Expression cloning of PIG-L, a candidate N-acetylglucosaminyl-phosphatidylinositol deacetylase.
Many eukaryotic cell surface proteins are bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Several genes involved in GPI anchor biosynthesis have been cloned using complementation of mutant mammalian cell lines and yeasts that are defective in its biosynthesis pathway. However, the gene involved in the second step of this pathway, in which N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is N-deacetylated to form glucosaminyl (GlcN)-PI, has not been cloned. In this study, we established a GPI anchor-deficient mutant of Chinese hamster ovary (CHO) cells defective in the second step. Complementation analysis with the known GPI anchor mutant cells demonstrated that it belonged to the same complementation group as the CHO cell mutant G9PLAP.85. Using the new mutant, we cloned a rat gene termed PIG-L (for phosphatidylinositol glycan class L) that is involved in this step. PIG-L encodes a 252-amino acid, endoplasmic reticulum membrane protein, most of which is in the cytoplasmic side. This orientation of PIG-L protein is consistent with the notion that the second step of GPI anchor biosynthesis occurs on the cytoplasmic side of the endoplasmic reticulum.
Nakamura
N
N
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka 565, Japan.
Inoue
N
N
Watanabe
R
R
Takahashi
M
M
Takeda
J
J
Stevens
V L
VL
Kinoshita
T
T
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018958:CD55 Antigens / Q000235:genetics / Q000378:metabolism; D018957:CD59 Antigens / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry; D005020:Ethyl Methanesulfonate / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009153:Mutagens / Q000378:metabolism; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000737:chemistry; D051381:Rats; D014162:Transfection
D018958:CD55 Antigens; D018957:CD59 Antigens; D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D009153:Mutagens; D012333:RNA, Messenger; D005020:Ethyl Methanesulfonate; D000581:Amidohydrolases; C087696:N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase