Ca2+/Calmodulin Causes Rab3A to Dissociate from Synaptic Membranes
1997; Elsevier BV; Volume: 272; Issue: 33 Linguagem: Inglês
10.1074/jbc.272.33.20857
ISSN1083-351X
AutoresJae Bong Park, Christopher C. Farnsworth, John A. Glomset,
Tópico(s)Retinal Development and Disorders
ResumoThe GTPase Rab3A has been postulated to cycle on and off synaptic membranes during the course of neurotransmission. Moreover, a Rab guanine nucleotide dissociation inhibitor has been shown to cause Rab3A to dissociate from synaptic membranes in vitro. We demonstrate here that Ca2+/calmodulin also can cause Rab3A to dissociate from synaptic membranes in vitro. Like Rab guanine nucleotide dissociation inhibitor, it forms a 1:1 complex with Rab3A that requires both the lipidated C terminus of Rab3A and the presence of bound guanine nucleotide. In addition, a synthetic peptide corresponding to the Lys62-Arg85 sequence of Rab3A can prevent the dissociating effect of each protein and disrupt complexes between each protein and Rab3A. However, Ca2+/calmodulin's effect differs from that of Rab guanine nucleotide dissociation inhibitor not only in being Ca2+-dependent but also in having a less stringent requirement for GDP as opposed to GTP and in involving a less complete dissociation of Rab3A. The functional significancein vivo of Ca2+/calmodulin's effect remains to be determined; it may depend in part on the relative amounts of Ca2+/calmodulin and Rab guanine nucleotide dissociation inhibitor that are available for binding to Rab3A in individual, activated nerve termini. The GTPase Rab3A has been postulated to cycle on and off synaptic membranes during the course of neurotransmission. Moreover, a Rab guanine nucleotide dissociation inhibitor has been shown to cause Rab3A to dissociate from synaptic membranes in vitro. We demonstrate here that Ca2+/calmodulin also can cause Rab3A to dissociate from synaptic membranes in vitro. Like Rab guanine nucleotide dissociation inhibitor, it forms a 1:1 complex with Rab3A that requires both the lipidated C terminus of Rab3A and the presence of bound guanine nucleotide. In addition, a synthetic peptide corresponding to the Lys62-Arg85 sequence of Rab3A can prevent the dissociating effect of each protein and disrupt complexes between each protein and Rab3A. However, Ca2+/calmodulin's effect differs from that of Rab guanine nucleotide dissociation inhibitor not only in being Ca2+-dependent but also in having a less stringent requirement for GDP as opposed to GTP and in involving a less complete dissociation of Rab3A. The functional significancein vivo of Ca2+/calmodulin's effect remains to be determined; it may depend in part on the relative amounts of Ca2+/calmodulin and Rab guanine nucleotide dissociation inhibitor that are available for binding to Rab3A in individual, activated nerve termini. The opening of voltage-gated Ca2+ channels in active zones of nerve terminals causes a brief, localized influx of Ca2+ followed by the secretion of neurotransmitters (1Zucker R.S. Biochem. Soc. Trans. 1993; 21: 395-401Crossref PubMed Scopus (21) Google Scholar, 2Ghosh A. Greenberg M.E. Science. 1995; 268: 239-246Crossref PubMed Scopus (1231) Google Scholar, 3Dunlap K Luebke J.I. Turner T.J. Trends Neurosci. 1995; 18: 89-98Abstract Full Text PDF PubMed Scopus (864) Google Scholar). The molecular basis of this effect is still unclear, but increased concentrations of intracellular Ca2+ may act at several levels to trigger fast fusion of pre-docked synaptic vesicles with the synaptic plasma membrane, promote endocytosis of the vesicle membranes and subsequent vesicle reformation, and mobilize additional vesicles to release sites (1Zucker R.S. Biochem. Soc. Trans. 1993; 21: 395-401Crossref PubMed Scopus (21) Google Scholar, 4Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1754) Google Scholar). Proteins that bind Ca2+ probably mediate many of these actions, and a number of candidate proteins have been identified. They include rabphilin (5Shirataki H. Kaibuchi K. Sakoda T. Kishida S. Yamaguchi T. Wada K. Miyazaki M. Takai Y. Mol. Cell. Biol. 1993; 13: 2061-2068Crossref PubMed Scopus (353) Google Scholar, 6Yamaguchi T. Shirataki H. Kishida S. Miyazaki M. Nishikawa J. Wada K. Numata S. Kaibuchi K. Takai Y. J. Biol. Chem. 1993; 268: 27164-27170Abstract Full Text PDF PubMed Google Scholar); the α-, β-II-, and γ isoforms of protein kinase C (7Tanaka C. Nishizuka Y. Annu. Rev. Neurosci. 1994; 17: 551-567Crossref PubMed Scopus (507) Google Scholar); and dynamin (8Liu J.-P. Powell K.A. Südhof T.C. Robinson P.J. J. Biol. Chem. 1994; 269: 21043-21050Abstract Full Text PDF PubMed Google Scholar), all of which show Ca2+-dependent binding to acidic phosphoglycerides. They also include calmodulin (CaM) 1The abbreviations used are: CaM, calmodulin; CaM kinase II, Ca2+/CaM-dependent kinase II; Rab GDI, Rab protein guanine nucleotide dissociation inhibitor protein; BS3, bis(sulfosuccinimidyl) suberate; DTT, dithiothreitol; REM, Rab3A-enriched membranes; MARCKS, myristoylated, alanine-rich protein kinase C substrate; GTPγS, guanosine 5′-O-(3-thiotriphosphate). 1The abbreviations used are: CaM, calmodulin; CaM kinase II, Ca2+/CaM-dependent kinase II; Rab GDI, Rab protein guanine nucleotide dissociation inhibitor protein; BS3, bis(sulfosuccinimidyl) suberate; DTT, dithiothreitol; REM, Rab3A-enriched membranes; MARCKS, myristoylated, alanine-rich protein kinase C substrate; GTPγS, guanosine 5′-O-(3-thiotriphosphate). (9Gnegy M.E. Annu. Rev. Pharmacol. Toxicol. 1993; 32: 45-70Crossref Google Scholar), synaptotagmin (10Brose N. Petrenko A.G. Südhof T.C. Jahn R. 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In the present investigation we examined the effects of Ca2+ and CaM on the behavior of Rab3A, a low molecular mass, di-geranylgeranylated, guanine nucleotide-binding protein that is attached to neurotransmitter-containing synaptic vesicles (22Fischer von Mollard G.F. Stahl B. Li C. Südhof T.C. Jahn R. Trends Biochem. Sci. 1994; 19: 164-168Abstract Full Text PDF PubMed Scopus (188) Google Scholar, 23Lledo P.M. Johannes L. Vernier P. Zorec R. Darchen F. Vincent J.D. Henry J.P. Mason W.T. Trends Neurosci. 1994; 17: 426-432Abstract Full Text PDF PubMed Scopus (62) Google Scholar). Previous investigators had shown that depolarization of rat brain synaptosomes causes a reduction in the contents of both Rab3A and a related guanine nucleotide-binding protein, Rab3C, in crude synaptic vesicles (Refs. 24Fisher von Mollard G.F. Südhof T.C. Jahn R. Nature. 1991; 349: 79-81Crossref PubMed Scopus (330) Google Scholar and 25Fischer von Mollard G.F. Stahl B. Khokhlatchev A. Südhof T.C. Jahn R. J. Biol. Chem. 1994; 269: 10971-10974Abstract Full Text PDF PubMed Google Scholar but see Ref. 26Bielinski D.F. Pyun H.Y. Linko-Stentz K. Macara I.G. Fine R.E. Biochim. Biophys. Acta. 1993; 1151: 246-256Crossref PubMed Scopus (60) Google Scholar for a conflicting view). Furthermore, action of a Rab guanine nucleotide dissociation inhibitor protein (Rab GDI) had been implicated because of its known ability to form a complex with Rab3A and cause it to dissociate from synaptic membranes in cell-free experiments (27Araki S. Kikuchi A. Hata Y. Isomura M. Takai