Proteomic and Molecular Genetic Investigation of Deubiquitinating Enzymes in the Budding Yeast Saccharomyces cerevisiae
Year: 2011
Type: dissertation
Abstract: 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\nmolecular genetic approaches.\nAs part of a large collaborative effort, interacting protein partners of the Ubps were\nisolated 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\ncomplex, along with a novel subunit, Sem1. As the human homologue of Sem1 was previously\nidentified 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.\niii\nI also found that purified Ubp2 interacted stably with the ubiquitin ligase Rsp5 and the\nprotein Rup1. UBP2 interacts genetically with RSP5, indicating a functional relationship, while\nRup1 facilitates the physical tethering of Ubp2 to Rsp5. Using the uracil permease Fur4, a Rsp5\nsubstrate, as a model reporter, I found that ubp2Δ cells exhibited a temporal stabilization of Fur4\nat the plasma membrane following the induction of endocytosis, implicating Ubp2 in protein\nsorting, 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.
Source: TSpace (University of Toronto)
Author: Mandy Hiu Yi Lam
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2755430209