regulated secretory events.
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Molecular biology and protein expression
The bacterial expression construct for STxB was provided by Y. Misumi (Fukuoka University School of Medicine, Fukuoka, Japan). Human Rab GAPs were identified by searching the GenBank/EMBL/DDBJ database using the TBC domain signature motifs defined by Rak et al. (2000). The human Rab GAPs (EVI-5, RN-tre [USP6NL], RUTBC1, RUTBC2, RabGAP-5 [RUTBC3], TBC1D1, TBC1D2, TBC1D3B, TBC1D4, TBC1D5, TBC1D6, TBC1D7, TBC1D8, TBC1D10A, TBC1D10B, TBC1D10C, TBC1D11 [GAPCenA], TBC1D12, TBC1D13, TBC1D14, TBC1D15, TBC1D16, TBC1D17, TBC1D18, TBC1D19, TBC1D20, TBC1D21, TBC1D22A, TBC1D22B, USP6, AK074305, KIAA1055, KIAA0676, KIAA0882, NP_060222, NP_060779, EVI5-like, KIAA0984, and KIAA1171) were then amplified from either human testis cDNA (Becton Dickinson) or HeLa cDNA using the pfu polymerase (Stratagene) and were cloned in pCRII-TOPO (Invitrogen). Point mutations were introduced using the QuikChange method (Stratagene). Constructs were confirmed by DNA sequencing (Medigenomix; Max Planck Institute biochemistry sequencing core facility). Mammalian expression constructs were made in pcDNA3.1+ (Invitrogen) modified to encode a myc epitope tag and the pEGFP-C2 vector (CLONTECH Laboratories, Inc.). For yeast two-hybrid assays, Rabs were inserted into the bait vector pFBT9, pGBT9 (CLONTECH Laboratories, Inc.) was modified to carry kanamycin resistance, and Rab GAPs and mutants thereof were inserted into the prey vector pACT2 (CLONTECH Laboratories, Inc.). Yeast two-hybrid assays were performed according to the yeast protocol handbook (CLONTECH Laboratories, Inc.) as described previously (Haas et al., 2005). Bacterial expression was performed using the T7 polymerase hexahistidine-GST expression vector pFAT2 for Rabs and the maltose-binding protein expression vector pMalC2 (New England Biolabs, Inc.) for Rab GAPs and the BL21(DE3) and JM109 strains, respectively. Fusion proteins were purified over nickel-nitrilotriacetic acid agarose (QIAGEN) or amylose resin (New England Biolabs, Inc.). Proteins were dialysed overnight against 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2 mM DTT, and aliquots were frozen in liquid nitrogen for storage at –80°C.
GTP hydrolysis assays
For Rab-loading reactions, 10 µl of assay buffer, 73 µl H2O, 10 µl 10 mM EDTA, pH 8.0, 5 µl of 1 mM GTP, 2 µl 
-[32P]GTP (10 mCi/ml; 5,000 Ci/mmol; GE Healthcare), and 100 pmol Rab protein were mixed on ice. After 15 min of incubation at 30°C, loaded Rabs were stored on ice. GTP binding was measured using a nitrocellulose filter-binding assay (Du and Novick, 2001). GAP reactions were started by the addition of 0.5 pmol Rab GAP as specified in the figures. A 2.5-µl aliquot of the assay mix was scintillation counted to measure the specific activity in counts per minute/picomole GTP. Reactions were then incubated at 30°C, taking 5-µl samples in duplicate at suitable time points as indicated in the figures. The 5-µl aliquots were immediately added to 795 µl of ice-cold 5% [wt/vol] activated charcoal slurry in 50 mM NaH2PO4, left for 1 h on ice, and centrifuged at 16,100 g in a benchtop microfuge (5417R; Eppendorf) to pellet the charcoal. A 400-µl aliquot of the supernatant was scintillation counted, and the amount of GTP hydrolyzed was calculated from the specific activity of the reaction mixture.
Cell culture and RNAi
HeLa cells were cultured at 37°C and 5% CO2 in growth medium (DME containing 10% FCS). HeLa cells plated on glass coverslips in a six-well plate at a density of 70,000 cells/well were used for plasmid transfection and at 25,000 cells/well for RNAi using conditions that were described previously (Haas et al., 2005). Rabs were targeted using siRNA duplexes obtained from Dharmacon. The sequences are listed in Fig. S5 B. For Western blotting, cells from three wells of a six-well plate were washed in 2 ml PBS and lysed in 70–80 µl of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% [wt/vol] Triton X-100. For each lane of a minigel, 10 µg of the protein lysate was loaded.
Shiga-like toxin cytotoxicity assays
Cytotoxicity was defined as a decrease in the ability of cells to incorporate [35S]methionine into acid-precipitable material after Shiga-like toxin 1 treatment using an established method (Spooner et al., 2004). HeLa cells were transiently transfected with either wild-type or RA mutant Rab GAPs for 9 h using Fugene-6 and the manufacturer's protocol (Roche Diagnostics) and were replated in 96-well plates at a density of 1.5 x 104 cells/well and left for another 15 h. After washing with PBS, cells were incubated for 1 h with 100 µl DME/FCS containing serial twofold dilutions from 0.05–50 ng/ml Shiga-like toxin 1. Subsequently, cells were washed with PBS and incubated in PBS containing 50 µCi/ml [35S]methionine for 30 min. Labeled proteins were precipitated with three washes in 5% [wt/vol] trichloroacetic acid, the wells were washed twice with PBS, 50 µl of scintillation fluid was added, and the amount of radiolabel incorporated was then determined in a counter (MicroBeta 1450 TriLux; PerkinElmer). For each condition, the IC50 for Shiga-like toxin 1 was calculated from the toxin titration performed in triplicate.
EGF uptake assays
EGF coupled to 200 µg/ml AlexaFluor488 or -555 (40x stock; Invitrogen) were stored as stock solutions in PBS at –20°C. For uptake assays, HeLa cells plated on glass coverslips at a density of 70,000 cells/well of a six-well plate were washed three times with serum-free growth medium 36 h after plating and were incubated in serum-free growth medium for 15–16 h at 37°C and 5% CO2. Coverslips were then washed three times in ice-cold PBS and placed on 40-µl drops of uptake medium (DME, 2% [wt/vol] BSA, and 20 mM Hepes-NaOH, pH 7.5) and 5 µg/ml EGF on an ice-cold metal plate covered in Parafilm (Pechiney Plastic Packaging). After 30 min of incubation, the coverslips were washed three times in ice-cold PBS to remove excess ligand. One coverslip was fixed to give the total bound ligand, whereas the remaining coverslips were transferred to a six-well plate containing prewarmed growth medium and were incubated at 37°C and 5% CO2. At the time points indicated in the figures, coverslips were fixed and processed for immunofluorescence microscopy.
Preparation of STxB and uptake assays
Recombinant STxB was prepared as described previously (Johannes et al., 1997; Sohda et al., 2005) and labeled on amine residues for 5 min at room temperature with an N-hydroxysuccinimidyl ester of Cy3 according to the manufacturer's instructions (GE Healthcare). These conditions lead to a stoichiometry of five Cy3 dye molecules per pentamer of STxB. The cell line and conditions used for uptake assays were the same as those described for EGF except that 0.7 µg/ml Cy3-STxB were used. For combined EGF and STxB assays, both proteins were mixed and bound simultaneously to the cell surface, and the standard procedure was followed.
Image acquisition
Cells to be imaged were fixed for 20 min in 3% [wt/vol] PFA, quenched for 10 min with 50 mM ammonium chloride, and permeabilized with 0.1% [vol/vol] Triton X-100 for 5 min to allow labeling of internal cell structures. For cell surface labeling, cells were not permeabilized. All solutions were made in PBS, and antibody staining was performed for 60 min using a 1,000-fold dilution of antiserum or purified antibody at a final concentration of 1 µg/ml. Coverslips were mounted in 10% [wt/vol] Moviol 4-88, 1 µg/ml DAPI, and 25% [wt/vol] glycerol in PBS. Images were collected at a room temperature of 22°C using a microscope (Axioskop-2; Carl Zeiss MicroImaging, Inc.) with a 63x plan Apochromat oil immersion objective (Carl Zeiss MicroImaging, Inc.) of NA 1.4, standard filter sets (Carl Zeiss MicroImaging, Inc.), a 1,300 x 1,030-pixel cooled CCD camera (CCD-1300-Y; Princeton Instruments), and Metavue software (Visitron Systems). Images were cropped in Photoshop 7.0 (Adobe) or CS2 software (Adobe) without contrast or other adjustments, sized, and placed using Illustrator 11.0 (Adobe) or CS2.
Online supplemental material
Fig. S1 shows that the interaction specificity of TBC1D11/GAPCenA with human Rab GTPases involves regions outside of the TBC domain. Fig. S2 shows the effect of Rab GAPs blocking Shiga toxin uptake on Golgi morphology. Fig. S3 shows the effect of Rab GAPs blocking Shiga toxin uptake on the Rab6 effector Bicaudal-D1. Fig. S4 shows that RabGAP-5 interacts with Rab5A-C but not other Rabs. Fig. S5 shows the specific depletion of target Rabs using RNAi. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200612068/DC1.
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Acknowledgments |
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This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (group of F.A. Barr). Work at the University of Warwick was supported by the National Institutes of Health grant 5 U01AI 65869.
Submitted: 13 December 2006
Accepted: 17 May 2007
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3). The corrected IC50 values for each condition are shown above each column, and the IC50 of empty vector–transfected control cells was 3.5 ng/ml. Error bars represent SD. Bar, 10 µm.
