engleski

Relative quantification of yeast cell wall proteins in logarithmic and stationary growth phase

Yeast Saccharomyces cerevisiae cell wall is an extracellular organelle important for protection of the cell and its communication with the environment. In the cell wall structure, mannoproteins are covalently or noncovalently attached to a polysaccharide network that mostly consists of β-1, 3-glucan, β-1, 6-glucan and chitin. The cell wall is continually remodeled as yeast cells undergo life cycle or growth cycle transitions, which are therefore expected to be coupled with changes in expression of genes encoding cell wall proteins. However, little is known about differences in cell wall protein levels during different growth phases, such as logarithmic and stationary phase. We therefore performed relative quantification of yeast cell wall proteins in whole protein extracts of logarithmic and stationary cultures. Cell wall proteins were tagged at their genomic loci with a C-terminal hemagglutinin tag and were analyzed in whole protein extracts by western blot. Since the cell wall represents the non-soluble fraction of the cellular extract, validity of this method for extraction and quantification of cell wall proteins was established by constructing a set of diploid strains in which one or both copies of BGL2 gene, encoding a β-glucanase non-covalently bound to the cell wall, were tagged. Whole protein extracts of these strains showed a clear difference in signal intensity of Bgl2-HA band, thereby demonstrating that this method is appropriate for rapid and efficient extraction of cell wall proteins and is sensitive enough to detect a two-fold difference in protein level.

yeast Saccharomyces cerevisiae, cell wall, growth phases, Scw, Bgl2

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