alpha-SNAP and NSF are required in a priming step during the human sperm acrosome reaction.
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 Ca2+-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 that alpha-soluble NSF-attachment protein (alpha-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 alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-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 Ca2+. These results suggest a key role for alpha-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.
Tomes
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CN
Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, CC 56, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina. ctomes@fcm.uncu.edu.ar
De Blas
G A
GA
Michaut
M A
MA
Farré
E V
EV
Cherhitin
O
O
Visconti
P E
PE
Mayorga
L S
LS
D000177:Acrosome / Q000737:chemistry / Q000378:metabolism; D020101:Acrosome Reaction / Q000502:physiology*; D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008297:Male; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013094:Spermatozoa / Q000737:chemistry / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology*; D020826:rab3A GTP-Binding Protein / Q000378:metabolism / Q000494:pharmacology
D000906:Antibodies; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D020826:rab3A GTP-Binding Protein; D002118:Calcium