Dissertation/Thesis Abstract

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Investigations of the functional interface between Env7 and TORC1 in <i>Saccharomyces cerevisiae</i>
by Shaoulian, Jonathan, M.S., California State University, Long Beach, 2016, 82; 10161773
Abstract (Summary)

In eukaryotic cells, deficiencies in proteins involved in lysosomal trafficking and function result in serious human disorders like Tay-Sach’s Disease or Alzheimer’s. The vacuole in Saccharomyces cerevisiae serves as a model for the mammalian lysosome. Our lab has identified ENV7, a novel gene involved in the late-endosome-to vacuole interface in Saccharomyces cerevisae. ENV7 (an ortholog of human STK16) encodes Env7, a vacuolar membrane kinase that negatively regulates vacuolar fusion. Tor1 is another conserved vacuolar membrane kinase, and has been proposed as a vacuolar fragmentation factor. Tor1, or its homolog Tor2, are the functional kinases of Tor Complex 1 (TORC1). Activated TORC1 stimulates cell growth and proliferation, as well as vacuolar fragmentation. In mammals, hyperactivity of TORC1 plays a major role in cancers and diabetes. Conversely, inhibition of TORC1 by starvation or treatment by the drug rapamycin, is associated with the initiation of autophagy, which itself involves a number of fusion events at the vacuole. Lastly, it has been shown that defects at various stages of vesicular trafficking play a role in disrupting Tor1 transport to the vacuole and perturbing the function of TORC1. Therefore, I hypothesized that there is a functional interface between Env7 and TORC1. Here I report that endogenously expressed Env7-GFP is localized to the vacuolar membrane, and upon inhibition of TORC1 by rapamycin, there is a significant mislocalization of Env7-GFP to diffuse cytoplasmic patterns. Overexpressed GFP-Env7 is also mislocalized when TORC1 activity is downregulated, but the mislocalization is to Golgi structures and to the plasma membrane. Additionally, while the absence of Tor1 is not sufficient to affect the localization of overexpressed GFP-Env7, when TORC1 is compromised by a deletion of TCO89 or by a more potent inhibition by rapamycin in both tor1Δ and tco89Δ cells, there is a significant mislocalization of GFP-Env7 to the plasma membrane. Taken together, my findings demonstrate that the mislocalization of Env7 is dependent on both the level of expression of Env7 and the state of TORC1 activity. Therefore, the results thus far support a possible functional interface between Env7 and TORC1. Furthermore, when TORC1 is inhibited by rapamycin treatment, unlike Env7-GFP, endogenously expressed Tor1-GFP remains on the vacuole, but forms numerous clusters at this location. In the course of testing my hypothesis, the effects of TORC1 inhibition on vacuolar morphology and onset of autophagy were also noted. I report that tor1Δ cells, when treated by rapamycin, display a higher percentage of enlarged vacuole morphology relative to wt, and that this enlarged vacuole morphology seems to be associated with slightly increased levels of autophagy. This suggests that the absence of Tor1, and presumably its substitution by Tor2, compromises TORC1 and makes the cell more sensitive to rapamycin-induced autophagy. Additionally, I also report that absence of Env7 has no significant effect on vacuolar enlargement and the onset of autophagy in response to rapamycin treatment, and thus, does not affect the autophagy regulation function of TORC1. The results of this study suggest a functional interface between Env7 and TORC1. Since both are conserved in humans, such an interface may also be conserved in humans.

Indexing (document details)
Advisor: Gharakhanian, Editte
Commitee: Fraser, Deborah, McAbee, Douglas
School: California State University, Long Beach
Department: Biological Sciences
School Location: United States -- California
Source: MAI 56/01M(E), Masters Abstracts International
Subjects: Molecular biology, Cellular biology, Biochemistry
Keywords: Autophagy, Endosome to vacuole interface, Rapamycin, Tor Complex 1, Vacuole, Yeast
Publication Number: 10161773
ISBN: 9781369166354