Pig-n, a mammalian homologue of yeast Mcd4p, is involved in transferring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol.
Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins. The core of the GPI anchor is conserved in all eukaryotes but is modified by various side chains. We cloned a mouse phosphatidylinositol glycan-class N (Pig-n) gene that encodes a 931amino acid protein expressed in the endoplasmic reticulum, which is homologous to yeast Mcd4p. We disrupted the gene in F9 embryonal carcinoma cells. In the Pig-n knockout cells, the first mannose in the GPI precursors was not modified by phosphoethanolamine. Nevertheless, further biosynthetic steps continued with the addition of the third mannose and the terminal phosphoethanolamine. The surface expression of Thy-1 was only partially affected, indicating that modification of the first mannose by phosphoethanolamine is not essential for attachment of GPI anchors in mammalian cells. An inhibitor of GPI biosynthesis, YW3548/BE49385A, inhibited transfer of phosphoethanolamine to the first mannose in mammalian cells but only slightly affected the surface expression of GPI-anchored proteins. Biosynthesis of GPI in the Pig-n knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug.
Hong
Y
Y
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
Maeda
Y
Y
Watanabe
R
R
Ohishi
K
K
Mishkind
M
M
Riezman
H
H
Kinoshita
T
T
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004983:Ethanolamines / Q000378:metabolism*; D005434:Flow Cytometry; D006023:Glycoproteins; D006031:Glycosylation / Q000187:drug effects; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology; D008358:Mannose / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010770:Phosphotransferases; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D013729:Terpenes / Q000494:pharmacology; D014162:Transfection
D004983:Ethanolamines; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D007783:Lactones; C118052:MCD4 protein, S cerevisiae; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013729:Terpenes; C109532:YW 3548; C005448:phosphorylethanolamine; D010770:Phosphotransferases; C403312:Pign protein, mouse; D008358:Mannose