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

MCD4 and GP17 are important for the addition of glycosylphosphatidylinosito l (GPI) anchors to proteins in the yeast Saccharomyces cerevisiae, Mutation s in these genes lead to a reduction of GPI anchoring and cell wall fragili ty. Gpi7 mutants accumulate a GPI lipid intermediate of the structure Man a lpha 1-2[NH2-(CH2)(2)-PO4-->]Man-alpha 1-2Man alpha 1- 6[NH2-(CH2)(2)-PO4-- >]Man alpha 1- 4GlcN alpha 1-6 [acyl-->] linositol-PO4-lipid, which, in com parison with the complete GPI precursor lipid CP2, lacks an HF-sensitive si de chain on the alpha 1-6-linked mannose, In contrast, mcd4-174 accumulates only minor amounts of abnormal GPI intermediates. Here we investigate whet her YLL031c, an open reading frame predicting a further homologue of GP17 a nd MCD4, plays any rob 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 inter mediates with the structures Man alpha 1-2Man alpha 1-2Man alpha 1-6[NH2-(C H2)(2)PO4-->]Man alpha 1- 4GlcN alpha 1-6[acyl-->]inositol-PO4-lipid and Ma n alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6[acyl-->] i nositol-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 ide a that YLL031c transfers the ethanolaminephosphate to the inner alpha 1-2-l inked mannose, ie. the group that links the GPI lipid anchor to proteins, w hereas Mcd4p and Gpi7p transfer ethanolaminephosphate onto the alpha 1-4- a nd alpha 1-6-linked mannoses of the GPI anchor, respectively.