Large-scale preparation of yeast strains expressing condensates derived from a glycolytic enzyme via controlled dissolved oxygen levels under hypoxia.

Under hypoxia, Saccharomyces cerevisiae forms cytoplasmic condensates composed of proteins, including glycolytic enzymes, that are thought to regulate cellular metabolism. However, the hypoxic conditions required for condensate formation remain unclear. In this study, we developed a 300-mL-scale culture method to produce condensate-forming cells by precisely controlling the dissolved oxygen (DO) level in the media. Using enolase as a model, a foci formation rate of more than 50% was achieved at ∼0.1% DO, and the results showed that the DO level affected the foci formation rate. The foci formation rates of the previously reported foci-deficient strains and strains with single amino acid substitutions in the endogenous enolase were examined, and the effect of these amino acid substitutions on glucose consumption and ethanol and glycerol production under hypoxia was evaluated. The results of this study contribute to the investigation of the mechanisms that regulate biomacromolecular condensates under hypoxia.

Condensate Formation by Metabolic Enzymes in Saccharomyces cerevisiae.

Condensate formation by a group of metabolic enzymes in the cell is an efficient way of regulating cell metabolism through the formation of "membrane-less organelles." Because of the use of green fluorescent protein (GFP) for investigating protein localization, various enzymes were found to form condensates or filaments in living Saccharomyces cerevisiae, mammalian cells, and in other organisms, thereby regulating cell metabolism in the certain status of the cells. Among different environmental stresses, hypoxia triggers the spatial reorganization of many proteins, including more than 20 metabolic enzymes, to form numerous condensates, including "Glycolytic body (G-body)" and "Purinosome." These individual condensates are collectively named "Metabolic Enzymes Transiently Assembling (META) body". This review overviews condensate or filament formation by metabolic enzymes in S. cerevisiae, focusing on the META body, and recent reports in elucidating regulatory machinery of META body formation.

Small-scale hypoxic cultures for monitoring the spatial reorganization of glycolytic enzymes in Saccharomyces cerevisiae.

At normal oxygen concentration, glycolytic enzymes are scattered in the cytoplasm of Saccharomyces cerevisiae. Under hypoxia, however, most of these enzymes, including enolase, pyruvate kinase, and phosphoglycerate mutase, spatially reorganize to form cytoplasmic foci. We tested various small-scale hypoxic culture systems and showed that enolase foci formation occurs in all the systems tested, including in liquid and on solid media. Notably, a small-scale hypoxic culture in a bench-top multi-gas incubator enabled the regulation of oxygen concentration in the media and faster foci formation. Here, we demonstrate that the foci formation of enolase starts within few hours after changing the oxygen concentration to 1% in a small-scale cultivation system. The order of foci formation by each enzyme is tightly regulated, and of the three enzymes, enolase was the fastest to respond to hypoxia. We further tested the use of the small-scale cultivation method to screen reagents that can control the spatial reorganization of enzymes under hypoxia. An AMPK inhibitor, dorsomorphin, was found to delay formation of the foci in all three glycolytic enzymes tested. These methods and results provide efficient ways to investigate the spatial reorganization of proteins under hypoxia to form a multienzyme assembly, the META body, thereby contributing to understanding and utilizing natural systems to control cellular metabolism via the spatial reorganization of enzymes.