Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane.

Abstract

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...

Genome‐wide identification of fungal GPI proteins

“…The ω region is followed by a less defined spacer region of about 10 amino acids followed by a hydrophobic tail. The length of the C-terminal signal peptide normally varies between 15 and 30 residues (Udenfriend and Kodukula, 1995) but, as was shown in S. cerevisiae for Gas1, signal peptides of 31 residues do occur (Nuoffer et al, 1991). In the set of S. cerevisiae GPI proteins so obtained, Asn or Gly predominated at the GPI attachment site (Caro et al, 1997).…”

“…The length and the hydrophobic character of the tail, rather than its specific sequence, therefore seem to be relevant for GPI anchoring (Ikezawa, 2002). Consistent with this, Coyne et al (1993) showed GPI modification of a truncated protein with a synthetic tail of at least 11 Leu residues, whereas introduction of a single charge in the middle of the hydrophobic tail blocks GPI anchoring (Nuoffer et al, 1991).…”

Loss of Function ofKRE5Suppresses Temperature Sensitivity of Mutants Lacking Mitochondrial Anionic Lipids

“…Interestingly, both Fks1p and Rho1p are also present in mitochondria (Sickmann et al, 2003). Gas1p, a putative ␤-1,3-glucan-remodeling enzyme (Popolo and Vai, 1999;Mouyna et al, 2000), the loss of which also results in reduced ␤-1,3-glucan in the cell wall (Popolo et al, 1993;Ram et al, 1998), is attached to the plasma membrane via a glycosyl-phosphatidylinositol anchor (Conzelmann et al, 1988;Nuoffer et al, 1991). Gas1p also is found in mitochondria (Grandier-Vazeille et al, 2001;Sickmann et al, 2003).…”

The GPI Transamidase Complex ofSaccharomyces cerevisiaeContains Gaa1p, Gpi8p, and Gpi16p

“…The accumulation of immature ER forms of GPI proteins such as Gas1p (Nuoffer et al, 1991) or Cwp1p (Shimoi et al, 1995) is a symptom of a delay or deficiency in the GPI anchor addition to newly synthesized proteins (Doering and Schekman, 1996). Western blotting shows an accumulation of the immature 105-kDa form of Gas1p and the immature 45-kDa form of Cwp1p after 5 and 15 h of depletion of Gpi16p ( Figure 6A), whereas there was no accumulation of immature carboxypeptidase Y (CPY) (not shown).…”