Glycosylphosphatidylinositol Biosynthesis Defects in Gpi11p- and Gpi13p-deficient Yeast Suggest a Branched Pathway and Implicate Gpi13p in Phosphoethanolamine Transfer to the Third Mannose

Abstract

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 thegpi11 deletion, but the rescued haploids are temperature sensitive. Abolition of Gpi11p or Pig-Fp function inGPI11 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 Man4-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 Man4-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.

Glycosylphosphatidylinositol (GPI) Proteins of Saccharomyces cerevisiae Contain Ethanolamine Phosphate Groups on the α1,4-linked Mannose of the GPI Anchor

“…The presence of these side chains on CP2 came as a surprise in as much as a previous analysis of the pool of the protein-linked GPI anchors of S. cerevisiae had failed to reveal EtN-P or other substituents on Man1 or Man2 (14). Candidate EtN-P transferase genes required for the attachment of these substituents have been identified in humans and yeast: PIG-N and MCD4 are involved in the transfer of EtN-P from phosphatidylethanolamine (PE) onto Man1 (13,15,16), GPI7 in the transfer onto Man2 (12), and PIG-O and GPI13 in the transfer of EtN-P from PE onto Man3 (17)(18)(19)(20)(21). These genes are homologous to each other, are found throughout the eukaryotic kingdom, possess an N-terminal globular domain facing the lumen of the ER or the extracellular space and multiple transmembrane domains in their C terminus.…”

Human Smp3p Adds a Fourth Mannose to Yeast and Human Glycosylphosphatidylinositol Precursors in Vivo

“…YPGal medium has the same composition as YPD but with 2% galactose in place of glucose. Inositol-free synthetic medium and synthetic medium containing 0.2% yeast extract and glycerol (SGlyYE), galactose (SGalYE) or glucose (SGlcYE) were prepared as described previously (19). Sporulation of diploid cells was performed on 1% potassium acetate agar containing 5 g/ml of each supplement required to complement strain auxotrophies.…”

“…(19). Because smp3::Kan R /pGAL-hSMP3-HA does not grow well in SGlyYE medium, SGalYE medium was used in its place.…”

“…Radiolabeled lipids were extracted from both yeast and mammalian cells in chloroform:methanol:water (10:10:3, v/v/v) and partitioned in 1-butanol as described previously (19). Radiolabeled lipids were analyzed by separation on silica 60 TLC plates (EM Science, Darmstadt, Germany).…”

“…Radiolabeled lipids were analyzed by separation on silica 60 TLC plates (EM Science, Darmstadt, Germany). TLC plates were pre-run in solvent A (chloroform:methanol: Enzyme Treatment of Human GPI Intermediates-[ 3 H]Mannose-labeled GPI mannolipids were treated with PI-phospholipase C as described previously (19), with 10 units of jack bean ␣-mannosidase (JB␣M; Sigma) for 20 h at 37°C in 3 M (NH 4 ) 2 SO 4 , 0.1 mM zinc acetate, pH 7.5, 0.5% Nonidet P-40 Substitute (Sigma), or in 10% fetal bovine serum as a source of GPI-phospholipase D at 37°C for 24 h in 50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 2.6 mM CaCl 2 , 0.1% Triton X-100, 0.01% Nonidet P-40 Substitute. After enzyme treatment, lipids were partitioned in 1-butanol prior to TLC separation.…”