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Proteinase A (PrA) is one of the most significant vacuolar proteinase in S. cerevisiae, and it plays an important role in S. cerevisiae physiology and metabolism, especially under unfavorable environment. In this study, the differences in pyruvate kinase (PYK) level under fructose-1,6-diphosphate (FDP) induction and ATP synthesis block among SC1 (the wild-type yeast that was industrial Saccharomyces cerevisiae WZ65), SC2 ( PEP4 partial deletion) and SC3 ( PEP4 complete deletion) were examined. Results showed that the induction caused by FDP clearly increased PYK expression no matter for which strain, but the increasing effect is more significant for SC2 ( P < 0.05). The comparative results of intracellular ATP accumulation showed that the induction by FDP may be affected at the presence of PrA. The block experiment of ATP synthesis showed that PYK activities in PEP4-modified strains are lower than that of the wild type, but the intracellular ATP levels in the wild-type one are generally higher than the PEP4-modified strains after rotenone treatment ( P < 0.01). This implies that the effect of PrA deficiency on intracellular ATP accumulation was much more pronounced than the effect of rotenone on oxidative phosphorylation. The cell morphology of three strains was comparatively examined by means of transmission electron microscopy (TEM). The PEP4-modified strains possessed more vacuoles, and cell structure were more integrated than the wild-type strain. Current data preliminarily indicated that the deletion of PEP4 gene in industrial S. cerevisiae WZ65 may not only affected PYK expression but also modulated the oxidative phosphorylation flux. [ABSTRACT FROM AUTHOR]

S. cerevisiae proteinase A (PrA) is a member of the aspartic proteinase superfamily. The roles of PrA in S. cerevisiae physiology and cell metabolism are controversial. The objective of the current study was to elucidate the effects of the absence of PrA on key enzymes with regard to the glycolytic flux in industrial S. cerevisiae. The observed activities of hexokinase (HK), phosphofructokinase (Pfk) and pyruvate kinase (PYKi) in PrA-modified S. cerevisiae strains (SC2 and SC3) were lower than that of the wild-type strain ( p < 0.01). Compared to other strains, SC3 revealed the lowest activity of three key glycolytic enzymes. Current results imply that PrA in industrial S. cerevisiae may control the glycolytic enzymes expression (HK, Pfk and PYKi) in the direct or indirect manner and thus lead to the delay of cell metabolism. The observed intracellular ATP levels between the wild-type strain and PrA-modified strains (SC1 and SC2) were significantly different ( p < 0.01). The pronounced differences of key glycolytic enzymes between the wild-type and PEP4-modified strains were as well characterized by SDS-PAGE. The intracellular protein concentration in the presence of PrA is higher than those of the PrA-modified strains. As a result, the interaction mode of PrA and the glycolytic enzymes was postulated in this work. The findings herein suggest that the glycolytic flux direction and rate may be regulated by vacuolar PrA in industrial S. cerevisiae. The present data obtained provide insights into understanding the roles of PrA in industrial S. cerevisiae. [ABSTRACT FROM AUTHOR]