Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of beta1,3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity - PubMed (original) (raw)

Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of beta1,3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity

A F Ram et al. J Bacteriol. 1998 Mar.

Abstract

Deletion of GAS1/GGP1/CWH52 results in a lower beta-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879-1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165-170, 1995). We show here that gas1delta cells release beta1,3-glucan into the medium. Western analysis of the medium proteins with beta1,3-glucan- and beta1,6-glucan-specific antibodies showed further that at least some of the released beta1,3-glucan was linked to protein as part of a beta1,3-glucan-beta1,6-glucan-protein complex. These data indicate that Gas1p might play a role in the retention of beta1,3-glucan and/or beta-glucosylated proteins. Interestingly, the defective incorporation of beta1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in beta1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.

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Figures

FIG. 1

FIG. 1

_gas1_Δ cells secrete more β-glucosylated proteins into the medium. Western analysis of medium proteins from wild-type (lanes 1 and 3) and _gas1_Δ (lanes 2 and 4 through 7) cells was performed with the affinity-purified β1,3-glucan antiserum (lanes 1 and 2), the β1,6-glucan antiserum (lanes 3 through 5), or anti-Cwp1p antiserum (lanes 6 and 7). In each lane, 2.5 μg of protein was applied. The sizes of standard molecular mass markers are given on the left. wt, wild type; Pabs, polyclonal antibodies.

FIG. 2

FIG. 2

Western analysis of SDS- and laminarinase-released cell wall proteins of wild-type (wt) cells (lanes 1 and 3), _gas1_Δ cells (lanes 2 and 4), and _fks1_Δ cells (lane 5) using affinity-purified β1,6-glucan antiserum. Equal cell equivalents of SDS-released proteins (equivalent to 1 mg [fresh weight] of cell walls) and equal cell equivalents of laminarinase-released proteins (equivalent to 5 mg [fresh weight] of cell walls) were loaded.

FIG. 3

FIG. 3

_gas1_Δ and _fks1_Δ cells have higher transcript levels of CWP1 than wild-type cells. The amounts of mRNA of CWP1 and TIP1, both encoding cell wall mannoproteins, are shown as percentages of wild-type levels after correction for actin.

FIG. 4

FIG. 4

Expression of Fks2p is elevated in _gas1_Δ cells. Shown are results of Western blot analysis of Fks1p and Fks2p in strains FY834 (wild type [wt]), AR104 (_gas1_Δ), and AR100 (_fks1_Δ) grown in YPD medium. Membrane samples (20 μg of protein) from the indicated strains were subjected to SDS-PAGE and Western blot analysis. Blots were separately probed with anti-Fks1p and anti-Fks2p antisera as previously described (35, 36).

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