Synapsin Is a Novel Rab3 Effector Protein on Small Synaptic Vesicles
2004; Elsevier BV; Volume: 279; Issue: 42 Linguagem: Inglês
10.1074/jbc.m403293200
ISSN1083-351X
AutoresSilvia Giovedı̀, Paola Vaccaro, Flavia Valtorta, François Darchen, Paul Greengard, Gianni Cesareni, Fabio Benfenati,
Tópico(s)Retinal Development and Disorders
ResumoSynapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals. Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals. Information transfer among neurons is controlled by neurotransmitters stored in synaptic vesicles (SV) 1The abbreviations used are: SV, synaptic vesicles; BSA, bovine serum albumin; CaMK, Ca2+/calmodulin-dependent protein kinase; DSS, disuccinimidyl suberate; GDI, GDP dissociation inhibitor; GST, glutathione S-transferase; mAb, monoclonal antibody; SASD, sulfosuccinimidyl 2-(p-azidosalicylamido)-ethyl-1,3′-dithioproprionate; SSV, synapsin-depleted synaptic vesicles; USV, untreated synaptic vesicles; GTPγS, guanosine 5′-3-O-(thio)triphosphate; MOPS, 4-morpholinepropanesulfonic acid; NTA, nitrilotriacetic acid; Ab, antibody; ELISA, enzyme-linked immunosorbent assay; h, human.1The abbreviations used are: SV, synaptic vesicles; BSA, bovine serum albumin; CaMK, Ca2+/calmodulin-dependent protein kinase; DSS, disuccinimidyl suberate; GDI, GDP dissociation inhibitor; GST, glutathione S-transferase; mAb, monoclonal antibody; SASD, sulfosuccinimidyl 2-(p-azidosalicylamido)-ethyl-1,3′-dithioproprionate; SSV, synapsin-depleted synaptic vesicles; USV, untreated synaptic vesicles; GTPγS, guanosine 5′-3-O-(thio)triphosphate; MOPS, 4-morpholinepropanesulfonic acid; NTA, nitrilotriacetic acid; Ab, antibody; ELISA, enzyme-linked immunosorbent assay; h, human. and released to the extracellular space by an efficient process of regulated exocytosis. Synaptic vesicles are organized in two distinct functional pools, a large reserve pool in which SV are restrained by the actin-based cytoskeleton, and a quantitatively smaller releasable pool in which SV contact the presynaptic membrane and eventually fuse with it upon stimulation (for review see Refs. 1Neher E. Neuron. 1998; 20: 389-399Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar and 2Brodin L. Low P. Gad H. Gustaffson J. Pieribone V.A. Shupliakov O. Eur. J. Neurosci. 1997; 9: 2503-2511Crossref PubMed Scopus (48) Google Scholar). Both SV trafficking and neurotransmitter release depend on a precise sequence of events that includes release from the cytoskeletal constraint, targeting to the active zone, docking, priming, fusion, and endocytotic retrieval of SV. These steps are believed to be mediated by a series of specific interactions among cytoskeletal, SV, presynaptic membrane, and cytosolic proteins that, by acting in concert, promote the spatial and temporal regulation of the exocytotic machinery (3Greengard P. Valtorta F. Czernik A.J. Benfenati F. Science. 1993; 259: 780-785Crossref PubMed Scopus (1118) Google Scholar, 4Benfenati F. Onofri F. Giovedì S. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1999; 354: 243-257Crossref PubMed Scopus (36) Google Scholar, 5Martin T.F. Neuron. 2002; 34: 9-12Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The synapsins are members of a multigene family of SV-specific phosphoproteins that are implicated in the regulation of neurotransmitter release and synapse formation (see Refs. 6De Camilli P. Benfenati F. Valtorta F. Greengard P. Annu. Rev. Cell Biol. 1990; 6: 433-460Crossref PubMed Scopus (251) Google Scholar and 7Hilfiker S. Pieribone V.A. Czernik A.J. Kao H.T. Augustine G.J. 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Lu B. Poo M.-M. Greengard P. Eur. J. Neurosci. 1995; 7: 261-270Crossref PubMed Scopus (66) Google Scholar). Modulation of SV and actin binding after synapsin phosphorylation by Ca2+/calmodulin-dependent protein kinase (CaMK) II (10Ceccaldi P. Grohovaz F. Benfenati F. Chieregatti E. Greengard P. Valtorta F. J. Cell Biol. 1995; 128: 905-912Crossref PubMed Scopus (134) Google Scholar, 13Schiebler W. Jahn R. Doucet J.-P. Rothlein J. Greengard P. J. Biol. Chem. 1986; 261: 8383-8390Abstract Full Text PDF PubMed Google Scholar, 14Bähler M. Greengard P. Nature. 1987; 326: 704-707Crossref PubMed Scopus (345) Google Scholar, 15Benfenati F. Valtorta F. Rubenstein J.L. Gorelick F.S. Greengard P. Czernik A.J. Nature. 1992; 359: 417-420Crossref PubMed Scopus (236) Google Scholar, 16Valtorta F. Greengard P. Fesce R. Chieregatti E. Benfenati F. J. Biol. Chem. 1992; 267: 11281-11288Abstract Full Text PDF PubMed Google Scholar, 17Stefani G. Onofri F. Valtorta F. Vaccaro P. Greengard P. Benfenati F. J. 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In living hippocampal neurons, synapsin I dissociates from SV and disperses into the axon during firing, whereas it reclusters upon return to the resting state, and the dispersion of the proteins depends on phosphorylation by CaMK and extracellular signal-regulated kinase (21Chi P. Greengard P. Ryan T.A. Nat. Neurosci. 2001; 4: 1187-1193Crossref PubMed Scopus (283) Google Scholar, 22Chi P. Greengard P. Ryan T.A. Neuron. 2003; 38: 69-78Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Injection of synapsin I into developing neurons stimulated the maturation of quantal neurotransmitter release and increased SV clustering (12Valtorta F. Iezzi N. Benfenati F. Lu B. Poo M.-M. Greengard P. Eur. J. Neurosci. 1995; 7: 261-270Crossref PubMed Scopus (66) Google Scholar, 23Lu B. Greengard P. Poo M.-M. Neuron. 1992; 8: 521-529Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 24Ghirardi M. Naretto G. Fiumara F. Vitiello F. Montarolo P.G. J. Neurosci. Res. 2001; 65: 111-120Crossref PubMed Scopus (9) Google Scholar). Conversely, neutralization of endogenous synapsins by the intracellular injection of anti-synapsin antibodies disrupted the clusters of SV, decreased neurotransmitter release evoked by high frequency stimulation in the lamprey giant reticulospinal neurons (11Pieribone V.A. Shupliakov O. Brodin L. Hilfiker-Rothenfluh S. Czernik A.J. Greengard P. Nature. 1995; 375: 493-497Crossref PubMed Scopus (418) Google Scholar), and induced the disappearance of post-tetanic potentiation, appearance of post-tetanic depression, and increased synaptic depression in Aplysia ganglion neurons (25Humeau Y. Dousseau F. Vitiello F. Greengard P. Benfenati F. Poulain B. J. Neurosci. 2001; 21: 4195-4206Crossref PubMed Google Scholar). Mutant mice lacking synapsin I, synapsin II, or both exhibited a decrease in the number of SV and in the maximal release of neurotransmitters, depression during high frequency stimulation, and increased recovery times after synaptic depression (26Li L. Chin L.-S. Shupliakov O. Brodin L. Sihra T.S. Hvalby Ø. Jensen V. Zheng D. McNamara J.O. Greengard P. Andersen P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9235-9239Crossref PubMed Scopus (296) Google Scholar, 27Takei Y. Harada A. Takeda S. Kobayashi K. Terada S. Noda T. Takahashi T. Hirokawa N. J. Cell Biol. 1995; 131: 1789-1800Crossref PubMed Scopus (147) Google Scholar, 28Rosahl T.W. Spillane D. Missler M. Herz J. Selig D.K. Wolff J.R. Hammer R.E. Malenka R.C. Südhof T.C. Nature. 