engleski

Binding of Pir proteins to glucan in the Saccharomyces cerevisiae cell wall

Yeasts have evolved three different ways of attaching proteins to cell wall glucan. Some proteins are bound to β -1, 3-glucan noncovalently, while others are attached covalently 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). 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 that reaction is dependent on the presence of divalent cations. Both wild type and mutants cells showed higher capacity for binding Ccw5p under increased osmotic pressure indicating that reaction might be under control of the HOG pathway. On the other hand some other shock conditions do not affect capability of cells to bind Ccw5p, showing that increased binding of Pir proteins is not generally caused by a shock. After disruption of all PIR genes (CCW5, CCW6, CCW7 and CCW8), 67kDa protein still remained in NaOH extract. SCW4 disruption in the ccw5ccw6ccw7ccw8 mutant resulted in disappearance of the 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 its role in the construction of the cell wall, yeast was transformed with a high copy number plasmid containing SCW4. Phenotypes of this strain as well as scw4, ccw5ccw6ccw7ccw8 and ccw5ccw6ccw7ccw8scw4 were examined.

yeast; cell wall; cell wall proteins; Pir-proteins

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