Sir Antagonist 1 (San1) Is a Ubiquitin Ligase

Dasgupta, Arindam; Ramsey, Kerrington L.; Smith, Jeffrey S.; Auble, David T.

doi:10.1074/jbc.m400894200

Cited by 50 publications

(46 citation statements)

References 68 publications

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“…Cells were grown at 37°C and induced for 1.5 h at an optical density at 600 nm of 0.6 to 0.7 with 0.3 mM IPTG (isopropyl-␤-D-thiogalactopyranoside). Cell lysates were made by sonication in a buffer containing 50 mM Tris-Cl, pH 8.0, and 1 mM EDTA, as described previously (11). Lysate (10 ml) was incubated for 1 h at 4°C with 1 ml prewashed GST-Sepharose beads in the presence of 1% Triton X-100 and protease inhibitors.…”

Section: Methodsmentioning

confidence: 99%

“…To determine whether the RING domain is important for the in vivo function of Rkr1, we mutated the first cysteine of the RING domain, changing it to an alanine (Rkr1-C1508A). Similar substitutions have been shown to disrupt the functions of other RING domain-containing proteins (11,80 RKR1 were transformed with plasmids expressing wild-type and C1508A forms of Rkr1. Growth assays showed that strains expressing wild-type Rkr1 grow on media lacking inositol, while strains expressing Rkr1-C1508A grow as poorly as rkr1⌬ strains ( Fig.…”

Section: Rkr1 Contains a Conserved Functionally Important Ring Domainmentioning

confidence: 96%

“…Several nuclear RING domain-containing ubiquitin-protein ligases have been characterized in yeast. These include Rad5 and Rad18, which direct the ubiquitylation of PCNA during DNA damage repair (27); San1, which is required for the degradation of certain misfolded nuclear proteins (11,19); Bre1, which ubiquitylates histone H2B K123 (31,83); and Not4, which is a component of the multifunctional Ccr4-Not complex (56).…”

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confidence: 99%

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Identification of Rkr1, a Nuclear RING Domain Protein with Functional Connections to Chromatin Modification in Saccharomyces cerevisiae

Braun

Costa

Crisucci

et al. 2007

Molecular and Cellular Biology

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Proper transcription by RNA polymerase II is dependent on the modification state of the chromatin template. The Paf1 complex is associated with RNA polymerase II during transcription elongation and is required for several histone modifications that mark active genes. To uncover additional factors that regulate chromatin or transcription, we performed a genetic screen for mutations that cause lethality in the absence of the Paf1 complex component Rtf1. Our results have led to the discovery of a previously unstudied gene, RKR1. Strains lacking RKR1 exhibit phenotypes associated with defects in transcription and chromatin function. These phenotypes include inositol auxotrophy, impaired telomeric silencing, and synthetic lethality with mutations in SPT10, a gene that encodes a putative histone acetyltransferase. In addition, deletion of RKR1 causes severe genetic interactions with mutations that prevent histone H2B lysine 123 ubiquitylation or histone H3 lysine 4 methylation. RKR1 encodes a conserved nuclear protein with a functionally important RING domain at its carboxy terminus. In vitro experiments indicate that Rkr1 possesses ubiquitin-protein ligase activity. Taken together, our results identify a new participant in a protein ubiquitylation pathway within the nucleus that acts to modulate chromatin function and transcription.Progression of the RNA polymerase II (Pol II) transcription cycle involves the coordinated functions of a large number of regulatory proteins. During transcription elongation, proteins that associate with Pol II assist it in overcoming obstacles to transcription, including DNA damage and condensed chromatin structure. Transcription elongation factors use a variety of mechanisms to facilitate movement of Pol II through a nucleosomal template and coordinate transcription with RNA processing. The Paf1 complex is a Pol II-associated factor that alters the state of the chromatin template during transcription elongation. Biochemical purification of the Saccharomyces cerevisiae Paf1 complex showed that it minimally contains five proteins: Paf1, Ctr9, Rtf1, Cdc73, and Leo1 (37,44,76). Chromatin immunoprecipitation experiments demonstrated that this complex is associated with the open reading frames (ORFs) of actively transcribed genes (37,59,72). Strains lacking members of the Paf1 complex are sensitive to the base analogs 6-azauracil and mycophenolic acid and exhibit altered RNA levels for a large number of genes (10,44,57,76). These results, combined with genetic and physical interactions with the elongation factors Spt4-Spt5 and Spt16-Pob3 (yFACT complex) (76), suggest that the Paf1 complex is important for transcription elongation. Members of the Paf1 complex are also required for proper 3Ј end formation of both polyadenylated and nonpolyadenylated transcripts (57, 68).It is now well established that the arrangements of posttranslational modifications on nucleosomal histones, along with interactions between histones and nonhistone proteins, coordinately affect chromatin structure. Histones can...

