Complexin/Synaptotagmin Interplay Controls Acrosomal Exocytosis

Abstract

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. Regulated secretion requires SNARE proteins and, in neurons, also synaptotagmin I and complexin. Recent reports suggest that complexin imposes a fusion block that is released by Ca2+ and synaptotagmin I. However, no direct evidence for this model in secreting cells has been provided and whether this complexin/synaptotagmin interplay functions in other types of secretion is unknown. In this report, we show that the C2B domain of synaptotagmin VI and an anti-complexin antibody blocked the formation of trans SNARE complexes in permeabilized human sperm, and that this effect was reversed by adding complexin. In contrast, an excess of complexin stopped exocytosis at a later step, when SNAREs were assembled in loose trans complexes. Interestingly, this blockage was released by the addition of the synaptotagmin VI C2B domain in the presence of Ca2+. We have previously demonstrated that the activity of this domain is regulated by protein kinase C-mediated phosphorylation. Here, we show that a phosphomimetic mutation in the polybasic region of the C2B domain strongly affects its Ca2+ and phospholipids binding properties. Importantly, this mutation completely abrogates its ability to rescue the complexin block. Our results show that the functional interplay between complexin and synaptotagmin has a central role in a physiological secretion event, and that this interplay can be modulated by phosphorylation of the C2B domain. Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. Regulated secretion requires SNARE proteins and, in neurons, also synaptotagmin I and complexin. Recent reports suggest that complexin imposes a fusion block that is released by Ca2+ and synaptotagmin I. However, no direct evidence for this model in secreting cells has been provided and whether this complexin/synaptotagmin interplay functions in other types of secretion is unknown. In this report, we show that the C2B domain of synaptotagmin VI and an anti-complexin antibody blocked the formation of trans SNARE complexes in permeabilized human sperm, and that this effect was reversed by adding complexin. In contrast, an excess of complexin stopped exocytosis at a later step, when SNAREs were assembled in loose trans complexes. Interestingly, this blockage was released by the addition of the synaptotagmin VI C2B domain in the presence of Ca2+. We have previously demonstrated that the activity of this domain is regulated by protein kinase C-mediated phosphorylation. Here, we show that a phosphomimetic mutation in the polybasic region of the C2B domain strongly affects its Ca2+ and phospholipids binding properties. Importantly, this mutation completely abrogates its ability to rescue the complexin block. Our results show that the functional interplay between complexin and synaptotagmin has a central role in a physiological secretion event, and that this interplay can be modulated by phosphorylation of the C2B domain. All types of intracellular membrane fusions, including regulated exocytosis (i.e. the stimulus-triggered fusion of secretory vesicles with the plasma membrane), require proteins from the SNARE 3The abbreviations used are: SNAREsoluble N-ethylmaleimide-sensitive factor attachment protein receptorAEacrosomal exocytosisBoNT/Bbotulinum neurotoxin type BPSAPisum sativum agglutininSNAP25synaptosome-associated protein of 25 kDaTPENN,N,N′,N′-tetrakis(2-pyridymethyl)ethylenediamineGTPγSguanosine 5′-O-3-(thio)triphosphatedansyl5-dimethylaminonaphthalene-1-sulfonylHSQC1H-15N heteronuclear single quantum coherencePKCprotein kinase CGSTglutathione S-transferaseFITCfluorescein isothiocyanatePBSphosphate-buffered salinePVPpolyvinyl pyrrolidoneTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineFRETfluorescence resonance energy transfer.3The abbreviations used are: SNAREsoluble N-ethylmaleimide-sensitive factor attachment protein receptorAEacrosomal exocytosisBoNT/Bbotulinum neurotoxin type BPSAPisum sativum agglutininSNAP25synaptosome-associated protein of 25 kDaTPENN,N,N′,N′-tetrakis(2-pyridymethyl)ethylenediamineGTPγSguanosine 5′-O-3-(thio)triphosphatedansyl5-dimethylaminonaphthalene-1-sulfonylHSQC1H-15N heteronuclear single quantum coherencePKCprotein kinase CGSTglutathione S-transferaseFITCfluorescein isothiocyanatePBSphosphate-buffered salinePVPpolyvinyl pyrrolidoneTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineFRETfluorescence resonance energy transfer. family (1Jahn R. Scheller R.H. Nat. Rev. Mol. Cell Biol. 2006; 7: 631-643Crossref PubMed Scopus (1909) Google Scholar). SNAREs are small membrane proteins that form stable hetero-oligomeric complexes consisting of a bundle of four parallel helices. Assembly of trans SNARE complexes between SNAREs from two opposing membranes is a key event for membrane fusion. SNAREs residing on the same membrane can form cis complexes that must be disassembled to render free SNAREs competent for membrane fusion. The exocytotic SNAREs involved in neurotransmission are syntaxin1A and SNAP25 in the plasmalemma, and synaptobrevin 2 (also called VAMP2) in secretory vesicles. These proteins are the targets of botulinum and tetanus toxins, a set of highly specific zinc-dependent endoproteases (2Schiavo G. Matteoli M. Montecucco C. Physiol. Rev. 2000; 80: 717-766Crossref PubMed Scopus (1063) Google Scholar). Only when not assembled in tight complexes are SNAREs susceptible to cleavage, making these toxins excellent tools for the diagnosis of SNARE assembly status. soluble N-ethylmaleimide-sensitive factor attachment protein receptor acrosomal exocytosis botulinum neurotoxin type B Pisum sativum agglutinin synaptosome-associated protein of 25 kDa N,N,N′,N′-tetrakis(2-pyridymethyl)ethylenediamine guanosine 5′-O-3-(thio)triphosphate 5-dimethylaminonaphthalene-1-sulfonyl 1H-15N heteronuclear single quantum coherence protein kinase C glutathione S-transferase fluorescein isothiocyanate phosphate-buffered saline polyvinyl pyrrolidone N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine fluorescence resonance energy transfer. soluble N-ethylmaleimide-sensitive factor attachment protein receptor acrosomal exocytosis botulinum neurotoxin type B Pisum sativum agglutinin synaptosome-associated protein of 25 kDa N,N,N′,N′-tetrakis(2-pyridymethyl)ethylenediamine guanosine 5′-O-3-(thio)triphosphate 5-dimethylaminonaphthalene-1-sulfonyl 1H-15N heteronuclear single quantum coherence protein kinase C glutathione S-transferase fluorescein isothiocyanate phosphate-buffered saline polyvinyl pyrrolidone N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine fluorescence resonance energy transfer. Additional proteins that are key for Ca2+-triggered neurotransmitter release include complexins and synaptotagmins (3Sudhof T.C. Annu. Rev. Neurosci. 2004; 27: 509-547Crossref PubMed Scopus (1870) Google Scholar). Complexins are small cytosolic proteins that bind with high affinity to SNARE complexes (4McMahon H.T. Missler M. Li C. Sudhof T.C. Cell. 1995; 83: 111-119Abstract Full Text PDF PubMed Scopus (389) Google Scholar), forming an antiparallel α helix that inserts into a groove between the synaptobrevin and syntaxin helices (5Chen X. Tomchick D.R. Kovrigin E. Arac D. Machius M. Sudhof T.C. Rizo J. Neuron. 2002; 33: 397-409Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Experiments where complexins were deleted or overexpressed have suggested that they play both activating and inhibitory roles, possibly to achieve synchronization of the fast component of neurotransmitter release (5Chen X. Tomchick D.R. Kovrigin E. Arac D. Machius M. Sudhof T.C. Rizo J. 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These proteins are Ca2+ sensors coupling cell stimulation to exocytosis (3Sudhof T.C. Annu. Rev. Neurosci. 2004; 27: 509-547Crossref PubMed Scopus (1870) Google Scholar). Synaptotagmins are transmembrane proteins with a cytoplasmic region that contains two conserved C2 domains capable of binding Ca2+ and negatively charged phospholipids (10Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G. Sudhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 11Davletov B.A. Sudhof T.C. J. Biol. Chem. 1993; 268: 26386-26390Abstract Full Text PDF PubMed Google Scholar, 12Shao X. Davletov B.A. Sutton R.B. Sudhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (294) Google Scholar). The synaptotagmin I C2A (membrane proximal) and C2B (membrane distal) domains have similar structure (10Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G. Sudhof T.C. Rizo J. 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Nature. 2002; 418: 340-344Crossref PubMed Scopus (258) Google Scholar). A recent report using proteins has that the cytoplasmic domain of synaptotagmin I can complexin from SNARE complexes in a in the presence of membranes J. Maximov A. Shin O.H. Dai H. Rizo J. Sudhof T.C. Cell. 2006; 126: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). interplay between complexin and synaptotagmin I in neurotransmitter release has been also from the that a of the fast component of release K. Mansour M. Varoqueaux F. McMahon H.T. Sudhof T.C. Brose N. Rosenmund C. Cell. 2001; 104: 71-81Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, M. Y. Li C. Sudhof T.C. Cell. Full Text PDF PubMed Scopus Google Scholar). A similar effect was a excess of complexin by a fusion protein in J. Maximov A. Shin O.H. Dai H. Rizo J. Sudhof T.C. Cell. 2006; 126: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). complexin was to block fusion and fusion by and synaptotagmin I released this block in a X. Shin J.A. Nat. Struct. Mol. Biol. 2006; 13: PubMed Scopus Google Scholar, Science. 2006; PubMed Scopus Google Scholar). However, no direct evidence has been provided that synaptotagmin can rescue the blockage in exocytosis by an excess of complexin in we to the interplay of synaptotagmin with complexin in the acrosomal a model to secretion C. R. of 2006; Scholar). are highly cells in a of functions with high R. E. The of Scholar). can fast by of a the of acrosomal and from the of of intracellular sperm are in a single membrane fusion the exocytosis of the acrosomal is when a intracellular Ca2+ is by of sperm that specific in the of the egg A. T. C. C. 2006; PubMed Scopus Google Scholar). on M. G. R. Sci. S. A. 2000; PubMed Scopus Google Scholar) and of the SNARE family M. G. P. Biol. 2002; PubMed Scopus Google Scholar). to a set of when Ca2+ the of in the of cis SNARE complexes Biol. 2005; PubMed Scopus Google Scholar). SNAREs in loose trans complexes an of Ca2+ synaptotagmin and membrane fusion Biol. 2005; PubMed Scopus Google Scholar). In this report, we show that in permeabilized human sperm, an model with membranes involved in physiological the functional interplay between complexin and synaptotagmin has a central role in interplay can be modulated by phosphorylation or by a phosphomimetic mutation in the polybasic region of the synaptotagmin C2B domain. was from of was from and an were from was from A was from kinase C was from was from and from All other were of and were from or the C2A and C2B domains of synaptotagmin VI to were provided by T. Sudhof of The and and and domain were J. M. Biol. 2005; PubMed Scopus Google Scholar). synaptotagmins were in cells and by affinity on Johnson PubMed Scopus Google Scholar). VI domains were by on in and The human complexin I and to in a were provided by J. of and and Davletov of the human was provided by P. and complexins were into was at with and proteins were on was used and with Biol. 2005; PubMed Scopus Google Scholar). The was from complexins and synaptotagmin domains by with of in and at 25 The of botulinum neurotoxin B and tetanus to were provided by T. was into E. and protein was at with A was into E. and protein at with of proteins was to The type and synaptotagmin VI C2B domains for were in with the were In phosphorylation synaptotagmin VI domains were in 2 and of activating for at the were with to and small in were from sperm were a in human with for at in an of was to and for at 2 was Biol. 2005; PubMed Scopus Google Scholar). were in 2 and in was in the The were was to the same for in the same All were at and free were in the of the of was on and in was by with Pisum sativum agglutinin that have an show with the at the acrosomal that have acrosomal exocytosis show no at the acrosomal region or to the of the cells were for acrosomal exocytosis were by the of in the of from and the a of the in the of The between and was where the was than were were on a at and the from to The were at in with vesicles and and and the of or A fluorescence but in the of protein was from the in the presence of protein (10Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G. Sudhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). were at on or with C2B domains in and Ca2+ binding was by 1H-15N heteronuclear coherence in the presence of or All were with F. S. G. J. A. J. 1995; PubMed Scopus Google Scholar) and with B.A. J. PubMed Scopus Google Scholar). of an anti-complexin antibody was to the of sperm and for at on in sperm were in The cells were with in for at with sperm were in at was blocked with in for at at in was used were with between cells were in and with at in by at in and with at were in and with an with a and a with was and were to render an or using and were in and proteins in and a for with and for at the sperm were by at for and used or at were on H. G. PubMed Scopus Google Scholar) and to membranes was blocked by for at with in were with the anti-complexin antibody in for at in was used antibody at and were by for in was with an and to for were using of The was used for were at the that complexin be of the membrane fusion involved in was in human sperm by the antibody not between I and the of sperm complexin that is the The protein to the acrosomal region has been in sperm of other P. J. J. PubMed Scopus Google Scholar, X. Li Mol. PubMed Scopus Google Scholar), with a role in the exocytosis of this into sperm, of a specific anti-complexin antibody the by Ca2+ in a that complexin is for secretion The effect of the antibody was was by with complexin Interestingly, addition of of complexin a fusion protein with or the I was complexin in exocytosis can in permeabilized sperm in the of Ca2+ G. M. R. A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), an effect that was also by an anti-complexin antibody and by complexin a role for this protein of the small These results that complexin is in human sperm and that a of this protein is for The C2B of VI the set to whether synaptotagmin the effect of complexin in In sperm, the C2A and C2B domains of synaptotagmin VI exocytosis