1995; 375: 488-493Crossref PubMed Scopus (617) Google Scholar, 29Ryan T.A. Li L. Chin L.-S. Greengard P. Smith S.J. J. Cell Biol. 1996; 134: 1219-1227Crossref PubMed Scopus (144) Google Scholar, 30Terada S. Tsujimoto T. Takei Y. Takahashi T. Hirokawa N. J. Cell Biol. 1999; 145: 1039-1048Crossref PubMed Scopus (68) Google Scholar). Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a large reserve pool of SV and in the regulation of short term synaptic plasticity, recent results indicate that the synapsins are also involved in some later step of exocytosis. The kinetics of release was slowed in the squid giant terminal after injection of a conserved synapsin COOH-terminal peptide (31Hilfiker S. Schweizer F.E. Kao H.T. Czernik A.J. Greengard P. Augustine G.J. Nat. Neurosci. 1998; 1: 29-35Crossref PubMed Scopus (129) Google Scholar), as well as in Aplysia ganglion neuron terminals after neutralization of endogenous synapsin by antibody injection (25Humeau Y. Dousseau F. Vitiello F. Greengard P. Benfenati F. Poulain B. J. Neurosci. 2001; 21: 4195-4206Crossref PubMed Google Scholar). However, there is no clear evidence, as yet, of an interaction of synapsins with presynaptic or SV proteins involved in the post-docking events of exocytosis. Synaptic vesicles docked or fused with the presynaptic membrane following electrical stimulation are only partially depleted of synapsins (11Pieribone V.A. Shupliakov O. Brodin L. Hilfiker-Rothenfluh S. Czernik A.J. Greengard P. Nature. 1995; 375: 493-497Crossref PubMed Scopus (418) Google Scholar, 32Torri-Tarelli F. Bossi M. Fesce R. Greengard P. Valtorta F. Neuron. 1992; 9: 1143-1153Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 33Bloom O. Evergren E. Tomilin N. Kjaerulff O. Low P. Brodin L. Pieribone V.A. Greengard P. Shupliakov O. J. Cell Biol. 2003; 161: 737-747Crossref PubMed Scopus (175) Google Scholar), and synapsin I staining is found on the membranes of vesicles fused with the plasma membrane (34Torri-Tarelli F. Villa A. Valtorta F. De Camilli P. Greengard P. Ceccarelli B. J. Cell Biol. 1990; 110: 449-459Crossref PubMed Scopus (99) Google Scholar). This raises the possibility that the putative post-docking effects of the synapsins are obtained via specific interactions with nerve terminal proteins playing a direct role in exocytosis. With the aim of uncovering potential synapsin partners, we have identified high affinity synapsin-binding peptides by phage display library analysis and have generated anti-peptide antibodies to identify nerve terminal proteins bearing the selected peptide motifs. By using this approach, we have observed that the small G protein Rab3A is one of the synapsin I interactors and that the binding of synapsin I to Rab3A occurs in purified SV and intact nerve terminals. 125I and 125I-labeled secondary antibodies were from Amersham Biosciences; the Renaissance enhanced chemiluminescence detection system was from PerkinElmer Life Sciences. Isoform-specific anti-synapsin polyclonal antibodies (G143, G211, G278, G281, G304, G306, G455, and RU316), anti-synapsin I (mAb 10.22 and 19.11), anti-synapsin II (mAb 19.21) monoclonal antibodies, and anti-Rab3A polyclonal antibodies raised against a 12-mer peptide corresponding to the NH2 terminus of rat Rab3A were made in our laboratories. Other antibodies were obtained from the following sources: rabbit anti-Rabphilin-3, Transduction Laboratories (Lexington, KY); goat anti-His6 and anti-glutathione S-transferase (GST), Amersham Biosciences; peroxidase-conjugated anti-mouse and