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Genes to Cells (2006) 11, 1023-1037. doi:10.1111/j.1365-2443.2006.00997.x
© 2006 Blackwell Publishing or its licensors
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Screening for target Rabs of TBC (Tre-2/Bub2/Cdc16) domain-containing proteins based on their Rab-binding activity

Takashi Itoh1, Megumi Satoh1, Eiko Kanno1 and Mitsunori Fukuda1,2,*

1 Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2 Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
It has recently been proposed that the TBC (Tre2/Bub2/Cdc16) domain functions as a GAP (GTPase-activating protein) domain for small GTPase Rab. Because of the large number of Rab proteins in mammals, however, most TBC domains have never been investigated for Rab-GAP activity. In this study we established panels of the GTP-fixed form of 60 different Rabs constructed in pGAD-C1, a yeast two-hybrid bait vector. We also constructed a yeast two-hybrid prey vector (pGBDU-C1) that harbors the cDNA of 40 distinct TBC proteins. Systematic investigation of 2400 combinations of 60 GTP-fixed Rabs and 40 TBC proteins by yeast two-hybrid screening revealed that seven TBC proteins specifically and differentially interact with specific Rabs (e.g. OATL1 interacts with Rab2A; FLJ12085 with Rab5A/B/C; and Evi5-like with Rab10). Measurement of in vitro Rab-GAP activity revealed that OATL1 and Evi5-like actually possess significant Rab2A- and Rab10-GAP activity, respectively, but that FLJ12085 do not display Rab5A-GAP activity at all. These results indicate that specific interaction between TBC protein and Rab would be a useful indicator for screening for the target Rabs of some TBC/Rab-GAP domains, but that there is little correlation between the Rab-binding activity and Rab-GAP activity of other TBC proteins.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Small monomeric GTPase Rab constitutes the largest family of putative membrane trafficking proteins that are conserved in all eukaryotic cells (reviewed in Pfeffer 2001; Segev 2001; Stenmark & Olkkonen 2001; Zerial & McBride 2001), and more than 60 distinct Rabs (i.e. approximately 40 different Rab subfamilies) are present in humans and mice (Bock et al. 2001; Pereira-Leal & Seabra 2001). Each Rab member cycles between the GDP-bound inactive state and GTP-bound active state, and the GTP-bound activated form mediates membrane transport through specific interaction with an effector molecule(s) (Pfeffer 2001; Segev 2001; Stenmark & Olkkonen 2001; Zerial & McBride 2001). Two key families of enzymes, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), are generally believed to control the GDP/GTP-cycling of Rabs (Segev 2001; Zerial & McBride 2001). In contrast to the Rab effector molecules, however, much less is known about the GEFs or GAPs of the mammalian Rab family, and only a small number of putative Rab-GEFs or Rab-GAPs have been identified. For example, Rabex-5 (Horiuchi et al. 1997), ALS2 (Otomo et al. 2003), RIN1–3 (Tall et al. 2001; Saito et al. 2002; Kajiho et al. 2003), and RME6 (Sato et al. 2005) function as Rab5 GEFs, Rab3-GEP (Wada et al. 1997) and GRAB (Luo et al. 2001) as Rab3 GEFs, and Rabin8 as a Rab8 GEF (Hattula et al. 2002). Since the putative GEF domains of Rab3-GEF, Rab5-GEF, and Rab8-GEF determined thus far do not exhibit any significant homology with each other, it is virtually impossible to screen for the unidentified GEFs of other Rabs by conventional homology search analysis. By contrast, all Rab-GAPs identified thus far (e.g. Rab5-GAP and Rab6-GAP) (Cuif et al. 1999; Lanzetti et al. 2000, 2004; Pei et al. 2002; Haas et al. 2005; Mîinea et al. 2005), except for Rab3A-GAP (Fukui et al. 1997), have contained the Tre2/Bub2/Cdc16 (TBC) domain, which has been conserved from yeast to mammals (reviewed in Bernards 2003).

The TBC domain is a conserved protein motif that consists of approximately 200 amino acids (Supplementary Fig. S1) and is known to be present in a variety of molecules in humans (Bernards 2003). Half of the human TBC domain-containing proteins (simply referred to as TBC proteins below) do not contain any additional known protein motifs, but the others contain a few other protein motifs, such as a PTB (phosphotyrosine binding) domain (van der Geer & Pawson 1995), an SH3 (Src homohogy) domain, a PH (pleckstrin homology) domain (Shaw 1996), a GRAM (glucosyltransferases, Rab-like GTPase activators and myotubularins) domain (Doerks et al. 2000) and a RUN (RPIP8, UNC-14, and NESCA) domain (Sakamoto et al. 2005) (Fig. 1). Since the number of TBC proteins (i.e. more than 50) in humans almost matches the number of Rab proteins (i.e. more than 60), the TBC proteins are expected to have distinct Rab-GAP activities (i.e. each TBC protein may have specific GAP activity toward one or two specific Rabs). Due to the large number of Rab and TBC proteins in mammals, however, no systematic genome-wide investigation of the Rab-GAP activity of TBC proteins toward all Rab proteins has ever been performed.


Figure 1
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  		Figure 1  Phylogenetic tree and structure of human or mouse TBC proteins used in this study. The phylogenetic tree of the human or mouse TBC domains (see also Table 1) used in this study was depicted by the CLUSTALX (version 1.8) program (Thompson et al. 1997). Schematic representations of proteins representative of each branch are shown on the right. The branches containing conserved domains are boxed with colors corresponding to the schematic representation on the right. The amino acid sequences used in the depiction of the phylogenetic tree are aligned in Supplementary Figure 1. TBC, Tre2, Bub2, and Cdc16; TLDc, TBC and LysM domain-containing; GRAM, glucosyltransferases, Rab-like GTPase activators and myotubularins domain; PTB, phosphotyrosine binding domain; UCH, ubiquitin carboxyl-terminal hydrolase; SMC, structural maintenance of chromosomes; RUN, RPIP8, UNC-14, and NESCA; SH3, Src homology domain 3; pkinase, protein kinase domain; RHOD, rodanese homology domain; and PH, pleckstrin homology domain.

