Dissecting the ER-Associated Degradation of a Misfolded Polytopic Membrane Protein

Nakatsukasa, Kunio; Huyer, Gregory; Michaelis, Susan; Brodsky, Jeffrey L.

doi:10.1016/j.cell.2007.11.023

Cited by 246 publications

(309 citation statements)

References 58 publications

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“…Both gain of catalytic conformation and degradation of RTA proceed from chaperone-bound states. This is highly reminiscent of the Hsc70-Hsp90 mediated regulation of signaling proteins (34), but may be unprecedented for a soluble protein that is dislocated from the ER lumen, because soluble ERAD substrates typically interact with other membrane-associated chaperone machineries (35), whereas cytosolic domains of misfolded membrane proteins typically interact with Hsc70 complexes (36). Our findings emphasize the central role of Hsc70-Hsp90 chaperone machineries in cytosolic protein triage.…”

Section: Discussionmentioning

confidence: 71%

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

Spooner

Hart

Cook

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states.Hsc70 ͉ Hsp90 ͉ ricin E ndoplasmic-reticulum (ER) associated protein degradation (ERAD) comprises coordinated disposal systems that recognize and remove misfolded and unassembled proteins in the ER, dislocating them across the ER membrane to the cytosol for proteasomal destruction. Degradation is normally facilitated by polyubiquitylation, usually on internal lysyl residues of the target protein. Both membrane-bound and soluble ER proteins can be disposed of by ERAD (1, 2).The plant cytotoxin ricin traffics to the ER lumen of mammalian cells where it is reduced to its RTA and RTB subunits before RTA dislocation (3). RTA does not penetrate the ER membrane directly; instead it exploits pre-existing proteinconducting channels as a nonnative species, mimicking ER proteins dispatched via ERAD (4, 5). Thus, it enters the cytosol in a form susceptible to proteolysis or aggregation. A proportion must evade these fates to gain a catalytic conformation that depurinates target ribosomes, stopping protein synthesis. The paucity of lysine residues in RTA may facilitate uncoupling from ERAD by reducing the potential for polyubiquitylation, thereby hampering proteasomal degradation (6). This, in turn, may provide opportunities for spontaneous or chaperone-assisted folding not normally sanctioned for ERAD substrates.We show an interaction of RTA with the cytosolic heat shock (cognate) protein Hsc70. From the chaperone-bound state, nonnative cytosolic RTA can achieve a catalytic conformation or can be inactivated. Its ultimate fate depends on the activities of ...

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Section: Discussionmentioning

confidence: 71%

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

Spooner

Hart

Cook

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Confirmation that ligase activity of Ubr1 is indeed required for substrate ubiquitination and not for other purposes came from ubiquitination assays. In wild-type cells, Ste6* was shown to be ubiquitinated at the membrane and thereafter extracted from the membrane in full length before its degradation by the proteasome (33). Therefore, after lysis of cells with glass beads, cytosolic and membrane fraction were separated via ultracentrifugation to distinguish between soluble and membrane-associated ubiquitinated species of Ste6*.…”

Section: Significancementioning

confidence: 99%

“…type cells. In this process, Ssa1 is thought to facilitate the integration of the substrate into the Doa10 ligase complex before its ubiquitination (17,33). Ssa1 also might mask hydrophobic regions of Ste6* after extraction from the ER membrane to prevent aggregation (33).…”

Section: Significancementioning

confidence: 99%

Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation

Stolz

Besser

Hottmann

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Quality control and degradation of misfolded proteins are essential processes of all cells. The endoplasmic reticulum (ER) is the entry site of proteins into the secretory pathway in which protein folding occurs and terminally misfolded proteins are recognized and retrotranslocated across the ER membrane into the cytosol. Here, proteins undergo polyubiquitination by one of the membrane-embedded ubiquitin ligases, in yeast Hrd1/Der3 (HMG-CoA reductase degradation/degradation of the ER) and Doa10 (degradation of alpha), and are degraded by the proteasome. In this study, we identify cytosolic Ubr1 (E3 ubiquitin ligase, N-recognin) as an additional ubiquitin ligase that can participate in ER-associated protein degradation (ERAD) in yeast. We show that two polytopic ERAD substrates, mutated transporter of the mating type a pheromone, Ste6* (sterile), and cystic fibrosis transmembrane conductance regulator, undergo Ubr1-dependent degradation in the presence and absence of the canonical ER ubiquitin ligases. Whereas in the case of Ste6* Ubr1 is specifically required under stress conditions such as heat or ethanol or in the absence of the canonical ER ligases, efficient degradation of human cystic fibrosis transmembrane conductance regulator requires function of Ubr1 already in wild-type cells under standard growth conditions. Together with the Hsp70 (heat shock protein) chaperone Ssa1 (stress-seventy subfamily A) and the AAA-type ATPase Cdc48 (cell division cycle), Ubr1 directs the substrate to proteasomal degradation. These data unravel another layer of complexity in ERAD.protein quality control | stress response | heat shock C onstantly occurring statistic folding errors, as well as misfolding due to stress such as heat, heavy metal ions, or oxygen require a rigorous protein quality control system in all cellular compartments. Irreversibly misfolded proteins are degraded by a selective proteolysis machinery, the ubiquitin proteasome system. In humans, impairment of the protein quality control and elimination system contributes to several severe diseases, including Parkinson disease, Alzheimer's disease, and CreutzfeldtJakob disease (1, 2), underscoring the importance of these quality control mechanisms. About one third of the cellular proteome consists of proteins passing the secretory pathway. Most of them are translocated into the endoplasmic reticulum (ER), where they are folded and permanently scanned for their functional structure. Only properly folded proteins are allowed to exit the ER and pass on to their site of action (3). Proteins that cannot fold properly are withdrawn from the secretory pathway, retrotranslocated across the ER membrane into the cytosol, polyubiquitinated and degraded by the 26S proteasome in a process termed ER-associated protein degradation (ERAD) (4).The eukaryotic model organism Saccharomyces cerevisiae has been a driving force in the discovery and elucidation of ERAD (4-10). It has been known for years that two polytopic ligases located in the ER membrane, Hrd1/Der3 (HMG-CoA reductas...

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“…The reason we prefer to screen in S. cerevisiae, rather than P. pastoris, is that cloning in P. pastoris is more time-consuming, as genes must be cloned into vectors, typically in E. coli, before their genomic integration. S. cerevisiae also has the added advantage that numerous localization studies have been carried out with functional GFP-fusions, and because S. cerevisiae has a highly regulated and extensively characterized quality-control systemin the endoplasmic reticulum 11,12 , assessing localization of fusions under overexpression conditions can be used as a good indication of functional expression. Of the highly expressed eukaryotic membrane proteins in S. cerevisiae, we find that more than half of those tested are targeted to the correct organelle and are monodisperse in more than one detergent 6 .…”

Section: Introductionmentioning

confidence: 99%

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

et al. 2008

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It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for highresolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 μGFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.

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Dissecting the ER-Associated Degradation of a Misfolded Polytopic Membrane Protein

Cited by 246 publications

References 58 publications

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

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