engleski

Investigation of processing of the native and mutated forms of Saccharomyces cerevisiae cell wall protein Scw4p

It was previously shown that S. cerevisiae cell wall protein Scw4p undergoes proteolytic processing with Kex2p. However, we observed that three forms of Scw4p with different molecular weights could be isolated from the cell wall. The fact that only one site for Kex2p processing is present in the Scw4p sequence indicates existence of an additional site for processing and an additional proteolytic enzyme involved in processing. According to the potential proteolytic processing sites found in the Scw4p sequence we presumed that aspartic proteases called yapsins might have a role in processing of Scw4p. In order to investigate the possible role of this enzymes in processing and to find the additional processing site, different mutations were introduced in SCW4 by which potential proteolytic sites where mutated. Native and mutated forms of Scw4p were expressed in wt, kex2 yeast strain and strain lacking all yapsin genes (5yps∆) and Scw4p processing was examined. 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 Scw4p forms isolated from the yeast cell wall were a consequence of its proteolytic processing with Kex2p and/or yapsin proteases using different processing sites in Scw4p sequence. In order to determine if there is a link between proteolytic processing and protein activity, mortality rate of yeast strains expressing different forms of Scw4p was estimated by methylene blue staining and MTT test.

Scw4p; Kex2p; yapsins

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