Modifications in the C Terminus of the Synaptosome-associated Protein of 25 kDa (SNAP-25) and in the Complementary Region of Synaptobrevin Affect the Final Steps of Exocytosis (2002)

Fusion proteins made of green fluorescent protein coupled to SNAP-25 or synaptobrevin were overexpressed in bovine chromaffin cells in order to study the role of critical protein domains in exocytosis. Point mutations in the C-terminal domain of SNAP-25 (K201E and L203E) produced a marked inhibition of secretion, whereas single (Q174K, Q53K) and double mutants (Q174K/Q53K) of amino acids from the so-called zero layer only produced a moderate alteration in secretion. The importance of the SNAP-25 C-terminal domain in exocytosis was also confirmed by the similar effect on secretion of mutations in analogous residues of synaptobrevin (A82D, L84E). The effects on the initial rate and magnitude of secretion correlated with the alteration of single vesicle fusion kinetics since the amperometric spikes from cells expressing SNAP-25 L203E and K201E and synaptobrevin A82D and L84E mutants had lower amplitudes and larger half-width values than the ones from controls, suggesting slower neurotransmitter release kinetics than that found in cells expressing the wild-type proteins or zero layer mutants of SNAP-25. We conclude that a small domain of the SNAP-25 C terminus and its counterpart in synaptobrevin play an essential role in the final membrane fusion step of exocytosis. Fusion proteins made of green fluorescent protein coupled to SNAP-25 or synaptobrevin were overexpressed in bovine chromaffin cells in order to study the role of critical protein domains in exocytosis. Point mutations in the C-terminal domain of SNAP-25 (K201E and L203E) produced a marked inhibition of secretion, whereas single (Q174K, Q53K) and double mutants (Q174K/Q53K) of amino acids from the so-called zero layer only produced a moderate alteration in secretion. The importance of the SNAP-25 C-terminal domain in exocytosis was also confirmed by the similar effect on secretion of mutations in analogous residues of synaptobrevin (A82D, L84E). The effects on the initial rate and magnitude of secretion correlated with the alteration of single vesicle fusion kinetics since the amperometric spikes from cells expressing SNAP-25 L203E and K201E and synaptobrevin A82D and L84E mutants had lower amplitudes and larger half-width values than the ones from controls, suggesting slower neurotransmitter release kinetics than that found in cells expressing the wild-type proteins or zero layer mutants of SNAP-25. We conclude that a small domain of the SNAP-25 C terminus and its counterpart in synaptobrevin play an essential role in the final membrane fusion step of exocytosis. soluble NSF attachment protein N-ethylmaleimide-sensitive fusion protein soluble NSF attachment protein receptor green fluorescent protein Herpes simplex virus synaptobrevin The SNAP1 (soluble NSF attachment protein) receptor (SNARE) hypothesis (1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2011) Google Scholar) has been crucial to our understanding of the molecular machinery responsible for exocytosis. The molecular events taking place during the exocytotic fusion of cellular and vesicular membranes should provide fundamental information about the mechanism of membrane fusion common to different membrane trafficking processes (2Bennett M. Scheller R. Proc. Natl. Acad. Sci. U. S. A. 1993; 0: 2559-2563Crossref Scopus (548) Google Scholar). The assembly of a ternary complex formed by the plasma membrane proteins syntaxin (3Bennett M.K. Calakos N. Scheller R.H. Science. 1992; 257: 255-259Crossref PubMed Scopus (1077) Google Scholar) and SNAP-25 (4Oyler G.A. Higgins G.A. Hart R.A. Battenberg E. Billigsley M. Bloom F.E. Wilson M.C. J. Cell Biol. 1989; 109: 3039-3052Crossref PubMed Scopus (690) Google Scholar) and the vesicle-associated protein synaptobrevin (5Trimble W.S. Cowan D.M. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 5: 4538-4542Crossref Scopus (451) Google Scholar) is considered to be one of the molecular events driving vesicle priming, involving maturation steps needed to promote the apposition and final fusion of membranes (6Südhof T.C. Nature. 1995; 75: 645-653Crossref Scopus (1770) Google Scholar). A heptad repeat structural motif typical of coiled-coils forming proteins is present and could be the basis for the formation of the core complex. This was confirmed by the observed increase in α-helix content following assembly of the complex (7Fasshauer D. Otto H. Eliason W.K. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 8Rice L.M. Brennwald P. Brunger A.T. FEBS Lett. 1997; 415: 49-55Crossref PubMed Scopus (60) Google Scholar). In recent years, precise structural studies on the nature of the complex (9Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar, 10Poirier M.A. Xiao W. Macosko J.C. Chan C. Shin Y.K. Bennett M.K. Nat. Struct. Biol. 1998; 5: 765-769Crossref PubMed Scopus (419) Google Scholar) have shown that single fragments of 60–70 residues of syntaxin and synaptobrevin together with two segments of SNAP-25 form helices in a four-stranded coil. Although most of the interactions between these helices are hydrophobic, there is a polar layer embedded in the middle of this rod-shaped structure formed by three glutamines and one arginine (zero layer), which is believed to be critical for SNARE complex formation. In addition, functional studies using specific neurotoxins (11Montecucco C. Schiavo G. Mol. Microbiol. 1994; 13: 1-8Crossref PubMed Scopus (496) Google Scholar), antibodies against critical domains (12Xu T. Rammer B. Margittal M. Artalejo A. Neher E. Jahn R. Cell. 1999; 99: 713-722Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), peptides imitating regions of SNAREs (13Gutiérrez L.M. Viniegra S. Rueda J. Ferrer-Montiel A.-V. Cànaves J. Montal M. J. Biol. Chem. 1997; 272: 2634-2639Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and overexpression of altered forms of these proteins (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar, 15Wei S. Xu T. Ashery U. Kollewe A. Matti U. Antonin W. Rettig J. Neher E. EMBO J. 2000; 19: 1279-1289Crossref PubMed Google Scholar) have revealed important details about the participation of SNAREs in the exocytotic process. These and other studies suggest that SNARE interaction results in a tighter structure reducing the repulsive energy barrier between vesicular and plasma membranes. Assembly of the complex may proceed from the distal N-terminal domains of SNAREs assembled in parallel and, in “zipper-like” fashion, end with the interaction of C-terminal domains relatively close to the fusion pore. Even though there is not direct evidence for this model, a number of authors suggest that SNAREs assembly is directly linked to membrane fusion (16Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar, 17Chen Y.A. Scales S.J. Patel S.J. Doung Y.C. Scheller R.H. Cell. 1999; 97: 165-174Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). We have overexpressed several mutants of SNAP-25 and synaptobrevin critical domains in adrenal chromaffin cells and studied their effect on catecholamine secretion. Our results indicate that specific residues at the SNAP-25 C-terminal domain and the equivalent synaptobrevin domain influence vesicle fusion kinetics, suggesting a role in the very last events of the exocytotic process. By contrast, zero layer amino acid mutants, despite their ability to negatively affect the formation and stability of SNAREs complexes assembled in vitro, did not produce strong alterations. To produce an in-frame fusion of SNAP-25 to the C terminus of GFP, the cDNA corresponding to the SNAP-25a isoform (18Bark I.C. Wilson M.C. Gene. 1994; 139: 291-292Crossref PubMed Scopus (131) Google Scholar) was cloned into the expression vector pEGFP-C3 (CLONTECH, Palo Alto, CA) as previously described (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar). The generation of the nine amino acids C-terminal deletion (Δ9) and the point mutation L203E has also been described (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar). The strategy for generating the other C-terminal mutants was based in the presence of a TaiI site at the 5′-end of the region containing the nucleotides to be mutated and a BamHI site at the 3′-end. Complementary oligonucleotides carrying the desired mutations and the mentioned restriction sites were annealed and cloned into the corresponding construct in place of the original sequence. The point mutation Q53K was generated by PCR of a fragment corresponding to the N terminus of SNAP-25 using the GFP-SNAP-25 construct as template, a sense primer (5′-CGGCATGGACGAGCTGTACA-3′) corresponding to the C terminus of GFP and close to the junction between GFP and SNAP-25 and an antisense primer, which included the sequence to be mutated (indicated in lower characters) The PCR was with and and to the equivalent fragment into the GFP-SNAP-25 which had been with the The point mutation was generated by PCR of a fragment corresponding to the C terminus of SNAP-25 with a sense primer containing the sequence to be mutated and an antisense primer of the C terminus of SNAP-25 The an close to the and a BamHI site at the 3′-end. These were to the original SNAP-25 sequence by the one in the GFP-SNAP-25 The double was by the single ones restriction and To produce an in-frame fusion of synaptobrevin to the N terminus of GFP, the region corresponding to synaptobrevin T. Jahn R. U. T.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar) was by PCR with the following and The PCR was with and BamHI and cloned into the sites of the expression vector (CLONTECH, Palo Alto, The strategy for generating the synaptobrevin mutants A82D and was based in the presence of an site at the 5′-end of the region containing the nucleotides to be mutated and the previously site at the 3′-end. sense carrying the desired mutations and the site as as the antisense primer with the BamHI site were in PCR The corresponding were to the original synaptobrevin sequence. the were to that only the desired mutations have been cells were from bovine adrenal by and from and by on as described (13Gutiérrez L.M. Viniegra S. Rueda J. Ferrer-Montiel A.