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Originally published In Press as doi:10.1074/jbc.M603808200 on August 21, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31823-31831, October 20, 2006
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Identification of EPI64 as a GTPase-activating Protein Specific for Rab27A*Formula

Takashi Itoh{ddagger}1 and Mitsunori Fukuda{ddagger}§2

From the {ddagger}Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198 and the §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

Received for publication, April 20, 2006 , and in revised form, July 27, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Small GTPase Rab27A plays a pivotal role in melanosome transport in melanocytes and in secretion by various secreting cells. Because the GTP- or GDP-locked mutant of Rab27A causes perinuclear aggregation of melanosomes, appropriate GTP-GDP cycling of Rab27A is essential for melanosome transport, and certain guanine nucleotide exchange factors and GTPase-activating proteins (GAPs) of Rab27A must be present in melanocytes. However, no such regulators of Rab27A have ever been identified. In this study we developed novel methods of rapidly screening 40 different TBC (Tre2/Bub2/Cdc16) proteins, putative Rab-GAPs, for Rab27A-GAP by: (i) searching for TBC proteins that induce melanosome aggregation in melanocytes; (ii) trapping GTP-Rab27A with a Rab27A effector domain (i.e. the SHD of Slac2-a) in cultured cells that express both Rab27A and TBC proteins; and (iii) measuring in vitro Rab27A-GAP activity. These methods allowed us to identify EPI64, previously characterized as an EBP50-binding protein that contains an orphan TBC domain, as a specific Rab27A-GAP. We further showed that mutations in the catalytic domain of EPI64 caused complete loss of its ability to induce melanosome aggregation. This is the first report of screening for Rab27A-GAP based on functional interactions, and our screening methods can be applied for other uncharacterized TBC proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Rab/Ypt small monomeric GTPases form the largest subfamily of the small GTPase superfamily, and they are thought to play important roles in intracellular membrane trafficking (reviewed in Refs. 1 and 2). Over 60 Rabs are present in mammals, and each Rab member is believed to be localized at a specific organelle(s) and to coordinate endocytosis, exocytosis, and inter-organelle vesicular transport. The same as members of other small GTPase subfamilies (e.g. Rho and Arf), Rab occurs in a GTP-bound active state and a GDP-bound inactive state, and cycling of these two states is essential for exertion of its function in membrane trafficking. Two classes of regulatory factors, guanine nucleotide exchange factors, which deprive Rabs of GDP and promote GTP loading, and GTPase-activating proteins (GAPs),3 which promote the GTPase activity of Rabs, accelerate Rab cycling (1). Despite the large number of Rabs in mammalian cells, however, only a few GAPs and guanine nucleotide exchange factors have been identified thus far.

The TBC (Tre/Bub2/Cdc16) domain has been proposed to be a GAP domain for the Rab family small GTPases, because several TBC domain-containing proteins (referred to as TBC proteins below) exhibit GAP activity toward certain Rabs (3). The first TBC/Rab-GAP reported was Gyp6 and was identified as a GAP of Ypt6 in Saccharomyces cerevisiae (4). The following TBC proteins in yeasts or mammals have been shown to exhibit GAP activity: Gyp1 exhibits GAP activity toward Sec4 and Ypt1 (5); GAP-CenA, toward Rab3, Rab4, and Rab6 (6); RN-Tre, toward Rab5 and Rab41 (7, 8); Prc17, toward Rab5 (9); Msb3/4, toward Sec4 (10); AS160, toward Rab2, 8, 10, and 14 (11); Rab-GAP-5, toward Rab5 (8); and TBC1D15, toward Rab7 and Rab11A (12).

Although the human genome project has revealed that over 50 putative Rab-GAPs (TBC proteins) are present in the human genome (3), only 6 of them have been characterized as specific Rab-GAPs, and the others remain orphan Rab-GAPs. Haas et al. (8) have recently reported an elegant method of screening for Rab5-GAPs based on the physical interaction between constitutively active Rabs and TBC proteins, and their method has shown that interaction between TBC proteins and specific Rabs is a good indicator for identifying the substrate of certain TBC proteins. In our preliminary experiments, however, some of the TBC proteins did not seem to need to physically or stably associate with its substrate Rab, and we were unable to identify any candidate Rab27A-GAPs by the above two-hybrid assay (50). Therefore, new screening methods not based on the protein-protein interactions were needed to identify the specific GAP of Rab27A.

Rab27A is localized on mature melanosomes in melanocytes, and it plays an essential role in melanosome transport through sequentially interacting with its effectors, Slac2-a/melanophilin and Slp2-a (13, 14). Melanosomes are produced and mature in the perinuclear region of melanocytes, and are then transported to the periphery of the cell to be translocated to the juxtaposed keratinocytes (15, 16). Slac2-a interacts with Rab27A, via its N-terminal Rab27A-binding domain (called the Slp homology domain, SHD), and with myosin Va, via its middle region, to form a tripartite complex that is essential for the melanosome transport along actin cables (17-19). Slp2-a simultaneously interacts with Rab27A, via its N-terminal SHD, and with phospholipids, via its C-terminal tandem C2 domains, to anchor the melanosomes just below the plasma membrane (13).

