Date of Award
Master of Science (MS)
Dr. Ellen France
Dr. Ashok Hegde
Dr. Michael Gleason
The 26S proteasome is a highly conserved proteolytic complex responsible for regulation of targeted degradation of proteins and cellular processes. It consists of two subassemblies: the 20S core particle (CP) and 19S regulatory particle (RP), which can further be divided into base and lid complexes. The base contains a heterohexameric ring (Rpt1-6) that interacts with the lid and CP. In Saccharomyces cerevisiae, the proteasome primarily localizes in the nucleus of proliferating cells. Inducing a cellular stress response such as carbon exhaustion causes the proteasome to shuttle from the nucleus to cytoplasmic compartments called proteasome storage granules (PSGs). Our study specifically focuses on the nucleocytoplasmic shuttling behavior of base subunit Rpt6. In this study, we observed under carbon exhaustion, Rpt6 moves from the nucleus to the cytoplasm into PSGs and colocalizes with Hsp42, a chaperone protein previously reported to localize to PSGs. A canonical nuclear localization signal does not appear to be present on Rpt6; therefore, we decided to truncate Rpt6 and observe its localization in order to identify the region required for Rpt6 nuclear shuttling. First, we attempted serial C-terminal truncations via PCR. However, C-terminal deletion caused a decrease in overall Rpt6 expression level and growth defect in a dominant-negative manner. Currently, we are exploring N-terminus region deletions using CRISPR-Cas9 technology, to target the first 44 amino acids of Rpt6 to be truncated. Identifying the specific region required for Rpt6 nuclear translocation will shed light on the mechanism of Rpt6 function and its role within the 26S proteasome in S. cerevisiae.
Groover, Des'ree, "Nucleocytoplasmic Shuttling of Rpt6 in Saccharomyces cerevisiae" (2020). Biology Theses. 12.
Available for download on Wednesday, July 21, 2021