No abstract

The temperature-sensitive Saccharomyces cerevisiae mutant sec59 accumulates inactive and incompletely glycosylated protein precursors in its endoplasmic reticulum at the restrictive temperature. O-mannosylation and glycosyl phosphatidylinositol membrane anchoring of protein are also abolished, consistent with a deficiency in dolichyl phosphate mannose. Membranes prepared from sec59 cells that had been shifted to the restrictive temperature, however, made normal amounts of dolichyl phosphate mannose when exogenous dolichyl phosphate was supplied, but dolichyl phosphate mannose synthesis was severely depressed in the absence of exogenous dolichyl phosphate. Quantitative measurements of dolichyl phosphate in secS9 cells showed that the levels were decreased to 48% ofwild type at the permssive temperature and to <10% at the restrictive temperature. Assays of enzymes from the dolichyl phosphate synthetic pathway, cis-prenyltransferase and dolichyl pyrophosphate phosphatase, gave wild-type levels. However, dolichol kinase activity was greatly decreased. When sec59 cells were transformed with a plasmid that overexpresses the wild-type gene, dolichol kinase activity increased 10-fold over wild-type levels. These results strongly suggest that the sec59 gene encodes dolichol kinase.Saccharomyces cerevisiae secretory (sec) mutants stop dividing and become enlarged and dense at the restrictive temperature, 370C (1), a property that allowed the selection of secretory mutants by density gradient centrifugation. The mutant secS9 was isolated in this manner and characterized as a class B secretory mutant, one which accumulates inactive and incompletely glycosylated secretory proteins at the restrictive temperature (2). Protein synthesis, as measured by radiolabeling with 35SO(4, remains normal for 2 hr while oligosaccharide synthesis as measured by [3H]mannose incorporation is decreased (2). The peptide forms accumulated in the endoplasmic reticulum (ER) bear fewer oligosaccharide chains (2) and these are also shorter on average than those attached at the permissive temperature (3).In addition to this effect on N-glycosylation, secS9 cells are also completely blocked in O-mannosylation and in the synthesis of glycosyl phosphatidylinositol (GPI) membrane anchors (4, 5), processes that require dolichyl phosphate mannose (Dol-P-Man) as donor. In these respects, secS9 cells have a very similar biochemical phenotype to that ofthe yeast class B mutant, secS, which is defective in phosphomannomutase, and hence in GDP-mannose supply (6).The gene complementing the secS9 mutation was cloned from a YEp13 yeast genomic library and sequenced (3). A highly hydrophobic 59-kDa protein was predicted, containing a sequence resembling the putative dolichol binding region of three glycosyltransferases, Leu-Phe-Val-Xaa-Phe-Xaa-XaaIle-Pro-Phe-Xaa-Phe-Tyr (7).The fact that the predicted SEC59 gene product contains a putative dolichol binding region, as well as the apparent decrease in the levels of Dol-P-Man, suggested that the deficiency in ...

No abstract