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

Abstract

During cell wall biogenesis in Saccharomyces cerevisiae, some glycosylphosphatidylinositol (GPI)-attached proteins are detached from GPI moieties and bound to ␤-1,6-glucan of the cell wall. The amino acid sequence requirement for the incorporation of GPI-attached proteins into the cell wall was studied by using reporter fusion proteins. Only the short -minus region composed of five amino acids, which is located upstream of the site for GPI attachment, determined the cellular localization of the GPI-associated proteins. Within the -minus region, amino acid residues at the -4 or -5 and -2 sites were important for the cell wall incorporation. Yap3p, a well characterized GPI-anchored plasma membrane aspartic protease, was localized in the cell wall when the -minus region was mutated to sequences containing Val or Ile at the -4 or -5 site and Val or Tyr at the -2 site.Mannoproteins, one of the components of the yeast cell wall, can be divided into three groups: SDS-extractable mannoproteins, reducing agent-extractable mannoproteins, and glucanase-extractable mannoproteins (1, 2). The glucanase-extractable mannoproteins are covalently bound to ␤-1,6-glucan of the cell wall and are released only by glucanase treatment (3-6). Many genes encoding glucanase-extractable cell wall mannoproteins have been isolated in Saccharomyces cerevisiae, and so far all of them have been identified as GPI-dependent 1 cell wall proteins (7-15).GPI-associated proteins have been isolated from various organisms from yeasts and protozoa to mammals (16 -18). These proteins are structurally related in that they all contain a signal sequence for secretion in the N terminus and a GPI signal for an attachment to a GPI in the C terminus. The GPI-signal region is composed of three domains: a GPI attachment region comprising , ϩ1, and ϩ2 sites; a spacer of 5-10 amino acids; and a hydrophobic stretch of 10 -15 amino acids. A protein containing the GPI-signal is cleaved at the site, and the resulting carboxyl terminus of the protein becomes covalently bound to a GPI moiety. This reaction occurs in the luminal face of the endoplasmic reticulum and, in yeast, requires GAA1 (19) and GPI8 (20) gene products. The GPI-attached proteins are then transported to the cell surface, where they are exposed on the extracytoplasmic face of the plasma membrane. During the transportation from the endoplasmic reticulum to the plasma membrane, the proteins are mannosylated and become GPI-anchored mannoproteins.In protozoa and mammals, the GPI-anchored mannoproteins remain on the plasma membrane and take biological functions related to cell-cell and cell-environment interactions (18,21,22). In addition to the above functions, some of the GPI-anchored mannoproteins in the yeast are further processed and are incorporated into the cell wall. The incorporation of GPIassociated proteins into the cell wall is thought to occur in two steps: detachment of a GPI moiety from the protein and linking of the GPI-detached protein to ␤-1,6-glucan of the cell wall (3). Our knowledge of t...

Genome‐wide identification of fungal GPI proteins

“…In order to predict which GPI proteins remain attached to the plasma membrane and which proteins are covalently attached to β-1,6-glucan in the cell wall, we have analysed the sequence requirements for the ω − region for efficient incorporation of proteins into the cell wall, as proposed by earlier studies on S. cerevisiae (Hamada et al, 1998b, Identification of fungal GPI proteins 795 1999; Vossen et al, 1997). First, we have analysed in known S. cerevisiae cell wall proteins the occurrence of V, I or L at positions 4 or 5 amino acids upstream of the ω site and of Y or N two amino acids upstream of the predicted ω sites, which has been shown to be positively correlated with cell wall incorporation (Hamada et al, 1999).…”

“…The presence of basic residues in the region immediately upstream of the GPI attachment site (ω − region) is favoured in proteins that are predominantly localized in the plasma membrane . On the other hand, the presence of V, I or L at 4 or 5 amino acids upstream of the ω site (ω-4, ω-5) and Y or N at ω-2 has been shown to act as a positive signal for cell wall localization (Hamada et al, 1998b(Hamada et al, , 1999. Thus, GPI protein localization seems at least partly to be determined by the amino acids in the ω − region.…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…If this region includes two basic amino acids, the protein will be mostly retained in the plasma membrane (Caro et al 1997;Frieman and Cormack 2003), but if basic residues are absent or replaced with hydrophobic ones, the predominant location is the wall (Hamada et al 1998b(Hamada et al , 1999Frieman and Cormack 2003). However, having two basic amino acids in the v(2) region does not guarantee membrane localization because the additional presence of a longer stretch of amino acids rich in Ser and Thr will override the dibasic motif and shift the protein to the wall (Frieman and Cormack 2004).…”

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1‐dependent cell wall integrity pathway

“…This skeletal layer is strengthened by coupling of chitin chains to non-reducing ends of b1,3-glucan chains. At the outside of this layer, two types of cell wall proteins (CWPs) have been identified that are covalently linked to b-glucan, namely the proteins with internal repeats (Pir)-CWPs, which are linked to the b-1,3-glucan network via an alkaline-sensitive linkage, and the glycosylphosphotidylinositol (GPI)-CWPs, which are linked to the b1,3-glucan network via short chains of b1,6-glucan (Kapteyn et al, 1997Mrsa et al 1997;Hamada et al, 1998). Collectively, these CWPs limit the cell wall permeability, although some of them are involved in specific cellular functions, including flocculation, pseudohyphal growth and sexual agglutination (Cid et al, 1995).…”