Identification of Two Mannoproteins Released from Cell Walls of a Saccharomyces cerevisiae mnn1 mnn9 Double Mutant by Reducing Agents

Abstract

In this report, we present the identification of the main polypeptides that are extracted from purified cell walls of aSaccharomyces cerevisiae mnn1 mnn9 strain by reducing agents. Treatment of the purified cell walls of this strain with β-mercaptoethanol releases several mannoproteins, of which three, with apparent sizes of 120, 45, and 40 kDa, are the most abundant. Analysis of the amino-terminal sequences revealed that the 120-kDa mannoprotein is Bar1p, the protease involved in the so-called barrier activity in yeast cells, and that the 45- and 40-kDa mannoproteins are the Kex2-unprocessed and Kex2-processed forms of the gene product of open reading frame (ORF) YJL158c, an ORF that belongs to the PIR (protein with internal repeats) family of genes, composed thus far of PIR1, PIR2/HSP150, andPIR3. Accordingly we have named this gene PIR4, and Pir4 denotes the 40-kDa Kex2-processed form of the mannoprotein. We have characterized Pir4 and have shown the feasibility of using it as a fusion partner for the targeting of recombinant proteins to the cell wall.

Dynamics of cell wall structure in Saccharomyces cerevisiae

“…PIR4/CIS3, on the other hand, is maximally expressed at the G 2 phase [117]. In agreement with this, Pir4/Cis3 is predominantly found in the wall of growing buds [27]. The GPI-CWP Crh1 shows a particularly interesting expression pattern.…”

“…Cell wall proteins are covalently linked to the L1,3-glucan^chitin network either indirectly through a L1,6-glucan moiety or directly (Sections 2.6 and 2.7). In addition, some proteins are di-sul¢de-bonded to other cell wall proteins [26,27].…”

“…They are all similarly organized (SP^Kex2^repeat(s)^CX(66)CX(16)CX(12)C), consisting of an N-terminal signal peptide, a Kex2 site, followed by a repeat-containing region with up to 11 repeats, and a highly conserved carboxy-terminal region with four cysteine residues in a conserved spacing pattern. Pir1, Pir2/ Hsp150, Pir3, and Pir4/Cis3 all have been localized to the cell wall immunologically [27,93,94]. Several additional proteins, such as Pau1 and its homologues, and Sps100, which is believed to contribute to maturation of the spore wall [95], and also Ygp1, which is induced by nutrient limitation [96], are predicted to have an N-terminal signal peptide, but not an addition signal for a GPI anchor.…”

“…In addition, several cell wall proteins such as Bar1, a protease [27], Aga2 [5,97], the active subunit of the sexual agglutinin complex in MATa cells, Pir4/Cis3 [27], and some known or potential cell wall glycanases such as Fig. 2.…”

Evidence for the attachment of Hsp150/Pir2 to the cell wall of through disulfide bridges

“…The proteins that are covalently bound to the structural glucan of the cell wall have themselves been classi¢ed in two groups: glycosylphosphatidylinositol (GPI) cell wall proteins, that are immobilized in the wall by linkage to L-1,6-glucan, and the Pir family of proteins, that are directly bound to the L-1,3-glucan [4,5]. However, Pir4, a member of the Pir family, has recently been described as a disul¢de-bound cell wall protein [6], and Pir2, another member of this family, is mostly secreted to the medium [7], only a fraction being retained in the cell wall. According to Kapteyn et al [5], this fraction of Pir2 would remain attached to the cell wall through direct linkages to the L-1,3-glucan.…”