Like most other eukaryotes, Saccharomyces cerevisiae harbors a GPI anchoring machinery and uses it to attach proteins to membranes. While a few GPI proteins reside permanently at the plasma membrane, a majority of them gets further processed and is integrated into the cell wall by a covalent attachment to cell wall glucans. The GPI biosynthetic pathway is necessary for growth and survival of yeast cells. The GPI lipids are synthesized in the ER and added onto proteins by a pathway comprising 12 steps, carried out by 23 gene products, 19 of which are essential. Some of the estimated 60 GPI proteins predicted from the genome sequence serve enzymatic functions required for the biosynthesis and the continuous shape adaptations of the cell wall, others seem to be structural elements of the cell wall and yet others mediate cell adhesion. Because of its genetic tractability S. cerevisiae is an attractive model organism not only for studying GPI biosynthesis in general, but equally for investigating the intracellular transport of GPI proteins and the peculiar role of GPI anchoring in the elaboration of fungal cell walls.

A 125-kDa glycoprotein exposed on the surface of Saccharomyces cerevisiae cells belongs to a class of eucaryotic membrane proteins anchored to the lipid bilayer by covalent linkage to an inositol-containing glycophospholipid. We have cloned the gene (GAS)) encoding the 125-kDa protein (Gaslp) and found that the function of Gaslp is not essential for cell viability. The nucleotide sequence of GAS) predicts a 60-kDa polypeptide with a cleavable N-terminal signal sequence, potential sites for N-and 0-linked glycosylation, and a C-terminal hydrophobic domain. Determination of the anchor attachment site revealed that the C-terminal hydrophobic domain of Gaslp is removed during anchor addition. However, this domain is essential for addition of the glycophospholipid anchor, since a truncated form of the protein failed to become attached to the membrane. Anchor addition was also abolished by a point mutation affecting the hydrophobic character of the C-terminal sequence. We conclude that glycophospholipid anchoring of Gaslp depends on the integrity of the C-terminal hydrophobic domain that is removed during anchor attachment.A number of eucaryotic membrane proteins are anchored to the lipid bilayer by a covalently linked glycosyl phosphatidylinositol (GPI) moiety (reviewed in references 20, 26, and 46). This particular mode of membrane attachment occurs in a wide variety of eucaryotic organisms. The modified proteins fall into diverse functional groups, including hydrolytic enzymes, cell adhesion molecules, protozoan coat proteins, and numerous cell surface antigens of unknown function.The complete structure of the GPI moiety has been determined for two forms of the variant surface glycoprotein of Trypanosoma brucei (25, 58) and the mammalian cell surface antigen . GPI anchors from these distantly related organisms share a common core structure, consisting of a phosphatidylinositol molecule linked to a linear tetrasaccharide composed of one nonacetylated glucosaminyl and three mannosyl residues. At its nonreducing end, the glycan is attached via a phosphodiester to ethanolamine, which is amide linked to the a-carboxyl group of the C-terminal amino acid of the mature protein.GPI-anchored proteins are commonly synthesized with a cleavable N-terminal signal sequence and a C-terminal domain composed predominantly of hydrophobic amino acids. This particular feature seems to be important in the mechanism of anchor addition. In all cases studied so far, addition of the GPI anchor involves the removal of 17 to 31 residues from the C terminus of a larger precursor (7,15,22,27,29,32,35,36,47,51,53,60,62,65,68). Since processing rapidly follows protein synthesis (2, 18, 24), it is believed that the GPI moiety is preassembled and transferred en bloc to the protein in the endoplasmic reticulum.Several lines of evidence suggest that a signal for GPI anchor attachment resides in the C-terminal domain of the proteins. However, the C-terminal sequences of GPI-anchored proteins do not exhibit any recognizable homology. Although the stru...

