Binding of PIR proteins to the cell wall of Saccharomyces cerevisiae

Saccharomyces cerevisiae cell wall contains more than 20 different mannoproteins. Some of them are noncovalently bound to the wall structural polysaccharides and can be extracted by hot SDS (Scw proteins-soluble cell wall proteins). The second group of proteins is covalently linked to the wall (Ccw proteins-covalently linked cell wall proteins) and can be divided into those extractable by NaOH, or by glucanases, respectively. Possible role of NaOH extractable proteins (members of the Pir protein family Ccw5p/Pir4p, Ccw6p/Pir1p, Ccw7p/Pir2p and Ccw8p/Pir3p) in the cell wall is still unknown. It has been suggested that Pir proteins get covalently linked enzimatically to cell walls by a transglutaminase-type of reaction. In order to get further insight in their binding mechanism capability of cells for binding Ccw5p protein added externally to growth medium was monitored. Successive mutations of PIR genes led to increased capability of cells to bind Ccw5p from growth medium and, similar to other phenotypes observed previously, it showed cummulative effect. The presence of EDTA in growth medium blocked the binding of Ccw5p, indicating that reaction is dependent on the presence of divalent cations. Both wild type and ccw mutant cells showed higher capacity for binding Ccw5p under increased osmotic pressure indicating that reaction is under control of the HOG pathway. Results observed by monitoring viability of ccw mutants showed increased fraction of dead cells in multiple ccw mutants (about 8% in exponentially, and about 25% in stationary phase), compared to wild type (less than 1%). Number of dead cells was significantly reduced in both growth phases when cells were grown in 1M sorbitol, indicating that increased mortality is caused by osmotic instability of mutant cells. These two results are in good correlation and indicate that Pir proteins are involved in osmotic stabilization of yeast cell wall. On the other hand some other shock conditions (addition of cell wall synthesis inhibitors Calcoflour White or Congo Red) do not affect capability of cells to bind Ccw5p, showing that increased binding level of Pir proteins is not generally caused by shock.