engleski

Synergistic and antagonistic behaviour of potential cell wall glucanases of Saccharomyces cerevisiae

Saccharomyces cerevisiae cell wall has complex structure composed mainly of glucan and more than 20 different mannoproteins. Cell wall proteins can be divided in two groups according to the mechanism by which they are attached to the wall. Some of them are noncovalntly 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 and can be divided into those extractable by 30 mM NaOH, or by glucanases, respectively. Physiological role of most cell wall proteins is unknown. Identification of noncovalently attached proteins revealed that they posses enzymatic activities (ß-egsoglucanase, Exg1p ; ß-endoglucanase, Bgl2p ; chitinase, Cts1p) or show significant homology to glucanases or transglucosidases, so they can have different roles in building, maintaining and modifying the wall itself. In order to assess possible physiological role of Scw proteins with significant homology to the already known cell wall glucanases, mutant strains were constructed by disruption of scw4, scw10, scw11, and bgl2 genes. Influence of these proteins on cell wall stability and protein secretion into the growth medium was examined. Double mutant, scw4scw10, triple mutant scw4scw10bgl2 and quadruple mutant scw4scw10scw11bgl2 secret less proteins in growth medium, while triple mutant scw4scw10scw11, as well as single mutants, show no differences in comparison to wild type cells. Furthermore, sensitivity of the cell wall to ß-1, 3-glucanase treatment was examined and scw4scw10scw11 exhibited a significant increase in resistance to ß-1, 3-glucanase treatment. After removal of the mannoprotein layer by proteinase K all mutants showed wild type level of sensitivity. Such results indicate that observed decreased sensitivity is probably connected with the structure of the mannan layer of the cell wall. In addition, viability of mutants was monitored. Mutant strains scw4scw10, scw4scw10 bgl2 and scw4scw10scw11bgl2 showed increased fraction of dead cells (4, 5-6 %) in the medium. Triple mutant scw4scw10scw11, in contrast to other mutants, has only 1% of dead cells. Results suggest the synergistic behavior of proteins Scw4p, Sw10p and Bgl2p, whereas the product of SCW11 acts antagonistically to those proteins.

yeast cell wall; mannoproteins; cell wall biosynthesis; glucanases

nije evidentirano