Spatial Reorganization of Saccharomyces cerevisiae Enolase To Alter Carbon Metabolism under Hypoxia

Abstract

c Hypoxia has critical effects on the physiology of organisms. In the yeast Saccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by an SNF1 knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-13 C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia, S. cerevisiae cells sense mitochondrial ROS and, by the involvement of SNF1/ AMPK, spatially reorganize metabolic enzymes in the cytosol via de novo protein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.

Glycolytic Enzymes Coalesce in G Bodies under Hypoxic Stress

“…Taken together, we confirmed G body localization of 31 out of 33 candidates identified by mass spectrometry, 29 of which were new G body components. Two factors (Fas1p and Fas2p) that we identified were also validated by colocalization by Miura et al, who also identified 8 unique factors by colocalization studies of their mass spectrometry candidates (Miura et al, 2013, S3D). …”

“…In this study, we characterize hypoxia-induced, non-membrane bound granules comprised of glycolytic enzymes that we refer to as glycolytic bodies,” or “G bodies,” in the budding yeast S. cerevisiae and in human hepatocarcinoma cells, confirming and expanding previous studies (Miura et al, 2013, Jang et al, 2016). Cells unable to form G bodies exhibit growth defects, specifically in hypoxia.…”

“…The mass spectrometry analysis was highly reproducible (Pearson Correlation Coefficient = 0.99, Figure S3B) and well-correlated in enrichment compared to normoxic or no-GFP tag controls (Figure S3C). Similar strategies, but omitting the enrichment steps by differential centrifugation, have been employed to purify glycolytic aggregates using tagged Eno2p as bait (Miura et al, 2013). …”

“…Altered protein localization and substrate channeling have also been proposed to regulate enzymatic and glycolytic activity (Kurganov et al, 1985, Menard et al, 2014). Recent work showing coalescence of certain glycolytic enzymes in yeast and C. elegans neurons under hypoxic stress suggests that changes in localization may be a stress response (Miura et al, 2013; Jang et al, 2016). …”

Spatial Organization of Metabolic Enzyme Complexes in Cells

“…32 Under hypoxic conditions, enolase in yeast cells was demonstrated to form punctate structures with glucokinase, glucose-6-phosphate isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, and pyruvate kinase. 33 In addition, F-actin was found to provide docking sites for the organization of hexokinase, glucose-6-phosphate isomerase, triose phosphate isomerase, glyceralde-hyde-3-phosphate dehydrogenase, phosphoglycerate mutase, and aldolase in yeast. 34 Particularly, their association with F-actin appears to increase individual enzyme activities while protecting against the inhibitory effects of trehalose in yeast cells.…”