The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5 extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer. hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins. The gene spans Ϸ25 kb of DNA on chromosome 1. Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins. Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase.

Every protein fated to receive the glycophosphatidylinositol (GPI) anchor post-translational modification has a C-terminal GPI-anchor attachment signal sequence. This signal peptide varies with respect to length, content, and hydrophobicity. With the exception of predictions based on an upstream amino acid triplet termed omega-->omega + 2 which designates the site of GPI uptake, there is no information on how the efficiencies of different native signal sequences compare in the transamidation reaction that catalyzes the substitution of the GPI anchor for the C-terminal peptide. In this study we utilized the placental alkaline phosphatase (PLAP) minigene, miniPLAP, and replaced its native 3' end-sequence encoding omega-2 to the C-terminus with the corresponding C-terminal sequences of nine other human GPI-anchored proteins. The resulting chimeras then were fed into an in vitro processing microsomal system where the cleavages leading to mature product from the nascent preproprotein could be followed by resolution on an SDS-PAGE system after immunoprecipitation. The results showed that the native signal of each protein differed markedly with respect to transamidation efficiency, with the signals of three proteins out-performing the others in GPI-anchor addition and those of two proteins being poorer substrates for the GPI transamidase. The data additionally indicated that the hierarchical order of efficiency of transamidation did not depend solely on the combination of permissible residues at omega-->omega + 2.

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...