In Saccharomyces cerevisiae, the GTP-binding Yptl protein (Yptlp) is essential for endoplasmic reticulumto-Golgi protein transport. By exploiting a GALIO-YPTI fusion to regulate YPTI expression, three multicopy suppressors, SLY2, SLY12, and SLY41, and a single-copy suppressor, SLYI-20, that allowed YPTi-independent growth were isolated. Wild-type Slylp is hydrophilic, is essential for cell viability, and differs from Slyl-20p by a single amino acid. SLY2 and SLY12 encode proteins with hydrophobic tails similar to synaptobrevins, integral membrane proteins of synaptic vesicles in higher eucaryotes. Sly4lp is hydrophobic and exhibits sequence similarities with the chloroplast phosphate translocator. SLY12 but not SLY41 is an essential gene. The SLY2 null mutant is cold and heat sensitive. The SLY gene products may comprise elements of the protein transport machinery.
In the budding yeast Saccharomyces cerevisiae, vacuoles are inherited by the projection of vesicles and tubules from the mother‐cell vacuole into the growing daughter cell during the S phase. These vesicles then fuse and form the daughter‐cell organelle. We have described previously in vitro reactions of the formation of vacuole‐derived segregation structures and of vacuole‐vacuole fusion. Homotypic vacuole fusion requires cytosol, ATP and a physiological temperature, and is sensitive to GTPase inhibitors. These reactions are divisible into early stages which require ATP and cytosol, and late stages which require neither. Here, we report that Ypt7p, a ras‐like GTPase implicated previously in endocytosis in yeast, is largely localized to the vacuole and is required on both partners during the in vitro vacuole fusion reaction. The in vitro fusion reaction is inhibited either by Gdi1p, which extracts the GDP‐bound form of ras‐like GTPases from membranes, or by antibodies specific for Ypt7p. The presence of anti‐Ypt7p during the early stages of the reaction inhibits the development of cytosol‐ and ATP‐independent intermediates. Although cytosol and ATP are no longer needed for the late stage of vacuole inheritance in vitro, the inhibition of this late stage by anti‐Ypt7p or Gdi1p requires the continued presence of ATP and cytosol. Ypt7p is the first GTPase for which a direct role in organelle inheritance has been established.
Abstract. The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52 % identity to rab5, YP/51 and YF/53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YF/~2, whose protein product exhibits a similar identity to tab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and a-factor, was inhibited in Aypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51yptS2yptS3 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.
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