<i>Saccharomyces cerevisiae</i> Ub-Conjugating Enzyme Ubc4 Binds the Proteasome in the Presence of Translationally Damaged Proteins

Chuang, Shiow-Shuh; Madura, Kiran

doi:10.1534/genetics.105.046888

Cited by 38 publications

(37 citation statements)

References 41 publications

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“…GST-C Rpn11 also enabled growth at the semipermissive temperature (30°C) on medium containing the translational inhibitors paromomycin and hygromycin-B. Both drugs generate high levels of damaged proteins and provide a rapid way to examine proteolytic proficiency (25). The partial suppression of the growth defect of rpn11-1 by GST-C Rpn11 was accompanied by a moderate recovery of proteasome function (see Fig.…”

Section: Proteasomes Purified With a Lid Subunit Confirmed An Assemblmentioning

confidence: 97%

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Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

Chandra¹,

Chen²,

Liang³

et al. 2010

Journal of Biological Chemistry

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A major fraction of intracellular protein degradation is mediated by the proteasome. Successful degradation of these substrates requires ubiquitination and delivery to the proteasome followed by protein unfolding and disassembly of the multiubiquitin chain. Enzymes, such as Rpn11, dismantle multiubiquitin chains, and mutations can affect proteasome assembly and activity. We report that different rpn11 mutations can affect proteasome interaction with ubiquitinated proteins. Moreover, proteasomes are unstable in rpn11-1 and do not form productive interactions with multiubiquitinated proteins despite high levels in cell extracts. However, increased levels of ubiquitinated proteins were found associated with shuttle factors. In contrast to rpn11-1, proteasomes expressing a catalytically inactive mutant (rpn11 AXA ) were more stable and bound very high amounts of ubiquitinated substrates. Expression of the carboxyl-terminal domain of Rpn11 partially suppressed the growth and proteasome stability defects of rpn11-1. These results indicate that ubiquitinated substrates are preferentially delivered to intact proteasome.

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Section: Proteasomes Purified With a Lid Subunit Confirmed An Assemblmentioning

confidence: 97%

“…Ten-fold serial dilutions were prepared and spotted on YPD agar plates and then incubated at either 23°C or 37°C. Aliquots were also spotted onto medium containing 0.8 mM paromomycin or 0.1 mM hygromycin-B (25).…”

Section: Measurement Of Llvy-amc Hydrolysis-protein Lysates (15mentioning

confidence: 99%

Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

Chandra¹,

Chen²,

Liang³

et al. 2010

Journal of Biological Chemistry

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“…15,16 The Ubc13/Mms2 heterodimer is a specialized E2 that forms Lys63-linked polyubiquitin 15 whereas Ubc4 and Ubc5 are considered prototypical E2 enzymes that form Lys48-linked polyubiquitin to target short-lived proteins to the proteasome. 17,18 However, despite this generalization, not all effects of Lys63-linked polyubiquitin conjugation are due to Ubc13/Mms2 activity. A substantial amount of Lys63-linked polyubiquitin depends on presence of Ubc4 and Ubc5.…”

Section: Introductionmentioning

confidence: 99%

Yeast Chfr homologs retard cell cycle at G₁and G₂/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination

Loring¹,

Christensen²,

Gerber³

et al. 2008

Cell Cycle

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Checkpoint with forkhead-associated and RING (Chfr) is a ubiquitin ligase (E3) that establishes an antephase or prometaphase checkpoint in response to mitotic stress. Though ubiquitination is essential for checkpoint function, the sites, linkages and ubiquitin conjugating enzyme (E2) specificity are controversial. Here we dissect the function of the two Chfr homologs in S. cerevisiae, Chf1 and Chf2, overexpression of which retard cell cycle at both G 1 and G 2 . Using a genetic assay, we establish that Ubc4 is required for Chf2-dependent G 1 cell cycle delay and Chf protein turnover. In contrast, Ubc13/Mms2 is required for G 2 delay and does not contribute to Chf protein turnover. By reconstituting cis and trans-ubiquitination activities of Chf proteins in purified systems and characterizing sites modified and linkages formed by tandem mass spectrometry, we discovered that Ubc13/Mms2-dependent modifications are a distinct subset of those catalyzed by Ubc4. Mutagenesis of Lys residues identified in vitro indicates that site-specific Ubc4-dependent Chf protein autoubiquitination is responsible for Chf protein turnover. Thus, combined genetic and biochemical analyses indicate that Chf proteins have dual E2 specificity accounting for different functions in the cell cycle.

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“…In yeast, two orthologs of UbcH5B exist, UBC4 and UBC5. These genes are redundant, but display 1 partially distinct functions in vivo (Seufert and Jentsch 1990;Chuang and Madura 2005).…”

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Modulation of Ubc4p/Ubc5p-Mediated Stress Responses by the RING-Finger-Dependent Ubiquitin-Protein Ligase Not4p in Saccharomyces cerevisiae

Mulder

Inagaki

Cameroni³

et al. 2007

Genetics

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The Ccr4-Not complex consists of nine subunits and acts as a regulator of mRNA biogenesis in Saccharomyces cerevisiae. The human ortholog of yeast NOT4, CNOT4, displays UbcH5B-dependent ubiquitinprotein ligase (E3 ligase) activity in a reconstituted in vitro system. However, an in vivo role for this enzymatic activity has not been identified. Site-directed mutagenesis of the RING finger of yeast Not4p identified residues required for interaction with Ubc4p and Ubc5p, the yeast orthologs of UbcH5B. Subsequent in vitro assays with purified Ccr4-Not complexes showed Not4p-mediated E3 ligase activity, which was dependent on the interaction with Ubc4p. To investigate the in vivo relevance of this activity, we performed synthetic genetic array (SGA) analyses using not4D and not4L35A alleles. This indicates involvement of the RING finger of Not4p in transcription, ubiquitylation, and DNA damage responses. In addition, we found a phenotypic overlap between deletions of UBC4 and mutants encoding single-aminoacid substitutions of the RING finger of Not4p. Together, our results show that Not4p functions as an E3 ligase by modulating Ubc4p/Ubc5p-mediated stress responses in vivo.

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Saccharomyces cerevisiae Ub-Conjugating Enzyme Ubc4 Binds the Proteasome in the Presence of Translationally Damaged Proteins

Cited by 38 publications

References 41 publications

Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

Yeast Chfr homologs retard cell cycle at G₁and G₂/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination

Modulation of Ubc4p/Ubc5p-Mediated Stress Responses by the RING-Finger-Dependent Ubiquitin-Protein Ligase Not4p in Saccharomyces cerevisiae

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Saccharomyces cerevisiae Ub-Conjugating Enzyme Ubc4 Binds the Proteasome in the Presence of Translationally Damaged Proteins

Cited by 38 publications

References 41 publications

Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

Yeast Chfr homologs retard cell cycle at G1and G2/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination

Modulation of Ubc4p/Ubc5p-Mediated Stress Responses by the RING-Finger-Dependent Ubiquitin-Protein Ligase Not4p in Saccharomyces cerevisiae

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Yeast Chfr homologs retard cell cycle at G₁and G₂/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination