Abstract
Quality control ubiquitin ligases promote degradation of misfolded proteins by the proteasome. If the capacity of the ubiquitin/proteasome system is exceeded, then misfolded proteins accumulate in aggregates that are cleared by the autophagic system. To identify components of the ubiquitin/proteasome system that protect against aggregation, we analyzed a GFP-tagged protein kinase, Ste11ΔN(K444R)-GFP, in yeast strains deleted for 14 different ubiquitin ligases. We show that deletion of almost all of these ligases affected the proteostatic balance in untreated cells such that Ste11ΔN(K444R)-GFP aggregation was changed significantly compared with the levels found in wild type cells. By contrast, aggregation was increased significantly in only six E3 deletion strains when Ste11ΔN(K444R)-GFP folding was impaired due to inhibition of the molecular chaperone Hsp90 with geldanamycin. The increase in aggregation of Ste11ΔN(K444R)-GFP due to deletion of UBR1 and UFD4 was partially suppressed by deletion of UBR2 due to up-regulation of Rpn4, which controls proteasome activity. Deletion of UBR1 in combination with LTN1, UFD4, or DOA10 led to a marked hypersensitivity to azetidine 2-carboxylic acid, suggesting some redundancy in the networks of quality control ubiquitin ligases. Finally, we show that Ubr1 promotes clearance of protein aggregates when the autophagic system is inactivated. These results provide insight into the mechanics by which ubiquitin ligases cooperate and provide feedback regulation in the clearance of misfolded proteins.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Azetidinecarboxylic Acid / pharmacology
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Benzoquinones / pharmacology
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Blotting, Western
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Gene Regulatory Networks*
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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HSP90 Heat-Shock Proteins / antagonists & inhibitors
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HSP90 Heat-Shock Proteins / genetics
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HSP90 Heat-Shock Proteins / metabolism
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Lactams, Macrocyclic / pharmacology
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MAP Kinase Kinase Kinases / chemistry
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MAP Kinase Kinase Kinases / genetics
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MAP Kinase Kinase Kinases / metabolism
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Microbial Viability / drug effects
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Microbial Viability / genetics
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Microscopy, Fluorescence
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Mutation
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Proteasome Endopeptidase Complex / genetics
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Proteasome Endopeptidase Complex / metabolism
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Protein Folding
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae / growth & development
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics*
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Saccharomyces cerevisiae Proteins / metabolism
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Ubiquitin / genetics
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Ubiquitin / metabolism
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Ubiquitin-Protein Ligases / genetics*
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Ubiquitin-Protein Ligases / metabolism
Substances
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Benzoquinones
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HSP90 Heat-Shock Proteins
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Lactams, Macrocyclic
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Saccharomyces cerevisiae Proteins
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Ubiquitin
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Green Fluorescent Proteins
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Azetidinecarboxylic Acid
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Ufd4 protein, S cerevisiae
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Ltn1 protein, S cerevisiae
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SSM4 protein, S cerevisiae
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UBR1 protein, S cerevisiae
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Ubiquitin-Protein Ligases
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Ubr2 protein, S cerevisiae
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MAP Kinase Kinase Kinases
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Ste11 protein, S cerevisiae
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Proteasome Endopeptidase Complex
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geldanamycin