A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.

Abstract

The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cellsurface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOHterminal processing of the resulting prominiPLAP to GPIanchored miniPLAP. Proteinase K protection assays verified that NH2-terminal-processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery.Posttranslational glycosylphosphatidylinositol (GPI) anchor addition serves as a general mechanism for linking a number of proteins to the outer leaflet of the cell-surface membrane (for review, see refs. 1 and 2). This mechanism is used by all eukaryotic cell types thus far studied and attaches proteins with a wide range of functions. Anchor incorporation into these proteins occurs in a concerted reaction in which preassembled GPI donors are substituted for COOH-terminal signal sequences in their nascent polypeptides following translocation of these polypeptides across the endoplasmic reticulum (ER) membrane.Most, if not all, of the intermediate steps in the biosynthesis of the GPI moiety have been elucidated (3-7). In the initial phase of the process, GlcN-acyl phosphatidylinositol (PI) is synthesized by assembly, deactylation, and acylation of GlcNAc-PI. In subsequent reactions, mannose (Man) and ethanolamine-phosphate (EthN-P) are added sequentially to this intermediate to produce the glycan core. Mutant cells defective in one or another of these biosynthetic steps do not exhibitThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.GPI proteins on their surfaces (8-10). In vitro translatio...

The Role of LORELEI in Pollen Tube Reception at the Interface of the Synergid Cell and Pollen Tube Requires the Modified Eight-Cysteine Motif and the Receptor-Like Kinase FERONIA

“…In yeast (Schizosaccharomyces pombe) and human cells, gpi8 mutants are defective in GPI attachment and cause a decrease in cell surface display of GPI-anchored proteins (Benghezal et al, 1996;Chen et al, 1996;Yu et al, 1997). The Arabidopsis genome contains a single gene (GPI8) that encodes a protein with high sequence similarity to yeast (43.8% identity) and human (45.7% identity) GPI8 and contains the postulated protease catalytic site that is conserved in C13 clade of cysteine proteases (Supplemental Figures 6A and 6B) (Zacks and Garg, 2006).…”

Thematic review series: Lipid Posttranslational Modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids

“…3A). Gaa1p and Gpi8p were the first to be identified through genetic approaches (143)(144)(145)(146) and were subsequently shown to form a complex (147); the other subunits were identified mainly because they specifically coimmunoprecipitated with the GPI8/Gpi8p-GAA1/Gaa1p complex (148)(149)(150)(151). GPITs from Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana are similar to the mammalian/ yeast enzyme.…”

Gaa1p and Gpi8p Are Components of a Glycosylphosphatidylinositol (GPI) Transamidase That Mediates Attachment of GPI to Proteins

“…A human mutant cell line termed class K that is defective in attachment of GPI (Mohney et al, 1994;Chen et al, 1996) is due to a defect in the human GPI8 gene (Yu et al, 1997). Microsomal membranes of class K cells did not have GPI transamidase activity (Chen et al, 1996;Yu et al, 1997). It was therefore suggested that Gpi8p is a component of the GPI transamidase (Benghezal et al, 1996;Yu et al, 1997).…”

“…GPI8 encodes a protein with homology to members of a family of cysteine proteases (Benghezal et al, 1996), one of which, a jack bean asparaginyl endopeptidase, showed transamidase activity in vitro (Abe et al, 1993). A human mutant cell line termed class K that is defective in attachment of GPI (Mohney et al, 1994;Chen et al, 1996) is due to a defect in the human GPI8 gene (Yu et al, 1997). Microsomal membranes of class K cells did not have GPI transamidase activity (Chen et al, 1996;Yu et al, 1997).…”