engleski

Cell wall protein Scw4 as a base for development of new homologous and heterologous expression system models

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). It was shown that three forms of Scw4 with different molecular weights could be isolated from the cell wall. The fact that only one site for Kex2 processing is present in the Scw4 sequence indicates existence of an additional site for processing and an additional proteolytic enzyme involved in processing. Given that group of broad specific aspartic proteases called yapsines process other cell wall proteins, it was presumed that this group of enzymes might have a role in processing of Scw4. In order to investigate the possible role of this enzymes in processing and to find the additional processing site, different mutations were introduced in HA-tagged SCW4 by which potential proteolytic sites where mutated. Native and mutated forms of HA-tagged-Scw4 were expressed in wt, kex2 yeast strain and strain lacking all yapsin genes (5yps∆) and Scw4 processing was examined by Western blot. Protein glycosilation was also examined in order to determine whether forms differ in this way. Results confirmed that the differences in the molecular weight of Scw4 forms isolated from the yeast cell wall were a consequence of its proteolytic processing with Kex2 and/or yapsin proteases using different processing sites in Scw4 sequence. Scw4 was first identified as one of the cell wall proteins non-covalently incorporated in the wall. Later, it has been found that a part of Scw4 could be extracted by mild alkalis from walls previously depleted of non-covalently attached proteins. In order to examine which forms of Scw4 where covalently linked to structures of cell wall, HA-tagged Scw4 was extracted by NaOH from cell wall preparations of w.t., kex2 and yapsin mutants previously depleted of non-covalently linked proteins. Mutants were grown either at pH 4, or at pH 7 in order to obtain all three forms of Scw4 and covalently attached part of the proteins was analysed by Western blot. Results showed that all three forms of Scw4 protein are covalently linked to wall, although smallest form of protein bounds to wall less efficiently. Additional experiments are required in order to determine which part of Scw4 is essential for covalent binding to wall. Given that Scw4 forms covalent link to cell wall, this protein is a good candidate for application in development of new homologous and heterologous expression systems. Future research will focus on identifying part of Scw4 sequence responsible for covalent binding and development of Scw4 based expression system models.

yeast, Saccharomyces cerevisiae, cell wall, mannoproteins

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