Abstract
The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant.
Highlights
Dolichol synthesis is a multistep process whose initial stagesfrom the acetyl-CoA precursor up to farnesyldiphosphate (FPP)are a common part of the mevalonate pathway leading to the synthesis of all isoprenoids
We have previously demonstrated that the perturbation of dolichol synthesis following the suppression of the RER2 gene alters the integrity of the cell wall and prevents hyphae formation in C. albicans
The Cwh8p protein is involved in DolPP/dolichyl phosphate (DolP) recycling, needed for the reutilization of the DolP in subsequent rounds of DolPP-oligosaccharide biosynthesis and protein N-glycosylation [4]
Summary
Dolichol synthesis is a multistep process whose initial stagesfrom the acetyl-CoA precursor up to farnesyldiphosphate (FPP)are a common part of the mevalonate pathway leading to the synthesis of all isoprenoids. It has been proposed that in addition to the de novo dolichol synthesis, the DolP pool is enriched by the DolP released from the DolPP-oligosaccharide precursor after each cycle of glycosylation [4]. When the precursor oligosaccharide is transferred from the lipid carrier to an appropriate AsnXSer/Thr sequence in the nascent polypeptide, DolPP is released in the ER lumen and partially dephosphorylated to give DolP. Since in a cwh8∆ yeast mutant the capacity to form DolPP-oligosaccharide was reduced, it was assumed that the de novo synthesis of DolP is not sufficient to support the cell requirement for DolP and, at least in some systems, the recycling of DolP after glycosylation is needed to ensure its correct level
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