The dynamics of SNARE assembly and disassembly during membrane recognition and fusion is a central issue in intracellular trafficking and regulated secretion. Exocytosis of sperm's single vesicle—the acrosome—is a synchronized, all-or-nothing process that happens only once in the life of the cell and depends on activation of both the GTP-binding protein Rab3 and of neurotoxin-sensitive SNAREs. These characteristics make acrosomal exocytosis a unique mammalian model for the study of the different phases of the membrane fusion cascade. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca2+ chelator we show that, in unactivated sperm, SNAREs are locked in heterotrimeric cis complexes. Upon Ca2+ entry into the cytoplasm, Rab3 is activated and triggers NSF/α-SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose trans complexes that are resistant to NSF/α-SNAP and differentially sensitive to cleavage by two vesicle-associated membrane protein (VAMP)–specific neurotoxins. Ca2+ must be released from inside the acrosome to trigger the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis.

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.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 3 family (1). 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 (2). Only when not assembled in tight complexes are SNAREs susceptible to cleavage, making these toxins excellent tools for the diagnosis of SNARE assembly status.Additional proteins that are key for Ca 2ϩ -triggered neurotransmitter release include complexins and synaptotagmins (3). Complexins are small cytosolic proteins that bind with high affinity to SNARE complexes (4), forming an antiparallel ␣ helix that inserts into a groove between the synaptobrevin and syntaxin helices (5). Experiments where complexins were deleted or overexpressed have suggested that they play ...