engleski

Binding of Pir proteins to the yeast cell wall

Saccharomyces cerevisiae cell wall has a bilayered structure. The inner part consists mainly of a network of linear ß-1, 3-glucan molecules, whereas the outer layer is composed mostly of mannoproteins. Yeasts have evolved three different ways of attaching proteins to the polysaccharide network. Some proteins are bound noncovalently while the others are covalently attached through glycosylphosphatidyl inositol (GPI) anchor and ß-1, 6-glucan, or directly to ß-1, 3-glucan (Pir – proteins with internal repeats). Pir proteins are soluble in mild alkali and do not have GPI anchor. Since these proteins are highly glycosylated their deletion changes the polysaccharide composition of the cell walls. Five PIR genes with varying number of repeating units (1-11) were identified, and the repeating unit seems to be responsible for the formation of an ester bond between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses. In order to get further insight in the binding mechanism, a novel, simple binding assay for Pir family proteins was developed in which externally added Ccw5p/Pir4p was bound to different cell wall mutant strains. It has been shown that pir, as well as scw4 and scw10 mutants can bind externally added Ccw5p to their cell wall. Further, we investigated the conditions under which these proteins were attached to cell wall. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild type and mutant cells showed enhanced binding in 0.6 M KCl. It was also shown that the native conformation of Ccw5p is required for its binding. A multiple disruption of genes coding for the Pir family proteins (ccw5 ccw6 ccw7 ccw8) was performed in order to investigate their potential role in the cell wall biogenesis. After disruption of all Pir family genes, 67kDa protein band still remained in the NaOH extract. It was eliminated by the disruption of SCW4, indicating that Scw4p could also be covalently linked to the cell wall by an alkali sensitive linkage.

yeast; cell wall; cell wall proteins; Pir proteins

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