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Genes to Cells (2009) 14, 41-52. doi:10.1111/j.1365-2443.2008.01251.x
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Identification and characterization of a novel Tre-2/Bub2/Cdc16 (TBC) protein that possesses Rab3A-GAP activity

 Koutaro Ishibashi, Eiko Kanno, Takashi Itoh and Mitsunori Fukuda*

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
 
The Tre-2/Bub2/Cdc16 (TBC) domain is a conserved protein motif that consists of approximately 200 amino acids and is thought to function as a specific Rab-GAP domain. Although more than 40 distinct TBC domain-containing proteins have been identified in humans, the GAP activity and specificity of most TBC proteins have never been determined. In this study we developed a novel method of screening for Rab3A-GAP and identified two TBC proteins (FLJ13130 and RN-tre) whose expression in PC12 cells was associated with exclusion of endogenous Rab3A molecules from dense-core vesicles. As expression of RN-tre caused fragmentation of the Golgi, which presumably resulted in the loss of dense-core vesicles themselves, we further characterized FLJ13130 as a candidate Rab3A-GAP. The results showed that expression of FLJ13130, but not of its catalytically inactive R134K mutant, greatly reduced the amount of GTP-Rab3A in living cells and promoted the GTPase activity of Rab3A in vitro. Unexpectedly, however, FLJ13130 also promoted the GTPase activity of Rab22A, Rab27A, and Rab35, but not of Rab2A or Rab6A. Based on these results, we propose that FLJ13130 is a novel type of Rab-GAP that exhibits broad GAP specificity and inactivates several distinct Rab isoforms, including Rab3A, just near the plasma membrane.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Intracellular membrane traffic, the transport of membrane-wrapped substances, is a fundamental biological activity in all eukaryotic cells, and a variety of membrane trafficking proteins are thought to be required to fine-tune intracellular membrane traffic. The small GTPase Rab family constitutes the largest family of membrane trafficking proteins conserved in all eukaryotic cells (reviewed in Pfeffer 2001; Zerial & McBride 2001), and more than 60 Rab members have been identified in humans and mice (Bock et al. 2001; Pereira-Leal & Seabra 2001; Itoh et al. 2006). Rab functions as a molecular switch by cycling between two nucleotide-binding states, a GTP-bound active state and a GDP-bound inactive state, and the GTP-Rab, together with its specific effector molecule, promotes intracellular membrane traffic (Grosshans et al. 2006; Schwartz et al. 2007; Fukuda et al. 2008). Switching of the two nucleotide-bound states is controlled by two regulatory enzymes, a guanine nucleotide exchange factor (GEF) and a GTPase-activating protein (GAP). In contrast to the Rab effectors, 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, Vps9 homology domain-containing proteins (e.g., Rabex-5) function as Rab5-GEFs (Carney et al. 2006; Horiuchi et al. 1997), DENN domain-containing Rab3-GEP (Wada et al. 1997) as a Rab3-GEF, and two Sec2 homology domain-containing proteins, GRAB and Rabin3, as a Rab3-GEF and a Rab8-GEF, respectively (Luo et al. 2001; Hattula et al. 2002). Although the putative GEF domains (i.e., Vps9, DENN, and Sec2) for distinct Rab isoforms do not resemble each other at the amino acid level, all Rab-GAPs identified thus far, except Rab3-GAP (Fukui et al. 1997), share a conserved Tre-2/Bub2/Cdc16 (TBC) domain (Bernards 2003). As the number of TBC domain-containing proteins (simply referred to as TBC proteins below) identified thus far in humans (more than 40) is almost the same as the number of Rab subfamily (approximately 40), TBC domains have been suggested to function as specific Rab-GAP domains (Bernards 2003). However, the GAP activity and specificity of most mammalian TBC proteins have never been investigated. Several methods of screening for the target Rabs of human TBC proteins have recently been developed: a yeast two-hybrid assay method that depends on the physical interaction between TBC proteins and their target Rabs (Haas et al. 2005; Itoh et al. 2006; Fuchs et al. 2007) and a method that depends on the inhibition of cellular functions, for example, melanosome transport, cilium formation, toxin transport, and maintenance of Golgi structure, by overexpression of TBC proteins and their catalytically inactive mutants in cultured cells (Itoh & Fukuda 2006; Fuchs et al. 2007; Haas et al. 2007; Yoshimura et al. 2007).

