Pleiotropic effects of Ubp6 loss on drug sensitivities and yeast prion are due to depletion of the free ubiquitin pool.

Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Mutation of the mouse Usp14 gene, encoding the homolog of yeast deubiquitinating enzyme Ubp6, causes ataxia. Here we show that deletion of the UBP6 gene in Saccharomyces cerevisiae causes sensitivity to a broad range of toxic compounds and antagonizes phenotypic expression and de novo induction of the yeast prion [PSI+], a functionally defective self-perpetuating isoform of the translation termination factor Sup35. Conversely, overexpression of ubiquitin (Ub) increases phenotypic expression and induction of [PSI+] in the wild type cells and suppresses all tested ubp6Delta defects, indicating that they are primarily due to depletion of cellular Ub levels. Several lines of evidence suggest that Ubp6 functions on the proteasome. First, Ub levels in the ubp6Delta cells can be partly restored by proteasome inhibitors, suggesting that deletion of Ubp6 decreases Ub levels by increasing proteasome-dependent degradation of Ub. Second, fluorescence microscopy analysis shows that Ubp6-GFP fusion protein is localized to the nucleus of yeast cell, as are most proteasomes. Third, the N-terminal Ub-like domain, although it is not required for nuclear localization of Ubp6, targets Ubp6 to the proteasome and cannot be functionally replaced by Ub. The human ortholog of Ubp6, USP14, probably plays a similar role in higher eukaryotes, since it fully compensates for ubp6Delta defects and binds to the yeast proteasome. These data link the Ub system to prion expression and propagation and have broad implications for other neuronal inclusion body diseases.
Mesh Terms:
Catalysis, Cell Nucleus, Cycloheximide, Cysteine Endopeptidases, Endopeptidases, Escherichia coli, Gene Deletion, Green Fluorescent Proteins, Humans, Immunoblotting, Kinetics, Luminescent Proteins, Microscopy, Fluorescence, Models, Genetic, Multienzyme Complexes, Mutation, Phenotype, Plasmids, Prions, Proteasome Endopeptidase Complex, Protein Structure, Tertiary, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Time Factors, Ubiquitin, Ubiquitin Thiolesterase
J. Biol. Chem. Dec. 26, 2003; 278(52);52102-15 [PUBMED:14559899]
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