Abstract
Mutations in Sir Antagonist 1 (SAN1) suppress defects in SIR4 and SPT16 in Saccharomyces cerevisiae. San1 contains a RING domain, suggesting that it functions by targeting mutant sir4 and spt16 proteins for degradation by a ubiquitin-mediated pathway. Consistent with this idea, mutant sir4 and spt16 proteins are unstable in SAN1 cells but are stabilized in san1Delta cells. We demonstrate that San1 possesses ubiquitin-protein isopeptide ligase activity in vitro, and the ubiquitin-protein isopeptide ligase activity of San1 is required for its function in vivo. Wild-type Sir4 has a half-life of about 21 min, and san1Delta increased Sir4 half-life to >90 min. In contrast, san1Delta did not affect the stability of wild-type Spt16, Sir3, Sir2, or the Spt16-associated proteins Pob3 and Nhp6. Loss of SAN1 also did not affect the stability of Ste6-166, a highly unstable protein in yeast. These results support the idea that San1 controls the turnover of a specific class of unstable nuclear proteins. Sir4 nucleates the assembly of silent chromatin at telomeres and the silent mating-type loci (HM) in S. cerevisiae. Sir4 can also affect silencing in the rDNA indirectly by sequestering limiting Sir2. Increasing the stability of wild-type Sir4 by deleting SAN1 had only subtle effects on silencing, suggesting that silent chromatin in yeast is robustly buffered against changes in Sir4 stability. Consistent with the idea that San1 participates as an accessory factor to regulate silent chromatin, including the silent mating-type loci, microarray analysis defined a small but statistically significant role for San1 in transcription of several mating pheromone-responsive genes.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Cell Cycle Proteins / metabolism
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DNA, Ribosomal / genetics
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DNA-Binding Proteins / metabolism
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Gene Expression
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Gene Silencing
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Histone Deacetylases / chemistry
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Histone Deacetylases / genetics
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Histone Deacetylases / metabolism
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Humans
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Minichromosome Maintenance Complex Component 4
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Molecular Sequence Data
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Protein Structure, Tertiary
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / cytology
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Saccharomyces cerevisiae / genetics
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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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Silent Information Regulator Proteins, Saccharomyces cerevisiae / chemistry
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Silent Information Regulator Proteins, Saccharomyces cerevisiae / genetics
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Silent Information Regulator Proteins, Saccharomyces cerevisiae / metabolism*
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Sirtuin 2
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Sirtuins / chemistry
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Sirtuins / genetics
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Sirtuins / metabolism
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Telomere / genetics
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Triticum / enzymology
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Ubiquitin / chemistry
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Ubiquitin / metabolism
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Ubiquitin-Protein Ligases / chemistry
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism*
Substances
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Cell Cycle Proteins
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DNA, Ribosomal
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DNA-Binding Proteins
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Recombinant Proteins
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SIR1 protein, S cerevisiae
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SIR4 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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Silent Information Regulator Proteins, Saccharomyces cerevisiae
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Ubiquitin
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Ubiquitin-Protein Ligases
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SIR2 protein, S cerevisiae
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Sirtuin 2
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Sirtuins
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Histone Deacetylases
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MCM4 protein, S cerevisiae
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Minichromosome Maintenance Complex Component 4