Production and Secretion of Saccharomyces cerevisiae β-Glucanases: Differences between Protoplast and Periplasmic Enzymes

SUMMARY: Cell-free extracts and culture medium from Saccharomyces cerevisiae S288C contained the two glucan hydrolases that are known to be periplasmically located in vegetative cells of this yeast. These were an endo-1,3-β-glucanase and a much more abundant exo-1,3-β-glucanase. Cell-free extracts of strains carrying two different mutant alleles of the EXB1 gene were totally deficient in the latter enzyme, whereas the former appeared in multiple heterogeneous forms, probably due to incomplete glycosylation. In contrast, protoplast lysates of wild-type and exb1 mutant strains were identical in their complement of glucanases, which consisted of two enzymes clearly distinguishable from the periplasmic glucanases. One was an exo-1,3-β-glucanase, which was active on pustulan, p-nitrophenyl β-d-glucoside, salicin and cellobiose, and was of higher M r than periplasmic exoglucanase. The other was a hydrolase acting on 1,3-β-glucan and 1,6-β-glucan but not the simple glucosides, which was not retained by DEAE-Biogel. Wild-type and exb1 mutant protoplasts secreted portions of the glucanases detected in protoplast lysates, when cultured in osmotically stabilized regeneration medium; the former also secreted the periplasmic exo-1,3-β-glucanase but the latter consistently failed to secrete it. It is concluded that the classically known glucanases of S. cerevisiae, located in the periplasmic space, must be formed as active enzymes, upon secretion, from inactive cytoplasmic precursors. On the other hand, the two new protoplast glucanases could be secreted by an alternative route that assures their incorporation into the wall structure, thus making their release into the culture medium more difficult.