Abstract
Yeast Ypt6p, the homologue of the mammalian Rab6 GTPase, is not essential for cell viability. Based on previous studies with ypt6 deletion mutants, a regulatory role of the GTPase either in protein retrieval to the trans-Golgi network or in forward transport between the endoplasmic reticulum (ER) and early Golgi compartments was proposed. To assess better the primary role(s) of Ypt6p, temperature-sensitive ypt6 mutants were generated and analyzed biochemically and genetically. Defects in N-glycosylation of proteins passing the Golgi and of Golgi-resident glycosyltransferases as well as protein sorting defects in the trans-Golgi were recorded shortly after functional loss of Ypt6p. ER-to-Golgi transport and protein secretion were delayed but not interrupted. Mis-sorting of the vesicular SNARE Sec22p to the late Golgi was also observed. Combination of the ypt6-2 mutant allele with a number of mutants in forward and retrograde transport between ER, Golgi, and endosomes led to synthetic negative growth defects. The results obtained indicate that Ypt6p acts in endosome-to-Golgi, in intra-Golgi retrograde transport, and possibly also in Golgi-to-ER trafficking.
Highlights
Intercompartmental transport of proteins in secretion and endocytosis is affected by a variety of vesicle populations, each having its own biochemical identity
The data obtained from genetic interactions and from experiments following the kinetics of protein transport and sorting as well as the state of protein glycosylation at permissive and nonpermissive conditions are best explained by Ypt6p acting both in recycling of proteins from endosomes to the Golgi and from late to early Golgi compartments
Previous studies on the role of Ypt6p in intracellular protein transport have led to two quite different models, one suggesting an involvement of this GTPase in recycling between endosome(s) and the trans-Golgi [7, 8], and the other postulating a regulatory role in anterograde transport between early Golgi compartments [11, 12]
Summary
Yeast transformations, mating, sporulation, and tetrad analyses were performed using standard techniques [19, 20]. C-Myc epitope tagging of ORFs was performed as described previously [21]. To produce pKS-YPT6-URA3 for site-directed mutagenesis, the 1.4-kb XbaI YPT6 gene fragment from pRS315-YPT6 was. MSUC-1A MSUC-3D ZLY1 ZLY2 ZLY2-K ZLY2-1A ZLY3 ZLY4 ZLY45 ZLY361 ZLY351 ZLY352 ZLY353 ZLY354 ZLY355 ZLY356 ZLY357 ZLY358 ZLY359 ZLY360 ZLY184 ZLY185 RPY18 RPY41 RPY108 RPY116 YXY12␣ YXY136 YLX15 BSH-1B CTY1-1A GWY67 MB7 YTX50 PC70 PC130 PC159 RH227-3A RH236-3A RH237-1A RH239-5A S27P4-9C
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