The N-glycosylation defect of cwh8Δ yeast cells causes a distinct defect in sphingolipid biosynthesis

Abstract

CWH8/YGR036c of Saccharomyces cerevisiae has been identified as a dolichylpyrophosphate (Dol-PP) phosphatase that removes a phosphate from the Dol-PP generated by the oligosaccharyltransferase (OST), while it adds N-glycans to nascent glycoproteins in the endoplasmic reticulum (ER). Lack of CWH8 was proposed to interrupt the so called dolichol (Dol) cycle by trapping Dol in the form of Dol-PP in the ER lumen. Indeed, cwh8Delta mutants display a severe deficiency in N-glycosylation. We find that cwh8Delta mutants have strongly reduced levels of inositolphosphorylceramide (IPC), whereas its derivative, mannosyl-(inositol-P)2-ceramide (M(IP)2C) is not affected. Microsomes of cwh8Delta contain normal ceramide synthase and IPC synthesis activities. Within a large panel of mutants affecting Dol dependent pathways such as N- or O-glycosylation, or glycosylphosphatidyl inositol (GPI)-anchoring, only the mutants having a deficiency of N-glycan addition show the defect in IPC biosynthesis. By mutating genes required for the addition of N-glycans or by treating cells with tunicamycin (Tm) one can similarly reduce the steady state level of IPC and exactly reproduce the phenotype of cwh8Delta cells. Some potential mechanisms by which the lack of N-glycans could lead to the sphingolipid abnormality were further explored.