engleski

Incorporation of alkali-extractable proteins in the Sacharomyces cerevisiae 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, and 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 to ß-1, 3-glucan noncovalently (Scw – soluble cell wall proteins ; extracted by hot SDS), while others are attached covalently (Ccw – covalently linked cell wall proteins ; extracted by glucanases) through glycosylphosphatidyl inositol (GPI) anchor and ß-1, 6-glucan, or directly to ß-1, 3-glucan by an alkali labile ester linkage between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir – proteins with internal repeats ; extracted by NaOH). The exact physiological roles for most of the cell wall proteins are still unknown. A multiple disruption of genes coding for the Pir family proteins (ccw5 ccw6 ccw7 ccw8) was performed in order to investigate their potential role. After disruption of the genes coding for all Pir family members, 67kDa protein band still remained in the NaOH extract. Disruption of SCW4 resulted in apparent disappearance of the 67kDa band, indicating that Scw4p could also be covalently linked to the cell wall in a similar manner like Pir proteins. In order to investigate the role of the Scw4p in the construction of the cell wall, wilde type yeast was transformed with a high copy number plasmid containing SCW4 gene. Phenotypes of this strain as well as scw4, ccw5ccw6ccw7ccw8 and ccw5ccw6ccw7ccw8scw4 were examined. In order to get further insight in the binding mechanism a novel, simple binding assay for Pir family proteins, was developed. It has been shown that pir, as well as scw4 and scw10 mutant cells, can bind externally added Ccw5p to their cell wall. A study of appropriate binding conditions revealed the requirement of the native conformation of Ccw5p. 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. Further experiments should provide answers to question regarding exact conditions required, as well as reveal if the binding occurs autocatalytically or require a particular enzyme in the wall.

yeast; cell wall; cell wall proteins; Pir proteins

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