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Date of Award
8-2024
Document Type
Restricted Thesis: Campus only access
Degree Name
Master of Science in Biology
Department
Biology
First Reader/Committee Chair
Nickerson, Daniel
Abstract
Rab GTPases (Rabs) are lipid-anchored signaling proteins in eukaryotic cells whose many functions include regulating the formation, budding, transport, tethering, and fusion of vesicles to the target membrane of organelles. Rab signaling is controlled by accessory proteins known as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that control Rab activation, signal termination, and localization. Proper regulation of Rab signaling is important to the health of cells and organisms. Therefore, it is crucial to get a better spatiotemporal understanding of GAP regulation on the activity of Rabs.
Ypt1 is an evolutionary conserved Rab protein in Saccharomyces cerevisiae (S. cerevisiae) that is similar to Rab1 in humans. Ypt1 regulates transport of vesicles between the ER to the Golgi and regulates autophagy, both bulk and selective. Autophagy is a process in which proteins and organelles are destroyed in the vacuole/lysosome. However, there are still many locations and cellular processes where the inactivation of Ypt1 signaling has yet to be characterized. A GAP in yeast cells, Gyp8, localizes to areas where Ypt1 signaling inactivation is unknown: mitochondria, peroxisomes, and ER.
Here I tested the regulatory impact of YPT1 and GYP8 mutations on pexophagy and mitophagy, the degradation of the peroxisomes and mitochondria, respectively. We hypothesized the absence and overexpression of GYP8 would have a negative or positive impact on Ypt1 signaling in pexophagy and mitophagy. A limiting dilution growth plate revealed strong growth defects for ypt1Q67L (slower GTP hydrolysis) and ypt1Q67L gyp8∆ cells compared to wild-type and gyp8∆ cells. A biochemical cleavage assay using Pex11-GFP as a marker demonstrated inefficient pexophagy for ypt1Q67L and ypt1Q67L gyp8∆ via western blot. Overexpression of GYP8 in ypt1Q67L cells did not rescue pexophagy. Also, both ypt1Q67L and ypt1Q67L gyp8∆ groups had a high percentage of petite (white and small) colonies on a plate, an indication of mitochondrial dysfunction.
Overall, the growth defect from the limiting dilution growth plate revealed meaningful dysfunction pathways in cells when Ypt1 is in its slower GTP hydrolysis state. Also, there was a meaningful significance that Gyp8 is not primarily required in autophagy. However, our preliminary data does support Gyp8 playing a secondary or cooperative role in autophagy.
Recommended Citation
Ramos, Dangie, "PHENOTYPIC ANALYSIS ON THE GAP GYP8 REGULATION OF RAB GTPASE YPT1 IN AUTOPHAGY PATHWAY IN S. CEREVISIAE" (2024). Electronic Theses, Projects, and Dissertations. 1993.
https://scholarworks.lib.csusb.edu/etd/1993