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Shortill, Mathew

University of British Columbia

Intracellular pH (pHi) is an important physiochemical property of the cell. This property is important for regulating biochemical reactions, protein folding and signal transduction. Eukaryotic cells have organelles with different pHs that allow for diversity of function and chemistry occurring within. In yeast, pHi is established and maintained by the plasma membrane proton pump Pma1. Pma1 is regulated by glucose availability, when glucose is scarce Pma1 is inhibited causing cytosolic acidification. Therefore, pHi is a potent metabolic signal and can be recognized by pH biosensors. Phosphatidic acid is a membrane lipid and pH biosensor that binds and sequesters, Opi1. When pHi is low, phosphatidic acid is protonated and loses affinity for Opi1, releasing it and allowing access to the nucleus to inhibit phospholipid biosynthesis. This links pHi to membrane biogenesis and suggests pH signaling occurs through the control of gene expression. To discover pathways responsive to pHi I utilized the pma1-007 hypomorphic allele with 50% reduced Pma1 protein expression. This strain is unable to maintain physiological pH when grown on acidic media and allows for a genetic model for cytosolic acidification. I grew WT and pma1-007 cells at pH 4 and performed RNA-seq on the cells to measure differentially expressed genes. I found a clustering of genes related to the electron transport chain, Krebs cycle and the degradation of allantoin (DAL). The DAL pathway is controlled by the Target of Rapamycin Complex 1 (TORC1) which is related to Pma1 function. The DAL pathway is regulated by activators, Gat1 and Gln3, and repressors, Dal80 and Gzf3. The pathway is comprised of the Dal1, Dal2 and Dal3 enzymes. Dal4 and Dal5 are transporters for the pathway allowing for nutrient import. I found that Gat1 and Gln3, were responsible for the pH dependent activation. Additionally, cells with downregulated Pma1 alone and in combination with deletion of GAT1, GLN3 or DAL80 were more sensitive to rapamycin, a TORC1 inhibitor, indicating TOR activity was reduced. I have discovered pH as a novel input for the DAL pathway and have provided more context for how Pma1 and TORC1 function together in regulating metabolic signalling.

Text

2025-02-14

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/90364

Cell and Developmental Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/