A connection between reversible tyrosine phosphorylation and SNARE complex-disassembly activity of N-ethylmaleimide-sensitive factor unveiled by the phosphomimetic mutant N-ethylmaleimide-sensitive factor-Y83E

RUETE, MARÍA CELESTE; DE PAOLA, MATILDE; MASONE, DIEGO; TOMES, CLAUDIA NORA; ZARELLI, VALERIA EUGENIA PAOLA; BUSTOS, DIEGO MARTÍN

MOLECULAR HUMAN REPRODUCTION.

Oxford University Press

Lugar: Oxford; Año: 2019 vol. 25 p. 344 - 358

1360-9947

N-ethylmaleimide-sensitive factor (NSF) disassembles fusion-incompetent cis soluble-NSF attachment protein receptor(SNARE) complexes making monomeric SNAREs available for subsequent trans pairing and fusion. In most cells the activity of NSF isconstitutive, but in Jurkat cells and sperm it is repressed by tyrosine phosphorylation; the phosphomimetic mutant NSF?Y83E inhibits secretionin the former. The questions addressed here are if and how the NSF mutant influences the configuration of the SNARE complex. Our modelis human sperm, where the initiation of exocytosis (acrosome reaction (AR)) de-represses the activity of NSF through protein tyrosinephosphatase 1B (PTP1B)-mediated dephosphorylation. We developed a fluorescence microscopy-based method to show that capacitationincreased, and challenging with an AR inducer decreased, the number of cells with tyrosine-phosphorylated PTP1B substrates in the acrosomaldomain. Results from bioinformatic and biochemical approaches using purified recombinant proteins revealed that NSF?Y83E bound PTP1Band thereupon inhibited its catalytic activity. Mutant NSF introduced into streptolysin O-permeabilized sperm impaired cis SNARE complexdisassembly, blocking the AR; subsequent addition of PTP1B rescued exocytosis. We propose that NSF?Y83E prevents endogenous PTP1Bfrom dephosphorylating sperm NSF, thus maintaining NSF?s activity in a repressed mode and the SNARE complex unable to dissociate. Thecontribution of this paper to the sperm biology field is the detection of PTP1B substrates, one of them likely being NSF, whose tyrosinephosphorylation status varies during capacitation and the AR. The contribution of this paper to the membrane traffic field is to have generateddirect evidence that explains the dominant-negative role of the phosphomimetic mutant NSF?Y83E.