Rab3A is one of the best-characterized Rab isoforms and is abundant on the secretory vesicles of neurons and endocrine cells, and it is involved in secretory vesicle exocytosis (Takai et al. 1996; Geppert & Südhof 1998; Fukuda 2008). Two key regulatory enzymes of Rab3A, Rab3-GEP and Rab3-GAP, have already been characterized (Fukui et al. 1997; Wada et al. 1997), but as the previously characterized Rab3-GAP lacks a TBC domain (unless otherwise stated, Rab3-GAP means non-TBC-type Rab3-GAP throughout the text), it would be interesting to determine whether TBC domain-containing Rab3A-GAP (hereafter referred to as TBC-type Rab3A-GAP to distinguish it from non-TBC-type Rab3-GAP) is also present in the human body. In this study we screened for TBC-type Rab3A-GAP by overexpressing 41 different TBC proteins in neuroendocrine PC12 cells and monitoring them for exclusion of endogenous Rab3A molecules from dense-core vesicles (i.e., inactivation of Rab3A; see Fig. 1). The results showed that FLJ13130 (official NCBI symbol: TBC1D10B), previously characterized as Rab27A-GAPβ (Itoh & Fukuda 2006), exhibited Rab3A-GAP activity both in vitro and in vivo. We also found that FLJ13130 exhibits GAP activity toward several distinct Rabs, including Rab3A, Rab22A, Rab27A, and Rab35. Based on these findings, we discuss the possible function and Rab-GAP specificity of FLJ13130.


Figure 1
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  		Figure 1  Scheme of the procedure to screen for TBC-domain-containing Rab3A-GAPs in PC12 cells (Rab3A exclusion assay). A GTP-bound active form of Rab3A (indicated by dots) is visible on dense-core vesicles in the distal portion of the neurites of PC12 cells. When GFP-tagged TBC-containing Rab3A-GAPs are overexpressed in PC12 cells, they inactivate endogenous Rab3A by promoting the GTPase activity of Rab3A. The resulting GDP-bound form of Rab3A is extracted from the vesicle membrane by GDI, and no Rab3A signals are observed in the neurites of PC12 cells in an immunofluorescence analysis (i.e., exclusion of Rab3A from dense-core vesicles).

 

   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Screening for TBC-type Rab3A-GAP by Rab3A exclusion assay in PC12 cells

We and others have reported two independent methods of screening for the target Rabs of TBC proteins: a yeast two-hybrid assay method that depends on the physical interaction between TBC proteins and their target Rabs (Haas et al. 2005; Itoh et al. 2006) and a method that depends on inhibition of cellular functions, for example, melanosome transport, by overexpression of TBC proteins (Itoh & Fukuda 2006; Fuchs et al. 2007; Haas et al. 2007; Yoshimura et al. 2007). However, the yeast two-hybrid screening method failed to identify TBC proteins that physically bound Rab3A (Itoh et al. 2006). Although Rab3A has been shown to be involved in the regulation of secretion by certain neuroendocrine cells, we were also unable to use the latter method to screen for TBC-type Rab3A-GAP by monitoring hormone secretion activity, because we previously found that Rab3A and Rab27A cooperatively regulate hormone secretion by PC12 cells (Tsuboi & Fukuda 2006). We therefore attempted to develop a novel screening method for TBC-type Rab3A-GAP (see scheme in Fig. 1). In nerve growth factor (NGF)-differentiated PC12 cells, endogenous Rab3A molecules are present on the dense-core vesicles and localized in the distal portion of their neurites (Fukuda et al. 2002). If green fluorescence protein (GFP)-tagged TBC proteins that possess Rab3A-GAP activity were transiently expressed in PC12 cells, the expressed TBC proteins would promote the GTPase activity of endogenous Rab3A on dense-core vesicles and the resulting, inactivated GDP-Rab3A would be dissociated from the vesicle membrane by a GDP dissociation inhibitor (GDI) (Seabra & Wasmeier 2004). This would make it possible to screen for TBC-type Rab3A-GAP by monitoring exclusion of Rab3A signals from the distal portion of the neurites of PC12 cells. To validate the use of this sequence of events as a screening method, we first expressed well-characterized non-TBC type Rab3-GAP (Fukui et al. 1997) in PC12 cells. As anticipated, Rab3A signals were almost completely lost from the distal portion of the neurites of the cells expressing GFP-Rab3-GAP (arrows in the middle panel of Fig. 2F), unlike the control cells expressing GFP alone (arrowheads in the middle panel of Fig. 2A). We then expressed each of the 41 different GFP-TBC proteins in PC12 cells and screened for TBC proteins whose expression resulted in exclusion of Rab3A from the distal portion of the neurites (summarized in Table 1). The results showed that only two TBC proteins, FJL13130 (also called Rab27A-GAPβ/TBC1D10B; Itoh & Fukuda 2006) and RN-tre, both of which were predominantly localized near the plasma membrane, had strong activity that reduced Rab3A signals in the neurites of PC12 cells (Fig. 2D,E, arrows), whereas PC12 cells that expressed other TBC proteins, including two closely related isoforms of FLJ13130, EPI64/TBC1D10A and mFLJ00332/TBC1D10C, had neurites containing strong Rab3A signals (Fig. 2B,C, arrowheads). To our surprise, however, RN-tre has previously been reported as Rab5-GAP (Lanzetti et al. 2000) and Rab41-GAP (Haas et al. 2007), and FJL13130 as Rab27A-GAPβ (Itoh & Fukuda 2006) and Rab35/22-GAP (Fuchs et al. 2007).


