Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER (original) (raw)
Related papers
Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein
The EMBO Journal, 2004
Aberrant polypeptides in the endoplasmic reticulum (ER) are retro-translocated to the cytoplasm and degraded by the 26S proteasome via ER-associated degradation (ERAD). To begin to resolve the requirements for the retro-translocation and degradation steps during ERAD, a cell-free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER-derived microsomes during the ERAD of a model, soluble polypeptide. As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system. Proficient ERAD was observed if wild-type cytosol was substituted with either purified yeast or mammalian proteasomes. Moreover, addition of only the 19S cap of the proteasome catalyzed ATPdependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle.
Journal of Biological Chemistry, 1999
The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human ␣ 1 -antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded ␣ 1 -antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded ␣ 1 -antitrypsin for degradation by the proteasome.
Disposing of misfolded ER proteins: A troubled substrate's way out of the ER
Molecular and Cellular Endocrinology, 2019
Secreted, plasma membrane, and resident proteins of the secretory pathway are synthesized in the endoplasmic reticulum (ER) where they undergo post-translational modifications, oxidative folding, and subunit assembly in tightly monitored processes. An ER quality control (ERQC) system oversees protein maturation and ensures that only those reaching their native state will continue trafficking into the secretory pathway to reach their final destinations. Those that fail must be recognized and eliminated to maintain ER homeostasis. Two cellular mechanisms have been identified to rid the ER of terminally unfolded and aggregated proteins. ER-associated degradation (ERAD) was discovered nearly 30 years ago and entails the identification of improperly matured secretory pathway proteins and their retrotranslocation to the cytosol for degradation by the ubiquitin-proteasome system. ER-phagy has been more recently described and caters to larger, more complex proteins and protein aggregates that are not readily handled by ERAD. This pathway has unique upstream components and relies on the same downstream effectors of autophagy used in other cellular processes to deliver clients to lysosomes for degradation. In this review, we describe the main elements of ERQC, ERAD, and ER-phagy and focus on recent advances in these fields.
Substrate selection by the proteasome during degradation of protein complexes
Nature chemical biology, 2009
The proteasome controls the turnover of most cellular proteins. Two structural features are typically required for proteins to be degraded: covalently attached ubiquitin polypeptides that allow binding to the proteasome, and an unstructured region in the targeted protein that initiates proteolysis. Here, we have tested the degradation of model proteins to further explore how the proteasome selects its substrates. Using purified yeast proteasome and mammalian proteasome in cell lysate, we have demonstrated that the two structural features can act in trans when separated onto different proteins in a multi-subunit complex. In such complexes, the location of the unstructured initiation site and its chemical properties determine which subunit is degraded. Thus, our findings reveal the molecular basis of subunit specificity in the degradation of protein complexes. In addition, our data provide a plausible explanation for how adaptor proteins can bind to otherwise stable proteins and target them for degradation. The ubiquitin proteasome system (UPS) controls the cellular concentrations of regulatory proteins, degrades aberrant or misfolded polypeptides, and produces peptides as part of the adaptive immune response1. Most proteins are targeted to the proteasome by the covalent attachment of many copies of the small protein ubiquitin2. This modification is recognized directly by the proteasome3 , 4 which then degrades its substrates by running sequentially along the substrates' polypeptide chain5. Many UPS substrates, such as cyclins, cyclindependent kinase (CDK) inhibitors, transcription regulators (e.g., IκBα), and transcription factors, are parts of multimeric complexes before degradation1. In some of these cases, the proteasome degrades specifically only one of the subunits to create a remodeled complex with a new composition and function. This remodeling activity of the proteasome was first demonstrated through the observation of subunit specificity in the degradation of tetrameric βgal substrates in vitro6 and in vivo7. Two well-known physiological examples of subunitspecific degradation are the proteolysis of cyclins from their complex with CDK8 and the proteolysis of the CDK inhibitor Sic1 while bound to the cyclin / CDK complex9. Until now, the specificity of degradation by the proteasome has been assumed to result directly from the specificity of the ubiquitination reaction. In other words, the protease has been thought to begin to degrade its substrate at point of the ubiquitin modification and, therefore, to degrade only the ubiquitinated subunit.
Endoplasmic reticulum-associated protein degradation—one model fits all?
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2004
The endoplasmic reticulum (ER) is the eukaryotic organelle where most secretory proteins are folded for subsequent delivery to their site of action. Proper folding of newly synthesized proteins is monitored by a stringent ER quality control system. This system recognizes misfolded or unassembled proteins and prevents them from reaching their final destination. Instead, they are extracted from the ER, polyubiquitinated and degraded by the cytosolic proteasome. With the identification of novel components and substrates, a more and more complex picture of this process emerges in which distinct pathways target different sets of substrates for destruction.
A method to selectively degrade proteins of the secretory pathway
2010
Proteins that enter the secretory pathway, either soluble or membrane bound, are subjected to ER quality control mechanisms: if protein folding is aberrant or delayed, proteins are subjected to additional folding cycles or selected for ERassociated degradation (ERAD). In this process, proteins that fail to reach their terminal folding, and thus selected for ERAD, are recognised and retrotranslocated to the cytosol for ubiquitinylation and subsequent degradation by the proteasome. The most characterised mammalian ubiquitin ligase is HRD1. For its activity HRD1 needs the interaction with SEL1L an ER resident type I transmembrane glycoprotein. SEL1L has been defined as an adaptor protein because, beside its interaction with the ubiquitin ligase HRD1, it interacts either with proteins involved in substrate recruitment or in the retro-translocation process. To induce degradation of specific targets we designed and constructed a new class of fusion molecules, termed degradins, where the N...
Journal of Cell Biology, 1999
Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and ...