Functional loss of Rab27A results in partial albinism in human Griscelli syndrome (20) and in a lighter coat color in ashen mice (21) because of the defect in melanosome transport to the periphery of the cell (22-24). The same melanosome defect has been induced by expression of the constitutively active or negative forms of Rab27A (23, 25, 26). We therefore assume that melanosome transport is a good model for initial screening for candidate Rab27A-GAPs by overexpressing various TBC proteins in melanocytes (i.e. expression of Rab27A-GAP in melanocytes should induce the melanosome aggregation).

In this study, we screened 40 human or mouse TBC proteins for putative Rab27A-GAPs by the above screening methods and identified EPI64/Rab27A-GAP{alpha} and its homologue FLJ13130/Rab27A-GAPbeta as Rab27A-GAPs by directly measuring in vitro Rab27A-GAP activity. We discuss the utility of our novel methods of screening for Rab-GAPs based on our findings.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Horseradish peroxidase (HRP)-conjugated anti-FLAG tag (M2) mouse monoclonal antibody was obtained from Sigma. HRP-conjugated anti-T7 tag mouse monoclonal anti-body was from Merck Biosciences Novagen (Darmstadt, Germany). Anti-Rab27A mouse monoclonal antibody and Alexa 594-conjugated anti-mouse IgG goat antibody were from BD Transduction Laboratories (Lexington, KY) and Molecular Probes (Eugene, OR), respectively. [{alpha}-32P]GTP was from Amersham Biosciences.

Plasmid Construction—The cDNAs encoding the human or mouse TBC proteins (summarized in Table 1) and Rab3-GAP were cloned as described elsewhere (50). The cDNAs of the TBC proteins were transferred to the pEGFP-C1 vector (Clontech, Palo Alto, CA), the pEF-T7 expression vector, or pEF-T7-GST expression vector (17, 27, 28) modified from pEF-BOS (29). Two EPI64 mutants, one with lysine substituted for arginine at amino acid position 160 (named EPI64-R160K), and the other with alanine substituted for glutamic acid at amino acid position 157 (named EPI64-D157A), were produced by two-step PCR techniques using the following mutagenic oligonucleotides having an artificial XhoI site (underlined), as described previously (30): 5'-TGTTTGTGTCTCGAGGGGGC-3' (TBC primer; sense) and 5'-GCCCCCTCGAGACACAAACATCTCATGGAATGGGAACTGCTTGTGCAG-3' (R160K primer; antisense) for EPI64-R160K, and 5'-TGTTTGTGTCTCGAGGGGGC-3' (TBC primer; sense) and 5'-GCCCCCTCGAGACACAAACATCTCATGGAATGGGAACTGCCGGTGCAGGGCACGCTC-3' (D157A primer; antisense) for EPI64-D157A. The mutant EPI64 fragments were then subcloned into the modified pEF-T7 expression vector or pEGFP-C1 vector. pEGFP-C1-EPI64+A (addition of alanine to the C-terminal end of EPI64) and pEGFP-C1-EPI64-{Delta}449 (deletion of the 59 C-terminal amino acids) were similarly constructed by conventional PCR techniques. The following pairs of nucleotides with a 19-base target site (ATCAATCTTCTGGCGGCTG) were used to generate small interfering RNA expression plasmids (named pSilencer 2.1-U6 neo-EPI64) (Ambion, Austin, TX) against mouse EPI64 mRNA as described previously (31): 5'-GATCCGCAGCCGCCGGAAGGTTGGTTTCAAGAGAATCAATCTTCTGGCGGCTGTTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAACAGCCGCCAGAAGATTGATTCTCTTGAAACCAACCTTCCGGCGGCTGCG-3'. Rab3A and Rab27A cDNAs (32) were subcloned into the pGEX-4T-3-gk vector modified from pGEX-4T-3 (Amersham Biosciences) by introducing a short Gly linker (PGISGGGGGT) just downstream of GST (glutathione S-transferase) (named pGEX-4T-3-gk-Rab3A and -Rab27A, respectively). Other expression plasmids (pEF-FLAG-Rab27A and pEF-T7-GST-Slac2-a-SHD) were prepared as described elsewhere (17, 32).


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  		TABLE 1
Summary of the melanosome aggregation activity of TBC proteins

Truncated forms containing the TBC domain, FLJ33929, KIAA0397, KIAA0608, KIAA0882, KIAA0984, KIAA1055, KIAA1108, KIAA1171, KIAA1941, USP6, and Vrp, were used for the screening. The other TBC proteins and Rab3-GAP used were full-length proteins, although AAH47400, Evi-5, FLJ10743-like, and MGC34741 contained alternative splicing. FLJ23725 and HSPC302 are the same gene products, but FLJ23725 contains an additional sequence at the N terminus.

 


Figure 1
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  		FIGURE 1.
Candidate Rab27A-GAPs. Melan-a cells expressing GFP alone (top panels), GFP-FLJ22474 (second panels from the top), GFP-Vrp (middle panels), GFP-EPI64 (second panels from the bottom), and GFP-FLJ13130 (bottom panels). GFP fluorescence is shown at the left, and the corresponding bright-field image is at the right. The arrowheads point to cells expressing each of the GFP-tagged TBC proteins and exhibiting the melanosome aggregation. Bar, 20 µm.

 
Screening for TBC Proteins Capable of Inducing the Melanosome Aggregation in Melanocytes—The immortalized melanocyte cell line melan-a (generous gift of Dorothy C. Bennett) was cultured as described previously (19, 33). Transfection of pEGFP-C1-TBC proteins (e.g. pEGFP-C1-EPI64) into melan-a cells with FuGENE 6 (Roche Molecular Biochemicals), and the melanosome aggregation assay were performed as described previously (19). The cells were examined for fluorescence and bright-field images with a confocal fluorescence microscope (Fluoview; Olympus, Tokyo, Japan). The melanosome distribution of the transfected cells ("aggregated" means accumulation of melanosomes in the perinuclear regions, as shown in the bottom right panel of Fig. 1, as opposed to the melanosomes in normal melan-a cells, which are localized at the periphery of the cell, as shown in the top right panel of Fig. 1) was evaluated as described previously (19).

GTP-Rab27A Pulldown Assay—Plasmids were transfected into COS-7 cells (7.5 x 105 cells/10-cm dish, the day before transfection) with Lipofectamine Plus (Invitrogen) according to the manufacturer's notes. COS-7 cells expressing FLAGtagged Rab27A (FLAG-Rab27A) and each T7-tagged TBC protein were homogenized in lysis buffer containing 50 mM HEPES-KOH (pH 7.2), 150 mM NaCl, 10% glycerol, 50 µM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 5 µM pepstatin A and then solubilized with 1% Triton X-100. After centrifugation, the supernatants were appropriately diluted with a lysis buffer so that the amounts of FLAG-Rab27A protein in the diluted samples were equal when immunoblotted with HRP-conjugated anti-FLAG tag antibody. The diluted samples were incubated for 1 h at 4°C with glutathi-one-Sepharose beads (Amersham Biosciences) coupled with the T7-GST-tagged SHD of Slac2-a (T7-GST-Slac2-a-SHD; i.e. GTP-Rab27A trapper (34)). After washing the beads three times, the GTP-Rab27A trapped by the beads was analyzed by 10% SDS-PAGE followed by immunoblotting with HRP-conjugated anti-FLAG tag antibody (1/5000 dilution) or HRP-conjugated anti-T7 tag antibody (1/5000 dilution).

Purification of GST-Rabs and In Vitro GAP Assay—Rab GST fusion proteins were expressed in Escherichia coli JM109 and purified by standard protocols (31). Thrombin digestion was performed as described previously (35). The GTP-loading protocol and the in vitro GAP assay were performed as described previously (36, 50). In this study 200 pmol of Rab proteins and 5 pmol of TBC proteins or Rab3-GAP proteins were used for the GAP assay.

Immunostaining of Melanocytes—The melan-a cell fixation and immunostaining with endogenous Rab27A protein were performed as described previously (19).

Sequence Analysis—Multiple sequence alignment and phylogenetic analysis of the TBC proteins were performed using the CLUSTAL_X program (37). We followed the amino acid sequences and positions of TBC domains in the NCBI data base to draw the phylogenetic tree (Fig. 3C).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Exhaustive Screening for Putative Rab27A-GAPs with Melanosome Aggregation in Melanocytes as the Indicator—For the initial screening of 40 distinct human or mouse TBC/Rab-GAP proteins (see Table 1) for putative Rab27A-GAPs we overexpressed each GFP (green fluorescent protein)-tagged TBC protein in cultured melanocytes, melan-a cells (33). As shown in the top right panel in Fig. 1, the normal melan-a cells were elongated (or dendritic), and melanosomes were localized at the periphery of the cells expressing GFP alone. If any of the TBC proteins inactivated Rab27A by promoting its GTPase activity, overexpression of that TBC protein would induce melanosome aggregation in the perinuclear region, the same as in the melanocytes of Griscelli syndrome patients or the corresponding mouse model, ashen (21, 26). As anticipated, only two of the 40 TBC proteins tested, EPI64 and FLJ13130, strongly induced typical melanosome aggregation and FLJ22474 and Vrp seemed to be associated with weak perinuclear aggregation (summarized in Table 1). The melan-a cells expressing GFP-tagged FLJ22474 (GFP-FLJ22474), GFP-Vrp, GFP-EPI64, or GFP-FLJ13130 exhibited melanosome aggregation, but the shape of the cells was unaffected (bottom four panels in Fig. 1). GFP-FLJ22474 was localized in the cytoplasm, whereas GFP-Vrp, GFP-EPI64, and GFP-FLJ13130 were localized at the periphery of the cells. FLJ22474 is an uncharacterized protein, and Vrp has been reported to be involved in angiogenesis (38). EPI64 was previously characterized as a binding protein of EBP50, which functions in actin bundling at the cell periphery, however, EPI64 itself is unlikely to be involved in actin bundling (39). FLJ13130 is a close homologue of EPI64, but nothing is known about its function or localization (see below). It should be noted that the Rab-GAP activity of these four TBC proteins has never been investigated.

Because the members of the Rab27 subfamily and Rab3 subfamily are closely related in the phylogenetic tree of the Rab family and share some effectors (e.g. rabphilin and Noc2) (40, 41), it is possible that Rab3-GAP, which has been established to be a GAP of Rab3 that does not contain a TBC domain (42), also acts as a Rab27A-GAP. When GFP-tagged Rab3-GAP was expressed in melan-a cells, however, Rab3-GAP did not induce melanosome aggregation at all (Table 1), indicating that a specific Rab27A-GAP exists for Rab27A.

Second Screening for Putative Rab27A-GAPs by GTP-Rab27A Pulldown Assay—To further investigate whether the four TBC proteins described above induce melanosome aggregation by directly inactivating Rab27A on melanosomes, we used the SHD of Slac2-a to perform a GTP-Rab27A pulldown assay. Because the Slac2-a SHD specifically recognizes the GTP-bound form of Rab27A, and does not recognize other GTP-Rabs (32, 34, 40), the amount of the active form of Rab27A in cell lysates can be estimated by quantitatively determining the Rab27A bound to the Slac2-a SHD column (i.e. GTP-Rab27A pulldown assay). Briefly, COS-7 cell lysates containing FLAG-Rab27A and T7-tagged TBC proteins were incubated with the T7-GST-Slac2-a-SHD beads, and the bound GTP-Rab27A was analyzed by immunoblotting with anti-FLAG tag antibody. The four TBC proteins capable of inducing melanosome aggregation (EPI64, FLJ13130, Vrp, and FLJ22474), and three TBC proteins that have no effect on melanosome distribution (MGC34741, Usp6, and Prc17) as negative controls, were tested in the GTP-Rab27A pulldown assay. When Rab27A was co-expressed with EPI64 or FLJ13130, virtually no active form of Rab27A was detected under our experimental conditions (lanes 2 and 3 in the second panel from the top in Fig. 2), whereas co-expression with FLJ22474 or Vrp had little effect or slightly decreased the amount of the active form of Rab27A (lanes 4 and 5 in the second panel from the top in Fig. 2), similar to the results observed when co-expressed with MGC34741, Usp6, or Prc17 (i.e. negative controls).


Figure 2
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  		FIGURE 2.
EPI64 is the most likely candidate Rab27A-GAP as revealed by the GTP-Rab27A pulldown assay. Total lysates of COS-7 cells expressing FLAG-Rab27A alone (control; lane 1), FLAG-Rab27A and T7-EPI64 (lane 2), FLAG-Rab27A and T7-FLJ13130 (lane 3), FLAG-Rab27A and T7-FLJ22474 (lane 4), FLAG-Rab27A and T7-Vrp (lane 5), FLAG-Rab27A and T7-MGC34741 (lane 6), FLAG-Rab27A and T7-Usp6 (lane 7), or FLAG-Rab27A and T7-Prc17 (lane 8) were incubated with glutathione-Sepharose beads that had been coupled with purified T7-GST-Slac2-a-SHD. Total lysates (input) and proteins trapped by the SHD column (IP) were analyzed by 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-FLAG tag antibody (second panel from the top) or HRP-conjugated anti-T7 tag antibody (bottom two panels). The lysates used as inputs (1/60 of the reaction mixture) were analyzed in the same manner with HRP-conjugated anti-FLAG tag antibody (top panel). An asterisk indicates TBC proteins that induced melanosome aggregation in melan-a cells (see Fig. 1).

 
The results of the GTP-Rab27A pulldown assay were highly consistent with the results obtained by the initial screening for the putative Rab27A-GAP by the melanosome aggregation assay. Both EPI64 and FLJ13130 induced melanosome aggregation in melanocytes and actually reduced the amount of GTP-Rab27A when co-expressed with Rab27A. By contrast, FLJ22474 and Vrp, which induced a weak aggregation phenotype in melanocytes, had little effect on the amount of GTP-Rab27A, suggesting that these two TBC proteins induce partial melanosome aggregation by mechanisms different from the down-regulation of GTP-Rab27A. We therefore selected EPI64 and FLJ13130 from the TBC protein family members as candidate Rab27A-GAPs and tentatively named them Rab27A-GAP{alpha} and Rab27A-GAPbeta, respectively.

In Vitro Rab27A-GAP Activity of EPI64/Rab27A-GAP{alpha} and FLJ13130/Rab27A-GAPbeta—A data base search revealed that EPI64/Rab27A-GAP{alpha}, FLJ13130/Rab27A-GAPbeta, and mFLJ00332 (which do not induce melanosome aggregation; see Table 1) form a subfamily of TBC proteins (shaded in Fig. 3C). These three proteins share the same domain structures: a TBC domain in the central region and a specific sequence at the C-terminal end (DTYL in EPI64, DAYF in FLJ13130, and DTRF in mFLJ00332; Fig. 3A) that interacts with a PDZ domain (43-45). Their overall sequences exhibit high similarity (61.9% identity between EPI64 and FLJ13130; 50.0% between EPI64 and mFLJ00332; and 48.9% between FLJ13130 and mFLJ00332), especially in the TBC domain (81.1% between EPI64 and FLJ13130; 60.2% between EPI64 and mFLJ00332; and 59.2% between FLJ13130 and mFLJ00332; Fig. 3, A and B). Because all three have a conserved arginine residue that has been shown to be essential for the GAP activity of yeast Rab-GAP, Gyp1 (46) (asterisk in Fig. 3B) and several mammalian TBC proteins (7, 9, 10), they are likely to have a certain Rab-GAP activity. Because the sequence of the TBC domain of mFLJ00332 is slightly different from that of EPI64 and FLJ13130, mFLJ00332 may act as a GAP of Rabs related to Rab27A (e.g. Rab3A, Rab26, or Rab37).


Figure 3
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  		FIGURE 3.
Domain structure of EPI64 family proteins. A, schematic representations of EPI64/Rab27A-GAP{alpha}, FLJ13130/Rab27A-GAPbeta, and mFLJ00332. TBC domains are shown as dark gray boxes, and the final four amino acids (i.e. putative recognition sequence by a PDZ domain) are shown at the right of the diagram. The percentages of identical amino acids in the TBC domain shared by each of two proteins are shown on the right. B, sequence alignment of the TBC domains of EPI64, FLJ13130, and mFLJ00332. The sequences of the TBC domains of EPI64 (amino acid residues 108-313), FLJ13130 (82-287), and mFLJ00332 (89-294) were aligned with the CLUSTAL_X (version 1.8) program. The conserved amino acids are shown against a gray background. An asterisk indicates the conserved arginine residue essential for the catalytic activity of the GAP domain of Gyp1. The arrowheads point to amino acid residues substituted in the EPI64 mutants (EPI64-D157A and EPI64-R160K). Amino acid numbers are shown at each end. C, phylogenetic tree of the human or mouse TBC domains used in this study. The phylogenetic tree was drawn with the CLUSTAL_X (version 1.8) program. Note that EPI64, FLJ13130, and mFLJ00332 form a small branch (i.e. subfamily of TBC proteins; shaded in gray).

 
To determine whether EPI64 and FLJ13130 indeed function as a Rab27A-GAP, we measured the in vitro GAP activity of purified recombinant EPI64 (i.e. T7-GST-EPI64), FLJ13130 (i.e. T7-GST-FLJ13130), and Rab27A (see "Experimental Procedures" for details). Consistent with the results of the melanosome aggregation assay (Fig. 1) and the GTP-Rab27A pulldown assay (Fig. 2), the purified recombinant EPI64 and FLJ13130 actually activated the GTPase activity of Rab27A significantly (Fig. 4, A and B), and the GAP activity of EPI64 seemed stronger than that of FLJ13130 (Fig. 4A). To further investigate the Rab-GAP specificity of EPI64, we measured the GAP activity toward Rab3A (the closest isoform of Rab27A), Rab2A, and Rab4A (40, 41, 47). It should be noted that EPI64 did not show any Rab-GAP activity toward these Rabs at all (Fig. 4C and data not shown). By contrast, Rab3-GAP only showed Rab3A-GAP activity, but not Rab27A-GAP activity (Fig. 4C). This result suggests that EPI64 and Rab3-GAP function as specific Rab27A-GAP and Rab3A-GAP, respectively.

Rab27A-GAP Activity of EPI64 Depends on Its Intact TBC Domain—Although we demonstrated that EPI64 and FLJ13130 actually have Rab27A-GAP activity by the two screening methods, it remained unknown whether the TBC domain of EPI64 or FLJ13130 itself possesses Rab27A-GAP activity, and whether the melanosome aggregation induced by EPI64 or FLJ13130 is caused by the inactivation of Rab27A in living cells. To address these issues, we constructed mutant forms of EPI64 by replacing the conserved arginine required for GAP activity with lysine (referred to as EPI64-R160K), or a conserved glutamic acid with alanine (referred to as EPI64-D157A), by the methods described in the previous studies (7, 10). The GTP-Rab27A pulldown GAP assay in COS-7 cells clearly indicated that neither EPI64-R160K nor EPI64-D157A reduced the amount of the active form of Rab27A, whereas wild-type EPI64 dramatically reduced the active form (compare lanes 6-8 in the upper panel of Fig. 5A). The lack of effect of the two EPI64 mutants was not attributable to their low expression level in the recombinant proteins in COS-7 cells, because they were expressed more stably than the wild-type EPI64 (lanes 2-4 in the middle panel of Fig. 5A). The lack of Rab27A-GAP activity of EPI64 mutants was further confirmed by in vitro GAP assay (i.e. both EPI64 mutant proteins failed to promote the GTPase activity of Rab27A) (Fig. 5B). We therefore concluded that the EPI64 TBC domain is a functional Rab27A-GAP domain.


Figure 4
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  		FIGURE 4.
In vitro GAP assay of EPI64. A, GAP activity of EPI64, FLJ13130, and Rab3-GAP toward bacterially produced Rab27A. B, time course of the GTP hydrolysis of Rab27A in the presence of bovine serum albumin (BSA) or the indicated amount of EPI64. The GTP hydrolysis by Rab27A was measured as described under "Experimental Procedures." The results are expressed as the amount of the GTP-bound form of Rab27A after the reaction as a percentage of the amount before the reaction, and the bars represent the mean ± S.D. of data from three independent experiments. *, p < 0.05 (Student's unpaired t test). C, Rab-GAP specificity of EPI64. The Rab-GAP activity of EPI64 or Rab3-GAP toward Rab3A (for 20 min) and Rab27A (for 40 min) is summarized. The GTPase activity of each Rab protein in the presence of BSA (white columns), EPI64, or Rab3-GAP (black columns) is shown. *, p < 0.05 (Student's unpaired t test). Note that EPI64 activated the GTPase activity of Rab27A, but not Rab3A, whereas Rab3-GAP showed Rab3A-GAP activity, but not Rab27A-GAP activity.

 
The phenotypes of melan-a cells expressing the EPI64 mutants (i.e. GFP-EPI64-R160K and GFP-EPI64-D157A) were quite consistent with the results of the GTP-Rab27A pulldown assay (Fig. 5C). Both the EPI64-R160K and EPI64-D157A mutants had completely lost the ability to induce melanosome aggregation, the same as GFP alone (Fig. 5D). It is noteworthy that both EPI64 mutants were localized at the periphery of the cell, the same as the wild-type EPI64 (Fig. 5C), indicating that both mutations (R160K and D157A) abolished only Rab27A-GAP activity and retained the other properties of EPI64 protein.

The C-terminal End of EPI64 Is Not Required for the EPI64 Function That Induces Melanosome Aggregation—EPI64 has been shown to be localized at the periphery of the cell through interaction with EBP50, which is involved in actin bundling (39). Because we previously showed that the interference with actin remodeling induces melanosome aggregation (19, 48), it is possible that EPI64 also affects melanosome distribution by impairing actin organization through interaction with EBP50, in addition to inactivating Rab27A. To rule out this possibility we prepared two C-terminal end mutants, EPI64+A and EPI64-{Delta}449 (see Fig. 6A for details), neither of which interacts with EBP50 (39). As anticipated, when overexpressed in melan-a cells both GFP-tagged mutants induced melanosome aggregation, the same as the wild-type EPI64 (Fig. 6B). These results indicated that induction of melanosome aggregation by EPI64 must be attributable to the inactivation of the Rab27A function, rather than to the impairment of actin filament bundling. Because the C-terminal EPI64 mutants are capable of targeting the actinrich cell periphery, the same as the wild-type EPI64 (Fig. 6B), additional factors other than EBP50 may determine the localization of EPI64 in melanocytes, in contrast to placental cells (39).

Expression of EPI64 in Melanocytes Induced Exclusion of Rab27A on Melanosomes—In general, the GDP-bound inactive form of Rabs is extracted from the membrane by a GDP-dissociation inhibitor for recycling of Rab proteins (49). Thus, it was expected that the GDP-bound form of Rab27A inactivated by EPI64 would also be extracted from the melanosome membrane, where Rab27A is localized to function, and be dispersed in the cytosol. As shown in Fig. 7, hardly any endogenous Rab27A signals were detected on the melanosomes of EPI64-expressing melanocytes (arrow), whereas Rab27A was present on the peripheral melanosomes of untransfected cells (arrowhead). We therefore concluded that EPI64 inactivates Rab27A in vivo by promoting the GTPase activity of Rab27A.


