VIEW ARTICLE    DOI: 10.1094/ASBCJ-53-0111

Characterization of Affinity-Purified Cell Wall-Binding Proteins of Saccharomyces cerevisiae: Possible Role in Flocculation. Robert J. Stewart (1), Inge Russell, and Graham G. Stewart (2), Labatt Breweries of Canada, 150 Simcoe Street, London, Ontario, Canada, N6A 4M3. (1) Author to whom correspondence should be addressed. (2) International Centre for Brewing and Distilling, Heriot Watt University, Riccarton, Edinburgh, Scotland, EH14 4AS. J. Am. Soc. Brew. Chem. 53(3):111-116, 1995. Accepted December 21, 1994.

The objective of this study was to isolate and characterize the cell wall protein or proteins that mediate flocculation directly from yeast cell extracts. A urea/mercaptoethanol extract of the yeast cell wall was prepared from flocculent yeast cells grown to stationary phase. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the extract revealed a broad range of proteins ranging in apparent relative molecular mass from 20 to 85 kilodaltons (kDa). Periodic acid Schiff's straining, after SDS-PAGE, revealed several glycoprotein bands in the range of 40-80 kDa. Interestingly, the extracted proteins were able to partially inhibit the flocculation of yeast cells, whereas the extraction treatment itself greatly reduced the flocculence of the yeast. Taken together, these data suggest that the protein or proteins that mediate flocculation of these yeast cells are present in the urea/mercaptoethanol extract. Further fractionation of the extract on an immobilized yeast cell column in the presence of calcium resulted in only a single fraction with ethylenediaminetraacetic acid (EDTA) elution. However, analysis of this fraction by SDS-PAGE yielded several distinct proteins with apparent relative molecular masses of 20-65 kDa, all of which are presumably binding to the yeast cells in a calcium-dependent manner. Notably, no larger proteins were detected, suggesting that perhaps the putative FLO1 protein (predicted molecular weight of 93-150 kDa) may have a regulatory role in flocculation, rather than mediating directly in the lectin-carbohydrate interaction. Keywords: Flocculation, Protein purification, Yeast