Synaptotagmin VI function in an acrosome reaction is regulated by phosphorylation

ROGGERO, CARLOS MARCELO; DE BLAS, GERARDO ANDRES; TOMES, CLAUDIA N.; MAYORGA, LUIS S.

San Carlos de Bariloche, Argentina

Congreso; XXXIX Reunión Anual Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB).; 2003

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 synchronize the calcium entry to exocytosis of vesicles, and share the same domain structure: an N-terminal single transmembrane domain, a spacer domain, two calcium and phospholipid binding domains (C2A and C2B), and a short carboxyl terminus. The ubiquitous synaptotagmin VI isoform was found in human sperm by Western blot. Immunocytochemistry localized the protein to the outer acrosomal membrane. Here we show that calcium-triggered acrosomal exocytosis in permeabilized sperm was completely inhibited by the C2A or C2B domains of synaptotagmin VI purified as fusion proteins with glutathione S-transferase. Surprisingly, phorbol esterdependent in vitro phosphorylation of these recombinant proteins abolished their inhibitory effect. When two Thr adjacent to the C2B effector domain were mutated to Ala, the effect of the protein became resistant to phosphorylation. In addition, when these two Thr were mutated to Glu, mimicking the negative charges accomplished by phosphorylation, the protein lost its effect on exocytosis. Our results strongly indicate that synaptotagmin VI is a key component in acrosomal exocytosis. Interestingly, its activity is regulated by phosphorylation.