Characterization of a novel ADP-ribosylation factor-like protein (yARL3) in Saccharomyces cerevisiae

ADP-ribosylation factors (ARFs) are highly conserved, similar to 20-kDa gua nine nucleotide-binding proteins that enhance the ADP-ribosyltransferase ac tivity of cholera toxin and have an important role in vesicular transport. Several cDNAs for ARF-like proteins (ARLs) have been cloned from human, Dro sophila, rat, and yeast, although the biological function(s) of ARLs is unk nown. We have identified a yeast gene (yARL3) encoding a protein that is st ructurally related (>43% identical) to the mammalian ARF-like protein ARP. Biochemical studies of purified recombinant yARL3 protein revealed properti es similar to those of ARF and ARL proteins, including the ability to bind and hydrolyze GTP. Like other ARLs, recombinant yARL3 did not stimulate cho lera toxin-catalyzed auto-ADP-ribosylation. Anti-yARL3 antibodies did not c ross-react with yARFs or yARL1. yARL3 was not essential for cell viability, but disruption of yARL3 resulted in cold-sensitive cell growth. At the non permissive temperature, processing of alkaline phosphatase and carboxypepti dase Y in arl3 mutant was slowed. yARL3 might be required for protein trans port from endoplasmic reticulum to Golgi or from Gels to vacuole at nonperm issive temperatures. On subcellular fractionation, unlike its mammalian hom ologue ARP, yARL3 was detected in the soluble fraction but not in the plasm a membrane. Indirect immunofluorescence analysis revealed that yARL3 when o verexpressed was associated in part with the endoplasmic reticulum-nuclear envelope, Thus, the structural and functional characteristics of yARL3 indi cate that it may have a unique role(s) in vesicular trafficking.