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:Eukaryotic cells maintain proteostasis by quality control (QC) degradation. These pathways can specifically target a wide variety of distinct misfolded proteins, and so are important for management of cellular stress. Although a number of conserved QC pathways have been described in yeast, the E3 ligases responsible for cytoplasmic QC are unknown. We now show that Ubr1 and San1 mediate chaperone-dependent ubiquitination of numerous misfolded cytoplasmic proteins. This action of Ubr1 is distinct from its role in the "N-end rule." In this capacity, Ubr1 functions to protect cells from proteotoxic stresses. Our phenotypic and biochemical studies of Ubr1 and San1 indicate that two strategies are employed for cytoplasmic QC: chaperone-assisted ubiquitination by Ubr1 and chaperone-dependent delivery to nuclear San1. The broad conservation of Ubr ligases and the relevant chaperones indicates that these mechanisms will be important in understanding both basic and biomedical aspects of cellular proteostasis.
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Project description:To study the pathway of degradation of the hepatitis B virus (HBV) middle envelope protein (M), human hepatoblastoma cells were transfected with a plasmid that specifies production of M in the absence of other viral proteins. When expressed in HepG2 cells, 90% of M protein was secreted into the culture media within a 24-h period. However, quite surprisingly, 10% of this protein remained cell associated and was only slowly degraded over a 24-48-h period. Treatment with inhibitors of the cytosolic proteasome complex resulted in the accumulation of full-length HBV M protein and M derived HBV-specific polypeptides of 20 and 17 kDa. Treatment with the endoglycosidases PNGase F and Endo H, confirmed that the two species were derived from a similar polypeptide with a N-linked glycan modification. Evidence that this peptide was derived from a proteolytic processing event was determined through the detection of the C-terminal fragment using a C-terminal tagged HA tagged construct. The hypothesis that the 20 and 17 kDa polypeptide species are intermediates of M degradation was reinforced by their detection in cells transfected with vectors specifying M secretion defective mutants that accumulate intracellular M. Moreover, deletion of a putative cleavage sites prevented the detection of the 20 and 17 kDa species, consistent with the notion that they are generated by the action of a cellular protease prior to proteasomal degradation. Thus, these results highlight an important way in which large protein aggregates, such as the HBsAg can be processed for efficient degradation via the proteasomes and allow for proper antigen presentation via the MHC I pathway.
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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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