Figure 2
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  		Figure 2  Screening for candidate Rab3A-GAPs. PC12 cells transiently expressing GFP alone (A, control), GFP-EPI64 (B), GFP-mFLJ00332 (C), GFP-FLJ13130 (D), GFP-RN-tre (E), or GFP-Rab3-GAP (F) were stained with anti-Rab3A antibody (red in the middle panels). The right panels show merged images of the left (GFP fluorescence) and middle panels. Note the loss of Rab3A signals in the neurites of PC12 cells expressing GFP-FLJ13130 (D), GFP-RN-tre (E), or GFP-Rab3-GAP (F) (arrows in the middle panels of D, E, and F, respectively). The arrowheads point to the distal portion of the neurites, where endogenous Rab3A protein was normally enriched in the control PC12 cells (A) and other GFP-TBC-expressing cells (B and C). Bars, 20 µm.

 

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  		Table 1  Summary of the results of the Rab3A-GAP screening
 
To determine whether the observed effects were secondary to dysfunction of the Golgi and/or inhibition of dense-core vesicle biogenesis, we co-stained the transfected cells with a Golgi marker (i.e., GM130) and a dense-core vesicle marker (i.e., synaptotagmin (Syt) I). Consistent with a previous report (Haas et al. 2007), the results showed that expression of RN-tre, but not of FJL13130 or Rab3-GAP, induced fragmentation of the Golgi (Fig. 3B, large arrowheads). In addition, in contrast to the control GFP-expressing cells (Fig. 3E) and Rab3-GAP-expressing cells (Fig. 3H), no Syt I signals (dense-core vesicle marker) were detected in the RN-tre-expressing cells (Fig. 3F, small arrowheads), suggesting that dense-core vesicle biogenesis is impaired in RN-tre-expressing cells. Expression of FLJ13130 had little effect on the Syt I signals in the neurites, although reduced Syt I signals were sometimes observed in cells expressing a high level of GFP-FLJ13130 (Fig. 3G, arrows). We therefore focused on FLJ13130 as a candidate TBC-type Rab3A-GAP for subsequent analysis.


Figure 3
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  		Figure 3  RN-tre induced fragmentation of the Golgi and disappearance of Syt I from the distal portion of the neurites. PC12 cells transiently expressing GFP alone (A and E, control), GFP-RN-tre (B and F), GFP-FLJ13130 (C and G), or GFP-Rab3-GAP (D and H) were stained with anti-GM130 antibody or anti-Syt I antibody (red in right panels). Note that expression of GFP-RN-tre induced fragmentation of the Golgi (large arrowheads in B), whereas expression of GFP-FLJ13130 or GFP-Rab3-GAP did not. In addition, expression of GFP-RN-tre resulted in the disappearance of Syt I from the distal portion of the neurites (small arrowheads in F), suggesting that RN-tre inhibits formation of dense-core vesicles from the Golgi. A reduced Syt I level was sometimes observed in PC12 cells with a high level of expression of GFP-FLJ13130 (arrows in G), whereas Syt I signals were clearly observed in PC12 cells with a normal level of expression of GFP-FLJ13130. Bars, 20 µm.

 
Expression of FLJ13130 reduced the amount of GTP-Rab3A in living cells

To determine whether the effect induced by expression of FLJ13130 was directly related to the decrease in GTP-Rab3A in living cells, we performed a GTP-Rab3A pull-down assay in COS-7 cells in which we used the Rab-binding domain (RBD) of Rim2 as a specific GTP-Rab3A trapper (Fukuda 2003, 2004). In brief, FLAG-Rab3A was co-expressed with T7-tagged Rab3-GAP, FLJ13130, EPI64, or mFLJ00332 in COS-7 cells, and lysates of the cells were then incubated with beads coupled with GST-Rim2-RBD. GTP-Rab3A trapped by the beads was detected by immunoblotting with anti-FLAG tag antibody (second panel from the top in Fig. 4A,B). As anticipated, FLJ13130, but not its homologue EPI64 or mFLJ00332, reduced the amount of GTP-Rab3A (lane 3 in Fig. 4B,D), the same as non-TBC-type Rab3-GAP did (Fig. 4A,C).


Figure 4
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  		Figure 4  GTP-Rab3A pull-down assay in COS-7 cells. (A) Expression of Rab3-GAP reduced the amount of GTP-Rab3A in COS-7 cells (lane 2 in the second panel from the top). (B) Expression of FLJ13130, but not its close homologue EPI64 or mFLJ00332, reduced the amount of GTP-Rab3A in COS-7 cells (lane 3 in the second panel from the top). The GTP-Rab3A pull-down assay in COS-7 cells was performed as described under ‘Experimental procedures’. Total lysates of COS-7 cells expressing FLAG-Rab3A alone (control; lane 1 in A and B), FLAG-Rab3A and T7-Rab3-GAP (lane 2 in A), FLAG-Rab3A and T7-EPI64 (lane 2 in B), FLAG-Rab3A and T7-FLJ13130 (lane 3 in B), or FLAG-Rab3A and T7-mFLJ00332 (lane 4 in B) were incubated with glutathione-Sepharose beads that had been coupled with purified T7-GST-Rim2-RBD. Proteins trapped by the RBD column (Bound) were analyzed by 10% SDS-PAGE and immunoblotting with HRP-conjugated anti-FLAG tag antibody (second panels from the top) or HRP-conjugated anti-T7 tag antibody (bottom two panels). The lysates used as inputs (1/80 volume of the reaction mixture) were analyzed in the same manner with HRP-conjugated anti-FLAG tag antibody (top panels). The results shown are representative of three independent experiments. The positions of the molecular mass markers (x10–3) are shown on the left. (C) and (D) Quantitative data of the pulled-down GTP-Rab3A. The amount of pulled-down Rab3A (second panels from the top in (A) and (B), respectively) was quantified and is expressed as the means ± SD of data from three independent experiments. *P < 0.01, Student's unpaired t test.

 
Catalytic Arg residue in the TBC domain of FLJ13130 is required for the reduction of GTP-Rab3A level in living cells

Next, to determine whether the reduction of GTP-Rab3A in living cells was related to the GAP activity of FLJ13130, we mutated the catalytic Arg residue at amino acid position 134 to Lys in the TBC domain of FLJ13130 according to the previous studies (Lanzetti et al. 2000; Gao et al. 2003) and named the mutant RK. As shown in Fig. 5A,B, the FLJ13130-RK mutant was completely devoid of any ability to exclude endogenous Rab3A molecules from the dense-core vesicles of PC12 cells. Moreover, the pull-down assay using GST-Rim2-RBD showed that the FLJ13130-RK mutant failed to reduce the amount of GTP-Rab3A in COS-7 cells (Fig. 5C,D). These results strongly indicated that the TBC domain of FLJ13130 is responsible for the reduction of the GTP-Rab3A level in living cells, most likely by promoting Rab3A-GAP activity.


Figure 5
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  		Figure 5  The catalytic Arg residue (Arg134) in the TBC domain of FLJ13130 is required for exclusion of Rab3A from dense-core vesicles (A and B) and for reduction of the amount of GTP-Rab3A in COS-7 cells (C and D). (A) PC12 cells transiently expressing GFP-FLJ13130-RK (green, top) were stained with anti-Rab3A antibody (red, bottom). Note that PC12 cells expressing GFP-FLJ13130-RK had no effect on the Rab3A localization in the neurites. The arrowheads point to the distal portion of the neurites. Bar, 20 µm. (B) Summary of the results of the Rab3A exclusion assay. Images of transfected cells were captured at random by using GFP fluorescence as a marker, and we judged whether the Rab3A signals had disappeared from the neurites based on the results of the immunofluorescence analysis. The results are expressed as percentages of cells exhibiting exclusion of Rab3A from the dense-core vesicles in their neurites, and the values are means ± SD of data from three independent experiments (n > 300). **P < 0.005, Student's unpaired t-test. (C) Expression of the FLJ13130-RK mutant failed to reduce the amount of GTP-Rab3A in COS-7 cells (lane 3 in the second panel from the top). The GTP-Rab3A pull-down assay in COS-7 cells was performed as described in the legend to Fig. 4. (D) Quantitative data from the pulled-down GTP-Rab3A shown in (C). The amounts of pulled-down Rab3A (second panels from the top in (C)) were quantified and are expressed as means ± SD of data from three independent experiments. *P < 0.01, Student's unpaired t test.

