Synaptotagmin VI participates in the acrosome reaction of human spermatozoa.
Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.
Michaut
M
M
Laboratorio de Biología Celular y Molecular, Istituto de Histologia y Embriologia (IHEM-CONICET), Universidad Nacional de Cuyo, Casilla de Correo 56, 5500 Mendoza, Argentina.
De Blas
G
G
Tomes
C N
CN
Yunes
R
R
Fukuda
M
M
Mayorga
L S
LS
D020101:Acrosome Reaction; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008562:Membrane Glycoproteins / Q000502:physiology*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000502:physiology*; D010703:Phorbol Esters / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism*; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010703:Phorbol Esters; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C494298:SYT6 protein, human; D050857:Synaptotagmins; D005982:Glutathione Transferase; D011493:Protein Kinase C; D002118:Calcium