engleski

Alkali-extractable proteins in Saccharomyces cerevisiae cell wall

The cell wall of yeast Saccharomyces cerevisiae contains more than 20 different manoproteins. They are considerd to play different roles in building, maintaining and modifying the wall itself when different cell cycle events and they are important for interactions of cells with their surrounding. Yeasts have evolved three different ways of attaching proteins to cell wall glucan. Some proteins are bound to β -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 GPI-anchor and β -1, 6-glucan, or directly to β -1, 3-glucan by 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). Disruption of genes coding for the Pir-proteins was performed to investigate their potential role. After disruption of all PIR genes, 67kDa protein still remained in NaOH extract. SCW4 disruption resulted in disappearance of a 67kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall simillar to Pir-proteins. Since it was reported previously that Scw4p was a noncovalently attached protein, this finding can be relevant for the role of Scw4p. In order to investigate the role of the Scw4p in the construction of the cell wall, yeast was transformed with a high copy number plasmid containing SCW4. 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.

yeast; cell wall; Pir proteins; CCW5; SCW4

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