engleski

Characterization of Saccharomyces cerevisiae cell wall proteins and their impact on yeast extracellular metabolism

Saccharomyces cerevisiae cell wall consists of a glucan network to which a number of mannoproteins are attached. There are four groups of proteins located externally to the plasma membrane: i) proteins secreted into the medium (e.g. Exg1p, Chs1p), ii) periplasmic proteins retained by the cell wall, probably by oligomerization into molecular structures too big to penetrate the wall, but not chemically attached to it (e.g. Pho3p, Pho5p, Suc2p), iii) proteins attached to cell wall by noncovalent linkages (e.g. Bgl2p), and iv) proteins covalently bonded to glucan (e.g. Aga1p, Aga1p). Localization mechanisms of extracellular proteins are still not completely understood. It seems that noncovalently linked cell wall proteins bind to glucan by hydrogen-bonding, while the attachment of some, but not all covalently linked proteins require the C-terminally located GPI-anchoring signal. In order to identify S. cerevisiae cell wall proteins, clone their genes an assess their physiological roles, the surface proteins of yeast cells were labeled by biotinylation, cell walls were purified, and labeled proteins were extracted either with hot SDS, or DTT, or released by NaOH (under mild beta-elimination conditions), or by glucanases, respectively. The first two procedures extracted noncovalently linked proteins, while the latter two released proteins covalently bound to wall polysaccharides. Nine SDS-extractable, five NaOH-extractable, and six glucanase-extractable proteins were identified. Purification and N-terminal sequencing of DTT-solubilized cell wall proteins revealed, as expected, endoglucanase (Bgl2p), exoglucanase (Exg1p), and chitinase (Chs1p), but also three so far unidentified proteins which showed homologies with the glucanase family. Mutation in one of the genes resulted in a clumpy phenotype, and a double mutant showed slow and aberrant growth. Four NaOH-extractable proteins were found to share very high sequence homologies. Two of their genes were previously suggested to code for "heat shock proteins", based on their high similarity with HSP150 coding for a heat induced protein secreted into the medium. Mutations in genes coding for NaOH-soluble wall proteins resulted in an increased sensitivity of cells to elevated temperatures, although their concentrations in the wall did not change upon the "heat shock". Two glucanase-extractable proteins were also identified. One has already been found among NaOH-extracted proteins, while the other was a so far unidentified, small but highly glycosylated one. The mutation of the corresponding gene did not bring about an apparent phenotype. The possible impact of yeast cell wall proteins on the extracellular metabolism will be discussed.

cell wall; extracellular proteins; glucanases

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