Acrosomal exocytosis is inhibited by a phosphomimetic NSF mutant(NSF-Y83E)

RUETE MC; ZARELLI VEP; LUCCHESI O; BUSTOS MA; TOMES CN

Mendoza

Congreso; XLVIII Reunión Anual Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2012

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

Acrosomal exocytosis (AE) is a regulated secretion essential forfertilization in mammals.Amember of the fusion machinery, NSF,is inactivated by tyrosine phosphorylation in resting sperm. Inconsequence, SNARE proteins are assembled in complexesinstead of cycling between this and monomeric configurations. NSFis derepressed when AE triggers activate PTP1B, a phosphatasethat dephosphorylates it. NSF does not bind directly to SNAREcomplexes but it is bridged to them via -SNAP. Here, we report thatthe phosphomimetic NSF-Y83E mutant blocks Ca -inducedexocytosis in streptolysin-O permeabilized human sperm. By meansof functional and indirect immunofluorescence assays we found thatNSFY-83E does not disassemble SNARE complexes as wild typeNSF does. We performed in vitro binding assays and found nodiminished affinity of the mutant for -SNAP. Furthermore, NSFY83Edissociated -SNAP from syntaxin as efficiently as did thewild type. We determined in functional assays that NSF-Y83E?sinhibitory effect was reversed by recombinant PTP1B. We suggestthat NSFY-83E inhibits secretion because it sequesters endogenousPTP1B, preventing the latter from dephosphorylating endogenousNSF. These data lend support to the notion that NSF activity is notconstitutive in all systems. Furthermore, it provides an explanationfor the ongoing puzzle about why this mutant inhibit secretion.