The N-glycosylation defect of cwh8Delta yeast cells causes a distinct defect in sphingolipid biosynthesis

CWH8/YGR036c of Saccharomyces cerevisiae has been identifiedas a dolichylpyrophosphate (Dol-PP) phosphatase that removesa phosphate from the Dol-PP generated by the oligosaccharyltransferase(OST), while it adds N-glycans to nascent glycoproteins in theendoplasmic reticulum (ER). Lack of CWH8 was proposed to interruptthe so called dolichol (Dol) cycle by trapping Dol in the formof Dol-PP in the ER lumen. Indeed, cwh8D mutants display a severedeficiency in N-glycosylation. We find that cwh8D mutants havestrongly reduced levels of inositolphosphorylceramide (IPC),whereas its derivative, mannosyl-(inositol-P)₂-ceramide (M(IP)₂C)is not affected. Microsomes of cwh8D contain normal ceramidesynthase and IPC synthesis activities. Within a large panelof mutants affecting Dol dependent pathways such as N- or O-glycosylation,or glycosylphosphatidyl inositol (GPI)-anchoring, only the mutantshaving a deficiency of N-glycan addition show the defect inIPC biosynthesis. By mutating genes required for the additionof N-glycans or by treating cells with tunicamycin (Tm) onecan similarly reduce the steady state level of IPC and exactlyreproduce the phenotype of cwh8D cells. Some potential mechanismsby which the lack of N-glycans could lead to the sphingolipidabnormality were further explored.