Yeast Gaalp Is Required for Attachment of a Completed GPI Anchor onto Proteins

Anchoring of proteins to membranes by glycosylphosphatidylinositols (GPIs) is ubiquitous among all eukaryotes and heavily used by parasitic protozoa. GPI is synthesized and transferred en bloc to form GPI-anchored proteins. The key enzyme in this process is a putative GPI:protein transamidase that would cleave a peptide bond near the COOH terminus of the protein and attach the GPI by an amide linkage. We have identified a gene, GAA/, encoding an essential ER protein required for GPI anchoring, gaa/ mutant cells synthesize the complete GPI anchor precursor at nonpermissive temperatures, but do not attach it to proteins. Overexpression of GAA/improves the ability of cells to attach anchors to a GPIanchored protein with a mutant anchor attachment site. Therefore, Gaalp is required for a terminal step of GPI anchor attachment and could be part of the putative GPI:protein transamidase. CI-IORINC of cell surface proteins with various functions to membranes by covalent attachment of glycosylphosphatidylinositol (GPI) ~ is used by all eukaryotes examined thus far. Even though all eukaryotes have GPI-anchored proteins, the use of GPI anchoring is of particular importance in protozoal pathogens. In Trypanosoma brucei, the variable surface glycoprotein that forms part of the protective coat around the bloodstream form of the parasite is GPI anchored. The malarial circumsporozoite protein is also believed to have a GPI anchor (Englund, 1993; McConville and Ferguson, 1993). For this reason, GPI anchoring has been considered as a possible target pathway for intervention in diseases caused by protozoa and possibly fungi. In yeast cells, GPI synthesis and/or anchoring are essential for viability (Leidich et al., 1994), whereas in certain mouse and human cell lines, GPI anchoring is not required for growth and division (Hyman, 1988; Hirose et al., 1992). Despite the apparent ability of some human cells to survive without GPI anchoring, a human somatic cell disease, paroxysmal nocturnal hemoglobinuria, has been found to be due to a defect in this process (Takeda et al., 1993; Bessler et al., 1994). Address correspondence to Howard Riezman, Biozentrum of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel.: (41) 61 267 2160. Fax: (41) 61 267 2148. E-mail [email protected]. The current address of 13. Hamburger is MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824. The current address of M. Egerton is ICI Pharmaceuticals, Mereside, Alderly Park, Macclesfield, Cheshire SK10 4TG, UK. 1. Abbreviations used in this paper: DAPI, 4,6-diamidino-2-phenylindole; GPI, glycosylphosphatidylinositol; JBAM, jack bean a-mannosidase; PIPLC, phosphoinositide phospholipase C; PLD, phospholipase D. The structures of GPI anchors from several organisms have been determined, and there is a core structure that is highly conserved among all characterized GPI anchors. This core includes an inositol, glucosamine, three mannoses, and a phosphoethanolamine linked in an amide linkage to the COOH terminus of the protein. Apart from the core structure, there is a wide variety of side chain modifications and variation in glycerolipid and ceramide structures that are attached to the inositol of GPI anchors (McConville and Ferguson, 1993; Englund, 1993; Conzelmann et al., 1992; Fankhauser et al., 1993). Despite the similarity in core structures among all organisms, the mode of GPI biosynthesis is not completely conserved. In trypanosomes, the mannose residues of the core structure are added onto GIcNH-PI (Menon et al., 1990), whereas in mammalian cells and yeast, they are added onto GIcNH-PI with an acylated inositol ring (Stevens, 1993; Leidich et al., 1994). GPI-anchored proteins are synthesized as precursors with a classic, cleavable signal sequence at the NH2 terminus and an additional hydrophobic region at the COOH terminus of the protein, which acts as a signal to direct GPI anchoring. After or during import into the ER, the COOH-terminal hydrophobic region is removed and replaced with a preformed, complete GPI anchor (McConville and Ferguson, 1993; England, 1993). Initial experiments defining GPI anchoring signals showed an apparent similarity between different organisms. The signal was proposed to comprise a COOHterminal hydrophobic region, a short "spacer" between it, and the cleavage/attachment site (oJ site), which must be an amino acid with a small side chain. The 2 amino acids after the ~0 site should also have small side chains, but this requirement seems to depend upon the context of the signal (Micanovic et al., 1990; Moran and Caras, 1991; Moran et al., © The Rockefeller University Press, 0021-9525/95/05/629/11 $2.00 The Journal of Cell Biology, Volume 129, Number 3, May 1995 629-639 629 on July 9, 2017 jcb.rress.org D ow nladed fom 1991; Gerber et al., 1992; Kodukula et al., 1993; Nuoffer et al., 1991, 1993). Despite this apparent consensus, recent experiments have shown that the signal requirements are not identical among protozoa and animal cells. Protozoan GPIanchoring signals do not function in animal ceils (Moran and Caras, 1994). Therefore, the protein recognizing this signal, presumably the GPI:protein transamidase, is a potential target for chemotherapy. In this study we have characterized a yeast mutant, gaa/, that is able to synthesize the entire GPI precursor but cannot transfer it to proteins. The corresponding essential protein, Gaalp, is a multi-membrane-spanning protein of the ER with a relatively large luminal domain. Overproduction of Gaalp can partially suppress the GPI anchoring defect found in a protein with a mutated anchor attachment site. These data show that Gaalp is required for a terminal step of GPI anchor attachment and suggest that it may be part of the putative GPI:protein transamidase. Materials and Methods