<p> The vacuole in <i>Saccharomyces cerevisiae</i> serves as a model for the mammalian lysosome. In a genome wide screen for mutants with severe growth <u>h</u>ypersensitivity to <u>h</u>ygromycin B, our lab identified 14 <i>HHY</i> genes. Each of the <i> hhy</i> mutants is defective in vacuolar trafficking and/or function and also sensitive to rapamycin and caffeine, suggesting a compromised target of rapamycin (TOR) kinase pathway. My research divides the <i>hhy</i> mutants into two groups based on quantitative growth analyses in the presence of hygromycin B. (1) a super affected group (<i>s-hhy's</i>) and (2) a dose-dependent group (<i>d-hhy's</i>). The <i>s-HHY</i> genes include <i>CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52,</i> and <i>VPS54</i>. Evaluation of the known functions of <i> s-HHY</i> gene products reveals vesicular trafficking function at the trans-Golgi and late endosome interface to be a common factor. In yeast, the TORC1 complex localizes to the vacuole. Because a compromised TORC1 complex signaling is suggested in <i>hhy</i> mutant strains due to their caffeine or rapamycin sensitivity, I hypothesized that compromised TORC1 signaling in <i>HHY's</i> may be due to defects in the vacuolar localization of Tor1 kinase. To assess Tor1 kinase localization, we utilized a strain expressing endogenously tagged Tor1-GFP and assayed localization to the vacuolar membrane in each of the s-hhy mutants using confocal microscopy. In wild-type cells, Tor1-GFP co-localizes with the vacuolar membrane marker FM4-64 while s-hhy deletion strains fail to localize Tor1-GFP to the vacuolar membrane when treated with hygromycin B. Our results implicate that Tor1p is transported to the vacuole membrane via the late endosome (CPY pathway) and not the ALP (Vam3) pathway. Additionally, <i>s-hhy</i> mutants are unable to recover growth after a 4-hour treatment with hygromycin B, similar to EG0 mutants, which fail to exit from G0 after treatment with the TORC1 inhibiting drug rapamycin. Based on our data, we propose a model in which the <i>s-HHY </i> gene functions in vesicular trafficking at the trans-Golgi/late endosome interface are involved in recruitment and subsequent transport of Tor1p to the vacuolar membrane, and that interface is hypersensitive to hygromycin B. We also propose that Tor1 kinase localization at the vacuole is essential for its cell cycle regulatory function.</p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:1527392 |
| Date | 08 April 2014 |
| Creators | Locken, Kristopher Michael |
| Publisher | California State University, Long Beach |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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