title: 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 Ca(2+) 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 Ca(2+). 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 Ca(2+) 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.
authors.affiliation: Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza 5500, Argentina.
authors.forename: Carlos M
authors.initials: CM
authors.lastname: Roggero
authors.forename: Gerardo A
authors.initials: GA
authors.lastname: De Blas
authors.forename: Han
authors.initials: H
authors.lastname: Dai
authors.forename: Claudia N
authors.initials: CN
authors.lastname: Tomes
authors.forename: Josep
authors.initials: J
authors.lastname: Rizo
authors.forename: Luis S
authors.initials: LS
authors.lastname: Mayorga
mesh_terms: D000177:Acrosome / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005306:Fertilization / Q000502:physiology; D006801:Humans; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D050863:Synaptotagmin I / Q000378:metabolism* / Q000494:pharmacology; D050857:Synaptotagmins / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050863:Synaptotagmin I; C494301:Syt6 protein, rat; C095992:complexin I; C095993:complexin II; D050857:Synaptotagmins; D002118:Calcium