ARF6 promotes membrane fusion by generating PIP2 and PA during acrosomal exocytosis

PELLETÁN LEONARDO, MAYORGA LUIS AND SILVIA A. BELMONTE

San Miguel de Tucumán. Tucumán. Argentina

Congreso; Sociedad Argentina de Investigación en Bioquímica y Biología Molecular, SAIB; 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular, SAIB

During sperm acrosome reaction (AR) the plasma membrane and outer acrosomal membrane fuse at multiple points releasing hybrid vesicles and causing the exposure of the inner acrosomal. AR is a special type of Ca–regulated exocytosis. Our previous results suggested that AR needs phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) after the opening of SOC channels. ADP-ribosylation factors (Arfs) are a family of monomeric GTP-binding proteins. In chromaffin cells, Arf6 activates both PI4P5Kinase and PLD1; both enzymes are thought to be coupled during signaling to support simultaneous increases in PIP2 and PA. We analyzed if this small GTPase was involved in AR regulation. By using Western blot and indirect immunofluorescence we demonstrated that Arf6 was present in human spermatozoa and localizes to the acrosomal region. Functional assays by using the permeabilized sperm model demonstrated that myristoylated and GTPgS loaded-Arf6 triggered AR and is absolutely required for calcium-regulated membrane fusion. Furthermore, we defined that Arf6-induced membrane fusion requires PIP2, PLD1 activity and consequently PA synthesis. By thin layer chromatography we demonstrated that Arf6-GTPgS increased PIP2 concentration. We propose a model where Arf6 regulates PLD1 and activates a kinase to maintain a pool of PIP2 in spermatozoa necessary for IP3-dependent acrosomal calcium release.