engleski

Incorporation of homologous and heterologous proteins in the Saccharomyces cerevisiae cell wall

Yeast cell wall contains proteins that are noncovalently (Scw-proteins) or covalently (Ccw-proteins) bound to β-1, 3-glucan, the latter either through GPI-anchors and β-1, 6-glucan, or by alkali labile ester linkages between γ-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins, extracted from the cell wall by mild alkali). Disruption of all four genes coding for Pir-proteins revealed that a 67kDa protein still remained in the NaOH extract. Disruption of SCW4, a gene coding for one of the most abundant Scw protein resulted in disappearance of the 67kDa band from the extract, indicating that Scw4p was partly also covalently linked to the cell wall by an apparently new alkali sensitive linkage. It was shown that noncovalently bound Scw4p (SDS released) undergoes the Kex2p proteolytic maturation. However, when alkali released proteins from wt and kex2 mutant were compared, covalently attached form of Scw4p had the same size indicating that only non-processed form of the protein was attached to β-1, 3-glucan. The SDS-extractable haemagglutinin-tagged Scw4p, from wt strain in which Scw4p was overproduced contained two forms, one processed by Kex2p and the other not. Scw4p protein released from the wall by glucanases or NaOH contained only the non-processed form. As expected, extraction of Scw4p by SDS from kex2 mutant also showed only one form of the protein demonstrating that the other form really was the product of Kex2p processing. Understanding of how yeast incorporates proteins in the cell wall can be used for biotechnological purposes to direct and immobilize heterologous proteins at the cell surface. In this way yeast cell itself can both serve as an insoluble matrix and perform the immobilization of the protein of interest, avoiding tedious chemical immobilization reactions. Present knowledge on the mechanisms for incorporation of both homologous and heterologous proteins in the model yeast Saccharomyces cerevisiae will be discused.

yeast; cell wall proteins; Scw4p; heterologous expression

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