J. M. Biol. 2005; PubMed Scopus Google Scholar). we whether the domains rescue the complexin block. these of both domains were to inhibitory of complexin Interestingly, the C2B domain In contrast, the C2A domain was exocytosis was used to whether the effect of the synaptotagmin VI domains on complexin was Ca2+ in no rescue was in the of Ca2+ with the C2B or C2A Ca2+ were used to the C2B not the domain the complexin block that the of effect in the presence of was not to a by an excess of the small These that the C2B domain of synaptotagmin VI is to the inhibitory effect of complexin in rescue is only in the presence of cytosolic Ca2+. and the VI C2B at of the of the of the complexin block by with the C2B domain of synaptotagmin VI to the in the exocytotic where complexin its In sperm, SNAREs are assembled in cis complexes to Biol. 2005; PubMed Scopus Google Scholar). bind to these complexes and exocytosis by with the of this sperm were with tetanus in the presence of complexin and with Ca2+. complexin SNAREs in cis complexes and synaptobrevin be from the of the a was to the and the C2B domain to the these exocytosis was that synaptobrevin was and complexin not with SNARE a that the activity of the was with tetanus no was in this addition of complexin not SNARE block exocytosis at a later the of synaptobrevin to toxins, sperm were with Ca2+ in the presence of complexin to secretion to the that is blocked by the this tetanus or were to the and for at the C2B domain was to the complexin block. these the was to but not to tetanus exocytosis when from the of the that they were these results suggest that complexin exocytosis at a assembled trans SNARE complexes to tetanus but to In a report J. M. Biol. 2005; PubMed Scopus Google Scholar), we have that an excess of C2B domain can we whether this blockage be by addition of complexin. in complexin was able to rescue the effect of to the of synaptobrevin to at the by an excess of the same used in but using C2B to and complexin to we that the process stopped at a in synaptobrevin was to tetanus and These suggest that synaptobrevin was not in loose trans complexes these but a or between and SNARE complexes. These suggest that at an of an excess of C2B the of loose this effect can be by adding complexin. a later an excess of complexin exocytosis when SNAREs are assembled in loose trans complexes. can be by adding synaptotagmin VI C2B domain. the Assembly of SNARE in suggest that complexin the formation of loose trans SNARE the C2B domain of synaptotagmin be to release complexin at a later to membrane fusion. A of this model is that the addition of C2B domain is an than the excess of complexin. We this using a Ca2+ that in the We have previously that this acrosomal exocytosis the formation of loose trans SNARE complexes Biol. 2005; PubMed Scopus Google Scholar). that when the was in the presence of the process to a that was to the addition of complexin but not to the addition of the synaptotagmin VI C2B domain of the model is that an excess of C2B domain the of complexin with the SNARE and the anti-complexin acrosomal exocytosis at the same The results with the are with this the C2B domain the anti-complexin antibody exocytosis the to the that requires the of from the and this more we the to at the blocked by the these was to in the inhibitory effect of the antibody be by complexin. to be and only was in a for both the antibody and complexin I and were in the that both proteins are in is that the of addition of these was from in where the antibody was with complexin adding to the exocytosis was in the presence of the anti-complexin synaptobrevin that in the of the exocytosis to a In a of the only were these not require addition of complexin to rescue stimulation in the presence of the antibody also the to a tetanus These the that synaptotagmin VI C2B domain and anti-complexin antibody the formation of tetanus SNARE an effect that can be by addition of complexin. The of the C2B by a in the to with the of synaptotagmin I with complexin for SNARE binding is and J. Maximov A. Shin O.H. Dai H. Rizo J. Sudhof T.C. Cell. 2006; 126: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). In a report, we have that phosphorylation of the polybasic region of the C2B domain of synaptotagmin VI its effect on J. M. Biol. 2005; PubMed Scopus Google Scholar). region has been in J.M. Y. Mol. Cell Neurosci. 