Glycosylphosphatidylinositol (GPI) substitution is now recognized to be a ubiquitous method of anchoring a protein to membranes in eukaryotes. The structure of GPI and its biosynthetic pathways are known and the signals in a nascent protein for GPI addition have been elucidated. The enzyme(s) responsible for GPI addition with release of a COOH-terminal signal peptide has been considered to be a transamidase but has yet to be isolated, and evidence that it is a transamidase is indirect. The experiments reported here show that hydrazine and hydroxylamine, in the presence of rough microsomal membranes, catalyze the conversion of the pro form of the engineered protein miniplacental alkaline phosphatase (prominiPLAP) to mature forms from which the COOH-terminal signal peptide has been cleaved, apparently at the same site but without the addition of GPI. The products, presumable the hydrazide or hydroxamate of miniPLAP, have yet to be characterized definitively. However, our demonstration of enzyme-catalyzed cleavage of the signal peptide in the presence of the small nucleophiles, even in the absence of an energy source, is evidence of an activated carbonyl intermediate which is the hallmark of a transamidase.

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...

Rough microsomal membranes from most mammalian cells, in the presence of a translation system, process nascent proteins with appropriate COOH-terminal signal peptides to their mature glycosylphosphatidylinositol (GPI)-linked forms. The present study, using preprominiplacental alkaline phosphatase as substrate, shows that as much as 10%Yo of the mature product is cleaved correctly but is not linked to GPI. Some of the factors that influence the relative proportions of GPI linked to free mini-placental alkaline phosphatase are the amounts ofGPI in the cells and the amino acid substituent at the o site of the nascent protein. A mechanism for explaining cleavage both with and without GPI addition is presented, which supports a transamidase type of enzyme as the catalyst.A nascent protein (preproprotein), prior to processing to a glycosylphosphatidylinositol (GPI) form, contains both NH2-and COOH-terminal signal peptides that are cleaved sequentially to yield the mature form. Cleavage of the NH2-terminal peptide follows translocation of the nascent protein into the endoplasmic reticulum and results in the proprotein. Cleavage of the COOH terminus from the latter, accompanied by attachment of the GPI moiety, yields the mature protein. We have investigated processing in cell-free systems using a shortened, engineered form of nascent placental alkaline phosphatase (PLAP) to which we have given the name preprominiPLAP (see Fig. 1). In studies with rough microsomal membranes (RMs) from normal and GPI-deficient cells, we presented evidence that GPI is an obligatory cosubstrate for COOH-terminal processing of nascent proteins (1). During those studies we noticed that, in addition to the major product of the reaction, mature GPI-linked miniPLAP, a minor product is also formed. From its migration on gels, we suggested that this could represent mature miniPLAP that was not GPI-linked. In this report, we present clear evidence that processing of preprominiPLAP in the endoplasmic reticulum to its mature GPI-linked form is accompanied by some cleavage at the w site (the amino acid in a nascent protein that accepts the GPI moiety and, after cleavage, becomes the COOH-terminal residue of the mature protein) without GPI addition. The significance of this finding with respect to the mechanism of cleavage/GPI addition is discussed.

Proteins anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) moiety are found in all eukaryotes. After NH2-terminal peptide cleavage of the nascent protein by the signal peptidase, a second COOH-terminal signal peptide is cleaved with the concomitant addition of the GPI unit. The proposed mechanism of the GPI transfer is a transamidation reaction that involves the formation of an activated carbonyl intermediate (enzyme-substrate complex) with the ethanolamine moiety of the preassembled GPI unit serving as a nucleophile. Other nucleophilic acceptors like hydrazine (HDZ) and hydroxylamine have been shown to be possible alternate substrates for GPI. Since GPI has yet to be purified, the use of readily available nucleophilic substitutes such as HDZ and hydroxylamine is a viable alternative to study COOH-terminal processing by the putative transamidase. As a first step in developing a soluble system to study this process, we have examined the amino acid requirements at the COOH terminus for the transamidation reaction using HDZ as the nucleophilic acceptor instead of GPI. The hydrazideforming reaction shows identical amino acid requirement profiles to that of GPI anchor addition. Additionally, we have studied other parameters relating to the kinetics of the transamidation reaction in the context of rough microsomal membranes. The findings with HDZ provide further evidence for the transamidase nature of the enzyme and also provide a starting point for development of a soluble assay.Glycosylphosphatidylinositol (GPI) anchoring is a mechanism used by all eukaryotic cells to tether proteins to the outer cell membrane (1-7). A nascent protein (preproprotein) that is destined to be GPI anchored contains an NH2-terminal signal sequence and a COOH-terminal signal sequence, both of which are cleaved sequentially first by the NH2-terminal signal peptidase and then by a putative transamidase to yield the mature GPI-linked protein. Cleavage at the COOH-terminal signal proceeds in a concerted manner in which the GPI moiety is incorporated at the newly exposed COOH terminus of the protein.The amino acid residue that condenses with the GPI moiety is designated the X residue (8,9). Analogous to the -1, -3 rule that has been described for NH2-terminal signal peptidase activity, an wc,w+2 rule for COOH-terminal processing has been described (8). (8) (Fig. 1A).In previous work with an in vitro translation system employing rough microsomal membranes (RM) (8), we demonstrated that the proform of an engineered version of human placental alkaline phosphatase (PLAP), miniPLAP, is processed to the mature COOH-terminal cleaved form. Processing of nascent preprominiPLAP (28 kDa) gives rise to prominiPLAP (27 kDa), in which the NH2-terminal signal sequence is removed. This proform is subsequently converted to mature miniPLAP (24.7 kDa), in which a 29-residue COOH-terminal signal sequence is cleaved and replaced by GPI. In addition to the formation of this anchor-containing product (Fig. 1B), cleavage of prominiPLAP...

No abstract