Cloning and characterization of seven novel Dictyostelium discoideum rac-related genes belonging to the rho family of GTPases

Abstract

Cellular processes including proliferation, organization of the actin cytoskeleton, vesicular traffic and secretion of proteins comprising the lysosomal/endosomal system are regulated by low-molecular-weight GTP-binding proteins of the Ras superfamily. However, to date only three Dictyostelium discoideum ras-like genes and two ypt-1/sec4-like genes have been identified and characterized. We report here the identification (using an oligodeoxyribonucleotide probe) of seven additional cDNAs coding for members highly related to the Rac proteins (Ras-related-C3 botulinum toxin substrate) which belong to the Rho (Ras homologous) family of GTPases. Three of these rac-related genes ( rac1A, rac1B and rac1C) predict proteins with > 90% amino acid (aa) sequence identity with each other and > 80% identity to the human rac1 gene product, whereas the other members ( racA, racB, racC and racD) predict proteins with 46–74% identity to the rac1 and rhoA gene products and to each other. The D. discoideum proteins were entirely conserved over the four regions known to be important for GTP binding and all contained the C-terminal CAAX aa motifs shared by other Rho proteins. Interestingly, the D. discoideum rac-related genes revealed unique patterns of expression during growth and development. For instance, the steady-state level of rac1 mRNA, encoded by three highly related genes, increased transiently during aggregation and then rapidly decreased. In contrast, the cellular abundance of mRNAs encoded by the other rac-like genes decreased at different rates and to different levels during development from the peak levels observed during growth. This suggests that the GTP-binding proteins encoded by these genes may play unique roles during the different stages of the D. discoideum life cycle. This study forms the foundation of future investigations to determine the role of these small GTPases in cell proliferation, organization of the actin cytoskeleton, vesicular trafficking during growth and development, and secretion of lysosomal hydrolases.