-V. Cànaves J. Montal M. J. Biol. Chem. 1997; 272: 2634-2639Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). were in using with and were at a of in of chromaffin cells were with a Herpes virus containing the described this were from the pEGFP-C3 or to the vector A. R. Proc. Natl. Acad. Sci. 1993; PubMed Scopus Google Scholar). The of the different deletion was as previously described F. D. P. P. S. PubMed Scopus Google Scholar). of virus was by fluorescent using of The virus containing of was in GFP was observed and at during the two following were during this To study from and cells expressing the different was by with the following acid and and the was to using a with and with were to catecholamine release from chromaffin in cells A. Viniegra S. Gutiérrez L.M. J. 1998; PubMed Scopus Google Scholar). were in close apposition to the using and membrane with an was with amperometric of an was using an of the catecholamine was with an and at using the J. for and of Scholar) on of the in a were in cells by with by by and a by the and the studied were studied using F. M.A. D. R. J. 2000; PubMed Scopus Google Scholar) by for and was at a of using an and at with than and the at the lower than were to to were by using the for in and cells was with were and in and included in cells in the for were for the number of cells in D. J. 2000; PubMed Scopus Google Scholar). The of in the was for SNAP-25 and synaptobrevin and with the values for the different was as from in a number of with cells from at three different cells were using a in during cells were with and in in the was using a the were and or were to study fusion proteins and synaptobrevin were produced by their in the vector J.E. PubMed Scopus Google Scholar). The different SNAP-25 and synaptobrevin mutants were to the site present in the initial of SNAP-25 or using an site the region of the in the proteins were from expressing for SNAP-25 and synaptobrevin and for expression with during at The were to of in and the proteins were by with by of this protein with of ternary complexes by the proteins at a and was in and in between and were and complex formation was by an of in was by forming complexes during in the described and during at from to of was by the to complexes were by of and forms of SNAP-25 in chromaffin cells is a its role in (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar, 15Wei S. Xu T. Ashery U. Kollewe A. Matti U. Antonin W. Rettig J. Neher E. EMBO J. 2000; 19: 1279-1289Crossref PubMed Google Scholar). In initial studies and in of than We to N. Cell. Full Text PDF Scopus Google Scholar). produced a of cells expressing of GFP-SNAP-25 as by their green We single to the ability of these cells to in to a spikes single fusion events were for a number of cells The was by the and of from to cells cells the GFP-SNAP-25 construct and cells expressing GFP A and produced about of the found in cells The close of GFP and GFP-SNAP-25 that kinetics were by expression of the GFP-SNAP-25 construct and that the in the number of observed in cells was to the than to a inhibition of the machinery by overexpression of SNAP-25. of the for SNAP-25 was confirmed by the catecholamine release in cells a GFP-SNAP-25 construct the mutation of to acid that had previously been shown to affect secretion kinetics (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar). The expression of this construct the of the A a in the with the GFP-SNAP-25 and initial rate of release about of the rate found for the GFP-SNAP-25 These results are with by (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar). results were observed using a construct the last nine C-terminal residues of this SNARE and equivalent to the form produced by the of A J. E. T. S. P. T.C. H. Jahn R. Nature. 1993; PubMed Scopus Google Scholar, G. A. J. F. Wilson M.C. C. FEBS Lett. 1993; PubMed Scopus Google Scholar). These results that the amino acid which is in this construct as as in the SNAP-25 by is critical for exocytosis. The was to the of cells expressing SNAP-25 proteins mutated at amino acids in critical has been that amino acid residues in the zero layer and of are critical for SNARE complex formation (9Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar, D. R.B. Brunger A.T. Jahn R. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The single mutation in a relatively small effect on secretion with the L203E magnitude and initial rate of catecholamine release were in to A effect was also observed with the Q53K mutation present in the zero layer forming of the SNAP-25 N-terminal which during not mutations in the previously mentioned glutamines produced lower inhibition in and initial rate of secretion, than that with Although these a role for the zero layer in secretion, to be important than the by at the SNAP-25 C To the importance of regions in molecular events to membrane fusion studied the role of residues at the SNAP-25 C-terminal domain in this process. To this end mutated and which have to the and of the in the fusion complex D. R.B. Brunger A.T. Jahn R. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), as as which is in between these The of cells expressing mutants and was with only the initial rate of vesicle release slower in the K201E had a effect on the number of during and the initial rate of vesicle release was also These results suggest that amino acids in the C-terminal domain of as and to a play a role in membrane fusion events to exocytosis and that interactions could also be in this process. structural suggest that several synaptobrevin amino acids as and could with of SNAP-25. We to these synaptobrevin residues in GFP linked to the synaptobrevin C-terminal which were also overexpressed following of bovine chromaffin cells with wild-type synaptobrevin as as the A82D and L84E mutants, were at similar in a suggesting their in chromaffin The of cells expressing these was studied using expressing wild-type synaptobrevin coupled to GFP had an exocytotic that observed in cells expressing GFP and the GFP construct coupled to wild-type SNAP-25 in that the kinetics the of secretion of was of had a relatively on the release of which a inhibition in and initial rate of release A82D and L84E had a effect on close to inhibition similar to that observed in the L203E of SNAP-25. These the that these synaptobrevin and SNAP-25 residues have a marked and influence on secretion in chromaffin cells different was by as described in of for the secretion was from and L84E The study of fusion kinetics could be in the the effects of SNAP-25 and synaptobrevin This is by the of single amperometric events R. C. J. Full Text PDF PubMed Scopus Google Scholar) in the of the A was using F. M.A. D. R. J. 2000; PubMed Scopus Google Scholar) that and and spikes with amplitudes and half-width values lower than The were to for from in which were for and similar to the ones shown for the in and in cells and cells expressing GFP-SNAP-25 or were by very similar from amplitudes of and were also similar in cells expressing single (Q174K, and and double mutants of the zero layer as as and mutants of the SNAP-25 C terminus the amplitudes with L203E and and K201E mutants were to of the amplitudes which were relatively in cells were in these These results are in with for C-terminal in chromaffin cells (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar). the other the alteration in was not with a in the which relatively in zero and C terminus mutants with values from to and By contrast, in the were observed for K201E and mutants of with values for wild-type SNAP-25 and and The half-width of wild-type SNAP-25 as as mutants and had a of events from to and values whereas cells expressing and mutants had and events the presence of spikes corresponding to with half-width values The amperometric events from synaptobrevin were also In this cells expressing A82D were by lower and half-width values The with to the were than observed with the SNAP-25 The effect was with the L84E despite a marked in secretion The from the wild-type construct from the of spikes from and mutants of SNAP-25 and synaptobrevin cells (Q174K/Q53K) (Δ9) using the of with the values and and using the of with the values and and values are as the from in a number of In the SNAP-25 and and using the of with the values and and in a The values are as the from in a number of In the SNAP-25 and and In only amino acids of SNAP-25 and in close to play a role in the membrane fusion events the release of The previously described forms of SNAP-25 and synaptobrevin together with syntaxin were to study the in stability of their ternary complexes in an to the molecular basis for the observed in exocytosis. complexes were formed using synaptobrevin the membrane and the different SNAP-25 mutants from the ability to form the complex at studies that the forms the complex formation with the wild-type not formed complexes were from to at of protein produced in the of complex in the of these complexes were also complexes to at complexes formed with the altered forms of SNAP-25 at lower to the of for complexes and and for the SNAP-25 and This indicate that mutations different effects on the of chromaffin as L203E and have a similar effect on complex formation in the Q53K mutation in the N terminus of had a of a direct between the ability to exocytotic and the in formation of SNARE complexes not to the of the complexes formed by A82D and L84E synaptobrevin mutants were by lower with complexes formed by wild-type synaptobrevin despite the found in their fusion kinetics the of the ternary complexes formed by these synaptobrevin mutants is very stability of the complexes formed with synaptobrevin were as described in the using the different synaptobrevin of ternary complex corresponding to synaptobrevin and to the mutants A82D and in the described in A for corresponding to three were as of the of formed complex and as The of has been crucial to the role by the SNAP-25 C terminus in the of chromaffin and cells A. Viniegra S. Gutiérrez L.M. J. 1998; PubMed Scopus Google Scholar, U. J. 1994; PubMed Scopus Google Scholar, A. J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. N. 1997; PubMed Scopus Google Scholar, T. T. H. Neher E. 1998; PubMed Scopus Google Scholar). the of containing in this domain (14Criado M. Gil A. Viniegra S. Gutiérrez L.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7256-7261Crossref PubMed Scopus (83) Google Scholar) has to protein segments different from by the of SNAP-25 by an overexpressed in which the last C-terminal amino acids are the single vesicle fusion kinetics by R. J. Full Text PDF PubMed Scopus Google Scholar), suggesting the of these residues in the very last events of exocytosis. The on our and into the exocytotic role of single amino acids from critical domains of SNAP-25 and Our was to the of these domains by these and with the of the SNAP-25 double mutants were to form complexes in vitro, and is to that also in as is by their effects on exocytosis. These effects were not and only mutants exocytosis in a present using mutants, is to the of the wild-type protein in the the mutated forms of SNAP-25 are is to that the role of the the that proteins may with mutated forms during exocytosis be this were the could one of two the protein could its of the of this not to be the since a effect is observed only with the expression of specific mutants could influence the protein the fusion and amperometric This complex between the and the proteins that be to be In our strategy of several mutants in the region and the of their effects on exocytosis the following the functional amperometric that L203E and K201E mutants as as the deletion of the SNAP-25 C terminus a strong inhibition of secretion and which is correlated to the altered kinetics of catecholamine release from These mutants, with expressing GFP-SNAP-25 or were by and amperometric slower single vesicle By contrast, the effect of single and Q53K) and double mutants of the zero layer as as the other C-terminal on secretion was and and the of the spikes these suggest that specific SNAP-25 C-terminal as and that had not been the of amino acids in the play a fundamental role in the very last membrane fusion steps of exocytosis and that the zero layer may in of this process. These are by a recent of S. Xu T. Ashery U. Kollewe A. Matti U. Antonin W. Rettig J. Neher E. EMBO J. 2000; 19: 1279-1289Crossref PubMed Google Scholar) in which the effects of the deletion and were studied by and was that the zero layer is critical for the formation of SNARE complexes in to the last nine C-terminal amino role in the between the two exocytotic The of and at the SNAP-25 C terminus was mutations in synaptobrevin residues as and which have been in their and the corresponding of the produced a very similar on and the vesicle fusion from their marked effect on complex in the presence of C forms of SNAP-25 as and L203E as as in their functional synaptobrevin A82D and and Q53K mutants of the SNAP-25 zero which did not affect secretion in a had a strong effect on the in formation and stability of the core complex. there is not a between the functional effects of the mutants in and their ability to the of the ternary complex in In this the most was the double mutation in the zero layer by which had a on the complex since could not its formation at which was to in since an between that of the and the most C-terminal These point to the that on the formation and stability of the fusion complex in should be with and with functional could be the role of and and synaptobrevin and amino acids during the final membrane fusion steps of previously these amino acids had not been to of the of amino acids to fusion complex formation. the nature of SNAP-25 and synaptobrevin and their in In addition, the importance of these residues may in their the SNAP-25 C terminus and the region of synaptobrevin the structural of the the C-terminal regions of the SNAREs J.E. Brennwald P. EMBO J. 1998; PubMed Scopus Google Scholar), form a of the complex and in its final and the membrane fusion by apposition of the membranes a critical and to the may be that its is the of the nature suggest an in interactions with other protein that may be present as of the machinery responsible for vesicle and at of the final membrane fusion hypothesis be that and SNAP-25 mutations and and synaptobrevin mutations the structure of the corresponding core complex this should be specific for these amino acids and not for in the as SNAP-25 and synaptobrevin In the described in this an important participation by specific amino acids of the SNAP-25 C-terminal domain and the corresponding synaptobrevin domain in the final membrane fusion steps of the exocytotic process. The of and is We and for the synaptobrevin and SNAP-25 and for the