Increased Association of Synaptosome-associated Protein of 25 kDa with Syntaxin and Vesicle-associated Membrane Protein following Acrosomal Exocytosis of Sea Urchin Sperm

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated integral membrane protein expressed almost exclusively in neuronal and neuroendocrine tissues. This protein forms a ternary complex with vesicle-associated membrane protein (VAMP) and syntaxin, which is thought to regulate the fusion of plasma and vesicle membranes during exocytosis. We report the identification of SNAP-25 expressed in sea urchin sperm. Sea urchin SNAP-25 shares greater identity with mammalian SNAP-25 than with mammalian SNAP-23, a ubiquitously expressed homologue believed to regulate membrane fusion in non-neuronal tissues. Sea urchin sperm contain a single exocytotic vesicle, the acrosomal vesicle, whose contents are exposed during the acrosome reaction. Fusion of the plasma membrane with the acrosomal vesicle membrane at multiple points (vesiculation) results in the release of SNAP-25 with the shed acrosome reaction vesicles. A complex containing SNAP-25, syntaxin, and VAMP is present in sperm, as detected by affinity chromatography and immunoprecipitation. Although this complex is present prior to the acrosome reaction, the amount of complex increases over 4-fold following acrosomal exocytosis. These findings support the involvement of SNAP-25 in the invertebrate sperm acrosome reaction, possibly through increased association with VAMP and syntaxin driving the fusion of plasma and acrosomal membranes. Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated integral membrane protein expressed almost exclusively in neuronal and neuroendocrine tissues. This protein forms a ternary complex with vesicle-associated membrane protein (VAMP) and syntaxin, which is thought to regulate the fusion of plasma and vesicle membranes during exocytosis. We report the identification of SNAP-25 expressed in sea urchin sperm. Sea urchin SNAP-25 shares greater identity with mammalian SNAP-25 than with mammalian SNAP-23, a ubiquitously expressed homologue believed to regulate membrane fusion in non-neuronal tissues. Sea urchin sperm contain a single exocytotic vesicle, the acrosomal vesicle, whose contents are exposed during the acrosome reaction. Fusion of the plasma membrane with the acrosomal vesicle membrane at multiple points (vesiculation) results in the release of SNAP-25 with the shed acrosome reaction vesicles. A complex containing SNAP-25, syntaxin, and VAMP is present in sperm, as detected by affinity chromatography and immunoprecipitation. Although this complex is present prior to the acrosome reaction, the amount of complex increases over 4-fold following acrosomal exocytosis. These findings support the involvement of SNAP-25 in the invertebrate sperm acrosome reaction, possibly through increased association with VAMP and syntaxin driving the fusion of plasma and acrosomal membranes. acrosome reaction vesicle synaptosome-associated protein of 25 kDa vesicle-associated membrane protein polymerase chain reaction polyethylene glycol. During fertilization in many animals, including sea urchins and mammals, outer investments of the egg trigger the exocytosis of a single exocytotic vesicle in sperm, the acrosomal vesicle (1Garbers D.L. Kopf G.S. Adv. Cyclic Nucleotide Res. 1980; 13: 251-306PubMed Google Scholar, 2Ward C.R. Kopf G.S. Dev. Biol. 1993; 158: 9-34Crossref PubMed Scopus (204) Google Scholar, 3Foltz K.R. Lennarz W.J. Dev. Biol. 1993; 158: 46-61Crossref PubMed Scopus (76) Google Scholar). This process, the acrosome reaction, exposes the sperm membrane that will fuse with the egg plasma membrane to initiate development. Exocytosis of the acrosomal vesicle during the acrosome reaction is unique in that the plasma and acrosomal membranes fuse at multiple points resulting in formation of acrosome reaction vesicles (ARVs),1 which are subsequently shed from sperm (4Yanagimachi R. Knobil E. Neill J. The Physiology of Reproduction. Raven Press, Ltd., New York1988: 135-185Google Scholar, 5Epel D. Vacquier V.D. Poste G. Nicolson G.L. Membrane Fusion. Elsevier/North-Holland Biomedical Press, Amsterdam1978: 1-63Google Scholar, 6Barros C. Bedford J.M. Franklin L.E. Austin C.R. J. Cell Biol. 1967; 34: C1-C5Crossref PubMed Google Scholar). ARVs can be collected and the associated proteins studied (7Schulz J.R. Wessel G.M. Vacquier V.D. Dev. Biol. 1997; 191: 80-87Crossref PubMed Scopus (46) Google Scholar).The sea urchin sperm acrosome reaction is triggered by interaction of a plasma membrane receptor (receptor for egg jelly) with a sulfated fucan in the egg jelly coat (8Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Crossref PubMed Scopus (135) Google Scholar). The activated receptor regulates ion channels, resulting in the influx of Ca2+ and exocytosis (9Darszon A. Lievano A. Beltran C. Curr. Top. Dev. Biol. 1996; 34: 117-167Crossref PubMed Google Scholar). This results in the exposure of bindin and the elongation of the acrosomal process. Acrosomal exocytosis can be induced by ionophores (10Podell S.B. Vacquier V.D. J. Cell Biol. 1984; 99: 1598-1604Crossref PubMed Scopus (41) Google Scholar), or by the addition of Ca2+ to digitonin-permeabilized sperm (11Castellano L.E. Godinez-Lopez J. Aldana G. Barrios-Rodiles M. Obregon A. de De la Torre L. Darszon A. Garcia-Soto J. Eur. J. Cell Biol. 1995; 67: 23-31PubMed Google Scholar), making sperm an interesting model for studying Ca2+-triggered exocytosis.To understand the mechanism of acrosomal exocytosis, we have identified homologues in sea urchin sperm of proteins believed to be key regulators of membrane fusion during exocytosis. Syntaxin, an intracellular protein integral to the plasma membrane (12Bennett M.K. Calakos N. Scheller R.H. Science. 1992; 257: 255-259Crossref PubMed Scopus (1065) Google Scholar, 13Bennett M.K. Garcia-Arraras J.E. Elferink L.A. Peterson K. Fleming A.M. Hazuka C.D. Scheller R.H. Cell. 1993; 74: 863-873Abstract Full Text PDF PubMed Scopus (586) Google Scholar), and VAMP (synaptobrevin; Refs. 14Trimble W.S. Cowan D.M. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4538-4542Crossref PubMed Scopus (449) Google Scholar and 15Baumert M. Maycox P.R. Navone F. De Camilli P. Jahn R. EMBO J. 1989; 8: 379-384Crossref PubMed Scopus (396) Google Scholar), which is integral to the vesicle membrane, are expressed in sea urchin sperm and are shed with the ARVs during the acrosome reaction. Previous work demonstrated that sperm syntaxin and VAMP increase their association following acrosomal exocytosis (7Schulz J.R. Wessel G.M. Vacquier V.D. Dev. Biol. 1997; 191: 80-87Crossref PubMed Scopus (46) Google Scholar). In neurons, these proteins form a ternary complex with SNAP-25 (16Oyler G.A. Higgins G.A. Hart R.A. Battenberg E. Billingsley M. Bloom F.E. Wilson M.C. J. Cell Biol. 1989; 109: 3039-3052Crossref PubMed Scopus (687) Google Scholar), which is postulated to regulate membrane fusion (17Rothman J.E. Protein Sci. 1996; 5: 185-194Crossref PubMed Scopus (145) Google Scholar). Here, we describe a sea urchin SNAP-25 homologue expressed in a non-neuronal cell type. Sperm SNAP-25 is found in a complex with syntaxin and VAMP and is also shed with the ARVs. The amount of complex of these three proteins increases following the acrosome reaction. This increase may represent the post-exocytotic state of these proteins in the absence of an endocytic membrane retrieval cycle common to most cells.DISCUSSIONSNAP-25 is an axonally transported protein in mammalian neurons where it localizes to nerve terminals (26Hess D.T. Slater T.M. Wilson M.C. Skene J.H. J. Neurosci. 1992; 12: 4634-4641Crossref PubMed Google Scholar, 27Loewy A. Liu W.S. Baitinger C. Willard M.B. J. Neurosci. 1991; 11: 3412-3421Crossref PubMed Google Scholar). At the presynaptic membrane, SNAP-25 regulates neurotransmission by forming a ternary complex with syntaxin and VAMP (28Söllner T. Whiteheart S.W. Brunner M. Erdjument B.H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2604) Google Scholar, 29Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1573) Google Scholar, 30Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (661) Google Scholar, 31Hayashi T. Yamasaki S. Nauenburg S. Binz T. Niemann H. EMBO J. 1995; 14: 2317-2325Crossref PubMed Scopus (227) Google Scholar). In this report, we have identified a sea urchin homologue of SNAP-25. This is the first report of SNAP-25 being expressed in the sperm of any animal. Phylogenetic analysis of SNAP-25 protein sequences suggests that the mammalian SNAP-25 homologue, SNAP-23 (23Ravichandran V. Chawla A. Roche P.A. J. Biol. Chem. 1996; 271: 13300-13303Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 32Wang G. Witkin J.W. Hao G. Bankaitis V.A. Scherer P.E. Baldini G. J. Cell Sci. 1997; 110: 505-513Crossref PubMed Google Scholar), arose by a gene duplication in the vertebrate lineage to function in non-neuronal cell types. In agreement with this idea, only a single isoform of SNAP-25 was amplified from a sea urchin testis cDNA library despite the use of degenerate primers designed to amplify both SNAP-25 and -23. This suggests that the sea urchin testis does not express a SNAP-23 homologue. The identification of SNAP-23 in mouse testis (32Wang G. Witkin J.W. Hao G. Bankaitis V.A. Scherer P.E. Baldini G. J. Cell Sci. 1997; 110: 505-513Crossref PubMed Google Scholar) raises the possibility that SNAP-23 has evolved to function in non-neuronal cell types in vertebrates, in agreement with the phylogenetic sequence analysis presented in Fig. 2.Sea urchin sperm SNAP-25 shares many properties with syntaxin and VAMP. All three proteins are shed from sperm with the ARVs during the acrosome reaction. These proteins can be isolated as a complex by immunoprecipitation and affinity chromatography prior to the acrosome reaction. Following acrosomal exocytosis, there is greater than a 4-fold increase in the amount of ternary complex present, as demonstrated by the changes in the sedimentation patterns of syntaxin, VAMP, and SNAP-25 and by the immunoprecipitation of gradient fractions. Interestingly, only a portion of SNAP-25 sediments into denser fractions following the acrosome reaction. The 6.9-fold molar excess of SNAP-25 over syntaxin in sperm extracts and ARVs suggests that SNAP-25 is not limiting for ternary complex formation. However, all the syntaxin present in ARVs completely shifts to denser fractions after the acrosome reaction. The estimated sedimentation coefficient for the syntaxin peak fractions following the acrosome reaction is 6.2 S (7Schulz J.R. Wessel G.M. Vacquier V.D. Dev. Biol. 1997; 191: 80-87Crossref PubMed Scopus (46) Google Scholar), which correlates with the size of the ternary complex based on the sedimentation of molecular weight standards.The presence of the ternary complex in ARVs may represent the post-exocytotic state of these proteins in the same lipid bilayer, and the increase in complex formation following the acrosome reaction may be an exocytotic intermediate formed in the absence a subsequent endocytic cycle.As the amount of ternary complex increases following acrosomal exocytosis, it will be of interest to determine what regulates complex formation prior to the acrosome reaction and how this correlates with the influx of Ca2+, which triggers acrosomal exocytosis. During fertilization in many animals, including sea urchins and mammals, outer investments of the egg trigger the exocytosis of a single exocytotic vesicle in sperm, the acrosomal vesicle (1Garbers D.L. Kopf G.S. Adv. Cyclic Nucleotide Res. 1980; 13: 251-306PubMed Google Scholar, 2Ward C.R. Kopf G.S. Dev. Biol. 1993; 158: 9-34Crossref PubMed Scopus (204) Google Scholar, 3Foltz K.R. Lennarz W.J. Dev. Biol. 1993; 158: 46-61Crossref PubMed Scopus (76) Google