Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae

Abstract

Three glucanase-extractable cell wall proteins from Saccharomyces cerevisiae were purified, and their Nterminal amino acid sequences were determined. With this information, we were able to assign gene products to three known open reading frames (ORFs). The N-terminal sequence of a 55-kDa mannoprotein corresponded with the product of ORF YKL096w, which we named CWP1 (cell wall protein 1). A 80-kDa mannoprotein was identified as the product of the TIP1 gene, and a 180-kDa mannoprotein corresponded to the product of the ORF YKL444, which we named CWP2. CWP1, TIP1, and CWP2 encode proteins of 239, 210, and 92 amino acids, respectively. The C-terminal regions of these proteins all consist for more than 40% of serine/threonine and contain putative glycosylphosphatidylinositol attachment signals. Furthermore, Cwp1p and Tip1p were shown to carry a ␤1,6-glucose-containing side chain. The cwp2 deletion mutant displayed an increased sensitivity to Congo red, calcofluor white, and Zymolyase. Electron microscopic analysis of the cwp2 deletion mutant showed a strongly reduced electron-dense layer on the outside of the cell wall. These results indicate that Cwp2p is a major constituent of the cell wall and plays an important role in stabilizing the cell wall. Depletion of Cwp1p or Tip1p also caused increased sensitivities to Congo red and calcofluor white, but the effects were less pronounced than for cwp2⌬. All three cell wall proteins show a substantial homology with Srp1p, which also appears to be localized in the cell wall. We conclude that these four proteins are small structurally related cell wall proteins.The two major components of the cell wall of the yeast Saccharomyces cerevisiae are glucan, which constitutes the inner layer of the cell wall, and mannoproteins, which are embedded in and cover this glucan layer. Chitin is a minor component of the cell wall (13, 18). The mannoproteins can be divided into two groups, the sodium dodecyl sulfate (SDS)-extractable mannoproteins and the glucanase-extractable mannoproteins, which are solubilized by glucanase digestion of the glucan layer. Several glucanase-extractable mannoproteins have been identified. These proteins have two characteristics in common: their C-terminal regions are rich in serine and threonine, and they all contain putative glycosylphosphatidylinositol (GPI) attachment signals. Two of these proteins, ␣-agglutinin (22) and the core subunit of a-agglutinin (33), are involved in mating. The third, which is involved in flocculation, is the product of the FLOI gene (42). Because of the high serine and threonine content of their C-terminal regions, these proteins are probably heavily O glycosylated, which could give the protein a rod-like structure (15). The presence of a GPI anchor has been demonstrated only for the intracellular precursor form of ␣-agglutinin (47). The glucanase-extractable mannoproteins are proposed to be covalently linked to glucan (29,38,45). Several groups have investigated which part of the protein is responsible for anchoring the protein...

Scw10p, a cell-wall glucanase/transglucosidase important for cell-wall stability in Saccharomyces cerevisiae

“…The resistance has been attributed to changes in the mannoprotein layer (Vink et al, 2002), with a specific function for some of the cell-wall proteins (Van Der Vaart et al, 1995). In mutant scw4scw10, zymolyase resistance is not lost after deletion of CHS3.…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…The glucan attachment sequence of PIR repeats is also found in the GPI-CWP Tip1, Tir1, Cwp1, and Cwp2 ( Van der Vaart et al 1995). In the case of Cwp1, the PIR repeat may be used as an additional wall anchorage point because the protein is attached to the wall by both an alkali-labile and a GPI-dependent linkage (Kapteyn et al 2001).…”

Changes in Aspergillus nidulans gene expression induced by bafilomycin, a Streptomyces-produced antibiotic

“…Its deduced amino acid sequence (BinB) is, near its Nterminus, similar (Fig. 5) to that of a cell-wall mannoprotein of Saccharomyces cerevisiae (Cwplp ;van der Vaart et al, 1995). The remaining two bafilomycininduced genes encode transcripts of 488 (binA) and 412 (binC) nucleotides.…”