Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes. The cleavage of ubiquitin chains from ubiquitinated proteins is performed by deubiquitinating enzymes, of which there are 16 in the Ubp (ubiquitin specific protease) group in the budding yeast Saccharomyces cerevisiae. The goal of my thesis has been to examine the biological roles and molecular functions of these enzymes using a combination of proteomic and
molecular genetic approaches.
As part of a large collaborative effort, interacting protein partners of the Ubps were
isolated through affinity purification of tagged proteins, followed by protein identification by mass spectrometry. Purification of tagged Ubp6 led to the identification of the 19S proteasome
complex, along with a novel subunit, Sem1. As the human homologue of Sem1 was previously
identified as being associated with a protein involved in the repair of DNA double-strand breaks, I examined the possible role of Sem1 in DNA damage repair. A deletion of Sem1 and other 19S subunits resulted in hypersensitivity to various DNA damaging drugs, implicating the 19S complex in the process of DNA repair.
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I also found that purified Ubp2 interacted stably with the ubiquitin ligase Rsp5 and the
protein Rup1. UBP2 interacts genetically with RSP5, indicating a functional relationship, while
Rup1 facilitates the physical tethering of Ubp2 to Rsp5. Using the uracil permease Fur4, a Rsp5
substrate, as a model reporter, I found that ubp2Δ cells exhibited a temporal stabilization of Fur4
at the plasma membrane following the induction of endocytosis, implicating Ubp2 in protein
sorting, specifically at the multivesicular body. In order to understand the role of Ubp2, I examined the effect of Ubp2 on Rsp5 function. I found that Rsp5, similar to its mammalian homologues, is auto-ubiquitinated in vivo, and that Ubp2 is able to directly deubiquitinate Rsp5 in vitro. Moreover, the presence of a substrate or Rup1 both resulted in increased autoubiquitination, implying an auto-inhibitory mechanism of Rsp5 regulation. Taken together, the data presented in this thesis implicate deubiquitinating enzymes in interesting and varied roles in the cell, and suggest a novel mechanism for the modulation of Rsp5-dependent trafficking processes.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/26364 |
| Date | 23 February 2011 |
| Creators | Lam, Mandy Hiu Yi |
| Contributors | Emili, Andrew |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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