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Section: Methodsmentioning

confidence: 99%

Section: Rkr1 Contains a Conserved Functionally Important Ring Domainmentioning

confidence: 96%

mentioning

confidence: 99%

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Identification of Rkr1, a Nuclear RING Domain Protein with Functional Connections to Chromatin Modification in Saccharomyces cerevisiae

Braun

Costa

Crisucci

et al. 2007

Molecular and Cellular Biology

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“…The lack of activity of the autophagy-lysosome system in the nucleus (9) suggests that nuclear PQC depends entirely on the UPS. Supporting this hypothesis, studies demonstrated that the San1 E3 ligase, which shows specificity for nuclear aberrant proteins, plays a pivotal role in nuclear PQC in the budding yeast Saccharomyces cerevisiae (10,11). Furthermore, recent studies in mammalian cells and primary neurons suggest that the UHRF-2 E3 ligase is an essential molecule for nuclear polyglutamine degradation as a component of the nuclear PQC machinery (12).…”

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confidence: 96%

Nuclear Protein Quality Is Regulated by the Ubiquitin-Proteasome System through the Activity of Ubc4 and San1 in Fission Yeast

Matsuo

Kishimoto

Tanae

et al. 2011

Journal of Biological Chemistry

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Eukaryotic cells monitor and maintain protein quality through a set of protein quality control (PQC) systems whose role is to minimize the harmful effects of the accumulation of aberrant proteins. Although these PQC systems have been extensively studied in the cytoplasm, nuclear PQC systems are not well understood. The present work shows the existence of a nuclear PQC system mediated by the ubiquitin-proteasome system in the fission yeast Schizosaccharomyces pombe. Asf1-30, a mutant form of the histone chaperone Asf1, was used as a model substrate for the study of the nuclear PQC. A temperature-sensitive Asf1-30 protein localized to the nucleus was selectively degraded by the ubiquitin-proteasome system. The Asf1-30 mutant protein was highly ubiquitinated at higher temperatures, and it remained stable in an mts2-1 mutant, which lacks proteasome activity. The E2 enzyme Ubc4 was identified among 11 candidate proteins as the ubiquitin-conjugating enzyme in this system, and San1 was selected among 100 candidates as the ubiquitin ligase (E3) targeting Asf1-30 for degradation. San1, but not other nuclear E3s, showed specificity for the mutant nuclear Asf1-30, but did not show activity against wild-type Asf1. These data clearly showed that the aberrant nuclear protein was degraded by a defined set of E1-E2-E3 enzymes through the ubiquitin-proteasome system. The data also show, for the first time, the presence of a nuclear PQC system in fission yeast.In eukaryotic cells, the ubiquitin-proteasome system (UPS) 3 has a pivotal role in multiple cellular events, including the cell cycle, signal transduction, and receptor-mediated endocytosis (1-3). This system is essential for the selective degradation of many cellular proteins. The substrate proteins destined for degradation are first recognized by the ubiquitination machinery, triggering the covalent attachment of a polyubiquitin chain to the target protein. Polyubiquitinated proteins are recognized and degraded by the 26 S proteasome. The formation of the polyubiquitin chain is accomplished by a series of enzymatic reactions catalyzed by three enzymes: an E1 (ubiquitin-activating enzyme), an E2 (ubiquitin-conjugating enzyme), and an E3 (ubiquitin ligase). The step catalyzed by the E3 is crucial in determining substrate selectivity and timing of degradation, which implies that the identification and understanding of E3s is important to elucidate the mechanisms of specific substrate selection.The UPS also contributes to cellular protein quality control (PQC) (4). Aberrant or misfolded proteins are produced in the cell by mutation or environmental stress. The intracellular accumulation of these proteins causes proteotoxic or harmful effects. In humans, for example, the accumulation of aberrant proteins is thought to be associated with diseases such as Alzheimer, Huntington, Parkinson, and Creutzfeldt-Jakob diseases (5). The removal of these harmful proteins and the maintenance of homeostasis are accomplished through the selective degradation of aberrant or misfolded proteins...

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“…Nuclear PQC in the budding yeast Saccharomyces cerevisiae is controlled by the San1 ubiquitin ligase (29,37). San1 contains highly disordered regions that identify misfolded substrates by binding to exposed hydrophobic stretches on the substrate (38 -43).…”

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confidence: 99%

The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control

Ibarra

Sandoval

Fredrickson

et al. 2016

Journal of Biological Chemistry

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Protein quality control (PQC) is a critical process wherein misfolded or damaged proteins are cleared from the cell to maintain protein homeostasis. In eukaryotic cells, the removal of misfolded proteins is primarily accomplished by the ubiquitin-proteasome system. In the ubiquitin-proteasome system, ubiquitin-conjugating enzymes and ubiquitin ligases append polyubiquitin chains onto misfolded protein substrates signaling for their degradation. The kinetics of protein ubiquitylation are paramount as a balance must be achieved between the rapid removal of misfolded proteins versus providing sufficient time for protein chaperones to attempt refolding. To uncover the molecular basis for how PQC substrate ubiquitylation rates are controlled, the reaction catalyzed by nuclear ubiquitin ligase San1 was reconstituted in vitro. Our results demonstrate that San1 can function with two ubiquitin-conjugating enzymes, Cdc34 and Ubc1. Although Cdc34 and Ubc1 are both sufficient for promoting San1 activity, San1 functions preferentially with Ubc1, including when both Ubc1 and Cdc34 are present. Notably, a homogeneous peptide that mimics a misfolded PQC substrate was developed and enabled quantification of the kinetics of San1-catalyzed ubiquitylation reactions. We discuss how these results may have broad implications for the regulation of PQC-mediated protein degradation.For cells to maintain proteostasis, a delicate balance must exist between protein biogenesis and degradation. The ubiquitin-proteasome system is a network of proteins and enzymes responsible for 70 -80% of intracellular protein degradation in eukaryotic cells (1). The signal for protein degradation is the assembly of a polyubiquitin chain onto protein substrate.Ubiquitylation occurs through the sequential action of three enzymes: E1 2 (ubiquitin-activating enzyme), E2 (ubiquitinconjugating enzyme), and E3 (ubiquitin ligase) (2-5). E1 activates ubiquitin, a highly conserved 76-amino acid protein forming a thioester bond between the C-terminal carboxyl group on ubiquitin and a cysteine residue located within the E1 active site. Next, ubiquitin is transferred from E1 to E2. The E2ϳubiquitin (ϳ is used to denote a thioester bond) is then recruited by an E3, which brings the E2ϳubiquitin and protein substrate into proximity. E3s may also participate in ubiquitylation by stimulating the ubiquitin transfer activity of E2s (6 -11). In most cases ubiquitin is transferred from E2ϳubiquitin to the protein substrate, forming an isopeptide bond between the ubiquitin C terminus and a lysine residue on the substrate. The dissociation of E2s from E3 and the binding of fresh E2ϳubiquitin complexes enables the formation of a polyubiquitin chain on the substrate. Typically, a chain of at least four ubiquitins is required for a substrate to be recognized by the 26S proteasome for degradation (12, 13); however, the modification of several lysine residues with a single ubiquitin on some substrates may also be sufficient (14, 15). PQC is a collection of critical pathways within the ...

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Sir Antagonist 1 (San1) Is a Ubiquitin Ligase

Cited by 50 publications

References 68 publications

Identification of Rkr1, a Nuclear RING Domain Protein with Functional Connections to Chromatin Modification in Saccharomyces cerevisiae

Identification of Rkr1, a Nuclear RING Domain Protein with Functional Connections to Chromatin Modification in Saccharomyces cerevisiae

Nuclear Protein Quality Is Regulated by the Ubiquitin-Proteasome System through the Activity of Ubc4 and San1 in Fission Yeast

The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control

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