Alpha-SNAP and NSF are required in a priming step during human sperm exocytosis

C TOMES, G DE BLAS, M MICHAUT, E FARRÉ, O CHERTIHIN, PE VISCONTI AND LS MAYORGA

MOLECULAR HUMAN REPRODUCTION.

Oxford University Press

Lugar: Oxford, UK; Año: 2005 vol. 11 p. 43 - 51

1360-9947

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca21-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that21-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show thatN-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that a-soluble NSF-attachment protein (a-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of a-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca21 and Rab3A-triggered ARs are inhibited by anti-a-SNAP antibodies. Third, bacterially expressed a-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for a-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca21. These results suggest a key role for a-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis-soluble NSF-attachment protein (a-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of a-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca21 and Rab3A-triggered ARs are inhibited by anti-a-SNAP antibodies. Third, bacterially expressed a-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for a-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca21. These results suggest a key role for a-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosisa-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca21 and Rab3A-triggered ARs are inhibited by anti-a-SNAP antibodies. Third, bacterially expressed a-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for a-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca21. These results suggest a key role for a-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis21 and Rab3A-triggered ARs are inhibited by anti-a-SNAP antibodies. Third, bacterially expressed a-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for a-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca21. These results suggest a key role for a-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosisa-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca21. These results suggest a key role for a-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis21. These results suggest a key role for a-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis