Troutman, Kayla
(2023)
A functional characterization of Pib2: a key TORC1 regulator in S. cerevisiae.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Cell growth and survival are dependent on the ability of cells to respond to their environment and the nutrients that are available to them. The target of rapamycin complex 1 (TORC1) is a highly conserved kinase that is critical for nutrient sensing and the control of cell growth. Dysregulation of TORC1 signaling, including both hyper- and hypo-activity, has been implicated in a variety of diseases; thus, understanding how TORC1 is regulated is crucial. In Saccharomyces cerevisiae, TORC1 subcellular localization and cellular nutrient availability are key for its activity. TORC1 localizes to both vacuolar and endosomal membranes, where distinct TORC1 signaling occurs. Nutrient availability is signaled to TORC1 by the combined actions of several signaling pathways and proteins. One notable regulator, Phosphatidylinositol 3-phosphate-binding protein 2 (Pib2), is necessary for TORC1 reactivation after starvation or pharmacological inhibition. Interestingly, Pib2 can both hinder and facilitate TORC1 activity, however, the regulatory mechanisms remain poorly understood. Through a systematic mutagenesis and functional dissection of conserved regions and domains within S. cerevisiae Pib2, we have identified regions and key residues involved in TORC1 regulation and Pib2 localization. We use rapamycin exposure assays and live-cell confocal imaging to show that two Pib2 regions, helical region E and the tail motif, are vital for TORC1 reactivation, whereas the N-terminal regions A and B have TORC1 inhibitory functions. Furthermore, we show that while the Pib2 PI3P-binding FYVE domain is critical for vacuolar localization, it is unexpectedly not required for recovery from rapamycin induced growth arrest. Using chimeric Pib2 targeting constructs, we show that endosomal Pib2 is not sufficient for TORC1 reactivation and cell growth following rapamycin exposure. Here we have demonstrated that Pib2 plays a unique role as a dual modulator of TORC1 activity in S. cerevisiae, and that each of the conserved Pib2 regions differentially contribute to TORC1 regulation. These findings highlight key factors that could be manipulated to control cell growth and could provide insight into TORC1 regulation mechanisms in other eukaryotes.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
9 May 2023 |
| Date Type: |
Publication |
| Defense Date: |
10 November 2022 |
| Approval Date: |
9 May 2023 |
| Submission Date: |
12 December 2022 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
157 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Cell Biology and Molecular Physiology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
TORC1, Pib2, yeast, cell growth, rapamycin, FYVE |
| Date Deposited: |
09 May 2023 18:51 |
| Last Modified: |
09 May 2023 18:51 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/43988 |
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