The Target of Rapamycin Complex 1 (TORC1) is a key and conserved regulator of cell growth and proliferation. The xenobiotic compound rapamycin is a potent inhibitor of TORC1 in yeast. The EGO complex, a non-essential activator of TORC1 is required for recovery of cells following rapamycin treatment. Why? Here, we find that rapamycin is in fact only a partial inhibitor of yeast TORC1; wild-type cells are able to maintain slow proliferation in the presence of high concentrations of the drug (i.e. concentrations multiple times the minimum inhibitory concentration). We find that this residual, rapamycin-insensitive, proliferation is dependent on the EGO complex and on TORC1 activity. We show that the ability of cells to maintain slow proliferation in the presence of rapamycin dictates their ability to recover. We find that rapamycin is not actively detoxified in yeast; instead, rapamycin is cleared by dilution-by-proliferation. The cell-associated intracellular pool of rapamycin is stable, decreasing only very slowly following washout of the drug and only diminishing at the rate of cell proliferation. The rapamycin-insensitive growth rate also persists long after rapamycin washout, indeed, until cells recover from the drug. The rapamycin-insensitive growth rate is not only able to quantitatively account for the observed kinetics of recovery from the drug in wild-type cultures, but also explains the severity of the ego- recovery defect. We contributed to a large-scale genetic screen seeking mutants that, like ego- mutants, fail to recover from rapamycin treatment. We find that loss of any one of 10 proteins identified results in a rapamycin recovery defect and a slow rapamycin-insensitive growth rate. Our data propose important or novel roles of the core HOPS/CORVET complex, threonine biosynthesis, Vps15p, Vsp34p, Ccr4p and Dhh1p activities in modulating the activity or efficiency of TORC1. Overall our results reveal that rapamycin is only a partial inhibitor of yeast TORC1, that persistence of the drug within the cell limits recovery and that rapamycin is not actively detoxified in yeast. Instead, recovery occurs due to dilution-by-proliferation and distribution of the drug among an increasing number of progeny cells. We also identify a set of potentially novel regulators of TORC1 activity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:643098 |
| Date | January 2015 |
| Creators | Evans, Stephanie Kaye |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/6168/ |
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