Ubr1 and Ubr2 function in a quality control pathway for degradation of unfolded cytosolic proteins.
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ABSTRACT:
SUBMITTER: Nillegoda NB
PROVIDER: S-EPMC2893976 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mutations causing protein misfolding and proteolysis are associated with many genetic diseases. The degradation of these aberrant proteins typically is mediated by protein-quality control pathways that recognize misfolded domains. Several E3 ubiquitin ligases have been shown to target cytosolic misfolded proteins to the proteasome. In this study, we characterized a panel of more than 20 cytosolic thermosensitive mutants from six essential genes in Saccharomyces cerevisiae. These wild-type proteins are stable at restrictive temperature. In contrast, we found that a large portion of the mutants is degraded at nonpermissive temperature in a proteasome-dependent manner. Approximately one-third of the assessed unstable mutants are targeted by the Ubr1 ubiquitin ligase. In two cases, efficient degradation of the thermosensitive mutants is abrogated in the absence of Ubr1 alone, whereas in a third case it is reliant on the dual deletion of Ubr1 and the nuclear E3 ligase San1. We found that the impairment of the degradation of these quality control substrates at the restrictive temperature is associated with the suppression of thermosensitive phenotype. This study confirms that Ubr1 plays an important role in the degradation of cytosolic misfolded proteins and indicates that degradation mediated by protein quality control is a major cause for the conditional lethality of mutated essential genes.
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Project description:Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes.
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Project description:Intracellular quality control systems monitor protein conformational states. Irreversibly misfolded proteins are cleared through specialized degradation pathways. Their importance is underscored by numerous pathologies caused by aberrant proteins. In the cytosol, where most proteins are synthesized, quality control remains poorly understood. Stress-inducible chaperones and the 26S proteasome are known mediators but how their activities are linked is unclear. To better understand these mechanisms, a panel of model misfolded substrates was analyzed in detail. Surprisingly, their degradation occurs not in the cytosol but in the nucleus. Degradation is dependent on the E3 ubiquitin ligase San1p, known previously to direct the turnover of damaged nuclear proteins. A second E3 enzyme, Ubr1p, augments this activity but is insufficient by itself. San1p and Ubr1p are not required for nuclear import of substrates. Instead, the Hsp70 chaperone system is needed for efficient import and degradation. These data reveal a new function of the nucleus as a compartment central to the quality control of cytosolic proteins.
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Project description:Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic "degron library" in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3 About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones.
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Project description:Chaperones can mediate both protein folding and degradation. This process is referred to as protein triage, which demands study to reveal mechanisms of quality control for both basic scientific and translational purposes. In yeast, many misfolded proteins undergo chaperone-dependent ubiquitination by the action of the E3 ligases Ubr1 and San1, allowing detailed study of protein triage. In cells, both HSP70 and HSP90 mediated substrate ubiquitination, and the canonical ATP cycle was required for HSP70's role: we have found that ATP hydrolysis by HSP70, the nucleotide exchange activity of Sse1, and the action of J-proteins are all needed for Ubr1-mediated quality control. To discern whether chaperones were directly involved in Ubr1-mediated ubiquitination, we developed a bead-based assay with covalently immobilized but releasable misfolded protein to obviate possible chaperone effects on substrate physical state or transport. In this in vitro assay, only HSP70 was required, along with its ATPase cycle and relevant cochaperones, for Ubr1-mediated ubiquitination. The requirement for the HSP70 ATP cycle in ubiquitination suggests a possible model of triage in which efficiently folded proteins are spared, while slow-folding or nonfolding proteins are iteratively tagged with ubiquitin for subsequent degradation.
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Project description:Damaged or redundant peroxisomes and their luminal cargoes are removed by pexophagy, a selective autophagy pathway. In yeasts, pexophagy depends mostly on the pexophagy receptors, such as Atg30 for Pichia pastoris and Atg36 for Saccharomyces cerevisiae, the autophagy scaffold proteins, Atg11 and Atg17, and the core autophagy machinery. In P. pastoris, the receptors for peroxisomal matrix proteins containing peroxisomal targeting signals (PTSs) include the PTS1 receptor, Pex5, and the PTS2 receptor and co-receptor, Pex7 and Pex20, respectively. These shuttling receptors are predominantly cytosolic and only partially peroxisomal. It remains unresolved as to whether, when and how the cytosolic pools of peroxisomal receptors, as well as the peroxisomal matrix proteins, are degraded under pexophagy conditions. These cytosolic pools exist both in normal and mutant cells impaired in peroxisome biogenesis. We report here that Pex5 and Pex7, but not Pex20, are degraded by an Atg30-independent, selective autophagy pathway. To enter this selective autophagy pathway, Pex7 required its major PTS2 cargo, Pot1. Similarly, the degradation of Pex5 was inhibited in cells missing abundant PTS1 cargoes, such as alcohol oxidases and Fox2 (hydratase-dehydrogenase-epimerase). Furthermore, in cells deficient in PTS receptors, the cytosolic pools of peroxisomal matrix proteins, such as Pot1 and Fox2, were also removed by Atg30-independent, selective autophagy, under pexophagy conditions. In summary, the cytosolic pools of PTS receptors and their cargoes are degraded via a pexophagy-independent, selective autophagy pathway under pexophagy conditions. These autophagy pathways likely protect cells from futile enzymatic reactions that could potentially cause the accumulation of toxic cytosolic products.Abbreviations: ATG: autophagy related; Cvt: cytoplasm to vacuole targeting; Fox2: hydratase-dehydrogenase-epimerase; PAGE: polyacrylamide gel electrophoresis; Pot1: thiolase; PMP: peroxisomal membrane protein; Pgk1: 3-phosphoglycerate kinase; PTS: peroxisomal targeting signal; RADAR: receptor accumulation and degradation in the absence of recycling; RING: really interesting new gene; SDS: sodium dodecyl sulphate; TCA, trichloroacetic acid; Ub: ubiquitin; UPS: ubiquitin-proteasome system Vid: vacuole import and degradation.
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