Ethanolaminephosphate side chain added to glycosylphosphatidylinositol (GPI) anchor by Mcd4p is required for ceramide remodeling and forward transport of GPI proteins from endoplasmic reticulum to golgi

Glycosylphosphatidylinositol (GPI) anchors of mammals as well as yeast contain ethanolaminephosphate side chains on the alpha 1-4- and the alpha 1-6-linked mannoses of the anchor core structure (protein-CO-NH-(CH2)(2)-PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH(2)-inositol-PO4- lipid). In yeast, the ethanolaminephosphate on the alpha 1-4-linked mannose is added during the biosynthesis of the GPI lipid by Mcd4p. MCD4 is essential because Gpi10p, the mannosyltransferase adding the subsequent alpha 1-2-linked mannose, requires substrates with an ethanolaminephosphate on the alpha 1-4-linked mannose. The Gpi10p ortholog of Trypanosoma brucei has no such requirement. Here we show that the overexpression of this ortholog rescues mcd4 Delta cells. Phenotypic analysis of the rescued mcd4 Delta cells leads to the conclusion that the ethanolaminephosphate on the alpha 1-4-linked mannose, beyond being an essential determinant for Gpi10p, is necessary for an efficient recognition of GPI lipids and GPI proteins by the GPI transamidase for the efficient transport of GPI-anchored proteins from the endoplasmic reticulum to Golgi and for the physiological incorporation of ceramides into GPI anchors by lipid remodeling. Furthermore, mcd4 Delta cells have a marked defect in axial bud site selection, whereas this process is normal in gpi7 Delta and gpi1. This also suggests that axial bud site selection specifically depends on the presence of the ethanolaminephosphate on the alpha 1-4-linked mannose.