anti-rabbit antibodies, Bio-Rad; and alkaline phosphatase-conjugated secondary antibodies, Santa Cruz Biotechnology (Santa Cruz, CA). The anti-RabGDI (GDP dissociation inhibitor) polyclonal antibody was a gift of Dr. M. Zerial (EMBL, Heidelberg, Germany). The pET3b HisGDI vector was a gift of Dr. W. E. Balch (Scripps Research Institute, La Jolla, CA). Glutathione-Sepharose, protein G-Sepharose, CH-Sepharose 4B, and pGEX-2T were from Amersham Biosciences; human recombinant Rab3A and bovine serum albumin (BSA) were from Calbiochem; Ni-NTA-agarose was from Qiagen (Valencia, CA); nitrocellulose membranes were from Schleicher & Schuell; sulfo-succinimidyl 2-(p-azidosalicylamido)-ethyl-1,3′-dithioproprionate (SASD) and disuccinimidyl suberate (DSS) were from Pierce. The pET-30a/pET-30c vectors and the S protein horseradish peroxidase conjugate were from Novagen Inc. (Madison, WI). Synapsin I was purified from bovine brain and subjected to cysteine-specific cleavage as described previously (13Schiebler W. Jahn R. Doucet J.-P. Rothlein J. Greengard P. J. Biol. Chem. 1986; 261: 8383-8390Abstract Full Text PDF PubMed Google Scholar, 14Bähler M. Greengard P. Nature. 1987; 326: 704-707Crossref PubMed Scopus (345) Google Scholar, 35Bähler M. Benfenati F. Valtorta F. Czernik A.J. Greengard P. J. Cell Biol. 1989; 108: 1841-1849Crossref PubMed Scopus (92) Google Scholar). Subcellular fractionation of rat forebrain from homogenate to purified SV was carried out through the step of controlled pore glass chromatography as described (36Huttner W.B. Schiebler W. Greengard P. De Camilli P. J. Cell Biol. 1983; 96: 1374-1388Crossref PubMed Scopus (888) Google Scholar). Purified SV containing endogenous synapsins (untreated SV (USV)) were quantitatively depleted of synapsin I by exposure to mild salt treatment (synapsin-depleted SV (SSV)) and reassociated in vitro with purified synapsin I (synapsin-rebound SV) as described previously (8Benfenati F. Valtorta F. Chieregatti E. Greengard P. Neuron. 1992; 8: 377-386Abstract Full Text PDF PubMed Scopus (124) Google Scholar). Synaptosomes were purified from forebrain P2 fractions by centrifugation on discontinuous Percoll gradients as described previously (37Dunkley P.R. Jarvie P.E. Heath J.W. Kidd G.J. Rostas J.A.P. Brain Res. 1986; 372: 115-129Crossref PubMed Scopus (388) Google Scholar). Bacterial cells (Escherichia coli BL21 strain) were transformed to ampicillin resistance by electroporation with constructs containing pGEX-2T alone or pGEX-2T in-frame with sequences encoding for wild-type rat Rab3A or rat Rabphilin-3-(1–206). Mutated forms of Rab3 carrying the mutation Q81L that abolishes the intrinsic GTPase activity or the mutations Q81L, V55E in the Rab effector domain were also expressed. Large scale cultures of Luria broth containing ampicillin (100 μg/ml) were inoculated with small overnight cultures, grown at 37 °C to log phase, and induced with isopropyl β-d-thiogalactopyranoside (100 μm) for 3–5 h. GST and GST-Rab3A fusion proteins were extracted from bacterial lysates, purified to homogeneity by affinity chromatography on glutathione-Sepharose, and dialyzed against 25 mm Tris-Cl, 50 mm NaCl, pH 7.4 (38Onofri F. Giovedì S. Kao H.-T. Valtorta F. Bongiorno Borbone L. De Camilli P. Greengard P. Benfenati F. J. Biol. Chem. 2000; 275: 29857-29867Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Purified GST-Rabphilin-3 was cleaved with thrombin and repurified. Rat Rab-GDI was expressed as a His6-tagged protein in BL21(DE3) pLysS strain and purified to homogeneity on NTA-agarose affinity columns. The DNAs corresponding to COOH-terminal truncations of rat synapsin Ia were amplified by PCR and subcloned into the pET-30c vector (38Onofri F. Giovedì S. Kao H.