Gpi7 was isolated by screening for mutants defective in the surface expression of glycosylphosphatidylinositol (GPI) proteins. Gpi7 mutants are deficient in YJL062w, herein named GPI7. GPI7 is not essential, but its deletion renders cells hypersensitive to Calcofluor White, indicating cell wall fragility. Several aspects of GPI biosynthesis are disturbed in ⌬gpi7. The extent of anchor remodeling, i.e. replacement of the primary lipid moiety of GPI anchors by ceramide, is significantly reduced, and the transport of GPI proteins to the Golgi is delayed. Gpi7p is a highly glycosylated integral membrane protein with 9 -11 predicted transmembrane domains in the C-terminal part and a large, hydrophilic N-terminal ectodomain. The bulk of Gpi7p is located at the plasma membrane, but a small amount is found in the endoplasmic reticulum. GPI7 has homologues in Saccharomyces cerevisiae, Caenorhabditis elegans, and man, but the precise biochemical function of this protein family is unknown. Based on the analysis of M4, an abnormal GPI lipid accumulating in gpi7, we propose that Gpi7p adds a side chain onto the GPI core structure. Indeed, when compared with complete GPI lipids, M4 lacks a previously unrecognized phosphodiesterlinked side chain, possibly an ethanolamine phosphate. Gpi7p contains significant homology with phosphodiesterases suggesting that Gpi7p itself is the transferase adding a side chain to the ␣1,6-linked mannose of the GPI core structure. Glycosylphosphatidylinositol (GPI)1 -anchored proteins represent a subclass of surface proteins found in virtually all eukaryotic organisms (1). The genome of Saccharomyces cerevisiae contains more than 70 open reading frames (ORFs) encoding for proteins that, as judged from the deduced primary sequence, can be predicted to be modified by the attachment of a GPI anchor (2, 3). In about 25 of them, the presence of an anchor has been confirmed biochemically. A majority of them lose part of the anchor and become covalently attached to the ␤1,6-glucans of the cell wall (4 -6). A minority of GPI proteins retain the GPI anchor in an intact form and stay at the plasma membrane (PM). For the biosynthesis of GPI anchors, phosphatidylinositol (PI) is modified by the stepwise addition of sugars and ethanolamine phosphate (EtN-P), thus forming a complete precursor lipid (CP) which subsequently is transferred en bloc by a transamidase onto newly synthesized proteins in the ER (7,8). The identification of genes involved in the biosynthesis of the CP and its subsequent attachment to proteins has been possible through the complementation of mammalian and yeast gpi Ϫ mutants, i.e. mutants being deficient in GPI anchoring of membrane proteins (7, 9 -20). In our laboratory, a series of recessive gpi Ϫ mutants (gpi4 to gpi10) has been obtained by screening for yeast mutants that are unable to display the GPI-anchored ␣-agglutinin (Sag1p) at the outer surface of the cell wall, although the synthesis and secretion of soluble proteins is normal (21,22).Here we report on the characterization of...

The anchors of mature glycosylphosphatidylinositol (GPI)-anchored proteins of Saccharomyces cerevisiae contain either ceramide or diacylglycerol with a C26:0 fatty acid in the sn2 position. The primary GPI lipid added to newly synthesized proteins in the ER consists of diacylglycerol with conventional C16 and C18 fatty acids. Here we show that GUP1 is essential for the synthesis of the C26:0-containing diacylglycerol anchors. Gup1p is an ER membrane protein with multiple membranespanning domains harboring a motif that is characteristic of membrane-bound O-acyl-transferases (MBOAT). Gup1⌬ cells make normal amounts of GPI proteins but most mature GPI anchors contain lyso-phosphatidylinositol, and others possess phosphatidylinositol with conventional C16 and C18 fatty acids. The incorporation of the normal ceramides into the anchors is also disturbed. As a consequence, the ER-to-Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. Gup1⌬ cells have fragile cell walls and a defect in bipolar bud site selection. GUP1 function depends on the active site histidine of the MBOAT motif. GUP1 is highly conserved among fungi and protozoa and the gup1⌬ phenotype is partially corrected by GUP1 homologues of Aspergillus fumigatus and Trypanosoma cruzi.

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430 -650-kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430 -650-kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate for the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact.