Ca2+/Calmodulin Causes Rab3A to Dissociate from Synaptic Membranes

Abstract

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 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 of causes a in the of both Rab3A and a guanine nucleotide-binding in synaptic vesicles von Mollard G.F. Südhof T.C. Jahn R. Nature. 1991; PubMed Scopus Google and von Mollard G.F. Stahl B. A. Südhof T.C. Jahn R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google but K. Biophys. 1993; PubMed Scopus Google for a Furthermore, of a Rab guanine nucleotide dissociation inhibitor protein had been of to a complex with Rab3A and cause it to dissociate from synaptic membranes in S. A. Y. M. Takai Y. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar, T. M. Takai Y. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, T. A. S. Y. M. S. Takai Y. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar). the that increased concentrations of Ca2+ the Rab3A dissociation we that Ca2+/CaM also can cause Rab3A to dissociate from synaptic of the of this effect and to that of Rab are this being in to a on a Mol. Scopus Google Scholar). this being in to a on a Mol. Scopus Google Scholar). CaM from and in for each from Science. from Rab3A and and the Rab by the of The CaM kinase II peptide from of in and to at concentrations of GTPγS, and Rab3A from Rab from as T. A. S. Y. M. S. Takai Y. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar), that all used the each of and and A. from and all at of Rab3A and CaM kinase II on the Rab dissociation of Rab3A from of of for at in of CaM, and concentrations of a Rab3A peptide the CaM kinase II The and as in of and and of the Rab3A the for each as a of the Rab3A in each of from at of of of for at in of that Rab and a of the peptide but The and as as as in A. are causes Rab3A to dissociate from synaptic of of in of for at with of CaM, and as the for at and the and to and with The Rab3A from and are for and CaM, and and CaM and that to the Ca2+/CaM-dependent dissociation of Rab3A from in this but that had effect in of on the of for at in of and CaM and for at to a The for at in the presence of the concentrations of with BS3, and by and molecular of proteins are is of of used in CaM binding kinase peptide sequence to are The to the peptide abbreviations used the and that the Rab3A and with to in a The peptide sequence to are The to the peptide abbreviations used the and that the Rab3A and with to used to from of from the of the at the of In of in each of and and a with of with and a a is at and on a a of the of von Mollard von Mollard G.F. Südhof T.C. Jahn R. Nature. 1991; PubMed Scopus Google and R. J. 1991; PubMed Scopus Google Scholar). In to be used for from of and The with a in with and in a of a Biochem. J. PubMed Scopus Google Scholar). a as von Mollard G.F. Südhof T.C. Jahn R. Nature. 1991; PubMed Scopus Google Scholar). and to a plasma a synaptic vesicle and a corresponding as P. P. J. Cell Biol. PubMed Scopus Google that to all The synaptic vesicle which Rab3A-enriched membranes in of and and at and The with a and and as of for at with in of DTT, and to the The for at in a The in of with of Rab3A, of each and and examined by and with the of of membranes and of used as of and and of and the of membranes used as and of The with a and the for the Rab protein to of of Rab proteins on the basis of the that The of each Rab protein from the as a of the Rab protein in the and shown in the In of by Rab3A, the number of by each is in the and dissociation and corresponding to of the sequence of Rab3A A. N. P. A. J. Biol. Chem. Full Text PDF PubMed Google as M. F. Sci. S. A. 1994; PubMed Scopus Google that a the peptide to to of the used for However, for in with Rab3A peptide a of a that had been Rab3A peptide The and each used at a also corresponding to of the sequence of Rab Y. A. S. Y. J. Y. Takai Y. Mol. Cell. Biol. PubMed Scopus Google Scholar). of this used for all by J. Biol. Chem. Full Text PDF PubMed Google Scholar, H. P. K. T. P. Biophys. 1994; PubMed Scopus Google followed by M. F. Sci. S. A. 1994; PubMed Scopus Google Scholar). In the of Rab3A, of of synaptic vesicle protein P. P. J. Cell Biol. PubMed Scopus Google and only of the In the of Rab GDI, of as P. P. J. Cell Biol. PubMed Scopus Google and to proteins of the that isoforms of Rab J. 1993; PubMed Scopus Google Scholar). The molecular of the complex by S. A. Y. M. Takai Y. J. Biol. 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J. 1991; PubMed Scopus Google Scholar). of the von Mollard G.F. Südhof T.C. Jahn R. Nature. 1991; PubMed Scopus Google and for at and in for and for at The of that had been by the of the at on a as the as by Nature. PubMed Scopus Google but with In to the Proteins from to membranes for at for of Rab3A for to of molecular as M. F. Sci. S. A. 1994; PubMed Scopus Google Scholar). Protein concentrations with the Biochem. PubMed Scopus Google for the concentrations in the presence of on the basis of binding Scholar). The of this investigation to Ca2+ and CaM the dissociation of Rab3A from synaptic this we from of the in and Rab3A-enriched membranes from the by we the in Ca2+ the for at the membranes from the by and the amounts of Rab3A in the and The of these that both Ca2+ and CaM, a Ca2+/CaM Rab3A to dissociate from the membranes but that CaM not The dissociation of Rab3A in the of and effects the concentrations of Ca2+ and CaM and dissociation of Rab3A the concentrations of Ca2+ and CaM and not the of the effect of we in the presence of that Ca2+/CaM and the and it by the of Rab3A in the we a of that had molecular of be from the of in with a from a The from the had a molecular and probably to a complex of Rab3A and Rab T. M. Takai Y. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). In subsequent we with the and the BS3, to we the by and with to Rab3A. In with the the the presence of that had molecular of The of these that Ca2+/CaM a 1:1 complex with Rab3A. additional this we with Ca2+/CaM that CaM, the and to we it by and by The that had molecular of and for the presence of a 1:1 complex of Rab3A and Ca2+/CaM is to complexes with many and to be Ca2+ to CaM, it a in CaM that binding sites for both and H. Annu. Rev. 1994; PubMed Scopus Google Scholar, P. T. Trends Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar, Science. 1993; PubMed Scopus Google Scholar). that the 1:1 complex of Rab3A and CaM we with Ca2+ and CaM, the and of it in the presence of concentrations of we the with and the by The that of the in the presence of the of the complex molecular and with the of the complex that be The binding of Ca2+ to CaM is to also and and both for the of a Rab3A can a complex with of the for at in of CaM and as and with by and protein with to all to to of the D. P. and Cell Scholar). in Rab3A peptide Lys62-Arg85 in of and CaM and as in that to each of the in and of to are the of in the we used we with the and of it in the presence of the we to the and the by with the complex not In the we Rab3A with BS3, and the The that complex of Rab3A with Ca2+/CaM be and that the lipidated C terminus of Rab3A is required for binding to the many proteins that complexes with Ca2+/CaM is CaM kinase II T. N. Science. PubMed Scopus Google Scholar). Furthermore, a and binding for Ca2+/CaM on this has been T. N. Science. PubMed Scopus Google Scholar, M.E. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Rab3A also contains a sequence that is in and the that the Rab3A Lys62-Arg85 sequence include a binding for we a peptide that to it and the effects of this peptide with of synthetic in the we with Ca2+/CaM in the presence of each peptide and the of Rab3A that to the The that a of the Rab3A Lys62-Arg85 peptide of a peptide corresponding to the CaM kinase II sequence the effect of Ca2+/CaM at concentrations of and In concentrations of that to the Rab3A terminus C terminus had with we the and of it with the Rab3A Lys62-Arg85 the CaM kinase II the Rab3A each and the of the Rab3A Lys62-Arg85 peptide and the CaM kinase II peptide the of complex that be but the Rab3A peptide had effect we the Rab3A with CaM in the presence of Ca2+ BS3, and the by The that the Lys62-Arg85 peptide a Ca2+-dependent complex with CaM but the Rab3A not the of these that Ca2+/CaM the dissociation of Rab3A from synaptic membranes by binding to the Rab3A Lys62-Arg85 shown that the Rab3A Lys62-Arg85 and CaM kinase II disrupt we examined the that of the complexes promote the of Rab3A to We this by with Ca2+/CaM to a the and of it with the of the in the presence of synaptic the we the membranes from the by and the of Rab3A in the and The that each peptide cause Rab3A to from the to the a peptide had effect Rab can a 1:1 complex with Rab3A and cause it to dissociate from synaptic membranes (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, von Mollard G.F. Südhof T.C. Jahn R. Nature. 1991; PubMed Scopus Google Scholar). the between this effect and that of we to the proteins bind to sites on Rab3A. In of we with Ca2+/CaM to a complex used the from a as a of we the complex for at with Rab the with the we the with the The of these that Ca2+/CaM and Rab with each for binding to Rab3A. In a of we with Rab in the presence of the Rab3A Lys62-Arg85 the CaM kinase II the Rab3A the Rab3A we the and the of Rab3A in the by The Rab3A Lys62-Arg85 and CaM kinase II the Rab dissociation of Rab3A from at concentrations of and In the Rab3A and In a of we with Rab GDI, the and it for at in the presence of the Rab3A Lys62-Arg85 peptide the Rab3A we to the and the The Rab3A Lys62-Arg85 peptide the of complex but the Rab3A peptide The between these and in the corresponding with Ca2+/CaM and with that Rab and Ca2+/CaM with binding sites the Rab3A Lys62-Arg85 The Rab dissociation of Rab3A from membranes is to be the of guanine S. A. Y. M. Takai Y. J. Biol. Chem. 265: Full Text PDF PubMed Google Scholar). guanine also the dissociation of Rab3A, we for at in the of guanine the with Ca2+/CaM Rab GDI, the of Rab3A that from the to the a to Ca2+/CaM Rab be with In Rab3A from that had been in the presence of GDP being with Ca2+/CaM Rab GDI, and GDP that Rab had a stringent requirement for GDP Ca2+/CaM The of these guanine effects remains to be determined. However, the of is to from that of C. A. 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Furthermore, it that Rab3A that contains bound GDP GTP has a guanine Rab3A and that this can be by The of Ca2+/CaM with Rab3A that of Rab with Rab3A in several proteins 1:1 complexes with Rab3A and cause it to dissociate from synaptic of each of the complexes requires both the lipidated C terminus of Rab3A and the presence of guanine Ca2+/CaM and Rab bind to sites the Rab3A Lys62-Arg85 The of Ca2+/CaM and Rab with Rab3A also in several the of Ca2+/CaM with Rab3A on In addition, effects of Ca2+/CaM on the dissociation of Rab3A from the dissociation of Rab3A from at concentrations of Ca2+/CaM that are the concentrations of Rab required to dissociate Rab3A from not The Ca2+/CaM-dependent dissociation of Rab3A from is less the Rab dissociation of Rab3A. the effect of Ca2+/CaM has a less stringent requirement for GDP that of Rab The of the effect of Ca2+/CaM remains to be but it is of that the Rab3A Lys62-Arg85 peptide contains a of are Furthermore, a of the sequence that the may be on of The CaM kinase II peptide has and P. 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