In vivo characterization of the GPI assembly defect in yeast mcd4-174 mutants and bypass of the Mcd4p-dependent step in mcd4Δ cells

Abstract

Yeast mcd4-174 mutants are blocked in glycosylphosphatidylinositol (GPI) anchoring of protein, but the stage at which GPI biosynthesis is interrupted in vivo has not been identified, and Mcd4p has also been implicated in phosphatidylserine and ATP transport. We report that the major GPI that accumulates in mcd4-174 in vivo is Man2-GlcN-(acyl-Ins)PI, consistent with proposals that Mcd4p adds phosphoethanolamine to the first mannose of yeast GPI precursors. Mcd4p-dependent modification of GPIs can partially be bypassed in the mcd4-174/gpi11 double mutant and in mcd4Δ; mutants by high-level expression of PIG-B and GPI10, which respectively encode the human and yeast mannosyltransferases that add the third mannose of the GPI precursor. Rescue of mcd4Δ; by GPI10 indicates that Mcd4p-dependent addition of EthN-P to the first mannose of GPIs is not obligatory for transfer of the third mannose by Gpi10p. © 2006 Federation of European Microbiological Societies.