We have reexamined the detection of the components in a β-mercaptoethanol and ammonium carbonate buffer extract of surface proteins of Candida albicans and the effects of postextraction manipulation of the extract on recovery of extract components. Following sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), preferential staining of some moieties was observed when bands detected by a commercial silver staining method or a Coomassie Brilliant Blue (CBB) staining method were compared. Additional protein bands that were either not detected or poorly detected by a single method alone were readily observed by a combined silver-CBB staining method. This method also detected alterations in the profile of extracted proteins from organisms grown in the presence of galactose or hemoglobin rather than glucose. Twodimensional electrophoresis (2-DE) gel analysis by double stain showed better detection of several acidic and basic protein spots. Less than 10% of the extract as determined by a dye-binding assay was lost following either or both lyophilization and dialysis. These manipulations of the extract did not change the protein profile following SDS-PAGE as determined by the combined staining or Western blot analysis of a 70 kDa protein. These observations suggest that soluble cell wall proteins are not unusually sensitive to procedures routinely used in protein purification. In addition, these studies suggest that a modified staining method that combines both silver stain and CBB stain provides improved detection of cell wall proteins compared to either method alone.
Non-covalently attached or soluble cell wall proteins of Saccharomyces cerevisiae were extracted using a high pH/2-mercaptoethanol procedure and were separated for peptide sequencing using 2D-PAGE. A partial N-terminal sequence of a major protein spot was obtained and showed high identity with enolase gene products. Western blotting with an anti-enolase antibody confirmed that enolase was present in the cell wall extract. Biotinylation of cells prior to protein extraction with a membrane impermeable biotinylating agent confirmed that the detection was not owing to cell lysis during extraction. Transmission immunoelectron microscopy showed enolase to be present in the cell wall. Enolase contains no known secretion signal that would localize it to the cell wall. Thus S. cerevisiae must have further mechanisms for targeting proteins to the cell wall.
mRNA transcript levels of 38 genes from Saccharomyces cerevisiae were investigated during attempted spheroplast regeneration. Many of the genes selected are involved in cell wall biosynthesis. Spheroplasts did not regenerate into osmotically competent cells during the experiment. However, at a mRNA level, the quantities of transcripts were altered between the experimental and control populations. KRE11, EGT2 and MSS10 had their transcript levels increased by more than 10-fold during attempted spheroplast regeneration. A further six genes, FLO1, TIR1, SED1, HKR1, YGR189 and MUC1, showed transcript level increases of at least 5-fold. Five genes showed a change in transcript levels from an undetectable level to a detectable level: SKT5, KRE1, KRE5, SEC53 and DHS1. PMT2 showed a rapid decrease in mRNA levels followed by an increase to the basal level. Thus, cell stress genes, biosynthetic genes and some glycosylphosphatidylinositol-anchored cell wall proteins have their transcript levels increased in regenerating spheroplasts, but their transcription was not sufficient to initiate the replacement of the cell wall in liquid medium. ß
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