Y. J. Biol. Chem. 1990; 265: 13007-13015Abstract Full Text PDF PubMed Google Scholar, 28Musha T. Kawata M. Takai Y. J. Biol. Chem. 1992; 267: 9821-9825Abstract Full Text PDF PubMed Google Scholar, 29Sasaki T. Kikuchi A. Araki S. Hata Y. Isomura M. Kuroda S. Takai Y. J. Biol. Chem. 1990; 265: 2333-2337Abstract Full Text PDF PubMed Google Scholar). While exploring the possibility that increased concentrations of Ca2+ might affect the Rab3A dissociation process, we discovered that Ca2+/CaM also can cause Rab3A to dissociate from synaptic membranes. Studies of the mechanism of this effect and its relation to that of Rab GDI are described below. 2While this paper was being revised in response to an initial review, a related paper appeared describing work based on a similar approach (30Park J.-B. Exp. Mol. Med. 1996; 28: 147-152Crossref Scopus (1) Google Scholar). 2While this paper was being revised in response to an initial review, a related paper appeared describing work based on a similar approach (30Park J.-B. Exp. Mol. Med. 1996; 28: 147-152Crossref Scopus (1) Google Scholar). CaM was obtained from Calbiochem and freshly dissolved in 50 mm HEPES, pH 7.4, for each experiment. CaCl2, Suprapur grade, was from EM Science. BS3was obtained from Pierce. Rab3A peptides Lys62-Arg85, Ala2-Asn18, and Glu177-Asp195 (Table I) and the Rab GDI peptide, Gly21-Ser45(GIMSVNGKKVLHMDRNPYYGGESSS), were synthesized by the University of Washington Biopolymer Facility. The CaM kinase II peptide Leu290-Ala309 (Table I) was from LC Laboratories. Stock solutions of peptides were prepared in Me2SO and then added to incubation mixtures at final Me2SO concentrations of <5%. GDP, GTPγS, and unprenylated Rab3A were from Calbiochem. Rab GDI was purified from bovine brain as described (29Sasaki T. Kikuchi A. Araki S. Hata Y. Isomura M. Kuroda S. Takai Y. J. Biol. Chem. 1990; 265: 2333-2337Abstract Full Text PDF PubMed Google Scholar), except that all buffers used after the ammonium sulfate precipitation step contained 10% glycerol, 0.25 mm phenylmethylsulfonyl fluoride, 2.5 μg/ml each of aprotinin and leupeptin, and 1 μg/ml pepstatin A. All other purchased chemicals were reagent grade from Sigma, and all procedures were performed at 4 °C unless otherwise indicated.Figure 5Effects of Rab3A and CaM kinase II peptides on the Ca2+/CaM- or Rab GDI-induced dissociation of Rab3A from REM. A, aliquots of REM (15 μg of protein) were incubated for 30 min at 30 °C in 50 μl of buffer B containing 100 μm CaCl2, 50 μm CaM, and 100 μm concentrations of a Rab3A peptide (Lys62-Arg85, Ala2-Asn18, or Glu177-Asp195) or the CaM kinase II peptide, Leu290-Ala309. The medium was then recovered and analyzed as described in Fig. 1. Following Western analysis of supernatant and pellet fractions and densitometry of the Rab3A containing band, the absorbance value obtained for each supernatant fraction was expressed as a percentage of the total Rab3A recovered in each sample. Error bars represent standard errors of mean values from at least two experiments; * denotes standard errors of 95% of the antibody-reactive material, was found. In the case of Rab GDI, 100 μg of nonhuman primate brain cytosol (9200 ×g av × 20 min supernatant as described (33Huttner W.B. Schieber W. Greengard P. DeCamilli P. J. Cell Biol. 1983; 96: 1374-1388Crossref PubMed Scopus (879) Google Scholar)) were similarly analyzed and found to contain two adjacent proteins (representing >90% of the antibody-reactive material) that most likely represented two charged isoforms of Rab GDI (39Steele-Mortimer O. Gruenberg J. Clague M.J. FEBS Lett. 1993; 329: 313-318Crossref PubMed Scopus (44) Google Scholar). The apparent molecular mass of the Rab3A·CaM complex was determined by sucrose gradient centrifugation (27Araki S. Kikuchi A. Hata Y. Isomura M. Takai Y. J. Biol. Chem. 1990; 265: 13007-13015Abstract Full Text PDF PubMed Google Scholar) modified as follows. Supernatants from Rab3A dissociation experiments with 90 μg of REM, 100 μmCaCl2, and 50 μm CaM in 250 μl of buffer B were overlaid onto a 5-ml, 5–25% continuous sucrose gradient containing 5 mm MgCl2, 1 mm DTT, 0.1 mm CaCl2, 50 mm HEPES, pH 7.4, and protease inhibitors as described for buffer B. After centrifugation for 7 h at 173,000 × g av in a Beckman SW50.1 rotor, fractions were analyzed for Rab3A with an immuno-dot blot assay. The high speed supernatant fraction from synaptosomal lysates (1 mg/250 μl) was similarly analyzed. For cross-linking experiments, REM (15–25 μg of protein) were washed twice by centrifugation in 50 mm HEPES, pH 7.4, containing 100 mm NaCl, 1 mm MgCl2, 1 mm DTT, and protease inhibitors as described for buffer B. The washed membranes were mixed with Ca2+/CaM in 25 μl of buffer B and incubated for 30 min at 30 °C as described in the figure legends. The incubation mixtures were centrifuged for 30 min at 100,000 ×g av; the supernatants were treated for 30 min at 30 °C with freshly prepared 1 mm BS3 (40Giedroc D.P. Keravis T.M. Staros J.V. Ling N. Wells J.N. Puett D. Biochemistry. 1985; 24: 1203-1211Crossref PubMed Scopus (26) Google Scholar), and the reactions were quenched with Tris buffer and analyzed as described above (41Taniuchi M. Clark H.B. Johnson Jr., E.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4094-4098Crossref PubMed Scopus (556) Google Scholar). A similar BS3 treatment procedure was used in all other cross-linking experiments. CaM (225 μg) in 50 μl of 50 mm HEPES buffer, pH 7.6, was modified by reaction with 0.5 mCi of 125I-labeled Bolton-Hunter reagent (2200 Ci/mmol; NEN Life Science Products) according to the manufacturer's directions. The125I-labeled CaM was then separated from unreacted reagent with the use of a Bio-Gel P-6 DG (Bio-Rad) spin column as described (42Yokoyama K. McGeady P. Gelb M.H. Biochemistry. 1995; 34: 1344-1354Crossref PubMed Scopus (113) Google Scholar). After centrifugation, a mixture of 50 μl of125I-labeled CaM (10 nmol) and 50 μl of REM (450 μg) was incubated for 45 min at 37 °C in the presence of 100 μm CaCl2 and 0.1 mm GDP. After incubation the mixture was centrifuged for 30 min at 100,000 ×g av, half of the supernatant was treated with BS3, and half was reserved as control. An aliquot of each half (50 μl) was then immunoprecipitated with 50 μl of anti-Rab3A IgG (1 μg) for 24 h at 0 °C. After immunoprecipitation, 40 μl of immobilized protein A on Trisacryl beads (Pierce; 50% slurry), which had been pretreated with bovine serum albumin, were added, and the sample was mixed for 20 h at 4 °C in a tube rotator. The protein A beads were then washed four times by a procedure that involved suspension in 50 mm HEPES, pH 7.4, containing 100 mm NaCl, 0.1 mm CaCl2, 0.1% Tween 20, 0.1 mm DTT, and 1 μg/ml each of leupeptin and aprotinin, followed by centrifugation for 4 min at 350 ×g av. The washed beads were boiled for 4 min in 30 μl of 2% SDS and prepared for SDS-PAGE analysis (described below). To generate Rab3A-depleted membranes, REM (30 μg) were incubated for 30 min at 30 °C with 1.6 μm Rab GDI in 50 μl of buffer B (but without Ca2+), pelleted by centrifugation for 30 min at 100,000 × g av, and suspended in 5 μl of buffer B (but without Ca2+). To study the transfer of Rab3A to these membranes, they were mixed with medium containing Rab3A·Ca2+/CaM complex (prepared by incubating REM with buffer containing 75 μm CaM and 100 μmCaCl2) and incubated for 30 min at 30 °C in the presence or absence of one of the peptides listed in Table I. Then the incubation mixtures were subfractionated by centrifugation and analyzed as described above. The ability of synaptosomes to secrete glutamate was measured after KCl-induced depolarization (24Fisher von Mollard G.F. Südhof T.C. Jahn R. Nature. 1991; 349: 79-81Crossref PubMed Scopus (330) Google Scholar) or treatment with 4-aminopyridine + 4β-phorbol dibutyrate (31Barrie A.P. Nicholls D.G. Sanchez-Prieto R. Sihra T.S. J. Neurochem. 1991; 57: 1398-1404Crossref PubMed Scopus (151) Google Scholar). A modification of the method previously described (24Fisher von Mollard G.F. Südhof T.C. Jahn R. Nature. 1991; 349: 79-81Crossref PubMed Scopus (330) Google Scholar) was used. Briefly, control and depolarized samples were incubated for 10 min at 37 °C and then placed in ice water for 2 min and centrifuged for 2 min at 13,000 × g av. The amount of NADPH that had been produced was determined by measuring the absorbance of the supernatant at 360 nm on a Beckman DU 640 spectrophotometer using 390 nm as the reference wavelength. SDS-PAGE was performed as described by Laemmli (43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar) but with 14% gels. In some cases, EGTA was added to the samples just before they were boiled. Proteins were transferred from SDS-PAGE gels to Immobilon P membranes for 30 min at 80 V for Western analysis of Rab3A or transferred for 60 min to identify cross-linked products of higher molecular mass. Immunoblots were performed as described (35Aepfelbacher M. Vauti F. Weber P.C. Glomset J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4263-4267Crossref PubMed Scopus (44) Google Scholar). Protein concentrations were determined with the Bradford method (44Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211946) Google Scholar) (Bio-Rad) or, for SDS-containing samples, the micro-BCA method (Pierce). Free calcium ion concentrations were varied in the presence of 1 mm EGTA, on the basis of established binding constants (45Martell A.E. Smith R.M. Critical Stability Constants. 1. Plenum Press, New York1974: 269Google Scholar). The initial aim of this investigation was to determine whether Ca2+ and CaM influence the dissociation of Rab3A from synaptic membranes. To examine this possibility, we isolated synaptosomes from samples of macaque cerebral cortex, lysed the synaptosomes in hypotonic medium, and prepared Rab3A-enriched membranes (REM) from the lysates by ultracentrifugation. Then we suspended the REM in medium containing Ca2+ and/or various other additives, incubated the mixtures for 30 min at 30 °C, separated the membranes from the medium by centrifugation, and separately measured the amounts of Rab3A recovered in the membrane and supernatant fractions. The results of these experiments demonstrated that medium containing both Ca2+ and CaM, i.e. a Ca2+/CaM complex, caused Rab3A to dissociate from the membranes but that medium containing either 100 μmCa2+ or 60 μm CaM alone did not (Fig.1). The dissociation of Rab3A occurred in the absence of added ATP, and half-maximal effects were observed when the concentrations of Ca2+ and CaM were about 0.5 and 20 μm, respectively. Maximal dissociation of Rab3A (approximately 65%) was obtained when the concentrations of Ca2+ and CaM were about 10 and 65 μm, respectively (data not shown). To
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