Glucose-Induced Sequential Processing of a Glycosyl-Phosphatidylinositol-Anchored Ectoprotein in Saccharomyces cerevisiae

Abstract

An increasing number of surface proteins are found anchored at the plasma membrane of various tissues and cell types by virtue of a glycosyl-phosphatidylinositol (GPI) structure instead of a transmembrane protein domain. This kind of membrane anchor typically contains, in addition to the myoinositol-harboring phospholipid, a carbohydrate core consisting of one nonacetylated glucosamine and three mannose residues in characteristic linkage. The latter may be branched and carry additional mannose, galactose, and/or phosphoethanolamine moieties. The preformed anchor structure is linked by a phosphodiester and an ethanolamine bridge to the carboxy terminus of the respective protein, thereby replacing a proteinaceous transmembrane domain present in the newly synthesized protein precursor (for reviews, see references 14, 17, 31, 52, and 56). The physiological significance of membrane anchorage by a glycolipidic structure has remained greatly obscure. Since GPI-anchored proteins have been detected recently also in lower eucaryotes, including the yeast Saccharomyces cerevisiae (9,35,43,54), the evolutionary conservation from S. cerevisiae to humans of the GPI anchor argues that it confers properties on the respective proteins that are not achieved by a proteinaceous transmembrane domain.It has been proposed that the spatial requirements, clustering behavior, and diffusion properties of GPI-anchored proteins in the outer leaflet of plasma membranes are the parameters critical for the use of either a transmembrane domain or a GPI moiety for membrane anchorage of a protein (7,13,61,62). In addition, it has been proposed, on the basis that, in situ, GPI anchors may be cleaved upon activation of endogenous (glycosyl)-phosphatidylinositol-specific phospholipases ([G]PIPLs), that GPI anchorage plays a role in regulating the concentration of certain proteins at the cell surface or in controlling protein localization and topology: in HeLa cells and in cultured bone marrow stromal cells, GPI-anchored decay-accelerating factor and heparan sulfate proteoglycan, respectively, are apparently removed constitutively from the plasma membrane and released into the culture medium by the action of an endogenous of type D (G)PI-PL ([G]PI-PLD) (32). In the yeast S. cerevisiae, expression of ␣-agglutinin at the cell wall depends on its synthesis as a GPI-anchored protein and occurs via an intermediate which lacks the myo-inositol moiety of the GPI anchor glycan (28, 59). However, other than (G)PI-PLs, which leave the myo-inositol residue attached to the glycan structure, processing enzymes of GPI anchors have not been identified. In addition to these cases of constitutive GPI anchor processing, in some vertebrate cells certain external signals, provided by serum factors like insulin and growth factors or by drugs, have been found to activate phospholipases which are capable of cleaving GPI anchors (20,26,40,48). In the case of the insulin-or sulfonylurea-induced lipolytic cleavage of the GPI anchors of lipoprotein lipase, 5Ј-nucleotidase, and ...

Cell Wall-linked Cryptococcal Phospholipase B1 Is a Source of Secreted Enzyme and a Determinant of Cell Wall Integrity

“…Only the latter fraction contained ␤-1,6-linked glucan, a sugar linkage that is unique to the fungal cell wall, indicating that there is a covalent association between Plb1 and cell wall ␤-1,6-linked glucan via either a mannose sugar within the Plb1 GPI anchor or an amino acid in the Plb1 C terminus (14,33). The source of the non-covalently bound Plb1, which lacked the ␤-1,6-linked glucan, is presumably the cell membrane.…”

“…The source of the non-covalently bound Plb1, which lacked the ␤-1,6-linked glucan, is presumably the cell membrane. Endogenous (G)PI-PLC activity has been associated with the release of GPI-anchored proteins from S. cerevisiae protoplasts (14).…”

“…Evidence from studies in the model yeast, S. cerevisiae and the pathogenic yeast, Candida albicans, demonstrate that GPI anchors direct the transport of the attached proteins to the plasma membrane (10,11) where they are rendered hydrophobic by attachment to the lipid membrane anchor (12). Some of these proteins are released as hydrophilic mannoproteins, for example, following cleavage by GPI-specific phospholipase C ((G)PI-PLC) (13,14), and routed to the cell wall (15) where they form much of its outer layer. In S. cerevisiae, C. albicans, and Aspergillus species these proteins attach specifically to ␤-1,6-linked glucans in the central layer of the cell wall.…”

Amino Acid Sequence Requirement for Efficient Incorporation of Glycosylphosphatidylinositol-associated Proteins into the Cell Wall of Saccharomyces cerevisiae

“…Treatment of Cell Walls with Laminarinase-Cell walls were isolated from yeast transformant cells as described previously (24,37,38). Yeast cells were broken with glass beads in LY buffer containing 2% SDS, and the cell lysate was centrifuged (in a microcentrifuge, 1 min, 10,000 rpm, room temperature).…”

“…From this structural information, it is possible that the GPI moiety may be removed by cleavage either among mannose residues or between the mannose and myoinositol residues, and then the resulting end is directly bound to ␤-1,6-glucan. Alternatively, as shown in the Gce1p protein (38), the GPI moiety may be lipolytically cleaved by an enzyme like PI-PLC. In this case, trimming of myo-inositol and glucosamine residues may be required before the binding of it to ␤-1,6-glucan, because the enzyme is expected to cleave between phosphatidylinositol and lipid.…”