 
In this study we cloned 60 different Rabs (almost the entire set of Rabs expressed in mice) and 40 different TBC domain-containing putative Rab-GAPs (Fig. 1 and Table 1), and established a novel yeast two-hybrid screening system to investigate the possible interaction between Rabs and putative Rab-GAPs, with GTP-fixed Rab mutants as bait and TBC proteins as prey. If certain TBC protein functions as a GAP for a specific Rab, the TBC protein should interact with its substrate, the GTP-fixed form of Rab. Actually, Haas et al. (2005) have recently identified a specific Rab5-GAP based on the physical interaction between Rab5 and RabGAP-5, a novel TBC domain-containing protein. We used this screening system to systematically test 2400 combinations of 60 GTP-fixed Rabs and 40 TBC proteins, and found that seven TBC proteins interact with certain Rabs in a highly specific manner. We also measured the in vitro GAP activity of these TBC proteins, and demonstrated that most of them indeed function as GAP for Rabs they interact. Based on our findings, we discuss the functional relationship between the GTP-Rab-binding activity of TBC proteins and their Rab-GAP activity.


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  		Table 1 Summary of the Rab-binding activity and Rab-GAP activity of TBC proteins
 

   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Establishment of panels of Rabs and TBC proteins

Since the Rab-GAP protein recognizes the GTP-bound form of Rab and promotes GTP hydrolysis, the TBC domains are likely to associate with the GTP-fixed form of the specific Rab isoform (i.e. mimic the substrate Rab), and interaction between the TBC domain and Rab may be a useful indicator for efficient screening for the target Rabs of various TBC domains (Haas et al. 2005). The first step in investigating all possible interactions between TBC domains and Rabs is to collect all Rab proteins expressed in mice and to establish panels of Rabs. To do so, we searched both the public mouse genome database and the mouse EST (expressed sequence tag) database, and found expression of 58 mouse Rabs at the mRNA level (Supplementary Table S1) and several pseudogenes in the mouse genome (data not shown). Comparison with the human Rabs reported thus far (Bock et al. 2001; Pereira-Leal & Seabra 2001) revealed that 56 of them are orthologues of human Rabs and that the other two are new members of the Rab family (designated Rab42 and Rab43, respectively). According to the phylogenetic tree of the mouse Rab family (Supplementary Fig. S2), these Rabs were classified into 41 distinct subfamilies (Rab1–43; numbers 16 and 31 are missing because Rab16 and Rab31 have been renamed Rab3D and Rab22B, respectively) (Pereira-Leal & Seabra 2001). Since we were unable to find expression of the mouse orthologues of human Rab6C and Rab40A in the public mouse database, we decided to include human Rab6C and Rab40A in our Rab panels. Mutant cDNAs encoding the GTP-fixed form of the Rabs were obtained by site-directed mutagenesis (glutamine-to-leucine substitution; see Experimental procedures for details), and then subcloned into the pGAD-C1, a yeast two-hybrid bait vector. In a similar manner, the cDNAs of 40 different TBC proteins (Fig. 1 and Table 1) were obtained and subcloned into the pGBDU-C1, a yeast two-hybrid prey vector.

Specific interactions between TBC proteins and Rabs

Since 60 combinations of a single TBC domain and Rab1–43 needed to be investigated at the same time, our classical biochemical approaches, such as co-immunoprecipitation assay in COS-7 cells (Fukuda 2003) and GST (glutathione S-transferase) pull-down assay (Kuroda et al. 2002), were inappropriate, and so we adopted the yeast two-hybrid system (bait, GTP-fixed Rab; and prey, TBC domain) to efficiently screen the target Rabs of the TBC domain (Haas et al. 2005). In the first set of experiments, we investigated 2400 combinations for possible interactions between 40 TBC proteins and 60 Rabs by the yeast two-hybrid screening system. Although most of the TBC proteins did not interact with any Rabs, seven of them specifically and differentially interacted with several Rab isoforms (boxed in Fig. 2 and summarized in Table 1). For example, OATL1, Evi5-like, and USP6 specifically interacted with a single Rab isoform, Rab2A, Rab10, and Rab33B, respectively. FLJ12085 and DJ1042K10.2 strongly recognized the Rab5 subfamily (Rab5A/B/C), and DJ1042K10.2 also very weakly interacted with Rab39B. FLJ12168 weakly, but specifically recognized the Rab5 subfamily, as did FLJ12085. FLJ38519 interacted with two phylogenetically related Rabs, Rab34 and Rab36 (Supplementary Fig. S2), and very weakly with Rab13.


Figure 2
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  		Figure 2  Specific interaction between TBC proteins and Rabs as revealed by the yeast two-hybrid assay. Yeast cells containing pGBDU plasmid expressing Rab-GAP protein (OATL1, Evi5-like, FLJ38519, USP6, DJ1042K10.2, FLJ12085, FLJ12168, or Rab3-GAP; indicated left of each panel) and pGAD plasmid expressing Rab protein (positions indicated in the bottom panels) were streaked on SC-AHLU and incubated at 30 °C. Positive patches are boxed. Although pGAD-Evi5-like induced a high background level, cells possessing pGBDU-Rab10 grew fastest of all (the 5-B position in the second left panel, boxed).

 
Since Haas et al. (2005) previously showed that a mutation of a putative catalytic arginine in the TBC domain (Rak et al. 2000) is necessary for stable interaction between RabGAP-5 and the GTP-fixed form of Rab5, the lack of interactions between the wild-type TBC protein and GTP-bound Rab in this study may be attributable to the lack of a catalytic arginine mutation in the TBC domain. To investigate this possibility, we prepared two types of catalytic arginine mutants of ten TBC proteins (i.e. Arg-to-Ala mutation and Arg-to-Lys mutation). We selected four TBC proteins (i.e. DJ1042K10.2, FLJ12085, FLJ38519, and OALT1; see Fig. 2) that interacted with specific Rabs and six TBC proteins (i.e. EPI64, FLJ10743, FLJ13130, GAP-CenA, MGC34741, and Vrp) that did not interact with any Rabs at all or interacted nonspecifically with most of the Rabs in our yeast two-hybrid screening. Interestingly, yeast two-hybrid screening revealed that introduction of the putative catalytic arginine mutation to alanine in most of the TBC proteins did not report the specific interaction between TBC(RA) mutant and Rab (boxed in Supplementary Fig. S3 and summarized in Table 1). For example, the EPI64(R155A), FLJ12085 (R417A), FLJ10743(R129A), FLJ13130(R160A), GAP-CenA(R540A), and MGC34741(K128A) mutants did not interact with any Rabs, although the wild-type FLJ12085 specifically interacted with Rab5A/B/C (Fig. 2) and GAP-CenA has been shown to exhibit Rab6-GAP activity (Cuif et al. 1999). The Vrp(R313A) mutant nonspecifically interacted with most of the Rabs, and the DJ1042K10.2(R165A) mutant showed reduced Rab5-binding activity and did not interact with Rab39B. By contrast, the OATL1(R294A) mutant strongly interacted with Rab2A and FLJ38519(R584A) strongly interacted with Rab34 and Rab36, the same as the wild-type proteins. Similar results were obtained for other TBC protein mutants with a catalytic arginine mutation to lysine (data not shown). The only exception was the interaction between FLJ38519(R584K) and Rab40B.

We found by database search that DJ1042K10.2 used in this study corresponds to RabGAP-5, a recently reported GAP specific for Rab5 (Haas et al. 2005; see above). In contrast to the study by Haas et al. (2005), however, the "wild-type" DJ1042K10.2/RabGAP-5 can interact with the GTP-fixed form of Rab5 regardless of the presence of a catalytic arginine mutation by our yeast two-hybrid screening (Fig. 2). We are not sure the exact reason for this discrepancy, but different yeast strains may differently affect interaction between DJ1042K10.2/RabGAP-5 and Rab5 in yeast. We therefore concluded that a catalytic arginine mutation to alanine is not always required for physical interaction between TBC protein and its substrate Rab by yeast two-hybrid screening.

Rab-GAP activity of OATL1 toward Rab2A

In the next set of experiments, we measured the GAP activity of OATL1, Evi5-like, FLJ38519, DJ1042K10.2, FLJ12085, and FLJ12168 in vitro (the GAP activity of USP6 could not be measured because of the very low yield of recombinant USP6 from COS-7 cells) to investigate whether the specific TBC Rab interactions described above correlated with the specific GAP activity of the TBC proteins. First, we focused on the GAP activity of OATL1 toward Rab2A, because OATL1 was found to specifically interact with Rab2A in our yeast two-hybrid screening (top left panel in Fig. 2). Rab-GAP assays were performed by using purified Rab and TBC proteins as described under Experimental procedures (unless otherwise specified, GST-Rab and GST-TBC proteins were used for GAP assays throughout the text). As anticipated, OATL1 actually exhibited the strong GAP activity toward Rab2A, whereas the other 11 TBC proteins that did not interact with Rab2A, and Rab3-GAP (Fukui et al. 1997) as a negative control, exhibited very little or no Rab2A-GAP activity (Fig. 3A). Measurement of the GAP activity of OATL1 toward four other Rabs, which did not interact with OATL1 in our yeast two-hybrid assay, showed that OATL1 also exhibited the significant GAP activity toward Rab13 and Rab34, but not toward Rab10 or Rab36 (Fig. 3B). However, since the Rab2A-GAP activity was stronger than the Rab13- and Rab34-GAP activity, OATL1 presumably prefers Rab2A as a substrate for GAP.


Figure 3
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  		Figure 3  Rab-GAP activity of OATL1. (A) Rab2A-GAP activity of 12 putative Rab-GAP/TBC proteins and Rab3-GAP. The GTPase activity of the putative Rab-GAPs indicated was measured as described under Experimental procedures. The results are expressed as the amount of the GTP-bound form of Rab2A after the reaction as a percentage of the amount before the reaction, and the bars represent means ± S.D. of data from three independent experiments. *P < 0.005; **P < 0.001 (Student's unpaired t-test). Note that OATL1 exhibited the strongest Rab2A-GAP activity among the putative Rab-GAPs tested. (B) Rab-GAP specificity of OATL1. The Rab-GAP activity of OATL1 toward Rab2A, Rab10, Rab13, Rab34, and Rab36 is summarized. The GTPase activity of each Rab protein in the presence of BSA (gray rectangle; see Figs. 4–8) or OATL1 (closed rectangle; see Figs. 4–8) is shown. *P < 0.005; **P < 0.001 (Student's unpaired t-test). Although OATL1 activated the GTPase activity of several other Rab proteins, Rab13 and Rab34, the Rab2A-GAP activity was most prominent, suggesting that OATL1 functions as a Rab2A-GAP.

 
Rab-GAP activity of Evi5-like toward Rab10

We next measured the GAP activity of Evi5-like toward Rab10, because Evi5-like preferentially interacted with Rab10 (left second panel in Fig. 2). Although the Rab10-GAP activity of Evi5-like was relatively weak, compared to the Rab2A-GAP activity of OATL1 described above, Evi5-like exhibited the strongest Rab10-GAP activity among the 13 TBC proteins and Rab3-GAP (Fig. 4A). Investigation of the specificity of the Rab-GAP activity of Evi5-like, however, revealed rather broad specificity (Fig. 4B). Evi5-like possessed weak GAP activity toward Rab4A, Rab7, and Rab10, but no activity toward Rab1A or Rab3A.


Figure 4
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  		Figure 4  Rab-GAP activity of Evi5-like. (A) Rab10-GAP activity of 13 putative Rab-GAP/TBC proteins and Rab3-GAP. The GTPase activity of the putative Rab-GAPs indicated was measured as described under Experimental procedures. The results are expressed as the amount of the GTP-bound form of Rab10 after the reaction as a percentage of the amount before the reaction, and the bars represent means ± S.D. of data from three independent experiments. *P < 0.005; **P < 0.001 (Student's unpaired t-test). Note that Evi5-like and some putative Rab-GAPs actually exhibited weak but significant GAP activity toward Rab10, and the Rab10-GAP activity of Evi5-like seemed to be the strongest. (B) Rab-GAP specificity of Evi5-like. The Rab-GAP activity of Evi5-like toward Rab1A, Rab3A, Rab4A, Rab7, and Rab10 is summarized. The GTPase activity of each Rab protein in the presence of BSA (Figure 4) or Evi5-like ({blacksquare}) is shown. *P < 0.005; **P < 0.001 (Student's unpaired t-test). Note that Evi5-like activated GTPase activity toward Rab10 most significantly.

 
Rab-GAP activity of FLJ38519 toward Rab22A, Rab34, and Rab39B

Since FLJ38519 interacted with three Rabs, Rab13, Rab34, and Rab36 (third left panel in Fig. 2), we first investigated the substrate specificity of the GAP activity. Unexpectedly, however, FLJ38519 exhibited the strongest GAP activity toward Rab22A, which did not interact with FLJ38519 in our yeast two-hybrid assay (Fig. 5A). FLJ38519 also exhibited mild GAP activity toward Rab34 and Rab39B, but no activity toward Rab2A, Rab5A, Rab10, Rab13, Rab36, or Rab40B (weak Rab13- and Rab36-GAP activity is observed in Fig. 5A, but it was not statistically significant under this experimental condition (2 pmol); but (see Fig. 7D) a large amount of FLJ38519 (10 pmol) can stimulate GTPase activity of Rab5A in vitro). Since FLJ38519 exhibited the strongest Rab22A-GAP activity, next we compared the Rab22A-GAP activity of ten TBC proteins and Rab3-GAP. It should be noted that FLJ38519 possessed the strongest Rab22A-GAP activity among the 11 putative Rab-GAPs tested (Fig. 5B), suggesting that FLJ38519 functions as a Rab22A-GAP, rather than as a Rab34-GAP. In contrast to OATL1 and Evi5-like described above, the Rab-binding activity of FLJ38519 was not correlated with its Rab-GAP activity.


Figure 5
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  		Figure 5  Rab-GAP activity of FLJ38519. (A) Rab-GAP specificity of FLJ38519. The GTPase activity of FLJ38519 was measured as described under Experimental procedures. The results are expressed as the amount of the GTP-bound form of each Rab protein after the reaction as a percentage of the amount before the reaction, and the bars represent means ± S.D. of data from three independent experiments. The GTPase activity of each Rab protein in the presence of BSA (Figure 5) or FLJ38519 ({blacksquare}) is shown. *P < 0.005; **P < 0.001 (Student's unpaired t-test). Note that FLJ38519 exhibited the strongest GAP activity toward Rab22A, which does not bind FLJ38519. (B) Rab22A- GAP activity of ten putative Rab-GAP/TBC proteins and Rab3-GAP. Although several TBC proteins activated the GTPase activity of Rab22A, FLJ38519 exhibited the strongest Rab22A-GAP activity. *P < 0.005; **P < 0.001 (Student's unpaired t-test). (C) Over-expression of GFP-FLJ38519 in NIH-3T3 cells resulted in a loss of the Rab5 effector EEA1 from endosomes. NIH-3T3 cells transiently expressing GFP-FLJ38519 (left panels) was stained with EEA1 (middle panels) as described under Experimental procedures. Right panels are the merged images between left and middle panels. Insets show magnification of the boxed areas, where the partial co-localization between GFP-FLJ38519 and EEA1 is apparent. Note that high expression level of GFP-FLJ38519 in NIH3T3 cells caused a loss of EEA1 signals (arrowheads in the top middle panel). Bar, 20 µm.

 

Figure 7
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  		Figure 7  Rab5A-GAP activity of FLJ12085 and FLJ12168, which specifically interact with Rab5A. Rab-GAP activity of TBC proteins (2 pmol) toward (A) GST-Rab5A and (B) untagged Rab5A. (C) Rab-GAP activity of DJ1042K10.2 (5 or 10 pmol) toward untagged Rab5A and Rab39B. (D) Rab5A-GAP activity of FLJ38519 (10 pmol). The GTPase activity of the TBC proteins indicated was measured as described under Experimental procedures. The results are expressed as the amount of the GTP-bound form of Rab5A after the reaction as a percentage of the amount before the reaction, and the bars represent mean ± S.D.  of data from three independent experiments. Except for the large amount of FLJ38519 (10 pmol), none of the TBC proteins tested showed significant GAP activity toward Rab5A under our experimental conditions. **P < 0.001 (Student's Unpaired t-text).

 
Because Rab22A has been suggested to control endosomal trafficking through interaction with EEA1, a well-known Rab5 effector (Christoforidis et al. 1999; Kauppi et al. 2002), we further investigated the effect of over-expression of FLJ38519 in NIH3T3 cells on endosomal localization of EEA1 (Fig. 5C). Green fluorescent protein (GFP)-tagged FLJ38519 seemed to be localized in various membrane structures, including nucleus, Golgi, and early endosomes (bottom middle panel Fig. 5C) and partial co-localization of FLJ38519 and EEA1 was clearly observed in cells showing low expression level of GFP-FLJ38519 (bottom right panel). It should be noted that EEA1 signals almost disappeared in cells showing high expression level of GFP-FLJ38519 (top middle panel Fig. 5C, arrowhead), but not in the control cells expressing GFP alone (data not shown) or untransfected cells, indicating that FLJ38519 acts as a GAP toward endosomal Rab (presumably Rab22A or Rab5A) in living cells.

Rab-GAP activity of DJ1042K10.2 toward Rab39B

Although DJ1042K10.2 strongly interacted with three Rab5 isoforms (top right panel in Fig. 2), it did not show any significant Rab5A-GAP activity under our experimental conditions (Fig. 6A). To our surprise, however, DJ1042K10.2 showed the strongest GAP activity toward Rab39B, which marginally interacted with DJ1042K10.2 in our two-hybrid assay. In addition, DJ1042K10.2 showed very little or no GAP activity toward the other Rabs tested (i.e. Rab1A, Rab2A, Rab3A, Rab4A, Rab5A, Rab7, Rab10, and Rab22A), suggesting that DJ1042K10.2 functions as a Rab39B-specific GAP. To further investigate this possibility, we compared the Rab39B-GAP activity of 11 putative Rab-GAPs, including DJ1042K10.2. As anticipated, DJ1042K10.2 most strongly activated the GTPase activity of Rab39B, while the others showed weak and non-significant GAP activity (Fig. 6B). These results strongly suggest that DJ1042K10.2 functions as a Rab39B-specific GAP among the TBC protein family.


Figure 6
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  		Figure 6  Rab-GAP activity of DJ1042K10.2. (A) Rab-GAP specificity of DJ1042K10.2. GTPase activity of DJ1042K10.2 was measured as described under Experimental procedures. The results are expressed as the amount of the GTP-bound form of each Rab protein after the reaction as a percentage of the amount before the reaction, and the bars represent means ± S.D. of data from three independent experiments. The GTPase activity of each Rab protein in the presence of BSA (Figure 6) or DJ1042K10.2 ({blacksquare}) is shown. **P < 0.001 (Student's unpaired t-test). Note that DJ1042K10.2 exhibited the strongest GAP activity toward Rab39B, which weakly interacts with DJ1042K10.2, whereas it lacked GAP activity toward Rab5A, which strongly interacts with DJ1042K10.2 (see also Fig. 7B,C). (B) Rab39B-GAP activity of ten putative Rab-GAP/TBC proteins and Rab3-GAP. Note that DJ1042K10.2 exhibited the strongest Rab39B-GAP activity. *P < 0.005; **P < 0.001 (Student's unpaired t-test).

 
As noted above, however, DJ1042K10.2 corresponds to RabGAP-5, which has recently been identified as a GAP specific for Rab5 (Haas et al. 2005), in contrast to our finding. Since we used GST-Rab5A for our GAP assay, the lack of Rab5A-GAP activity of DJ1042K10.2 in this study may be attributable to the fusion of Rab with GST (i.e. fusion of Rab with GST may inhibit Rab-GAP activity or alter Rab-GAP specificity of TBC proteins). To further investigate this possibility, we removed GST by thrombin digestion and prepared "untagged" Rab5A and Rab39B. As shown in Fig. 7B,C, however, intrinsic GTPase activity of untagged Rab5A was not different from that of GST-Rab5A, and DJ1042K10.2 still did not show any GAP activity toward untagged Rab5A even though five times greater amount of DJ1042K10.2 (10 pmol) was used for the assay. In contrast, DJ1042K10.2 did show strong Rab39B-GAP activity (Fig. 7C), indicating that fusion of Rab with GST is unlikely to alter Rab-GAP activity or specificity. At present, we are not sure the exact reason for the discrepancy between the study by Haas et al. (2005) and ours, but the only difference may be the source of recombinant DJ1042K10.2 protein (i.e. bacteria and COS-7 cells, respectively). It is possible that certain post-translational modifications of DJ1042K10.2 may modulate the Rab-GAP activity or specificity. Further work is necessary to elucidate this issue.

Lack of Rab5A-GAP activity of FLJ12085 and FLJ12168

The results of the two-hybrid assay indicated that both FLJ12085 and FLJ12168 specifically interacted with Rab5A/B/C (right second and third panels in Fig. 2, respectively). To our surprise, however, neither TBC protein showed any Rab-GAP activity toward GST-Rab5A (Fig. 7A) or untagged Rab5A (Fig. 7B) under our experimental conditions, the same as DJ1042K10.2 described above (Fig. 7C). Although FLJ10743 and FLJ12168-like, Rab5A-unbound TBC proteins (up to 10 pmol), still did not show GAP activity toward untagged Rab5A (data not shown), large amount of FLJ38519 (10 pmol, but not 2 pmol) showed significant GAP activity toward both GST-Rab5A and untagged Rab5A (Fig. 7D). This observation was not surprising because we characterized FLJ38519 as a putative Rab22A-GAP in this study (Fig. 5) and Rab22A is a most closely related isoform of Rab5A (Supplementary Fig. S2). We also investigated their GAP activity toward several other Rabs (e.g. Rab2A, Rab10, Rab22A, and Rab39B), which did not interact with FLJ12085 or FL12168 in our two-hybrid assay, but, unfortunately, we did not detect any Rab-GAP activity at all. Although we have not yet identified the actual target Rab for FLJ12085 and FL12168, there is no relation between the Rab-binding activity and Rab-GAP activity of these two Rab-GAPs, and they presumably activate the GTPase activity of unidentified target Rabs without stably or strongly interacting with their targets.

