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Title: Proteasome-associated deubiquitinating enzymes in fission yeast
Author: Stone, Miranda Lucy
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2002
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Two putative proteasome-associated DUBs were identified in S. pombe. Uch2 had previously been suggested to copurify and colocalise with the proteasome (Li et al., 2000). A second DUB, Ubp6, was identified in a sequence homology search for S. pombe proteins containing a ubiquitin-like (Ubl) domain (C. Semple and C. Gordon, unpublished). This domain has been shown to mediate interactions with the proteasome, implying that Ubp6 might also be proteasome-associated (Wilkinson et al., 2001). In the first part of this study, both Uch2 and Ubp6 are shown to be proteasome-associated DUBs. The S. pombe proteasome is purified and Uch2 and Ubp6 are both demonstrated to be present. In support of this finding, immunofluorescence microscopy reveals that Uch2 and Ubp6 colocalise with the proteasome at the nuclear periphery (Wilkinson et al., 1998). Construction of uch2 and ubp6 null mutants and the uch2ubp6 double mutant is described. These mutants do not have any obvious phenotype, suggesting that other redundant DUBs may be present at the proteasome. ubp6 is shown to be synthetically lethal with the mts1, mts2 and mts3 proteasome mutants, however uch2 is not synthetically lethal with any proteasome mutant tested (Gordon et al., 1993; Gordon et al., 1996; Penney et al., 1998; Wilkinson et al., 1997; Wilkinson et al., 2000, C. Gordon, unpublished). The DUBs function to cleave the αNH-peptide and the εNH-isopeptide bonds between ubiquitin and other species (Chung and Baek, 1999; Wilkinson, 2000). Purified 26S proteasomes and recombinant Uch2 are shown to cleave peptide linked ubiquitin using an in vitro assay and Uch2 is identified as the major ubiquitin hydrolase of the proteasome. Both Uch2 and Ubp6 are demonstrated to be capable of cleaving isopeptide-linked ubiquitin in vitro.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available