CLONING AND CHARACTERIZATION OF 5 NOVEL DICTYOSTELIUM-DISCOIDEUM RAB-RELATED GENES

Low-M(r), GTPases belonging to the Ras superfamily are known to regulate a wide range of cellular processes including cell proliferation, actin cytoskeleton organization, and vesicular trafficking along the secretory and endosomal/lysosomal pathway. We are studying the regulation of lysosomal and endosomal vesicular trafficking in the simple eukaryote, Dictyostelium discoideum. Using an oligodeoxyribonucleotide (oligo) encoding one of the most highly conserved amino acid (aa) regions found in the low-M(r) GTPases (important in CTP binding), we have cloned 18 new cDNAs encoding proteins belonging to the Ras superfamily. In this report, we describe the characterization of five of these cDNAs coding for proteins belonging to the Ypt1/Sec4/Rab family; mammalian members of this family have been shown to function in the regulation of vesicular trafficking. Two of the cDNAs, rab1A and rab1B, code for proteins highly homologous to mammalian Rab1. An additional cDNA, rabA, codes for a protein that is only 60% identical to Rab1 at the aa sequence level and probably represents a new member of the rab gene family. Finally, two cDNAs, rabB and RabC, code for novel proteins belonging to the Rab gene family that are no greater than 50% identical in aa sequence to any previously described member. Southern blot analysis indicated that rab1A and rab1B belong to a small Dictyostelium family of at least five related genes, while rabA belongs to a different and smaller family of related genes. In contrast, rabB and rabC appear to be represented by single genes. Northern blot analysis of RNA purified at different times during growth and development revealed that four of the genes were expressed during growth with no change associated with differentiation.