Vengayil, V. | Search bioRxiv

bioRxiv2022

Phosphate budgeting to mitochondria controls glucose-mediated mitochondrial repression

Many cells in high glucose repress mitochondrial respiration, as observed in the Crabtree and Warburg effects. Our understanding of biochemical constraints for mitochondrial activation is limited. Using a Saccharomyces cerevisiae screen, we identified the conserved deubiquitinase Ubp3 (Usp10), as necessary for mitochondrial repression. Ubp3 mutants have increased mitochondrial activity despite abundant glucose, along with decreased glycolytic enzymes, and a rewired glucose metabolic network with increased trehalose production. Utilizing{Delta} ubp3 cells, along with orthogonal approaches, we establish that the high glycolytic flux in glucose continuously consumes free Pi. This restricts mitochondrial access to inorganic phosphate (Pi), and prevents mitochondrial activation. Contrastingly, rewired glucose metabolism with enhanced trehalose production and reduced GAPDH (as in{Delta} ubp3 cells) restores Pi. This collectively results in increased mitochondrial Pi and derepression, while restricting mitochondrial Pi transport prevents activation. We therefore suggest that glycolytic-flux dependent intracellular Pi budgeting is a key constraint for mitochondrial repression.

biochemistry↗

bioRxiv2019

The E3 ubiquitin ligase Pib1 regulates effective gluconeogenic shutdown in S. cerevisiae.

Cells use multiple mechanisms to regulate their metabolic states depending on changes in their nutrient environment. A well-known example is the response of cells to glucose availability. In S. cerevisiae cells growing in glucose-limited medium, the re-availability of glucose leads to the downregulation of gluconeogenesis, the activation of glycolysis, and robust glucose repression. However, our knowledge of the initial mechanisms mediating this glucose-dependent downregulation of the gluconeogenic transcription factors is incomplete. We used the gluconeogenic transcription factor Rds2 as a candidate with which to discover regulators of early events leading to glucose repression. Here, we identify a novel role for the E3 ubiquitin ligase Pib1 in regulating the stability and degradation of Rds2. Glucose addition to glucose-limited cells results in rapid ubiquitination of Rds2, followed by its proteasomal degradation. Through in vivo and in vitro experiments, we establish Pib1 as a ubiquitin E3 ligase that regulates Rds2 ubiquitination and stability. Notably, this Pib1 mediated Rds2 ubiquitination, followed by proteasomal degradation, is specific to the presence of glucose. Pib1 is required for complete glucose repression, and enables cells to optimally grow in competitive environments when glucose becomes re-available. Our results reveal the existence of a Pib1 E3-ubiquitin ligase mediated regulatory program that mediates glucose-repression when glucose availability is restored.

biochemistry↗