Rab-GAP activity and Rab-binding activity of Rab3-GAP

The results for the GAP-activity of the TBC proteins described above show that Rab-binding activity is not always correlated with the Rab-GAP activity of the TBC domain (e.g. FLJ38519 in Fig. 5A). In the final set of experiments, we investigated the relationship between the Rab-binding activity and Rab-GAP activity of Rab3-GAP, which does not contain a TBC domain (i.e. distinct class of Rab-GAP) (Fukui et al. 1997). The two-hybrid assay described above revealed that Rab3-GAP strongly interacted with Rab39A and Rab39B, and weakly with Rab22A, but not at all with its known substrate, Rab3A (Fig. 2, bottom right panel). Since the Rab-GAP activity of Rab3-GAP toward Rab22A and Rab39 has never been elucidated, we used Rab3-GAP-binding Rabs (i.e. Rab22A and Rab39B) and non-binding Rabs (i.e. Rab1A, Rab2A, Rab3A, Rab4A, and Rab10) to investigate the substrate specificity of Rab3-GAP. As shown in Fig. 8, Rab3-GAP activated the GTPase activity of Rab3A most strongly, consistent with the previous report (Fukui et al. 1997), and the Rab-GAP activity toward Rab22A and Rab39B, which interact with Rab3-GAP, was relatively weak. We therefore concluded that there is no relation between the Rab-binding activity and Rab-GAP activity of Rab3-GAP.


Figure 8
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  		Figure 8  Rab-GAP specificity of Rab3-GAP. The GTPase activity of Rab3-GAP was measured as described under Experimental procedures. The results are expressed as the amount of the GTP-bound form of each Rab protein after the reaction as a percentage of the amount before the reaction, and the bars represent means ± S.D. of data from three independent experiments. The GTPase activity of each Rab protein in the presence of BSA (Figure 8) or Rab3-GAP ({blacksquare}) is shown. *P < 0.005; **P < 0.001 (Student's unpaired t-test). Consistent with the previous report (Fukui et al. 1997), Rab3-GAP showed the strongest GAP activity toward Rab3A.

 

   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In the present study we attempted to identify the target Rab proteins of orphan TBC domains by yeast two-hybrid screening, and we succeeded in identifying OATL1, Evi5-like, FLJ38519, and DJ1042K10.2 as a Rab2A-GAP, Rab10-GAP, Rab22A-GAP, and Rab39B-GAP, respectively. Our large-scale screening for the target Rabs of TBC proteins yielded several important findings about the Rab-GAP activity of TBC proteins. First, our initial assumption that the TBC domain specifically recognizes and interacts with a substrate Rab (i.e. the GTP-bound form of Rab) is not true of most TBC proteins, because 33 of the 40 TBC proteins tested did not interact with any Rab isoforms. Since the previously characterized Rab5-GAP (RN-Tre and PRC17) (Lanzetti et al. 2000; Pei et al. 2002), Rab6-GAP (GAP-CenA) (Cuif et al. 1999), or Rab7-GAP (FLJ12085/TBC1D15) (Zhang et al. 2005) also did not interact with their substrate Rabs (Table 1), we are compelled to conclude that there is no correlation of the Rab-binding activity with the Rab-GAP activity of most TBC proteins. Second, the Rab-GAP specificity of TBC proteins is not very high, and every TBC protein tested in this study showed significant GAP-activity toward several Rab isoforms in vitro (e.g. OATL1 for Rab2A, Rab13, and Rab36 in Fig. 3), although the extent of their GTPase activation differed (e.g. OATL1 prefers Rab2A). Similar observations have also been made in recent studies by other groups (Cuif et al. 1999; Mîinea et al. 2005; Zhang et al. 2005). Therefore, it is still possible that certain TBC proteins which were not tested in this study have stronger Rab2A-GAP (or Rab22A-GAP) activity than OATL1 (or FLJ38519), and that such TBC proteins may function as a Rab2A-GAP (or Rab22A-GAP) under physiological conditions. This possibility is also true of the Rab-GAPs previously characterized, because no one has investigated all Rabs in mice to determine their specificity. Another screening system is required to determine the actual target Rab of TBC proteins, but it seemed to us that measurement of the Rab-GAP activity of a single TBC domain toward 60 Rab isoforms one-by-one may be the best way to identify the actual target Rab, because we already possess 60 different Rabs. Third, since several TBC proteins can interact with specific Rab isoforms (e.g. FLJ12085 with Rab5A/B/C) and lack putative catalytic arginine residue (e.g. threonine instead of arginine in USP6; see asterisk in supplementary Fig. 1), we speculate that TBC domains may have other functions, e.g. as a Rab effector, in addition to Rab-GAP activity (Lanzetti et al. 2004). It would be interesting to determine whether over-expression of FLJ12085 affects the function of Rab5A in endosomal trafficking.

In summary, for the first time, we established a nearly complete set of Rab panels and panels of 40 TBC proteins, investigated interactions by 2400 combinations of the 40 TBC domains and the 60 Rabs by yeast two-hybrid screening, and identified several Rab-GAPs. Further study of the intracellular localization and function of these Rab-GAPs at the cellular level is necessary to determine whether they function as actual Rab-GAPs in vivo. Although screening for the target Rabs of the TBC proteins by the two-hybrid assay was found to be limited, we think that the Rab panels established in this study are very powerful tools for determining the specificity of Rab effectors or other Rab-binding proteins, because Rab effectors, such as rabphilin and rabenosyn-5 (Fukuda 2003, 2005; Fukuda et al. 2004; Eathiraj et al. 2005), have recently been shown to interact with several Rab isoforms. Our Rab panels should greatly accelerate our understanding of the roles of Rab proteins in membrane trafficking in the near future.


