Rabconnectin-3, a Novel Protein That Binds Both GDP/GTP Exchange Protein and GTPase-activating Protein for Rab3 Small G Protein Family*

Rab3A, a member of the Rab3 small G protein family, regulates Ca2+-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). It remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated. We isolated here a novel protein that was co-immunoprecipitated with Rab3 GEP and GAP by their respective antibodies from the crude synaptic vesicle fraction of rat brain. The protein, named rabconnectin-3, bound both Rab3 GEP and GAP. The cDNA of rabconnectin-3 was cloned from a human cDNA library and its primary structure was determined. Human rabconnectin-3 consisted of 3,036 amino acids and showed a calculated M r of 339,753. It had 12 WD domains. Tissue and subcellular distribution analyses in rat indicated that rabconnectin-3 was abundantly expressed in the brain where it was enriched in the synaptic vesicle fraction. Immunofluorescence and immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles at synapses. These results indicate that rabconnectin-3 serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles.

Rab3A is a member of the Rab3 family, along with Rab3B, -3C, and -3D, and plays a key regulatory role in Ca 2ϩ -dependent exocytosis of neurotransmitter (for reviews, see Refs. [1][2][3]. The process of Ca 2ϩ -dependent exocytosis of neurotransmitter consists of several steps: translocation of synaptic vesicles from the reserve pool to the active zone of the presynaptic plasma membrane where a Ca 2ϩ channel localizes, docking of the vesicles to the active zone, transition from the docking to the priming of the vesicles in the readily releasable pool, and fusion of the vesicles with the membrane induced by Ca 2ϩ influx (1)(2)(3). The Rab3A gene knockout mouse analysis has revealed two actions of Rab3A: it facilitates the translocation and docking of synaptic vesicles to the presynaptic plasma membrane (4), and it prevents Ca 2ϩ -triggered fusion of the vesicles with the plasma membrane (5).
The Rab3 family members are regulated by three regulators: Rab GDI 1 and Rab3 GEP and GAP (1)(2)(3). Rab3 GEP and GAP are specific for the Rab3 family members, but Rab GDI is active on all the Rab family members. The cyclical activation and inactivation of Rab3A by the action of these regulators are essential for Ca 2ϩ -dependent exocytosis of neurotransmitter. A current model for the mode of action of these regulators is as follows: GDP-Rab3A is kept in the cytosol in a complex with Rab GDI. This complex is recruited to synaptic vesicles where GDP-Rab3A is activated to GTP-Rab3A by the action of Rab3 GEP with the help of another unidentified molecule, such as GDI displacement factor (6) or Rab recycling factor (7) proposed for other vesicle trafficking systems. GTP-Rab3A binds to its two downstream effectors: rabphilin-3 and Rim localized on the vesicles and the active zone, respectively. Before or after the fusion step, GTP-Rab3A in a complex with the effectors is inactivated to GDP-Rab3A by the action of Rab3 GAP. GDP-Rab3A forms a complex with Rab GDI, resulting in the translocation from the vesicles to the cytosol. Rat Rab3 GEP consists of 1,602 aa and shows a calculated M r of 177,982 (8). Human Rab3 GAP consists of one catalytic subunit (p130) and one noncatalytic subunit (p150): the catalytic and noncatalytic subunits consist of 981 and 1,393 aa and show calculated M r values of 110,521 and 156,081, respectively (9,10). In this model, however, it still remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated.
To address these issues, we attempted here to isolate a protein(s) that was co-immunoprecipitated with Rab3 GEP and/or GAP by their respective Abs from CSV of rat brain and identified a novel protein, named rabconnectin-3, which bound both Rab3 GEP and GAP. We describe here the isolation and characterization of rabconnectin-3 and discuss its possible function in Ca 2ϩ -dependent exocytosis of neurotransmitter. * The work at Osaka University was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2001). The work at Tokushima University was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2001). 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF389880.
