The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratus sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical gr anules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocyto sis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regul ate a number of cellular processes, including organization of the actin cyt oskeleton, We report here that a crude membrane fraction from homogenates o f Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which A DP-ribosylates specifically Rho proteins) and [P-32]NAD, displayed an [P-32 ]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [P-32]ADP-rib osylated Rho from sea urchin sperm; ii) identical mobility in isoelectro fo cusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses ind icated that RhoA was localized preferentially to the cortical granules; thi s was confirmed by experiments of [P-32]ADP-ribosylation with C3 in isolate d cortical granules, Rho was secreted and retained in the fertilization mem brane after insemination or activation with A23187. It was observed that th e Rho protein present in the sea urchin sperm acrosome was also secreted du ring the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated ex ocytosis or actin reorganization that accompany the egg activation.