Characterization of Ccw12p, a major key player in cell wall stability of Saccharomyces cerevisiae

Abstract

The GPI-anchored mannoprotein Ccw12p is a crucial structural component of the cell wall of Saccharomyces cerevisiae. Compared to wild-type, the mutant ccw12 grows more slowly, is highly sensitive to Calcofluor white and contains 2.5 times more cell wall chitin. In this study, electron microscopy of ccw12 cell walls revealed that, with respect to wild-type, the inner glucan layer is thicker with irregular depositions of wall material, whereas the outer mannan layer is less condensed. Biochemical analyses of cell wall glucan suggest that in the absence of Ccw12p, GPI-anchored cell wall proteins are transferred preferentially to chitin and random deposition of cell wall material reinforces the inner glucan-chitin layer, thereby enhancing the overall stability of the cell wall. To further elucidate the role of Ccw12p, structure-function analysis was performed. We demonstrate that Ccw12p is highly N-glycosylated. However, loss of N-glycans does not affect Ccw12p functionality. In contrast, deletion of the repeated amino acid motive TTEAPKNGTSTAAP in the C-terminal part of the protein affects Ccw12p function.

Modulation of Yeast Sln1 Kinase Activity by the Ccw12 Cell Wall Protein

“…The ccw12 mutant exhibits reduced agglutination and mating (35) possibly due to inaccessibility of agglutinins at the cell surface or to other changes in the structure of the cell wall. Increased sensitivity to Calcofluor White, Congo Red (35), hygromycin B, and low doses of zymolyase 5 (40) suggest extensive alterations in cell wall composition and structure in the ccw12 mutant.…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…Strains lacking one or more of these GPI-CWP have wall defects, and expression of some of these proteins can vary with cell cycle stage or be induced during mating or sporulation in response to cell wall stress or when oxygen levels are low. In general, GPI-CWPs have a collective role in maintaining cell wall stability (Lesage and Bussey 2006;Ragni et al 2007c).…”

“…Ccw12 is likely abundant because its gene has a very high codon adaptation index (Klis et al 2010). Ccw12 has a major role in the wall, because cells lacking it are hypersensitive to CFW and other wall stressing agents and rounder than wild type, with thick, disorganized walls, lysis-prone buds, and elevated levels of chitin (Mrša et al 1999;Hagen et al 2004;Ragni et al 2007c;Shankarnarayan et al 2008). Man-to-Glc ratios in ccw12D cells are unchanged, but levels of alkali-soluble relative to alkali-insoluble glucan are higher, indicating altered organization and cross-linkage of wall components (Ragni et al 2007c).…”

“…Ccw12 has a major role in the wall, because cells lacking it are hypersensitive to CFW and other wall stressing agents and rounder than wild type, with thick, disorganized walls, lysis-prone buds, and elevated levels of chitin (Mrša et al 1999;Hagen et al 2004;Ragni et al 2007c;Shankarnarayan et al 2008). Man-to-Glc ratios in ccw12D cells are unchanged, but levels of alkali-soluble relative to alkali-insoluble glucan are higher, indicating altered organization and cross-linkage of wall components (Ragni et al 2007c). Ccw12 localizes to sites of active wall synthesis, including the future bud site, the septum, the lateral walls of enlarging daughter cells, as well as the tips of mating projections, but then turns over, suggesting that it may stabilize walls as daughter cells and that mating projections are being formed .…”

“…Ccw12 localizes to sites of active wall synthesis, including the future bud site, the septum, the lateral walls of enlarging daughter cells, as well as the tips of mating projections, but then turns over, suggesting that it may stabilize walls as daughter cells and that mating projections are being formed . Loss of Ccw12 alone activates the CWI pathway-mediated chitin stress response (Ragni et al 2007c see Chitin synthesis in response to cell wall stress), but deletion of additional GPI-CWP genes forces cells over a threshold that leads to triggering of a new compensatory response to loss of multiple GPI-CWP that depends on Sed1 and the non-GPI-CWP Srl1 (see File S7).…”