ABSTRACT
Pir proteins are unique proteins with internal repeat sequences that are reported to be present in the cell wall of Saccharomyces cerevisiae. They are covalently attached to the cell wall and can be released by mild alkali treatment. In this study the biotinylated cell wall preparations from Candida albicans and S. cerevisiae were extracted by alkali and beta-1,3 glucanase and analyzed in parallel. Among the four bands detected by streptavidin, two proteins were recognized by the antibody to the S. cerevisiae Pir protein Hsp150. The antibody also detected a high molecular mass protein secreted in the growth medium of C. albicans. Using S. cerevisiae HSP150/PIR2 gene as a probe, Southern and Northern hybridizations were performed with DNA and RNA of C. albicans. Hybridization with DNA digested with different restriction enzymes showed more than one hybridized fragment. An increased level of mRNA was found in heat shocked cells (37 degrees C for 45 min compared to 25 degrees C). Hybridization of ScHSP150 gene to mRNAs from cells grown in different media was also determined. Two transcripts of size approximately 3.5 kb and 2.0 kb were detected in mRNAs from cells grown in defined medium with glucose as carbon source or in the same medium supplemented with hemoglobin. The lower transcript of size 2.0 kb was absent in cells grown in medium with galactose as carbon source. A single band was also observed when cells were grown in rich medium. Together these results demonstrated the existence of beta1,3 glucan linked proteins in C. albicans, which are related to Pir family proteins of S. cerevisiae.
Subject(s)
Candida albicans/chemistry , Fungal Proteins/analysis , Glycoproteins/analysis , Membrane Proteins/analysis , Saccharomyces cerevisiae Proteins , Biotinylation , Blotting, Northern , Blotting, Southern , Candida albicans/genetics , Candida albicans/growth & development , Cell Wall/chemistry , Electrophoresis, Polyacrylamide Gel , Fungal Proteins/genetics , Glycoproteins/genetics , Heat-Shock Proteins/genetics , Membrane Proteins/genetics , Molecular Weight , RNA, Messenger/genetics , Saccharomyces cerevisiae/geneticsABSTRACT
We have reexamined the detection of the components in a beta-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 sulfate-polyacrylamide 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. Two-dimensional 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.