Scholar). This process, the acrosome reaction, exposes the sperm membrane that will fuse with the egg plasma membrane to initiate development. Exocytosis of the acrosomal vesicle during the acrosome reaction is unique in that the plasma and acrosomal membranes fuse at multiple points resulting in formation of acrosome reaction vesicles (ARVs),1 which are subsequently shed from sperm (4Yanagimachi R. Knobil E. Neill J. The Physiology of Reproduction. Raven Press, Ltd., New York1988: 135-185Google Scholar, 5Epel D. Vacquier V.D. Poste G. Nicolson G.L. Membrane Fusion. Elsevier/North-Holland Biomedical Press, Amsterdam1978: 1-63Google Scholar, 6Barros C. Bedford J.M. Franklin L.E. Austin C.R. J. Cell Biol. 1967; 34: C1-C5Crossref PubMed Google Scholar). ARVs can be collected and the associated proteins studied (7Schulz J.R. Wessel G.M. Vacquier V.D. Dev. Biol. 1997; 191: 80-87Crossref PubMed Scopus (46) Google Scholar). The sea urchin sperm acrosome reaction is triggered by interaction of a plasma membrane receptor (receptor for egg jelly) with a sulfated fucan in the egg jelly coat (8Vacquier V.D. Moy G.W. Dev. Biol. 1997; 192: 125-135Crossref PubMed Scopus (135) Google Scholar). The activated receptor regulates ion channels, resulting in the influx of Ca2+ and exocytosis (9Darszon A. Lievano A. Beltran C. Curr. Top. Dev. Biol. 1996; 34: 117-167Crossref PubMed Google Scholar). This results in the exposure of bindin and the elongation of the acrosomal process. Acrosomal exocytosis can be induced by ionophores (10Podell S.B. Vacquier V.D. J. Cell Biol. 1984; 99: 1598-1604Crossref PubMed Scopus (41) Google Scholar), or by the addition of Ca2+ to digitonin-permeabilized sperm (11Castellano L.E. Godinez-Lopez J. Aldana G. Barrios-Rodiles M. Obregon A. de De la Torre L. Darszon A. Garcia-Soto J. Eur. J. Cell Biol. 1995; 67: 23-31PubMed Google Scholar), making sperm an interesting model for studying Ca2+-triggered exocytosis. To understand the mechanism of acrosomal exocytosis, we have identified homologues in sea urchin sperm of proteins believed to be key regulators of membrane fusion during exocytosis. Syntaxin, an intracellular protein integral to the plasma membrane (12Bennett M.K. Calakos N. Scheller R.H. Science. 1992; 257: 255-259Crossref PubMed Scopus (1065) Google Scholar, 13Bennett M.K. Garcia-Arraras J.E. Elferink L.A. Peterson K. Fleming A.M. Hazuka C.D. Scheller R.H. Cell. 1993; 74: 863-873Abstract Full Text PDF PubMed Scopus (586) Google Scholar), and VAMP (synaptobrevin; Refs. 14Trimble W.S. Cowan D.M. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4538-4542Crossref PubMed Scopus (449) Google Scholar and 15Baumert M. Maycox P.R. Navone F. De Camilli P. Jahn R. EMBO J. 1989; 8: 379-384Crossref PubMed Scopus (396) Google Scholar), which is integral to the vesicle membrane, are expressed in sea urchin sperm and are shed with the ARVs during the acrosome reaction. Previous work demonstrated that sperm syntaxin and VAMP increase their association following acrosomal exocytosis (7Schulz J.R. Wessel G.M. Vacquier V.D. Dev. Biol. 1997; 191: 80-87Crossref PubMed Scopus (46) Google Scholar). In neurons, these proteins form a ternary complex with SNAP-25 (16Oyler G.A. Higgins G.A. Hart R.A. Battenberg E. Billingsley M. Bloom F.E. Wilson M.C. J. Cell Biol. 1989; 109: 3039-3052Crossref PubMed Scopus (687) Google Scholar), which is postulated to regulate membrane fusion (17Rothman J.E. Protein Sci. 1996; 5: 185-194Crossref PubMed Scopus (145) Google Scholar). Here, we describe a sea urchin SNAP-25 homologue expressed in a non-neuronal cell type. Sperm SNAP-25 is found in a complex with syntaxin and VAMP and is also shed with the ARVs. The amount of complex of these three proteins increases following the acrosome reaction. This increase may represent the post-exocytotic state of these proteins in the absence of an endocytic membrane retrieval cycle common to most cells. DISCUSSIONSNAP-25 is an axonally transported protein in mammalian neurons where it localizes to nerve terminals (26Hess D.T. Slater T.M. Wilson M.C. Skene J.H. J. Neurosci. 