FIGURE 4.

Eng1 and Exg8 are responsible for the majority of extracellular glucanase activity produced by yeasts. A, extracellular glucanase activity in the soluble fraction or associated with yeast cells. Culture supernatants were separated from yeast cells, and both fractions were tested for hydrolysis of laminarin to determine the activity in culture filtrates (dark bars) or activity associated with the yeast cells (light bars) of wild-type (gfp-RNAi) or glucanase-deficient strains (ENG1-RNAi, EXG8-RNAi, and ENG1/EXG8-RNAi strains). Data represent the glucose equivalents (micromoles) of reducing sugars released after 90 min of incubation with laminarin. Dashed line represents the limit of detection of the assay, and bars represent the mean reducing sugars released ± S.D. among replicate assays (n = 3). B, localization of Exg8 in extracellular fractions. Soluble and cell-associated fractions were tested for Exg8-FLAG protein by immunoblot of the FLAG epitope. Proteins in the culture filtrate, the soluble yeast cell lysate, and proteins extracted by SDS + DTT or by zymolyase + chitinase digestion of the insoluble cell wall fraction were separated by electrophoresis and probed for the FLAG epitope. Protein samples were derived from equivalent numbers of yeast cells. Numbers represent the molecular mass (kDa) of protein standards. n.s., not significant. *, p < 0.05; ***, p < 0.001.