2005; PubMed Scopus Google Scholar) and C. M. Davletov Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar, C.A. Shin Reist N.E. Mol. Biol. Cell. 2006; PubMed Scopus Google Scholar) functions of two in the of the polybasic region are to or to the of this protein are strongly the effect of the on be modulated by phosphorylation and the not exocytosis J. M. Biol. 2005; PubMed Scopus Google Scholar). whether these the Ca2+ and binding of the C2B and proteins were fusion proteins and the was binding of these domains to was by a energy between the protein and into (10Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G. Sudhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). The type synaptotagmin VI C2B domain a addition of Ca2+ the protein to in a A similar was with the In contrast, the with the was not by that this not interact with in a the of the type and synaptotagmin VI C2B we used a to Ca2+ binding to proteins X. Davletov B.A. Sutton R.B. Sudhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (294) Google Scholar). The 1H-15N of the type and C2B domains that they are that both the and in the that from the of of the a with the Importantly, Ca2+ in the 1H-15N of the type and C2B domains and but not in the the is able to bind the mutation Ca2+ in with the of the C2B to the whether phosphorylation or the phosphomimetic mutation the ability of the C2B domain to rescue the inhibitory effect of complexin. this we or C2B domains in with inhibitory of complexin. type C2A and with and in the polybasic region of this domain J. M. Biol. 2005; PubMed Scopus Google Scholar) were in the Only the type C2B domain and the were able to The type protein and the were to with complexin of the C2A exocytosis in the presence of complexin In phosphorylation of the C2B domain or of a phosphomimetic mutation in the polybasic region of this protein completely its ability to rescue blocked by an excess of complexin. The for the effect of complexin in exocytosis are only The recent that synaptotagmin the Ca2+ in with complexin for SNARE binding J. Maximov A. Shin O.H. Dai H. Rizo J. Sudhof T.C. Cell. 2006; 126: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar) a between complexin and Ca2+ for an interplay between complexin and synaptotagmin I has been in cell Science. 2006; PubMed Scopus Google Scholar) and X. Shin J.A. Nat. Struct. Mol. Biol. 2006; 13: PubMed Scopus Google Scholar) fusion. However, no direct evidence for this model using membranes in a physiological regulated secretion has been is whether the complexin/synaptotagmin interplay is a of neurotransmitter release or also functions in other regulated is an complexins have been in cell A. G. T. R. G. J. Cell Sci. 2004; PubMed Scopus Google Scholar) and cell S. Nakanishi M. Hirashima N. J. Cell Sci. 2005; 118: 2239-2246Crossref PubMed Scopus (53) Google Scholar) In of a and role for the functional we report that complexin is for acrosomal exocytosis in human sperm, that exocytosis is blocked by excess and that the C2B domain of synaptotagmin VI this is to that the C2A domain in with the that this domain complexin from SNARE complexes in H. N. Arac D. Rizo J. J. Mol. Biol. PubMed Scopus Google Scholar). In permeabilized sperm, both complexin and the synaptotagmin VI C2B domain are inhibitory and both can rescue the blockage by the These to the of an interplay between these proteins in acrosomal the results with and the to that an excess of C2B domain is inhibitory at an the assembly of loose trans SNARE an excess of complexin at a later when tetanus complexes have All these can be by the between complexin and the synaptotagmin C2B domain for binding to SNARE complexes using proteins in H. N. Arac D. Rizo J. J. Mol. Biol. PubMed Scopus Google Scholar). In an complexin has an activating role in the formation of trans complexes. this an excess of C2B complexin from the or the of complexin an inhibitory render the trans complexes and in with a that be to the formation of the loose trans the release of Ca2+ from the the of membrane fusion that require the of complexin from the SNARE complexes. this the C2B domain is no but can rescue a blockage by an excess of complexin In sperm, complexin exocytosis at a where synaptobrevin was to tetanus but to neurotoxin has been in the cell fusion using SNAREs Science. 2006; PubMed Scopus Google Scholar). In the complexin fusion at the of X. Shin J.A. Nat. Struct. Mol. Biol. 2006; 13: PubMed Scopus Google this is with loose trans complexes is at (1Jahn R. Scheller R.H. Nat. Rev. Mol. Cell Biol. 2006; 7: 631-643Crossref PubMed Scopus (1909) Google Scholar). Ca2+ is essential for the complexin/synaptotagmin interplay in fusion X. Shin J.A. Nat. Struct. Mol. Biol. 2006; 13: PubMed Scopus Google Scholar, Science. 2006; PubMed Scopus Google Scholar). is also for the synaptotagmin of complexin from SNARE complexes J. Maximov A. Shin O.H. Dai H. Rizo J. Sudhof T.C. Cell. 2006; 126: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). In sperm, we show the C2B domain can rescue the complexin block only when exocytosis is with Ca2+. when the Ca2+ and phospholipids binding of the C2B domain are by a phosphomimetic mutation or by phosphorylation with the domain the to we that phosphorylation of synaptotagmin a key process that its functional with complexin process be of for sperm, a have a single to release the acrosomal at the and to an We M. and A. for and T. S. J. E. P. D. and T. C. for and