-T. Valtorta F. Bongiorno Borbone L. De Camilli P. Greengard P. Benfenati F. J. Biol. Chem. 2000; 275: 29857-29867Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The resulting expression plasmids were then verified by sequencing and transformed into E. coli BL21(DE3) Lys E. The following mutants, containing His6 and S tags at the NH2 terminus, were made: DE peptide (syn-(413–704)), D peptide (syn-(413–652)), D1 peptide (syn-(413–617)), D2 peptide (syn-(413–585)), D3 peptide (syn-(413–556)), and D4 peptide (syn-(413–538)). Recombinant proteins were extracted from bacterial lysates and purified to homogeneity on NTA-agarose columns. Truncation mutants were detected in immunoblotting assays by either anti-His6 antibodies, protein S, or mAb 10.22 recognizing distinct sites on their NH2 terminus and detecting all mutants with the same sensitivity (39Vaccaro P. Dente L. Onofri F. Zucconi A. Martinelli S. Valtorta F. Greengard P. Cesareni G. Benfenati F. Mol. Brain Res. 1997; 52: 1-16Crossref PubMed Scopus (32) Google Scholar). We have used a previously described nonapeptide phage display library (40Felici F. Castagnoli L. Musacchio A. Jappelli R. Cesareni G. J. Mol. Biol. 1991; 222: 301-310Crossref PubMed Scopus (390) Google Scholar) containing ≈108 independent clones. Random oligonucleotides were inserted into the PC89 phagemid at the 5′ end of the gene encoding for the major coat protein pVIII. The resulting chimeric pVIII protein was incorporated into phage particles by introduction of the phagemid into E. coli cells subsequently superinfected with M13-K07 helper phage. Selection of the phages displaying peptides that bound to purified synapsin I immobilized on polystyrene beads (6.4 mm diameter; Precision Plastic Balls, Chicago, IL) was performed by the standard panning method as described previously (41Parmley S.F. Smith G.P. Gene (Amst.). 1988; 73: 305-318Crossref PubMed Scopus (734) Google Scholar). Generally, two or three cycles of panning were needed to achieve a reliable enrichment of phage clones. Synapsin-positive clones, identified by immunoscreening analysis, were isolated, and single-stranded genome DNAs were purified and sequenced (39Vaccaro P. Dente L. Onofri F. Zucconi A. Martinelli S. Valtorta F. Greengard P. Cesareni G. Benfenati F. Mol. Brain Res. 1997; 52: 1-16Crossref PubMed Scopus (32) Google Scholar, 42Cesareni G. Castagnoli L. Dente L. Iannolo G. Vetriani C. Felici F. Luzzago A. Monaci P. Nicosia A. Cortese R. Zegers N.D. Boersna W.J.A. Claasson E.E. Immunological Recognition of Peptides in Medicine and Biology. CRC Press, Inc., Boca Raton, FL1995: 43-58Google Scholar). The deduced amino acid sequences were aligned, and consensus sequences were derived by using Wisconsin Sequence Analysis Software package (Genetics Computer Group, Madison, WI). The relative affinity of interaction of the selected phage clones with native synapsin I was tested by ELISAs as described previously (42Cesareni G. Castagnoli L. Dente L. Iannolo G. Vetriani C. Felici F. Luzzago A. Monaci P. Nicosia A. Cortese R. Zegers N.D. Boersna W.J.A. Claasson E.E. Immunological Recognition of Peptides in Medicine and Biology. CRC Press, Inc., Boca Raton, FL1995: 43-58Google Scholar). Briefly, polystyrene beads coated with synapsin I (1 μg/ml) were incubated with the most reactive phage clones (50 μl of supernatant) for 1 h at 37 °C on a rocking platform. After washing the wells were incubated for 1 h at room temperature with an anti-pIII monoclonal antibody and revealed using alkaline phosphatase-conjugated secondary rat antibodies and the alkaline phosphatase detection system, and then reading the absorbance at 405–620 nm using a Labsystem reader. The site-specific binding of the selected phage clones was assayed by far Western overlay. Holosynapsin I and its cysteine-specific cleavage fragments (7.5 μg of total protein) (35Bähler M. Benfenati F. Valtorta F. Czernik A.J. Greengard P. J. Cell Biol. 1989; 108: 1841-1849Crossref PubMed Scopus (92) Google Scholar) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated for 1 h at room temperature in TBS (150 mm NaCl, 25 mm Tris-Cl, pH 7.4) containing 5% (w/v) nonfat dry milk (blocking buffer), rinsed with TBS, and incubated overnight at 4 °C in blocking buffer supplemented with 0.1% (v/v) Triton X-100 containing 1010 phage/ml. The membranes were then washed and incubated for 2 h at room temperature with a rat anti-pIII monoclonal antibody and revealed using alkaline phosphatase-conjugated secondary antibodies and the alkaline phosphatase detection system. Peptides corresponding to the sequences of the most reactive clones were synthesized at the Rockefeller University Oligomer Facility. Peptides were coupled to thyroglobulin in 10 mm KPO4, 150 mm NaCl, pH 7.0, by dropwise addition of glutaraldehyde to a final concentration of 0.1% (v/v), incubated for 2 h at 4 °C, and used to immunize rabbits in complete Freund's adjuvant. Sera were collected at various time intervals after immunization, and the antipeptide antibodies were affinity-purified using columns made by coupling synthetic peptides to CH-Sepharose 4B beads following the manufacturer's instructions (5-ml bed volume; 20–40 ml of serum). After serum loading, the columns were washed sequentially with 50 mm Tris-Cl, 150 mm NaCl, 0.1% Tween 20, pH 7.4; 25 mm sodium borate, 0.5 m NaCl, 0.1% Tween 20, pH 8.3; 50 mm sodium acetate, 0.5 m NaCl, 0.1% Tween 20, pH 5.5; 50 mm Tris-Cl, 150 mm NaCl, pH 7.4. The antibodies were eluted with 100 mm glycine, pH 2.5, immediately neutralized with 1 m Tris-Cl, pH 9.0, dialyzed against 10 mm MOPS, 150 mm NaCl, pH 7.5, and concentrated to about 1 mg/ml. The COOH-terminal fragment of synapsin I, generated by cysteine-specific cleavage, was conjugated in the dark with SASD (1.4 mm) for 40 min at room temperature and subsequently iodinated on the reagent moiety as described previously (15Benfenati F. Valtorta F. Rubenstein J.L. Gorelick F.S. Greengard P. Czernik A.J. Nature. 1992; 359: 417-420Crossref PubMed Scopus (236) Google Scholar). The modified, 125I-labeled COOH-terminal fragment (100–400 nm final concentration) was incubated for 30 min on ice in the dark with synapsin-depleted SV (10–30 μg of total protein). After high speed sedimentation, SV pellets were photolyzed for 2 min at 254 nm using an ultraviolet lamp (UVP, San Gabriel, CA) and subjected to SDS-PAGE under reducing conditions. Cross-linking of synaptosomal proteins was performed by incubating intact synaptosomes at 37 °C for 45 min with the cell-permeable cross-linker DSS dissolved in dimethyl sulfoxide and used at a final concentration of 5 mm (43Stahl B. Chou J.H. Li C. Südhof T.C. Jahn R. EMBO J. 1996; 15: 1799-1809Crossref PubMed Scopus (113) Google Scholar). After quenching the reaction with 100 mm glycine for 30 min, synaptosomes were osmotically lysed and fractionated by differential centrifugation, and the resulting crude SV fraction was subjected to