Immunoprecipitation and Determination of the aa Sequences of p340 (Rabconnectin-3)-To identify a Rab3 GEP-and/or GAP-binding protein(s), CSV was incubated at 4°C for 90 min in Buffer A (20 mM Tris/Cl at pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 0.8% n-octylglucopyranoside) or Buffer B (20 mM Tris/Cl at pH 8.0, 1 mM EDTA, 150 mM NaCl, and 1% Nonidet P-40), followed by centrifugation at 100,000 ϫ g for 60 min. The supernatant in Buffer A or B (4 mg of protein each) was incubated at 4°C overnight with the anti-Rab3 GEP or anti-Rab3 GAP p150 Ab immobilized on protein A-Sepharose beads (40 l of wet volume), respectively. After the beads were extensively washed with the respective buffers, the bound proteins were eluted by boiling the beads in an SDS sample buffer (60 mM Tris/Cl at pH 6.7, 3% SDS, 2% (v/v) 2-mercaptoethanol, and 5% glycerol). The sample was subjected to SDS-PAGE, followed by protein staining with silver.
The protein band corresponding to p340 (rabconnectin-3) was cut out from the gel and digested with a lysyl endopeptidase, and the digested peptides were separated by C18 reverse phase high pressure liquid column chromatography as described previously (13). The aa sequences of the peptides were determined with a peptide sequencer (Hewlett-Packard G1005A protein sequencing system).
Molecular Cloning of p340 (Rabconnectin-3)-Three sets of oligonucleotide primers were designed as follows: AGA TTA CAT CCT GAT CCT TTC CTG CGT/TTC ATC TAA GGC ACT GTC GTG ATC TTC, GTG TGG CAT GTG AAG TAT TTG GAT GAA/ATC CAC TAC AGC TTG ATA GAG TCT CAG, and ATG CAT CTG CAT CAG GTC CTC/ CAA AAG ACA GTC TTC TGG TAA. Three cDNA fragments were amplified with these primers from a human brain cDNA (CLONTECH). A human brain cDNA library in ZAPII (Stratagene) was screened by use of these cDNA fragments as probes. DNA sequencing was performed by the dideoxynucleotide termination method using a DNA sequencer (Applied Biosystems PRISM 3100 Genetic Analyzer, PE Biosystems).
Binding of Rabconnectin-3 to Rab3 GEP and GAP-The extract of CSV (0.5 mg of protein) was prepared with Buffer A containing 150 mM NaCl and mixed with recombinant Rab3 GEP, His 6 -tagged Rab3 GAP p130, or p150 (10 pmol each). The sample mixed with Rab3 GEP was then incubated at 4°C for 120 min with the anti-rabconnectin-3 Ab2 immobilized on protein A-Sepharose beads (20 l of wet volume) through a goat anti-rat IgG Fc Ab (Chemicon) in Buffer A containing 150 mM NaCl. The sample mixed with Rab3 GAP p130 or p150 was similarly incubated with the anti-rabconnectin-3 Ab2 immobilized on protein G-Sepharose beads (20 l of wet volume). After the beads were extensively washed with the same buffer, the bound proteins were eluted by boiling the beads in the SDS sample buffer. The sample was subjected to SDS-PAGE, followed by Western blotting.
Miscellaneous Procedures-SDS-PAGE (16), subcellular fractionation of rat brain (15), immunofluorescence microscopy of frozen sections (17) and cultured cells (18), and immunoelectron microscopy (19) were performed as described. Protein concentrations were determined with bovine serum albumin as a reference protein as described previously (20).

RESULTS
A Protein Co-immunoprecipitated with Rab3 GEP and GAP by Their Abs-To first identify a Rab3 GEP-binding protein(s), Rab3 GEP was immunoprecipitated by its Ab from the extract of CSV of rat brain and the immunoprecipitate was subjected to SDS-PAGE, followed by protein staining. Four major proteins with molecular mass values of 340 kDa (p340), 200 kDa (p200), 160 kDa (p160), and 60 kDa (p60) were immunoprecipitated (Fig. 1Aa). Western blot analysis showed that of the four proteins, p200 was Rab3 GEP. The protein staining did not reveal the presence of Rab3A in the immunoprecipitate, because the IgG light chain disturbed the detection of Rab3A (data not shown). However, Western blot analysis showed that Rab3A was faintly co-immunoprecipitated with Rab3 GEP. The amount of Rab3A immunoprecipitated was estimated to be less than 10% of that of Rab3 GEP immunoprecipitated.
To then identify a Rab3 GAP-binding protein(s), Rab3 GAP was immunoprecipitated by the anti-Rab3 GAP p150 Ab from the same fraction, and the immunoprecipitate was subjected to SDS-PAGE, followed by protein staining. Four major proteins with molecular mass values of 340 kDa (p340), 150 kDa (p150), 130 kDa (p130), and 90 kDa (p90) were immunoprecipitated (Fig. 1Ab). Western blot analysis showed that of the four proteins, p150 and p130 were the noncatalytic and catalytic subunits of Rab3 GAP, respectively. Rab3A was not detected in the immunoprecipitate (data not shown).
The p340 protein co-immunoprecipitated with Rab3 GEP and the p340 co-immunoprecipitated with Rab3 GAP showed apparently a similar molecular mass. The bands of these two proteins were separately cut out from the gel and digested with a lysyl endopeptidase. The peptides of each protein were separated by C18 reverse phase high pressure liquid column chromatography. The same peptide peaks were observed for both the p340 proteins, and the aa sequences of the seven peptides of each p340 protein were determined. The same aa sequences were obtained. These results indicated that the same protein FIG. 1. Binding of rabconnectin-3
Molecular Cloning of p340 (Rabconnectin-3)-Computer search with these aa sequences of p340 against GenBank TM data bases revealed that two of the seven peptides were included in the aa sequence deduced from a human brain cDNA fragment (KIAA0856, GenBank TM accession number AB020663). All of them were included in the aa sequence deduced from two overlapping BAC clones of the human genome (GenBank TM accession numbers AC020892 and AC066613). On the basis of this information, we screened a human brain cDNA library and isolated overlapping clones 1-3 (see Fig. 2B). These three clones with the KIAA0856 cDNA were aligned, and the complete nucleotide sequence was determined. The sequence contained an initiation codon downstream of an in-frame stop codon in the 5Ј region and an in-frame stop codon in the 3Ј region, indicating that this sequence encoded the entire coding region. All the aa sequences of the peptides were included in this sequence. The complete nucleotide sequence encoded a protein with 3,036 aa and a calculated M r of 339,753 (Gen-Bank TM accession number AF389880) ( Fig. 2A). We named this protein rabconnectin-3. Rabconnectin-3 contained 12 WD domains (Fig. 2B). WD domains are found in a variety of proteins and likely to be responsible for protein-protein interactions (21). Rabconnectin-3 showed a domain structure similar to that of DMXL-1, which has 10 WD domains. DMXL-1 has been identified as a WD domain protein, but its function is unknown (22).
Binding of Rabconnectin-3 to Both Rab3 GEP and GAP-When rabconnectin-3 was immunoprecipitated from the extract of CSV by the anti-rabconnectin-3 Ab, neither Rab3 GEP, Rab3 GAP p130, p150, nor Rab3A was detected in the immunoprecipitate (data not shown). This might be just due to the low amounts of Rab3 GEP and GAP in this fraction (see Fig.  3B). We then mixed the recombinant sample of Rab3 GEP, Rab3 GAP p130, or p150 with the extract of CSV, and rabconnectin-3 was immunoprecipitated. Rab3 GEP and Rab3 GAP p150, but not Rab3 GAP p130, were co-immunoprecipitated with rabconnectin-3 (Fig. 1B). These results indicate that rabconnectin-3 binds both Rab3 GEP and Rab3 GAP p150.
Tissue and Subcellular Distribution of Rabconnectin-3-Western blot analysis of various rat tissues revealed that rabconnectin-3 was abundantly expressed in the brain (Fig. 3A). The subcellular distribution analysis in rat brain revealed that rabconnectin-3 was highly concentrated in CSV (Fig. 3B). On the other hand, both Rab3 GEP and GAP were mainly recovered in the synaptic soluble fraction, whereas rabphilin-3 was highly concentrated in CSV. This subcellular distribution of these proteins is consistent with our previous results (10,11,19). Immunofluorescence microscopy revealed that rabconnectin-3 was concentrated in the synaptic regions of mouse hippocampus and primary cultured rat hippocampal neurons (Fig.  4, Aa and Ab). Immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles of primary cultured rat hippocampal neurons (Fig. 4B). These results indicate that rabconnectin-3 is associated with synaptic vesicles at synapses. DISCUSSION We have identified here a novel protein that binds both Rab3 GEP and GAP and named it rabconnectin-3. Tissue distribution analysis of rabconnectin-3 in rat has revealed that it is abundantly expressed in the brain. Subcellular distribution analysis of rabconnectin-3 in rat brain and immunofluorescence and immunoelectron microscopy of mouse hippocampus and primary cultured rat hippocampal neurons have revealed that it is associated with synaptic vesicles at synapses. Rabconnectin-3 has no transmembrane segment and is extracted from the vesicles by a detergent (data not shown), suggesting that it is a peripheral membrane protein of synaptic vesicles. Taken together, rabconnectin-3 may serve as a scaffold protein A, tissue distribution. The homogenates of various rat tissues (40 g of protein each) were subjected to SDS-PAGE (6% polyacrylamide gel), followed by Western blotting with the anti-rabconnectin-3 Ab1. B, subcellular distribution in rat brain. The homogenate of rat cerebra was subjected to subcellular fractionation. An aliquot of each fraction (10 g of protein each) was subjected to SDS-PAGE (6 or 8% polyacrylamide gel), followed by Western blotting with the anti-rabconnectin-3 Ab1, the anti-Rab3 GEP, anti-Rab3 GAP p130, anti-Rab3 GAP p150, or antirabphilin-3 Ab. Rc, rabconnectin-3; Rp, rabphilin-3; Ho, the homogenate fraction; P1, the nuclear pellet fraction; P2, the crude synaptosome fraction; P3, the microsome fraction; S, the soluble cytosol fraction; P2A, the myelin fraction; P2B, the endoplasmic reticulum and Golgi complex fraction; P2C, the synaptosome fraction; P2D, the mitochondria fraction; SS, the synaptic soluble fraction; CSV, the crude synaptic vesicle fraction; CSM, the crude synaptic membrane fraction. These results are representative of three independent experiments. of Rab3 GEP and GAP on synaptic vesicles.
In contrast to rabconnectin-3, subcellular distribution analysis indicates that Rab3 GEP and GAP are mainly recovered in the synaptic soluble fraction and partly associated with synaptic vesicles. The exact reason for the different distribution of these proteins is currently unknown, but when Rab3 GEP and GAP activate and inactivate Rab3A, respectively, in the process of Ca 2ϩ -dependent exocytosis of neurotransmitter, these regulators may be translocated from the cytosol to synaptic vesicles through their binding to rabconnectin-3. After Rab3 GEP and GAP finish their functions, these regulators may be released from rabconnectin-3, resulting in the translocation from the vesicles to the cytosol. The binding of Rab3 GEP and GAP to rabconnectin-3 may be regulated by modifications of some of these three proteins, such as phosphorylation.
It is of crucial importance to know whether rabconnectin-3 affects Rab3 GEP or GAP activity. To address this issue, we attempted to make recombinant proteins of the full length of rabconnectin-3 in E. coli, Sf9 cells, and COS7 cells, but we have not yet succeeded in preparing any recombinant protein. We cannot conclude from the present immunoprecipitation analysis whether Rab3 GEP and GAP directly or indirectly bind to rabconnectin-3, because we do not have the pure sample of rabconnectin-3.
We have shown here that Rab3A is co-immunoprecipitated by the anti-Rab3 GEP Ab, but not by the anti-Rab3 GAP p150 or anti-rabconnectin-3 Ab, suggesting that Rab3A binds to Rab3 GEP. Thus, rabconnectin-3 directly or indirectly binds Rab3A and its regulators, raising a possibility that rabconnectin-3 additionally binds Rab GDI, rabphilin-3, and Rim directly or indirectly and functions as a core protein scaffolding Rab3A and its related molecules. Rabconnectin-3 may also bind a putative GDI displacement or Rab recycling factor for Rab3A. It is important to clarify the molecular linkage among rabcon-nectin-3, Rab3A, and its related molecules in more detail for our understanding of Ca 2ϩ -dependent exocytosis of neurotransmitter.