Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase

Abstract

One of the major proteins of the Saccharomyces cerevisiae cell wall, a 13-glucanase (BGL2 gene product), has been isolated and purified to homogeneity under conditions for preserving enzyme activity. The study of enzyme properties of the protein revealed that it is an endo-0-1,3-glucanase and not an exoglucanase as reported previously (F. Klebl and W. Tanner, J. Bacteriol. 171:6259-6264, 1989 (5,6,11). Both corresponding gene products are transported through the cell membrane, with the BGLJ product being mainly secreted into the medium (11) while the BGL2 product is incorporated into the cell wall (5). The lack of either protein did not result in a phenotype (5, 11). The BGLI gene has been identified by selecting for overproducers (6), and it seems to code for the main exo-pglucanase activity of yeast cells (11). The BGL2 gene showed a significant sequence similarity to plant j3-glucanases (5).The BGL2 gene product was originally purified as a dominant cell wall protein with an apparent molecular mass of 29 kDa. Although the purification included a heat step, the purified protein still showed minor 13-1,3-glucanase activity (5). To study this protein in more detail, it was purified to homogeneity under conditions for preserving enzyme activity. The enzyme is not, as previously described (5), an exoglucanase but an endo-,3-1,3-glucanase. Physicochemical and enzymatic properties of the protein have been studied. MATERILS AND METHODSThe following strains were used: S. cerevisiae X-2180-1A (MATa SUC2 malgal2 CUPJ) and SEY2101 (MATa ura3-52 leu2-3,112 ade2-1 suc2-A9 gal2) (3) peptone, 2% D-glucose) to the late logarithmic phase, harvested by centrifugation, washed with water, and resuspended in 5 ml of 20 mM sodium phosphate buffer, pH 7.0. Cells were disrupted mechanically in a Braun MSK homogenizer, and cell debris were centrifuged at 20,000 x g and washed with buffer. Zymolyase 100T (Seikagaku Kogyo Co. Ltd., Tokyo, Japan) was added to the suspension containing cell walls at a concentration of 6 jug/ml, and the mixture was incubated for 1 h at 370C. Cell walls were then centrifuged and the supernatant was dialyzed against 10 mM sodium phosphate buffer, pH 6.2. The enzyme preparation was applied to a DEAE-Sepharose CUB column (1.6 by 5 cm) and eluted by a linear 0 to 400 mM NaCl gradient at a flow rate of 20 ml/h. Three-milliliter fractions were collected and analyzed for the 29-kDa protein by immunoblot and for the p-nitrophenylglucosidase activity. The activity againstp-nitrophenylglucoside was found to be completely separated from the 29-kDa protein peak (data not shown). The 29-kDa glucanase fractions were collected and applied to a Mono Q high-performance liquid chromatography (HPLC) column (Hi 5/5; Pharmacia) in 10 mM sodium phosphate buffer, pH 6.2, and eluted with a 0 to 500 mM NaCl gradient at 1 ml/min. The peak containing the glucanase was applied to a TSK 2000 HPLC column (Ultropack column TSK G2000SW; LKB) equilibrated with 20 mM sodium phosphate buffer, pH 6.2, containing 0.1 M NaCl and was eluted wi...

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“…We also disrupted another glucanase encoded by BGL2 to check the specificity of suppression. Bgl2p is a non-GPI type endoglucanase, and the expression is not regulated by Ace2p (38). Mutation in BGL2 did not suppress the temperature sensitivity of gpi7 cells (Fig.…”

Amyloid-like properties ofSaccharomyces cerevisiaecell wall glucantransferase Bgl2p

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“…Bgl2p is a conserved protein with homologous counterparts described for a wide range of yeast species. 17,20,21 …”

Extraction of cell surface‐associated proteins from living yeast cells

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