   Experimental procedures
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Molecular cloning of mouse and human Rab and TBC protein cDNAs

cDNA encoding the mouse Rabs (Rab1B, Rab2B, Rab4B, Rab5B, Rab5C, Rab6B, Rab9B, Rab11B, Rab22A, Rab22B, Rab33B, Rab39A, Rab39B, Rab40C, Rab41, Rab42/Rab7-like and Rab43/Rab39-like) and the human Rabs (Rab6C and Rab40A) was amplified from Marathon-Ready adult mouse or human brain cDNA (BD Clontech, Palo Alto, CA, USA) by PCR with specific oligonucleotides (see Supplementary Table S2 for details) as previously described (Fukuda et al. 1999). cDNA encoding the human or mouse TBC proteins (summarized in Table 1) and Rab3-GAP was similarly amplified by PCR using human or mouse brain cDNA or KIAA clones obtained from Kazusa DNA Research Institute (Chiba, Japan) as a template (Fukuda et al. 1999). Specific oligonucleotides with an additional restriction enzyme site were designed according to the sequences in the public database (see also GeneID in Table 1), and the sequence information for the oligonucleotides used is available from the authors on request. Purified PCR products were directly inserted into the pGEM-T Easy vector (Promega, Madison, WI, USA) and verified by DNA sequencing as previously described (Fukuda et al. 1999). The Rab cDNAs were transferred to the pEF-FLAG tag mammalian expression vectors (modified from pEF-BOS) (Mizushima & Nagata 1990; Fukuda et al. 1994, 1999). The cDNA of the TBC proteins was transferred to the pEF-T7 tag or pEF-T7-GST tag mammalian expression vectors (Fukuda et al. 1999, 2002b). The cDNA of FLJ38519 was also subcloned into the pEGFP-C1 vector (BD Clontech). Other FLAG-Rab expression vectors were prepared as previously described (Kuroda et al. 2002; Fukuda et al. 2002b; Fukuda 2003). Plasmid DNA for transfection into COS-7 cells was prepared by using Qiagen (Chatsworth, CA, USA) maxiprep kits according to the manufacturer's notes. The mouse or human Rab1–43 cDNA was also subcloned into the pGEX-4T-3 vector (Amersham Biosciences, Buckinghamshire, UK). Rab5A and Rab39B cDNAs were subcloned into the pGEX-4T-3-gk vector modified from pGEX-4T-3 by introducing a short Gly linker (PGISGGGGGT) just downstream of GST (named pGEX-4T-3-gk-Rab5A and -Rab39B, respectively). The nomenclature of the Rabs is according to Pereira-Leal and Seabra (Pereira-Leal & Seabra 2001) (see Supplementary Fig. S2 and Table S1).

Sequence analysis

Multiple sequence alignment and phylogenetic analysis of the Rab family and the TBC proteins were performed using the ClustalW program (<http://www.ddbj.nig.ac.jp/search/clustalw-j.html>) and CLUSTALX program (Thompson et al. 1997), respectively, set at the default parameters.

Site-directed mutagenesis

GTP-fixed mutants of Rab were produced by replacement of a conserved glutamine required for GTP hydrolysis with leucine by conventional or two-step PCR techniques as previously described (Fukuda et al. 1995; Fukuda & Kuroda 2002; Fukuda 2002, 2003). The sequences of the mutagenic oligonucleotides used are available from the authors on request. The following Rab mutants were used in this study: Rab1A(Q70L), Rab1B(Q67L), Rab2A(Q65L), Rab2B(Q65L), Rab3A(Q81L), Rab3B(Q81L), Rab3C(Q89L), Rab3D(Q81L), Rab4A(Q72L), Rab4B(Q67L), Rab5A(Q79L), Rab5B(Q93L), Rab5C(Q80L), Rab6A(Q72L), Rab6B(Q72L), Rab6C(Q72L), Rab7(Q67L), Rab8A(Q67L), Rab8B(Q67L), Rab9A(Q66L), Rab9B(Q66L), Rab10(Q68L), Rab11A(Q70L), Rab11B(Q70L), Rab12(Q100L), Rab13(Q67L), Rab14(Q70L), Rab15(Q67L), Rab17(Q77L), Rab18(Q67L), Rab19(Q76L), Rab20(R59L), Rab21(Q76L), Rab22A(Q64L), Rab22B(Q64L), Rab23(Q68L), Rab24(S67L), Rab26(Q57L), Rab27A(Q78L), Rab27B(Q78L), Rab28(Q72L), Rab29(Q67L), Rab30(Q68L), Rab32(Q83L), Rab33A(Q95L), Rab33B(Q92L), Rab34(Q111L), Rab35(Q67L), Rab36(Q116L), Rab37(Q89L), Rab38(Q69L), Rab39A(Q72L), Rab39B(Q68L), Rab40A(Q73L), Rab40B(Q72L), Rab40C(Q73L), Rab41(Q75L), Rab42(Q67L) and Rab43(Q72L). Since Rab20, Rab24, and Rab25 lack the conserved glutamine residue responsible for GTP hydrolysis and its position is occupied by arginine, serine, and leucine, respectively, we used Rab20(R59L), Rab24(S67L), and wild-type Rab25 as GTP-fixed mutants, respectively, in this study.

TBC protein mutants with a mutation of the putative catalytic arginine residue to alanine or lysine (DJ1042K10.2(R165A), DJ1042K10.2(R165K), EPI64(R155A), EPI64(R155K), FLJ10743(R129A), FLJ10743(R129K), FLJ12085(R417A), FLJ12085(R417K), FLJ13130(R160A), FLJ13130(R160K), FLJ38519(R584A), FLJ38519(R584K), GAP-CenA(R540A), GAP-CenA(R540K), MGC34741(K128A), MGC34741(K128R), OATL1(R294A), OATL1(R294K), Vrp(R313A) and Vrp(R313K)) were also produced by conventional or two-step PCR techniques as previously described (Fukuda et al. 1995). The sequences of the mutagenic oligonucleotides used are also available from the authors on request.

Two-hybrid assay

cDNA of the GTP-fixed mutants of Rab (or wild-type Rab25) was subcloned into the pGAD-C1 vector (James et al. 1996). The cDNA of the TBC proteins (or Rab3-GAP) was also subcloned into the pGBDU-C1 vector (James et al. 1996). The yeast strain, medium, culture conditions, and transformation protocol are described in James et al. (1996). The materials used for the two-hybrid assay in this study were: yeast strain pJ69–4 A; plasmid pGAD-C1 for expression of activating domain fusion protein; plasmid pGBDU-C1 for expression of DNA-binding domain fusion protein; and a synthetic complete medium lacking adenine, histidine, leucine, and uracil (SC-AHLU, 0.67% yeast nitrogen base w/o amino acids, 2% glucose, 2% Bacto agar, 0.01% tryptophan, 0.0125% lysine, and 0.01% methionine) for selection medium. All reagents used were analytical grade or the highest grade commercially available.

Preparation of GST-fusion proteins

GST-fusion proteins of the Rabs were expressed in Escherichia coli JM109 and purified by standard protocols (Kuroda & Fukuda 2005), and thrombin digestion was performed as previously described (Fukuda et al. 2002a). Protein concentrations were determined by using the Bio-Rad protein assay kit (Hercules, CA, USA) and bovine serum albumin (BSA) as a reference. COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin G, and 100 µg/mL streptomycin, at 37 °C under 5% CO2. pEF-T7-GST-TBC protein vectors (4 µg of plasmids) were transfected into COS-7 cells (7.5 x 105 cells, the day before transfection/10 cm-dish) by using LipofectAmine Plus reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's notes. Three days after transfection, cells were harvested and homogenized, and total cell lysates were prepared as previously described (Fukuda & Kanno 2005). Recombinant T7-GST-TBC proteins were affinity-purified with glutathione-Sepharose beads (Amersham Biosciences) and eluted from the beads with a buffer containing 5 mM glutathione and 50 mM Tris-HCl, pH 8.0, as previously described (Kuroda & Fukuda 2005). Purified proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) followed by staining with Coomassie Brilliant Blue R-250, and protein concentrations were determined on SDS-polyacrylamide gels, using BSA as a reference. Most of the recombinant TBC proteins used in this study were purified as a single band on SDS-polyacrylamide gel without contaminating by degradation products (Supplementary Fig. S4).

Assays for GTP loading of Rab and GAP activity

The GTP-loading protocol was adapted from the Rab GEF assay described in Otomo et al. (2003). A 200 pmol amount of purified Rab protein (unless otherwise specified, GST-Rab proteins were used for Rab-GAP assay) was incubated with 6.7 pmol of [{alpha}-32P]GTP (Amersham Biosciences) and 800 pmol of cold GTP (Sigma Chemical Co., St. Louis, MO, USA) in 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2.5 mM EDTA, and 0.5 mg/mL BSA for 10 min at 30 °C. After addition of MgCl2 (final concentration, 10 mM), the mixture was passed through a PD-10 column (Amersham Biosciences) filled with Sephadex G-25 (Amersham Biosciences), and the 3.0–3.5-mL eluate fractions were collected. The GTPase activity reaction was initiated by adding 2 pmol (or 10 pmol for Fig. 7B–D) of GST-TBC protein, or BSA as a control, to 10 µL aliquots of the fraction (equivalent of 2 pmol of Rab protein), and incubating at 30 °C. The reaction time was varied according to the Rab isoform tested (Rab1A for 1 h; Rab2A, 1 h; Rab3A, 15 min; Rab4A, 1 h; Rab5A, 12 min; Rab7, 1 h; Rab10A, 1 h; Rab13A, 1 h; Rab22A, 1 h; Rab34, 1 h; Rab36, 1 h; Rab39B, 40 min; and Rab40B, 40 min), because the intrinsic GTPase activity of the Rab family members differs. The reaction was halted by addition of an equal volume of stop buffer (20 mM EDTA and 0.4% SDS), and incubation for 5 min at 70 °C. A 2 µL sample was dropped on a TLC plate (Merck Biosciences, Darmstadt, Germany) and developed in 0.5 M LiCl and 1 M formic acid. The amounts of GTP and GDP were determined with a BAS-2500 Bioimage analyzer (FUJIFILM, Tokyo, Japan) and an imaging plate.

Immunostaining of NIH3T3 cells

NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin G, and 100 µg/mL streptomycin at 37 °C under 5% CO2. pEGFP-C1-FLJ38519 vector was transfected into NIH3T3 cells by using FuGENE6 (Roche, Basel, Switzerland) according to the manufacturer's instructions. At 48–72 h after transfection, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton-X-100 for 2 min, and blocked with the blocking buffer (1% BSA and 0.1% Triton-X-100 in PBS) for 1 h. The cells were then immunostained with anti-EEA1 mouse monoclonal antibody (BD Biosciences PharMingen, San Diego, CA, USA) followed by Alexa Fluor secondary IgG (1/5000 dilution, Molecular Probes). Fluorescence images were acquired and pseudocolored with a confocal laser scanning microscope (Fluoview, OLYMPUS), and the images were processed with Adobe Photoshop software (version 7.0).


   Acknowledgements
 
This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan Grants 17657067, 18022048, 18050038, 18057026, 18207015 (to M. F.), by the Kato Memorial Bioscience Foundation (to M. F.), by the Sumitomo Foundation (to M. F.), and by the Nakatomi Foundation (to M. F.). We thank Dr Takahiro Nagase for kindly donating KIAA cDNA clones, Dr Akio Toh-e for a yeast strain and plasmids for yeast two-hybrid assay, and Drs Gustav E. Lienhard and Hiroyuki Sano for useful discussions during the course of yeast two-hybrid screening of Rab-GAPs.


   Footnotes
 
Communicated by: Akihiko Nakano

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankä/EBI Data Bank with accession number(s) AB232583-AB232642.

* Correspondence: E-mail: nori{at}mail.tains.tohoku.ac.jp.


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Received: 21 February 2006
Accepted: 1 June 2006




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