Inorganic phosphate \((P_i)\) is an essential nutrient whose intracellular levels are maintained by the PHO pathway in Saccharomyces cerevisiae. \(P_i\) limitation triggers upregulation of the PHO genes whose gene products primarily function to counterbalance the \(P_i\) deficiency. Despite a growing catalogue of genes that are involved in signaling of the PHO pathway, little is known about how cells actually sense \(P_i\) limitation. To better characterize the \(P_i\) sensing mechanism, I exploited two comprehensive and orthogonal approaches: 1) genome-wide genetic screening to identify novel genes involved in signaling \(P_i\) limitation through the PHO pathway and characterization of genetic interactions among these genes and 2) liquid chromatography /mass spectrometry (LC/MS)-based metabolic profiling to characterize the metabolomic response to changes in \(P_i\) availability. In genome-wide screening, I found that the aah1 mutant constitutively activated the PHO pathway and showed that AAH1 is involved in regulating PHO pathway activity. Moreover, I identified several novel genetic interactions of genes involved in inositol polyphosphate metabolism with those involved in purine metabolism and mitochondrial fatty acid biosynthesis.Through metabolomic profiling, I showed that all adenine nucleotides were downregulated in the constitutively induced ado1, adk1, and aah1 mutants in high \(P_i\) as well as in the wild type strain in low \(P_i\). These observations led to the hypothesis that downregulation of adenine nucleotides triggers activation of the PHO pathway. However, I find that decreases in adenine nucleotides appear to be the consequence of downregulation of glycolysis and of the pentose phosphate pathway rather than an activation signal for the PHO pathway.Among all the detected metabolites, S-adenosyl-L-homocysteine (SAH) responded the most quickly and significantly to changes in \(P_i\) concentration. It was known that SAH is an inhibitor of de novo synthesis of phosphatidylcholine (PC). I showed that overall PC levels were downregulated in low \(P_i\), suggesting that phospholipid metabolism is downregulated in low \(P_i\) conditions. Furthermore, I observed that exogenous SAH induces activation of the PHO pathway in high \(P_i\) implying a possible role of SAH as an initiating activation signal of the PHO pathway. / Chemistry and Chemical Biology
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/11125111 |
| Date | 07 June 2014 |
| Creators | Choi, Joonhyuk |
| Contributors | O'Shea, Erin K |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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