Substrate Recognition in Nuclear Protein Quality Control Degradation Is Governed by Exposed Hydrophobicity That Correlates with Aggregation and Insolubility

Fredrickson, Eric K.; Gallagher, Pamela; Candadai, Sarah V. Clowes; Gardner, Richard G.

doi:10.1074/jbc.m112.406710

Cited by 48 publications

(61 citation statements)

References 53 publications

(31 reference statements)

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“…A recent report from the Gardner laboratory demonstrates that San1p can recognize substrates via continuous stretches of at least five hydrophobic amino acids (82,83). However, NBD2* does not appear to contain any obvious San1p recognition motifs (data not shown).…”

Section: Discussionmentioning

confidence: 98%

“…Two previously studied CytoQC substrates, ⌬ssCPY* and ⌬ssPrA, show a strong dependence on San1p (26,27), and because both are mislocalized substrates, they probably expose otherwise buried hydrophobic motifs, which are recognized by San1p. To this end, San1p contains natively unfolded regions at its N and C termini (82,83) that may be sufficient to mediate its association with unfolded protein substrates. Thus, we were surprised to discover a strong Ssa1p/Hsp70 dependence on the interaction between San1p and NBD2* (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Hsp70 Targets a Cytoplasmic Quality Control Substrate to the San1p Ubiquitin Ligase

Guerriero

Weiberth

Brodsky

2013

Journal of Biological Chemistry

109

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Background: San1p is a nuclear ubiquitin ligase that helps degrade misfolded cytoplasmic proteins in yeast. Results: The Hsp70, Ssa1p, facilitates an interaction between a novel misfolded substrate and San1p. Conclusion: Chaperones play a direct role in bridging aberrant cytoplasmic proteins to ubiquitin ligases. Significance: Understanding how chaperones select and target cytoplasmic proteins will help define how diseases associated with proteotoxic stress might be treated.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Hsp70 Targets a Cytoplasmic Quality Control Substrate to the San1p Ubiquitin Ligase

Guerriero

Weiberth

Brodsky

2013

Journal of Biological Chemistry

109

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“…San1 localizes to the nucleus under stress and nonstress conditions [74, 75], and ubiquitylates a large variety of misfolded substrates, including destabilized endogenous mutant proteins, short synthetic polypeptides, exogenous model misfolded proteins, as well as misfolded ER substrates lacking their ER-targeting sequences [74, 76–81]. The subcellular localizations of these polypeptides are diverse, where they are found in the nucleus but, perhaps more surprisingly, also in the cytoplasm as soluble or ER membrane-associated states (more on this below).…”

Section: Ups In the Nucleusmentioning

confidence: 99%

How the Nucleus Copes with Proteotoxic Stress

Shibata

Morimoto

2014

Current Biology

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Summary The proper folding of proteins is continuously challenged by intrinsic and extrinsic stresses, and the accumulation of toxic misfolded proteins is associated with many human diseases. Eukaryotic cells have evolved a complex network of protein quality control pathways to protect the proteome, and these pathways are specialized for each subcellular compartment. While many details have been elucidated for how the cytosol and endoplasmic reticulum counteract proteotoxic stress, relatively little is known about the pathways protecting the nucleus from protein misfolding. Here, we offer a conceptual framework for how proteostasis is maintained in this organelle. We define the particular requirements that must be considered for the nucleus to manage proteotoxic stress, summarize the known and implicated pathways of nuclear protein quality control, and identify the unresolved questions in the field. Proper maintenance of nuclear proteostasis has important implications in preserving genomic integrity, as well as for aging and disease.

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“…These are regions that are normally hidden in the native structure and are exposed upon structure perturbation. Such degrons include hydrophobic domains [5][6][7] and intrinsically disordered segments 8 .…”

Section: Introductionmentioning

confidence: 99%

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Cohen

Geffen

Ravid

et al. 2014

JoVE

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Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways. Video LinkThe video component of this article can be found at

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Substrate Recognition in Nuclear Protein Quality Control Degradation Is Governed by Exposed Hydrophobicity That Correlates with Aggregation and Insolubility

Cited by 48 publications

References 53 publications

Hsp70 Targets a Cytoplasmic Quality Control Substrate to the San1p Ubiquitin Ligase

Hsp70 Targets a Cytoplasmic Quality Control Substrate to the San1p Ubiquitin Ligase

How the Nucleus Copes with Proteotoxic Stress

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

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