Figure 5
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  		FIGURE 5.
The intact TBC domain is required for the Rab27A-GAP activity of EPI64. A, the mutations in the TBC domain of EPI64 abolished the Rab27A-GAP activity of EPI64. The lysates of COS-7 cells expressing FLAG-Rab27A alone (control; lanes 1 and 5), FLAG-Rab27A and T7-EPI64 (lanes 2 and 6), FLAG-Rab27A and T7-EPI64-R160K (lanes 3 and 7), or FLAG-Rab27A and T7-EPI64-D157A (lanes 4 and 8) were incubated with glutathione-Sepharose beads that had been coupled with the purified T7-GST-Slac2-a-SHD. Total lysates (Input, lanes 1-4) and proteins trapped by the SHD (IP, lanes 5-8) were analyzed by 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-FLAG tag antibody (top panel) or HRP-conjugated anti-T7 tag antibody (bottom two panels). Under our experimental conditions, FLAG-Rab27A was often detected as doubled bands probably because of the absence and presence of C-terminal geranylgeranylation. B, in vitro Rab27A-GAP activity of EPI64 mutants. The ratios of the GTP-bound Rab27A after a 40-min incubation with BSA, EPI64, EPI64-R160K, or EPI64-D157A are shown. Note that only the wild-type EPI64, but not its mutants, showed significant Rab27A-GAP activity. ***, p < 0.001, one-way analysis of variance with Bonferroni multiple comparisons test for post hoc comparisons. C, the TBC domain mutants of EPI64 induced hardly any melanosome aggregation. Melan-a cells were transfected with a vector encoding GFP-EPI64 (top row), GFP-EPI64-R160K (middle row), or GFP-EPI64-D157A (bottom row). The cells were then fixed and examined by confocal microscopy for GFP fluorescence (green in the right column). Bright-field images (left column) show the melanosome distribution in the cells. Arrowheads point to the melan-a cells exhibiting melanosome aggregation. Bar:20 µm. D, summary of the results of the melanosome distribution assay. Images of transfected cells were captured at random by using GFP fluorescence as a marker, and we judged whether melanosomes had aggregated in the perinucleus based on the corresponding bright-field images as described previously (19). The results are expressed as percentages of cells exhibiting perinuclear melanosome aggregation, and the values are mean ± S.D. of data from three independent experiments (n > 150). Note that only the wild-type EPI64, but not its mutants, induced perinuclear aggregation of melanosomes in melan-a cells. ***, p < 0.001, one-way analysis of variance with Bonferroni multiple comparisons test for post hoc comparisons.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A new method of rapid screening for putative Rab-GAPs based on the physical interaction between Rab and TBC protein has recently been reported (8). Whereas this method is useful for screening some TBC proteins (e.g. RabGAP-5 and RN-Tre), it cannot be applied to the EPI64/Rab27A-GAP{alpha} and FLJ13130/Rab27A-GAPbeta identified in the present study, because neither of them (or the EPI64-R160A mutant) can stably interact with GTP-locked Rab27A in the yeast two-hybrid assay or co-immunoprecipitation assay (50). We have therefore developed two novel screening methods based on the functional interaction between Rab and TBC proteins. The first screening method we developed is based on impairment of the specific intracellular localization of organelles (e.g. melanosomes) or Rab proteins (e.g. Rab27A) in cultured cells. If the candidate Rab-GAP inactivates a specific Rab, a defect in specific organelle transport (e.g. melanosome aggregation; Fig. 1) or decrease in the immunoreactivity of Rab protein on the specific organelle by the function of GDP-dissociation inhibitor (i.e. inactive GDP-Rab is extracted from the membrane) is observed (Fig. 7). Because Rab proteins are thought to be localized on distinct compartments within the cell (1), this screening method should be applicable to most other Rab proteins in addition to Rab27A. The advantage of this screening method is its ability to identify candidate RabGAPs at the single cell level without having to produce a large amount of recombinant proteins or achieve high transfection efficiency. Its drawback may be the difficulty of excluding indirect effects of TBC proteins on the localization of organelles or Rab proteins. Indeed, we identified four TBC proteins as candidates for Rab27A-GAPs, but two of them (i.e. FLJ22474 and Vrp) lacked Rab27A-GAP activity in vitro (Figs. 1 and 2), indicating this method alone is not sufficient to identify specific Rab-GAPs.


Figure 6
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  		FIGURE 6.
The C-terminal EBP50-binding region of EPI64 is not required for melanosome aggregation activity. A, schematic representations of EPI64 C-terminal mutants. EPI64+A contains an additional alanine residue at its C-terminal end that abrogates the interaction with EBP50. EPI64-{Delta}449 lacks 59 amino acids at its C-terminal end. B, typical images of cells expressing wild-type EPI64 or the C-terminal mutants of EPI64. Melan-a cells expressing GFP-EPI64 (top panels), GFP-EPI64+A (middle panels), or GFP-EPI64-{Delta}449 (bottom panels) are shown. GFP fluorescence is shown on the left, and the corresponding bright-field images are on the right. Bar, 20 µm.

 


Figure 7
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  		FIGURE 7.
EPI64 not only induced the melanosome aggregation but Rab27A exclusion from the melanosomes. Melan-a cells transfected with a vector encoding GFP-EPI64. After fixing the cells and permeabilizing them with 0.3% Triton X-100, they were stained with anti-Rab27A antibody and Alexa 594-conjugated antimouse IgG antibody. The cells were examined for Rab27A fluorescence (red in left) and GFP-EPI64 fluorescence (green in middle) by confocal microscopy. Bright-field image (right) showing the melanosome distribution in the cells. No Rab27A signals were detected in the cell expressing GFP-EPI64 (arrow), whereas Rab27A was present on the melanosomes of the untransfected control cell (arrowhead). Bar, 20 µm.

 
We then developed the second screening method, which is based on GTP-Rab pulldown with a specific effector domain (e.g. SHD for Rab27A) that specifically recognizes the GTP-bound form of Rab (Fig. 2). In the present study we used the Slac2-a SHD to specifically pull down the GTP-bound form of Rab27A (34) and succeeded in identifying two candidate Rab27A-GAPs, EPI64/Rab27A-GAP{alpha} and FLJ13130/Rab27A-GAPbeta. This method enables more direct evaluation of the GAP activity of TBC proteins than the first method, even though an indirect effect of TBC proteins on the amount of GTP-Rab cannot be completely ruled out. However, it can be easily ruled out by testing mutant TBC proteins that contain specific mutations in the TBC domain (e.g. D157A and R160K in EPI64). In reality, we did not observe any Rab27A-GAP activity by the EPI64 TBC mutants (Fig. 5). The only drawback of this method seemed to be its limitation to Rabs whose effectors have already been identified. Because the Rab-GAPs of most well characterized Rabs (e.g. Rab1A, Rab2A, Rab4A, and Rab11A) have never been identified, our GTP-Rab pulldown assay will be useful for many of the Rabs whose effectors have been reported. Identification of other Rab-GAPs by using the known effector domains is now in progress in our laboratory.

If EPI64/Rab27A-GAP{alpha} is a functional Rab27A-GAP in vivo, melan-a cells should endogenously express EPI64 (and/or FLJ13130/Rab27A-GAPbeta). Actually, we found that EPI64 is endogenously expressed in melan-a cells, in which Rab27A is abundantly expressed (supplemental materials Fig. 1A). Therefore, it is highly possible that EPI64 functions as a Rab27A-GAP in melanocytes and is involved in the control of Rab27A-mediated melanosome transport (13, 17, 18). In our preliminary experiment, however, small interfering RNA-mediated knockdown of endogenous EPI64 had no effect on melanosome distribution in melan-a cells (supplemental materials Fig. 1B). However, it is possible that FLJ13130/Rab27A-GAPbeta is also expressed in melan-a cells and compensates for the function of EPI64 in melanosome transport. Alternatively, the GAP activity of EPI64 may not be required for microtubule- or actin-based melanosome transport, but it may be required for the later stage of melanosome transport, e.g. transfer of melanosomes to adjacent keratinocytes. Further work is necessary to determine the precise regulatory mechanism of Rab27A-GAP activity of EPI64 in vivo.

In summary, we succeeded in isolating two candidate Rab27A-GAPs, EPI64 and FLJ13130/Rab27A-GAPbeta, by novel screening methods based on functional interactions between Rabs and TBC domains without testing their physical interactions. Our new screening methods should greatly accelerate the identification of the target Rabs of orphan TBC proteins and our understanding of the regulatory mechanisms of small GTPase Rabs.


   FOOTNOTES
 
* This work was supported in part by Ministry of Education, Culture, Sports, and Technology of Japan Grants 17657067, 18022048, 18050038, 18057026, and 18207015 (to M. F.), the Kato Memorial Bioscience Foundation (to M. F.), the Sumitomo Foundation (to M. F.), and the Nakatomi Foundation (to M. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 Supported by the Special Postdoctoral Researchers Program of RIKEN. Back

2 To whom correspondence should be addressed: 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. Tel.: 81-22-795-7731; Fax: 81-22-795-7733; E-mail: nori{at}mail.tains.tohoku.ac.jp.

3 The abbreviations used are: GAP, GTPase-activating protein; GFP, green fluorescent protein; GST, glutathione S-transferase; HRP, horseradish peroxidase; SHD, Slp homology domain; Slp, synaptotagmin-like protein; TBC, Tre/Bub2/Cdc16. Back


   ACKNOWLEDGMENTS
 
We thank Eiko Kanno and Megumi Satoh for technical assistance, Dr. Dorothy C. Bennett for the kind gift of the melan-a cell line, Dr. Takahiro Nagase for kindly donating KIAA cDNA clones, and members of the Fukuda Initiative Research Unit for valuable discussions.



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 DISCUSSION
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K. Ishibashi, E. Kanno, T. Itoh, and M. Fukuda
Identification and characterization of a novel Tre-2/Bub2/Cdc16 (TBC) protein that possesses Rab3A-GAP activity
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