The fluxes through glycolytic enzymes in Saccharomyces cerevisiae are predominantly regulated at posttranscriptional levels

Abstract

Metabolic fluxes may be regulated "hierarchically," e.g., by changes of gene expression that adjust enzyme capacities (V(max)) and/or "metabolically" by interactions of enzymes with substrates, products, or allosteric effectors. In the present study, a method is developed to dissect the hierarchical regulation into contributions by transcription, translation, protein degradation, and posttranslational modification. The method was applied to the regulation of fluxes through individual glycolytic enzymes when the yeast Saccharomyces cerevisiae was confronted with the absence of oxygen and the presence of benzoic acid depleting its ATP. Metabolic regulation largely contributed to the approximately 10-fold change in flux through the glycolytic enzymes. This contribution varied from 50 to 80%, depending on the glycolytic step and the cultivation condition tested. Within the 50-20% hierarchical regulation of fluxes, transcription played a minor role, whereas regulation of protein synthesis or degradation was the most important. These also contributed to 75-100% of the regulation of protein levels.

Fig. 1.

Cellular levels and in vivo fluxes of S. cerevisiae glycolysis cultivated in glucose-limited chemostats in aerobiosis or anaerobiosis with or without benzoic acid. (A) In vivo fluxes, calculated from measured external metabolites. (B) Specific enzyme activities (V_max). (C) Transcript levels. (D) Protein levels of S. cerevisiae cultivated in glucose-limited anaerobic versus aerobic chemostats. White bars represent aerobic cultures, gray bars represent anaerobic cultures, and black bars represent anaerobic cultures with benzoate, except in D, where gray bars represent the ratio of anaerobic to aerobic expression. Asterisks indicate significant changes as compared with aerobic chemostats (Student's t test, P = <0.01). Triangles indicate significant changes between anaerobic cultures with and without benzoic acid (Student's t test, P = <0.01).