Saccharomyces cerevisiae sec59 cells are deficient in dolichol kinase activity.

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 ...

Dissecting the role of dolichol in cell wall assembly in the yeast mutants impaired in early glycosylation reactions

“…The sec 59-1 strain displays a growth phenotype at non-permissive temperature resulting from a very low pool of DolP (Heller et al,1992). The RER2 gene expressed from the PMA1 promoter Table 4.…”

“…Quantitative measurements showed that the level of dolichyl phosphate in sec 59-1 cells was decreased to 48% of wild-type at the permissive temperature, and to below 10% after the shift of the cells to the restrictive temperature. Analysis of the activity of enzymes from the dolichyl phosphate biosynthetic pathway revealed that the cells are defective in CTP-dependent dolichol kinase activity at both the permissive and the restrictive temperature (Heller et al, 1992). A gene complementing the sec 59 mutation was cloned and sequenced (Bernstein et al, 1989).…”

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP

“…Various models have been proposed for these final steps in dolichol biosynthesis (Chojnacki & Dallner, 1988). For example, Heller et al (1992) proposed that once the appropriate isoprenoid chain length has been attained, the a-unsaturated pyrophosphate derivative is dephosphorylated to the monophosphate, followed by a-saturation to yield Dol-P. However, based on [ 3 H]mevalonate labelling studies in membrane fractions, Ekström et al (1987) proposed that terminal isoprene condensation and a-saturation occur cooperatively.…”

“…Allen et al (1978Allen et al ( , 1982 first showed CTP-dependent dolichol phosphorylation in mammalian tissues. The CTP-dependent dolichol kinase (DK) in yeast is encoded by sec59 (Heller et al, 1992) which, through complementation studies, led to the cloning of human DK (Fernandez et al, 2002;Shridas & Waechter, 2006). Recently, mutations in DK have been associated with autosomal recessive forms of dilated cardiomyopathy in paediatric patients, extending the genes in dolichol biosynthesis associated with CDGs (Denecke & Kranz, 2009;Kapusta et al, 2013;Kranz et al, 2007;Lefeber et al, 2011).…”

N-Linked Protein Glycosylation in the Endoplasmic Reticulum

“…The a-saturated isoprene is then phosphorylated to form dolichyl phosphate (Dol-P). In yeast, SEC59 encodes the CTP-dependent dolichol kinase and complementation of the yeast sec59-1 conditional mutant with cDNA allowed the identification of the human homolog of SEC59, hDK1 (Heller et al 1992;Fernandez et al 2002).…”