SDS-PAGE and immunoblot assay. Affinity Purification of Rab3A-binding Proteins from Brain Synaptosomes—Wild-type or mutated GST-Rab3A fusion proteins (30 μg) were loaded with either GDP or GTPγS (500 μm) in buffer containing 20 mm Tris-Cl, 25 mm NaCl, 4 mm EDTA, pH 7.4 (loading buffer), for 10 min at 30 °C. After the incubation, the samples were chilled at 4 °C, and MgCl2 was added to a final concentration of 5 mm. When the loading procedure was carried out with a trace amount of either [35S]GTPγS or [3H]GDP, the average nucleotide binding stoichiometry was 0.22 ± 0.05 mol of nucleotide/mol of GST-Rab3A. Preloaded GST-Rab3A or GST alone was coupled to glutathione-Sepharose (30 μl of settled beads) by an overnight incubation at 4 °C under gentle rotation in binding buffer (10 mm Hepes, 150 mm NaCl, 1% (v/v) Triton X-100, 2 mg/ml BSA, pH 7.4) and 5mm MgCl2. Samples were then extensively washed with 20 volumes of binding buffer and mixed with 1 ml of a 1% (v/v) Triton X-100 extract of Percoll-purified synaptosomes (1 mg of protein/ml) (37Dunkley P.R. Jarvie P.E. Heath J.W. Kidd G.J. Rostas J.A.P. Brain Res. 1986; 372: 115-129Crossref PubMed Scopus (388) Google Scholar). After a 3–5-h incubation at 4 °C under gentle rotation, samples were extensively washed and eluted with Laemmli sample buffer (44Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206658) Google Scholar). The eluted proteins were separated by SDS-PAGE and analyzed by immunoblotting. Rab3A-Synapsin I Binding Assays—The binding of wild-type and mutated GST-Rab3A to purified synapsin I or to truncation mutants of the COOH-terminal region of synapsin I was assessed by co-precipitation experiments as follows. GST-Rab3A was loaded with either GDP or GTPγS as described above. Preloaded GST-Rab3A fusion proteins or GST alone was coupled to glutathione-Sepharose (0.04 nmol of fusion protein per μl of settled beads) in binding buffer containing 5 mm MgCl2. After extensive washing, protein-coupled beads (15 μl) were incubated for 3–5 h at 4 °C with increasing concentrations of either purified synapsin I (62.5–2000 nm) or synapsin truncation mutants (400 nm) in 500 μl of binding buffer. After the incubation, the beads were pelleted by centrifugation, extensively washed with binding buffer and detergent-free binding buffer, resuspended in Laemmli sample buffer, and boiled for 2 min. Binding was analyzed by SDS-PAGE followed by quantitative immunoblotting. The recovery of recombinant bait proteins in the pellets was routinely assessed by protein stain of the gels or of the corresponding blots. Protein concentrations were determined using the Bradford (Bio-Rad) assay with BSA as standard. One- and two-dimension gel electrophoresis (SDS-PAGE and nonequilibrium pH gradient electrophoresis) was performed according to Laemmli (44Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206658) Google Scholar) and O'Farrell et al. (45O' Farrell P.Z. Goodman H.M. O' Farrell P.H. Cell. 1977; 12: 1133-1142Abstract Full Text PDF PubMed Scopus (2582) Google Scholar). Proteins in the gels were electrophoretically transferred to nitrocellulose membranes and analyzed by immunoblotting by using either chemiluminescence or 125I-labeled secondary antibodies as detection system. Quantitative immunoblotting was performed either by laser scanning densitometry (Ultroscan XL, Amersham Biosciences) of the films obtained in the linear range of the emulsion response or by direct radioactivity counting, followed by interpolation of the valu
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