Glycosylphosphatidylinositol biosynthesis defects in Gpi11p-and Gpi13p-deficient yeast suggest a branched pathway and implicate Gpi13p in phosphoethanolamine transfer to the third mannose

Glycosylphosphatidylinositols (GPIs) are critical for membrane anchoring an d intracellular transport of certain secretory proteins. GPIs have a conser ved trimannosyl core bearing a phosphoethanolamine (EthN-P) moiety on the t hird mannose (Man-3) through which the glycolipid is linked to protein, but diverse GPI precursors with EthN-Ps on Man-1 and Man-2 have also been desc ribed. We report on two essential yeast genes whose products are required l ate in CPI assembly. GPI11 (YDR302w) encodes a homologue of human Mg-Fp, a protein implicated in the addition of EthN-P to Man-3. PIG-F complements th e gpi11 deletion, but the rescued haploids are temperature sensitive. Aboli tion of Gpi11p or rig-Fp function in GPI11 disruptants blocks GPI anchoring and formation of complete GPI precursors and leads to accumulation of two GPIs whose glycan head groups contain four mannoses but differ in the posit ioning and number of side chains, probably EthN-Ps. The less polar GPI bear s EthN-P on Man-2, whereas the more polar lipid has EthN-P on Man-3. The la tter finding indicates that Gpi11p is not required for adding EthN-P to Man -3. Gpi13p (YLL031cp), a member of a family of phosphoryltransferases, is a candidate for the enzyme responsible for adding EthN-P to Man-3. Depletion of Gpi13p in a Gpi11p-defective strain prevents formation of the GPI beari ng EthN-P on Man-3, and Gpi13p-deficient strains accumulate a Man(4)-GPI is oform that bears EthN-P on Man-1. We further show that the lipid accumulati on phenotype of Gpi11p-deficient cells resembles that of cells lacking Gpi7 p a sequence homologue of Gpi13p known to add EthN-P to Man-2 of a late-sta ge GPI precursor. This result suggests that in yeast a Gpi11p-deficiency ca n affect EthN-P addition to Man-2 by Gpi7p, in contrast to the Pig-Fp defec t In mammalian cells, which prevents EthN-P addition to Man-3. Because Gpi1 1p and Pig-Fp affect EthN-P transfer to Man-2 and Man-3, respectively, thes e proteins may act in partnership with the GPI-EthN-P transferases, althoug h their involvement in a given EthN-P transfer reaction varies between spec ies. Possible roles for Gpi11p in the supply of the EthN-P donor are discus sed. Because Gpi11p- and Gpi13p-deficient cells accumulate isoforms of Man( 4)-GPIs with EthN-P on Man-2 and on Man-1, respectively, and because the GP Is that accumulate in Gpi11p-defective strains are likely to have been gene rated independently of one another, we propose that the yeast GPI assembly pathway is branched.