1992; 12: 4634-4641Crossref PubMed Google Scholar, 27Loewy A. Liu W.S. Baitinger C. Willard M.B. J. Neurosci. 1991; 11: 3412-3421Crossref PubMed Google Scholar). At the presynaptic membrane, SNAP-25 regulates neurotransmission by forming a ternary complex with syntaxin and VAMP (28Söllner T. Whiteheart S.W. Brunner M. Erdjument B.H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2604) Google Scholar, 29Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1573) Google Scholar, 30Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (661) Google Scholar, 31Hayashi T. Yamasaki S. Nauenburg S. Binz T. Niemann H. EMBO J. 1995; 14: 2317-2325Crossref PubMed Scopus (227) Google Scholar). In this report, we have identified a sea urchin homologue of SNAP-25. This is the first report of SNAP-25 being expressed in the sperm of any animal. Phylogenetic analysis of SNAP-25 protein sequences suggests that the mammalian SNAP-25 homologue, SNAP-23 (23Ravichandran V. Chawla A. Roche P.A. J. Biol. Chem. 1996; 271: 13300-13303Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 32Wang G. Witkin J.W. Hao G. Bankaitis V.A. Scherer P.E. Baldini G. J. Cell Sci. 1997; 110: 505-513Crossref PubMed Google Scholar), arose by a gene duplication in the vertebrate lineage to function in non-neuronal cell types. In agreement with this idea, only a single isoform of SNAP-25 was amplified from a sea urchin testis cDNA library despite the use of degenerate primers designed to amplify both SNAP-25 and -23. This suggests that the sea urchin testis does not express a SNAP-23 homologue. The identification of SNAP-23 in mouse testis (32Wang G. Witkin J.W. Hao G. Bankaitis V.A. Scherer P.E. Baldini G. J. Cell Sci. 1997; 110: 505-513Crossref PubMed Google Scholar) raises the possibility that SNAP-23 has evolved to function in non-neuronal cell types in vertebrates, in agreement with the phylogenetic sequence analysis presented in Fig. 2.Sea urchin sperm SNAP-25 shares many properties with syntaxin and VAMP. All three proteins are shed from sperm with the ARVs during the acrosome reaction. These proteins can be isolated as a complex by immunoprecipitation and affinity chromatography prior to the acrosome reaction. Following acrosomal exocytosis, there is greater than a 4-fold increase in the amount of ternary complex present, as demonstrated by the changes in the sedimentation patterns of syntaxin, VAMP, and SNAP-25 and by the immunoprecipitation of gradient fractions. Interestingly, only a portion of SNAP-25 sediments into denser fractions following the acrosome reaction. The 6.9-fold molar excess of SNAP-25 over syntaxin in sperm extracts and ARVs suggests that SNAP-25 is not limiting for ternary complex formation. However, all the syntaxin present in ARVs completely shifts to denser fractions after the acrosome reaction. The estimated sedimentation coefficient for the syntaxin peak fractions following the acrosome reaction is 6.2 S (7Schulz J.R. Wessel G.M. Vacquier V.D. Dev. Biol. 1997; 191: 80-87Crossref PubMed Scopus (46) Google Scholar), which correlates with the size of the ternary complex based on the sedimentation of molecular weight standards.The presence of the ternary complex in ARVs may represent the post-exocytotic state of these proteins in the same lipid bilayer, and the increase in complex formation following the acrosome reaction may be an exocytotic intermediate formed in the absence a subsequent endocytic cycle.As the amount of ternary complex increases following acrosomal exocytosis, it will be of interest to determine what regulates complex formation prior to the acrosome reaction and how this correlates with the influx of Ca2+, which triggers acrosomal exocytosis. SNAP-25 is an axonally transported protein in mammalian neurons where it localizes to nerve terminals (26Hess D.T. Slater T.M. Wilson M.C. Skene J.H. J. Neurosci. 1992; 12: 4634-4641Crossref PubMed Google Scholar, 27Loewy A. Liu W.S. Baitinger C. Willard M.B. J. Neurosci. 1991; 11: 3412-3421Crossref PubMed Google Scholar). At the presynaptic membrane, SNAP-25 regulates neurotransmission by forming a ternary complex with syntaxin and VAMP (28Söllner T. Whiteheart S.W. Brunner M. Erdjument B.H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2604) Google Scholar, 29Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1573) Google Scholar, 30Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (661) Google Scholar, 31Hayashi T. Yamasaki S. Nauenburg S. Binz T. Niemann H. EMBO J. 1995; 14: 2317-2325Crossref PubMed Scopus (227) Google Scholar). In this report, we have identified a sea urchin homologue of SNAP-25. This is the first report of SNAP-25 being expressed in the sperm of any animal. Phylogenetic analysis of SNAP-25 protein sequences suggests that the mammalian SNAP-25 homologue, SNAP-23 (23Ravichandran V. Chawla A. Roche P.A. J. Biol. Chem. 1996; 271: 13300-13303Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 32Wang G. Witkin J.W. Hao G. Bankaitis V.A. Scherer P.E. Baldini G. J. Cell Sci. 1997; 110: 505-513Crossref PubMed Google Scholar), arose by a gene duplication in the vertebrate lineage to function in non-neuronal cell types. In agreement with this idea, only a single isoform of SNAP-25 was amplified from a sea urchin testis cDNA library despite the use of degenerate primers designed to amplify both SNAP-25 and -23. This suggests that the sea urchin testis does not express a SNAP-23 homologue. The identification of SNAP-23 in mouse testis (32Wang G. Witkin J.W. Hao G. Bankaitis V.A. Scherer P.E. Baldini G. J. Cell Sci. 1997; 110: 505-513Crossref PubMed Google Scholar) raises the possibility that SNAP-23 has evolved to function in non-neuronal cell types in vertebrates, in agreement with the phylogenetic sequence analysis presented in Fig. 2. Sea urchin sperm SNAP-25 shares many properties with syntaxin and VAMP. All three proteins are shed from sperm with the ARVs during the acrosome reaction. These proteins can be isolated as a complex by immunoprecipitation and affinity chromatography prior to the acrosome reaction. Following acrosomal exocytosis, there is greater than a 4-fold increase in the amount of ternary complex present, as demonstrated by the changes in the sedimentation patterns of syntaxin, VAMP, and SNAP-25 and by the immunoprecipitation of gradient fractions. Interestingly, only a portion of SNAP-25 sediments into denser fractions following the acrosome reaction. The 6.9-fold molar excess of SNAP-25 over syntaxin in sperm extracts and ARVs suggests that SNAP-25 is not limiting for ternary complex formation. However, all the syntaxin present in ARVs completely shifts to denser fractions after the acrosome reaction. The estimated sedimentation coefficient for the syntaxin peak fractions following the acrosome reaction is 6.2 S (7Schulz J.R. Wessel G.M. Vacquier V.D. Dev. Biol. 1997; 191: 80-87Crossref PubMed Scopus (46) Google Scholar), which correlates with the size of the ternary complex based on the sedimentation of molecular weight standards. The presence of the ternary complex in ARVs may represent the post-exocytotic state of these proteins in the same lipid bilayer, and the increase in complex formation following the acrosome reaction may be an exocytotic intermediate formed in the absence a subsequent endocytic cycle. As the amount of ternary complex increases following acrosomal exocytosis, it will be of interest to determine what regulates complex formation prior to the acrosome reaction and how this correlates with the influx of Ca2+, which triggers acrosomal exocytosis. We thank J. Armstrong, S. Conner, and C. Francis for helpful discussions. We thank S. Conner for the glutathione S-transferase-syntaxin (sea urchin) construct.