2 GTPASE ISOFORMS, YPT31P AND YPT32P, ARE ESSENTIAL FOR GOLGI FUNCTION IN YEAST

In eukaryotic cells, monomeric GTPases of the Ypt/Rab family function as regulators at defined steps of vesicular transport in exo- and endo cytosis, Here we report on the isolation and characterization of two g enes (YPT31 and YPT32) of the yeast Saccharomyces cerevisiae which enc ode members of the Ypt family exhibiting >80% sequence identity, Where as the disruption of one of the two genes was phenotypically neutral, the disruption of both YPT31 and YPT32 led to lethality, Depletion of wild-type Ypt31p or of a short-lived ubiquitin-Ypt31p in a ypt32 null background led to a massive accumulation of Golgi-like membranes, an i nhibition of invertase secretion and defects in vacuolar protein matur ation, Similar alterations were observed in a conditional-lethal ypt31 -1 mutant at 30 min after shift to the non-permissive temperature. Acc ording to subcellular fractionation, a significant part of Ypt31p appe ared to be located in Golgi-enriched membrane fractions. In accordance with this, indirect immunofluorescence using affinity-purified anti-Y pt31p antibodies gave a punctate staining similar to that observed wit h Golgi-located proteins, From the phenotypic alterations observed in ypt31 and ypt32 mutants, it seems likely that the two GTPases are invo lved in intra-Golgi transport or in the formation of transport vesicle s at the most distal Golgi compartment.