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 and intracellular transport of certain secretory proteins. GPIs have a conserved trimannosyl core bearing a phosphoethanolamine (EthN-P) moiety on the third 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 described. We report on two essential yeast genes whose products are required late in GPI assembly. GPI11 (YDR302w) encodes a homologue of human Pig-Fp, a protein implicated in the addition of EthN-P to Man-3. PIG-F complements the gpi11 deletion, but the rescued haploids are temperature sensitive. Abolition of Gpi11p or Pig-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 positioning and number of side chains, probably EthN-Ps. The less polar GPI bears EthN-P on Man-2, whereas the more polar lipid has EthN-P on Man-3. The latter 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 bearing EthN-P on Man-3, and Gpi13p-deficient strains accumulate a Man(4)-GPI isoform that bears EthN-P on Man-1. We further show that the lipid accumulation phenotype of Gpi11p-deficient cells resembles that of cells lacking Gpi7p, a sequence homologue of Gpi13p known to add EthN-P to Man-2 of a late-stage GPI precursor. This result suggests that in yeast a Gpi11p-deficiency can affect EthN-P addition to Man-2 by Gpi7p, in contrast to the Pig-Fp defect in mammalian cells, which prevents EthN-P addition to Man-3. Because Gpi11p and Pig-Fp affect EthN-P transfer to Man-2 and Man-3, respectively, these proteins may act in partnership with the GPI-EthN-P transferases, although their involvement in a given EthN-P transfer reaction varies between species. Possible roles for Gpi11p in the supply of the EthN-P donor are discussed. 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 GPIs that accumulate in Gpi11p-defective strains are likely to have been generated independently of one another, we propose that the yeast GPI assembly pathway is branched.
Taron
C H
CH
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Wiedman
J M
JM
Grimme
S J
SJ
Orlean
P
P
D000595:Amino Acid Sequence; D001692:Biological Transport; D002240:Carbohydrate Sequence; D004983:Ethanolamines / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D005804:Genes, Lethal; D005816:Genetic Complementation Test; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D008055:Lipids / Q000737:chemistry; D008358:Mannose / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D004983:Ethanolamines; D005656:Fungal Proteins; C411468:GPI11 protein, S cerevisiae; D017261:Glycosylphosphatidylinositols; D008055:Lipids; D008565:Membrane Proteins; D011134:Polysaccharides; D029701:Saccharomyces cerevisiae Proteins; C005448:phosphorylethanolamine; C413542:GPI13 protein, S cerevisiae; D017853:Phosphotransferases (Alcohol Group Acceptor); D008358:Mannose