The Regulation of GTP-Binding Proteins during Fertilization and Zygote Differentiation in Dictyostelium discoideum

Abstract

The developmental changes in GTP-binding proteins and the regulation of their appearance by calcium ions were investigated during early sexual development in Dictyostelium discoideum. GTPγS strongly inhibited gamete cell fusion, while GDPβS slightly augmented it, suggesting that G-proteins have a critical role in cell fusion. A 52-kDa protein recognized by an anti-GTP-binding site-specific immune serum, was abundant during calcium-dependent early sexual development but decreased in amount concomitant with cell fusion. This protein remained at high levels in Ca 2+-deficient cultures, suggesting that its down-regulation is linked to the events of sexual development. Analysis of substrates for cholera and pertussis toxin-mediated [ 32P]ADP-ribosylation in D. discoideum extracts determined that the 52-kDa protein is a G-α subunit similar to mammalian Gs. The 52-kDa protein was also detected in vegetative, asexual amoebae, but diminished rapidly within the first 2 h of starvation. Together these data indicate that the 52-kDa protein functions during the growth phase and is lost upon entry into either the sexual or asexual developmental programs. The amounts of several lower molecular weight GTP-binding proteins, ranging from 21- to 28 kDa, increased during the stage of zygote differentiation and their increases were calcium dependent. These data provide the first analysis of G-proteins during sexual development of D. discoideum and lay the foundation for continued analysis of the signal transduction events mediating cell fusion and zygote differentiation.