 
Unexpected broad Rab-GAP specificity of FLJ13130 in vitro

Finally, we directly measured the Rab3A-GAP activity of FLJ13130 in vitro by using purified recombinant proteins (Fig. 6A). Although FLJ13130 had almost the same ability to reduce both the Rab3A signals in living PC12 cells and the GTP-Rab3A level in COS-7 cells, the same as Rab3-GAP did (Figs 4,5B), the in vitro Rab3A-GAP activity of FLJ13130 was unexpectedly weak in comparison with that of Rab3-GAP (Fig. 6A,B). The weaker Rab3A-GAP activity of FLJ13130 in comparison with the control sample was statistically significant (P < 0.05), and the FLJ13130-RK mutant did not display any significant Rab3A-GAP activity (Fig. 6A), consistent with the results of the cellular assays described above (Fig. 5). We then investigated the Rab-GAP specificity of FLJ13130 (Fig. 6C,D) and to our surprise discovered that FLJ13130 also showed significant GAP activity toward three distinct Rabs, that is, Rab22A, Rab27A, and Rab35, but not toward Rab2A or Rab6A, suggesting that FLJ13130 exhibits rather broad GAP specificity. The Rab3A-GAP activity of FLJ13130 was comparable to its Rab35-GAP activity but weaker than its Rab27A-GAP activity (Fig. 6D).


Figure 6
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  		Figure 6  In vitro GAP assay of FLJ13130. (A) GAP activity of FLJ13130, FLJ13130-RK, and Rab3-GAP toward Rab3A. GTP hydrolysis by Rab3A was measured as described previously (Itoh et al., 2006). The results are expressed as the amount of the GTP-bound form of Rab3A after the reaction as a percentage of the amount before the reaction, and the bars represent the means ± SD of data from three independent experiments. *P < 0.05; **P < 0.01 (Student's unpaired t-test). (B) Dose-dependent Rab3A-GAP activity of FLJ13130. The GTPase activity of FLJ13130 was measured as described above. The results are expressed as the amount of the GTP-bound form of Rab3A after the reaction as a percentage of the amount before the reaction. (C) Rab-GAP specificity of FLJ13130. The Rab-GAP activity of FLJ13130 toward Rab2A, Rab3A, Rab6A, Rab22A, Rab27A, and Rab35 is summarized. The GTPase activity of each Rab protein in the presence of BSA (white columns) or FLJ13130 (black columns) is shown. Note that FLJ13130 significantly activated the GTPase activity of Rab3A, Rab22A, Rab27A, and Rab35, but not of Rab2A or Rab6A. *P < 0.05; **P < 0.01 (Student's unpaired t test). (D) Time course of the GTP hydrolysis of Rab2A, Rab3A, Rab6A, Rab22A, Rab27A, and Rab35 in the presence of BSA or FLJ13130. The GTP hydrolysis by each Rab was measured as described in (A). The results are expressed as the amount of the GTP-bound form of each Rab after the reaction as a percentage of the amount before the reaction, and the bars represent the means ± SD of data from three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Based on the Rab-GAP function of yeast TBC proteins, it has been suggested that their mammalian homologues function as specific Rab-GAPs (Bernards 2003). Because of the large number of Rab isoforms in mammals, however, no target Rab of most of the mammalian TBC proteins has been identified. In the present study, we tried screening for TBC proteins that exhibit Rab3A-GAP activity by monitoring PC12 cells for exclusion of endogenous Rab3A molecules from their dense-core vesicles (Fig. 1). Of the 41 TBC proteins tested by transiently expressing them in PC12 cells, only two, FLJ13130 and RN-tre, had the ability to exclude Rab3A from dense-core vesicles (Figs 2,5B). As the exclusion of Rab3A from the dense-core vesicles by RN-tre is likely to be secondary to Golgi dysfunction (Fig. 3) (Haas et al. 2007), the only candidate for a TBC-type Rab3A-GAP was FLJ13130, although we cannot completely rule out the possibility that an additional TBC-type Rab3A-GAP whose GAP activity is strictly regulated by extracellular stimuli (e.g., secretagogues) is present in humans. We then showed by GTP-Rab3A pull-down assay in COS-7 cells that expression of FLJ13130 actually reduced the amount of GTP-Rab3A to the same extent as Rab3-GAP did (Fig. 4), and the reduction was found to be completely restored by substitution of the catalytic Arg residue of FLJ13130 (Fig. 5). In contrast to the Rab3A-GAP activity of FLJ13130 in living cells, however, the in vitro Rab3A-GAP activity of purified FLJ13130 was unexpectedly weaker than that of Rab3-GAP (Fig. 6A). This discrepancy may be explained by a difference in the local concentration of FLJ13130: FLJ13130 is predominantly localized near the plasma membrane (i.e., high local concentration), whereas Rab3-GAP is present throughout the cytosol (compare Fig. 2D,F). Alternatively, the weak in vitro Rab3A-GAP activity of FLJ13130 may be attributable to instability of the purified sample (e.g., inactivation of the sample during the purification procedure) or to the lack of an additional factor in the purified sample that is required for full activation of FLJ13130. Further work will be necessary to resolve this issue.

The most unexpected result in this study was that FLJ13130 showed GAP activity toward several distinct Rab isoforms, including Rab3A, Rab22A, Rab27A, and Rab35 (Fig. 6B). This property is in sharp contrast to EPI64 and mFLJ00332, both of which belong to the same subfamily as FLJ13130 in the phylogenetic tree (TBC1D10 subfamily; Itoh & Fukuda 2006). EPI64 displays GAP activity toward Rab27A, but not toward Rab3A or Rab35 (Itoh & Fukuda 2006 and unpublished data), and mFLJ00332 displays GAP activity toward Rab35, but not toward Rab3A or Rab27A (Patino-Lopez et al. 2008). Thus, it seems likely that FLJ13130 has a combination of the functions of EPI64 and mFLJ00332. As the TBC domain of the members of the TBC1D10 subfamily is highly conserved (Itoh & Fukuda 2006), it would be interesting to attempt to identify the mechanism by which the TBC domain of the TBC1D10 subfamily determines Rab-GAP specificity, by performing a sequence comparison combined with site-directed mutagenesis. Although clear Rab22A- and Rab35-GAP activities of FL13130 have also recently been reported (Fuchs et al. 2007), they were not as strong under our experimental conditions, in comparison with its Rab3A-GAP activity. This discrepancy may be attributable to the difference in source of the recombinant FLJ13130 protein, that is, bacteria in the earlier study (Fuchs et al. 2007) as opposed to mammalian cells in the present study. Actually, post-translational modification of certain TBC proteins (e.g., phosphorylation of AS160) has been reported to affect GAP activity (Sano et al. 2003). Further work is necessary to determine whether post-translational modification(s) of FLJ13130 occurs and modulates its GAP activity and/or specificity.

As FLJ13130 is mainly localized just near the plasma membrane (Fig. 2D), the target Rab(s) of FLJ13130 would also be expected to be localized near the cell periphery, and consistent with this expectation, both Rab3A and Rab27A are mainly localized on the dense-core vesicles docked to the plasma membrane of PC12 cells (Tsuboi & Fukuda 2006). It is therefore tempting to speculate that FLJ13130 promotes the GTPase activity of both Rab3A and Rab27A to retrieve Rab proteins after exocytosis of dense-core vesicles from the plasma membrane (Kondo et al. 2006). As mFLJ00332 (a homologue of FLJ13130) exhibits Rab35-GAP activity and Rab35 is also localized on the plasma membrane and regulates the endocytic recycling pathway (Kouranti et al. 2006), FLJ13130 is also likely to function as a Rab35-GAP and that may impair recycling of dense-core vesicle proteins (e.g., Syt I in Fig. 3G). Although the functional relationship between endosomal Rab22A and FLJ13130 is unknown, we suspect that FLJ13130 is a novel type of Rab-GAP with broad Rab-GAP specificity and that it inactivates several distinct Rab isoforms (e.g., Rab3A, Rab27A, and Rab35) just near the plasma membrane. Immunoblot analysis using anti-FLJ13130-specific antibody indicated that PC12 cells endogenously express FL13130 protein (Fig. S1 in Supporting Information). However, because it was impossible to use the antibody for an immunofluorescence analysis (data not shown), we were unable to determine the exact localization of endogenous FLJ13130 protein in PC12 cells. Generation of an additional antibody against FLJ13130 that can be used for an immunofluorescence analysis will be necessary to resolve this issue.

In summary, we have established a novel method of screening for Rab3A-GAP, that is, a Rab3A exclusion assay in PC12 cell that can be applied to screen for other unidentified Rab-GAPs. The results obtained by using this method in the present study revealed that FLJ13130 was the only candidate Rab3A-GAP among the human TBC proteins tested. However, FLJ13130 is not a specific Rab3A-GAP and exhibits broad Rab-GAP specificity in vitro, in contrast to the other Rab-GAPs previously characterized. In other words, no Rab3-specific TBC-type Rab-GAP is present in humans. We therefore suspect that not all human TBC proteins function as specific Rab-GAPs and that some TBC proteins have several target Rabs (i.e., broad Rab-GAP specificity) or lack Rab-GAP activity because of a mutation of the catalytic Arg residue (Frittoli et al. 2008).


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Materials

Horseradish peroxidase (HRP)-conjugated anti-FLAG tag (M2) mouse monoclonal antibody was obtained from Sigma–Aldrich Corp. (St. Louis, MO). HRP-conjugated anti-T7 tag mouse monoclonal antibody was from Merck Biosciences Novagen (Darmstadt, Germany). Anti-Rab3 mouse monoclonal antibody and anti-GM130 mouse monoclonal antibody were from BD Transduction Laboratories (Lexington, KY). Anti-Syt I (SYA148) and Alexa 594-conjugated anti-mouse IgG goat antibody were from StressGen Biotechnologies Corp. (Victoria BC, Canada) and Invitrogen Corp. (Carlsbad, CA), respectively.

Plasmid construction

pEGFP-C1 vectors (BD Biosciences Clontech, Mountain View, CA) harboring cDNAs of 41 different human or mouse TBC proteins and mouse Rab3-GAP were prepared as described previously (Itoh & Fukuda 2006; Itoh et al. 2006). cDNA encoding FLJ20337, FLJ32666, or mKIAA1055 was amplified from Marathon-Ready human or mouse brain cDNA (BD Biosciences Clontech) by PCR with the following pairs of oligonucleotides containing a restriction enzyme (underlined) site or a stop codon (boldface) as described previously (Fukuda et al. 1999): 5'-GGATCCATGGCCGCTGAGAACAGCAA-3' (FLJ20337-Met primer, sense) and 5'-CTATCGGCGGTAGTGATTTG-3' (FLJ20337-stop primer, antisense); 5'-AGATCTATGACTGAG GACTCTCAGAG-3' (FLJ32666-Met primer, sense) and 5'-TCAGCTTGAATGGACCGGGG-3' (FLJ32666-stop primer, antisense); 5'-AGATCTATGCCGGGGGCCGGGGACGG-3' (mKIAA1055-Met primer, sense) and 5'-GCTGCTGCCTG ATGATGTCG-3' (mKIAA1055-C1 primer, antisense); and 5'-AGCAGTAGTGATCCTTTGCT-3' (mKIAA1055-N1 primer, sense) and 5'-TCAAGTGTCTTCCTCCTCAT-3' (mKIAA1055-stop primer, antisense). The purified PCR products were inserted directly into the pGEM-T Easy vector (Promega, Madison, WI) and were sequenced completely. The cDNA inserts were then subcloned into the pEGFP-C1 vector (BD Biosciences Clontech). As several forms of FLJ13130 were found in the public database, presumably as a result of alternative splicing at the N-terminal domain, the 533 C-terminal amino acids (Itoh & Fukuda 2006), which include the entire TBC domain, were used in this study. The short form of TBC1D8B (AB449891) was used for a Rab3A exclusion assay (see below). The nucleotide sequences of the 41 TBC proteins and Rab3-GAP used in this study have been deposited in the GenBank/EBI Data Bank under the accession numbers AB449874–916.

A FLJ13130 mutant carrying an Arg-to-Lys mutation at amino acid position 134 (named RK) was produced by using conventional PCR techniques and the following mutagenic oligonucleotide containing an artificial XhoI site (underlined) and substituted nucleotides (italics) as described previously (Fukuda et al. 1995): 5'-CTCGAGCAGCAAACATCTCGTGGAAAGGGAACTG TTTGTGCAG-3' (FLJ13130-RK primer, antisense). The mutant FLJ13130 fragment was then subcloned into the pEF-T7 expression vector modified from pEF-BOS (Fukuda et al. 1994, 1999) or pEGFP-C1 vector. Other expression plasmids (pEF-T7-FLJ13130, pEF-T7-EPI64, pEF-T7-mFLJ00332, pEF-T7-RN-tre, pEF-T7-Rab3-GAP, pEF-FLAG-Rab3A, pGEX-4T-3-Rab2A, pGEX-4T-3-Rab6A, pGEX-4T-3-Rab3A, pGEX-4T-3-Rab22A, pGEX-4T-3-Rab27A, pGEX-4T-3-Rab35, and pEF-T7-GST-Rim2-RBD) were prepared as described elsewhere (Kuroda et al. 2002; Fukuda 2004; Itoh & Fukuda 2006; Itoh et al. 2008).

Immunofluorescence analysis

PC12 cells were cultured on collagen type IV-coated 35-mm dishes in Dulbecco's modified Eagle's medium containing 10% horse serum and 10% fetal bovine serum at 37 °C under 5% CO2. Two micrograms of pEGFP-C1-TBC, pEGFP-C1-Rab3-GAP, or pEGFP-C1 (a vector control) was transfected into PC12 cells by using Lipofectamine 2000 reagent (Invitrogen Corp.) according to the manufacturer's instructions. Thirty-six hours after transfection, the cells were treated with 100 ng/mL NGF (Merck Biosciences Calbiochem, Darmstadt, Germany). One day after NGF treatment, the cells were fixed with 4% paraformaldehyde (Wako Pure Chemicals, Osaka, Japan) 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-Rab3 antibody (1/50 dilution), anti-GM130 antibody (1/400 dilution), or anti-Syt I antibody (1/100 dilution) followed by Alexa-Fluor 594-labeled secondary IgG (1/5000 dilution). The cells were examined for fluorescence with a confocal laser-scanning microscope (Fluoview 500, Olympus, Tokyo, Japan), and the images were processed with Adobe Photoshop software (version 7.0). For the Rab3A exclusion assay shown in Fig. 2, Rab3A signals in the neurites of the transfected cells were examined for fluorescence with the confocal fluorescence microscope (more than 100 cells/dish, three independent dishes for each plasmid), and the number of cells with reduced Rab3A signals was counted.

GTP-Rab3A pull-down assay in COS-7 cells

Plasmids were transfected into COS-7 cells (7.5 x 105 cells/10-cm dish, the day before transfection) by using Lipofectamine Plus (Invitrogen Corp.) according to the manufacturer's notes. COS-7 cells expressing FLAG-Rab3A and T7-FLJ13130 (T7-FLJ13130-RK, T7-EPI64, T7-mFLJ00332, or T7-Rab3-GAP) were homogenized in a homogenization buffer containing 50 mM HEPES–KOH (pH 7.2), 150 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 10 µM pepstatin A, and then solubilized with 1% Triton X-100 for 1 h. After centrifugation, the supernatants were appropriately diluted with a lysis buffer so that the amounts of FLAG-Rab3A protein in the diluted samples would be equal when immunoblotted with HRP-conjugated anti-FLAG tag antibody. The diluted samples were incubated for 1 h at 4 °C with glutathione-Sepharose beads (GE Healthcare Ltd., Buckinghamshire, UK) coupled with the T7-GST-tagged RBD of Rim2 (T7-GST-Rim2-RBD; i.e., GTP-Rab3A trapper (Fukuda 2004)). After washing the beads three times, the GTP-Rab3A 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). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; GE Healthcare Ltd.).

In vitro GAP assay

GST-Rabs were prepared from bacteria and frozen in liquid nitrogen for storage at –80 °C. T7-GST-FLJ13130 (or T7-GST-Rab3-GAP) was prepared from COS-7 cells (Fig. S2 in Supporting Information) as described previously (Kuroda & Fukuda 2005; Itoh et al. 2006) and immediately used for a GAP assay, because the freeze-thawed FLJ13130 sample was unable to promote GTPase activity of Rab3A and Rab27A (data not shown). A 200 pmol of purified GST-Rab protein was incubated for 10 min at 30 °C with 6.7 pmol of [{alpha}-32P]GTP (Muromachi Yakuhin Kaisha Ltd., Tokyo, Japan) and 800 pmol of cold GTP (Sigma-Aldrich Corp.) in 25 mM Tris–HCl (pH 7.5), 50 mM NaCl, 2.5 mM EDTA, and 0.5 mg/mL bovine serum albumin (BSA). After addition of MgCl2 (final concentration, 10 mM), the mixture was passed through a PD-10 column (GE Healthcare Ltd.) filled with Sephadex G-25 (GE Healthcare Ltd.), and the 3.0–3.5-mL eluate fractions were collected. The GTPase activity reaction was initiated by adding 8 pmol (1, 10, and 15 pmol in Fig. 6B) 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. Because the intrinsic GTPase activity of Rab family members differs, the reaction time varied according to the Rab isoform tested: Rab2A, 20 min; Rab3A, 20 min; Rab6A, 20 min; Rab22A, 40 min; Rab27A, 40 min; and Rab35, 40 min. 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) and developed in 0.5 M LiCl and 1 M formic acid. The amounts of GTP and GDP were determined with a FLA-3000 fluorescent and radioisotope imaging analyzer (FUJIFILM, Tokyo, Japan) and an imaging plate.


   Acknowledgements
 
Authors thank Dr Takahiro Nagase for kindly donating KIAA cDNA clones and members of the Fukuda Laboratory for valuable discussions. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants 18207015, 19044003, 20022004, and 20054001 to M. F., and 19790242 to T. I.), by the Naito Foundation, by the Takeda Science Foundation, by the Gushinkai Foundation, by the Uehara Memorial Foundation (to M. F.), and by the Cosmetology Research Foundation (to T. I.).


   Footnotes
 
Communicated by: Kozo Kaibuchi

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank with accession number(s) AB449874–916.

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


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 10 August 2008
Accepted: 5 October 2008





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