YLL031c belongs to a novel family of membrane proteins involved in the transfer of ethanolaminephosphate onto the core structure of glycosylphosphatidylinositol anchors in yeast

Abstract

MCD4 and GPI7 are important for the addition of glycosylphosphatidylinositol (GPI) anchors to proteins in the yeast Saccharomyces cerevisiae. Mutations in these genes lead to a reduction of GPI anchoring and cell wall fragility. Gpi7 mutants accumulate a GPI lipid intermediate of the structure Manα1-2[NH2-(CH2)2-PO4]Man-α1-2Manα1-6[NH2-(CH2)2-PO4] Manα1-4GlcNα1-6 [acyl]inositol-PO4-lipid, which, in comparison with the complete GPI precursor lipid CP2, lacks an HF-sen-sitive side chain on the α1-6-linked mannose. In contrast, mcd4-174 accumulates only minor amounts of abnormal GPI intermediates. Here we investigate whether YLL031c, an open reading frame predicting a further homologue of GPI7 and MCD4, plays any role in GPI anchoring. YLL031c is an essential gene. Its depletion results in a reduction of GPI anchor addition to GPI proteins as well as to cell wall fragility. YLL031c-depleted cells accumulate GPI intermediates with the structures Manα1-2Manα1-2Manα1-6[NH2-(CH2)2-PO4]Manα1-4GlcNα1-6[a cyl]inositol-PO4-lipid and Manα1-2Manα1-2Manα1-6Manα1-4GlcNα1-6[acyl] inositol-PO4-lipid. Subcellular localization studies of a tagged version of YLL031c suggest that this protein is mainly in the ER, in contrast to Gpi7p, which is found at the cell surface. The data are compatible with the idea that YLL031c transfers the ethanolaminephosphate to the inner α1-2-linked mannose, i.e. the group that links the GPI lipid anchor to proteins, whereas Mcd4p and Gpi7p transfer ethanolaminephosphate onto the α1-4- and α1-6-linked mannoses of the GPI anchor, respectively.