Increased mortality of Saccharomyces cerevisiae cell wall protein mutants

Abstract

The yeast cell wall contains an unusually high number of different mannoproteins. The physiological role of most of them is unknown and gene disruptions leading to depletion of different proteins do not affect major functions of the wall. In this work the phenotype of different single and multiple cell wall protein mutants was observed at the level of individual cells. It was found that the lack of the non-covalently bound wall proteins Scw4p, Scw10p and Bgl2p increases the mortality of Saccharomyces cerevisiae cells grown exponentially under standard laboratory conditions, as assayed by methylene blue staining. Mutation of SCW11, however, suppressed the phenotype of scw4scw10, or scw4scw10bgl2, indicating that Scw4p, Scw10p and Bgl2p act synergistically while Scw11p has an activity antagonistic to that of the other three proteins. Mutants lacking major covalently bound proteins, either all four described Pir-proteins or the five most abundant glycosylphosphatidylinositol (GPI)-anchored proteins (Ccw12p, Ccw13p/Dan1p, Ccw14p/Icwp1p, Tip1p and Cwp1p), also had increased mortalities, the first somewhat more and the latter less than that of scw4scw10bgl2. In all cases the observed phenotype was suppressed by the addition of an osmotic stabilizer to the growth medium, indicating that cells died due to decreased osmotic stability. If cells were grown to stationary phase, Scw-mutants showed only slightly increased mortality, but mutants lacking Pir-or GPI-anchored proteins had significantly increased sensitivity, suggesting that their physiological function is primarily expressed in stationary-phase cells. In many cases structures consisting of a living ccw5ccw6ccw7ccw8 (multiple Pir-protein mutant) mother with two methylene blue-stained daughters could be seen.

Binding assay for incorporation of alkali‐extractable proteins in the Saccharomyces cerevisiae cell wall

“…Scw11p was previously described as an antagonist of Scw4p and Scw10p, since it was observed that the mutation of SCW11 suppressed the scw4scw10 phenotype (Teparić et al, 2004). The results of the binding experiments corroborated previous findings.…”

“…These results do not suggest a common physiological role of Scw4p and Pir proteins. Overproduction of Scw4p increased sensitivity to zymolyase only slightly (Figure 2A), but it Results reported previously showed that the mutation of PIR genes caused increased mortality of cells in the culture, a feature more pronounced in stationary cells (Teparić et al, 2004). In the same report, only a small increase in the mortality of scw4 was detected.…”

“…The cell suspension was mixed with an equal volume of methylene blue dye and incubated for 5 min at room temperature. The dead (blue-coloured) cells were scored (Teparić et al, 2004).…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…pir1D pir2D pir3D pir4D mutants show a loss of viability that is suppressed in osmotically supported medium (Teparic et al 2004). These findings suggest a collective role for PIR proteins in maintenance of a normal wall.…”

Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography–tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p

“…Deletion of CIS3 results in a mild growth defect compared to wild type cells and substantial changes in cell morphology resulting in increased cell clumping [45,46].…”