In vivo iodination of a misfolded proinsulin reveals co-localized signals for Bip binding and for degradation in the ER.

Schmitz, Anton; Maintz, M.; Kehle, T.; Herzog, Volker

doi:10.1002/j.1460-2075.1995.tb07092.x

Cited by 49 publications

(31 citation statements)

References 46 publications

(25 reference statements)

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“…We show further that BiP is required to export p␣F from the ER and thus is involved in the recognition of ERAD substrates and/or directly in the export process. In support of this first hypothesis, other reports indicated that mammalian BiP recognizes aberrantly folded or unassembled polypeptides in the ER and targets them for degradation (24,34,35). A direct role for BiP in the export process is more difficult to imagine unless it regulates the translocation channel to permit access of ERAD substrates to the cytoplasm.…”

Section: Discussionmentioning

confidence: 72%

“…Although the nature of this substrate selectivity is not completely clear, structural motifs that are buried when proteins are correctly folded and assembled may be recognized by the ERAD machinery if protein folding is hindered (22)(23)(24). Two ER lumenal molecular chaperones, BiP and calnexin, are known to be involved in protein maturation and "quality control" in the ER and are likely candidates to play a role in ERAD.…”

mentioning

confidence: 99%

“…First, iodination of a misfolded proinsulin polypeptide in vivo indicated co-localized signals for BiP binding and ERAD (24). Second, the dissociation of BiP from unassembled Ig chains correlated with the kinetics of Ig degradation (34).…”

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confidence: 99%

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The Requirement for Molecular Chaperones during Endoplasmic Reticulum-associated Protein Degradation Demonstrates That Protein Export and Import Are Mechanistically Distinct

Brodsky¹,

Werner²,

Dubas³

et al. 1999

Journal of Biological Chemistry

258

172

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Polypeptide import into the yeast endoplasmic reticulum (ER) requires two hsp70s, Ssa1p in the cytosol and BiP (Kar2p) in the ER lumen. After import, aberrant polypeptides may be exported to the cytoplasm for degradation by the proteasome, and defects in the ER chaperone calnexin (Cne1p) compromise their degradation. Both import and export require BiP and the Sec61p translocation complex, suggesting that import and export may be mechanistically related. We now show that the cne1⌬ and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-␣ factor is defective in kar2 mutant microsomes. Pulse-chase analysis indicates that A1PiZ, another substrate for degradation, is stabilized in the kar2 strains at the restrictive temperature. Because two of the kar2 mutants examined are proficient for polypeptide import, the roles of BiP during ER protein export and import differ, indicating that these processes must be mechanistically distinct. To examine whether Ssa1p drives polypeptides from the ER and is also required for degradation, we assembled reactions using strains either containing a mutation in SSA1 or in which the level of Ssa1p could be regulated. We found that pro-␣ factor and A1PiZ were degraded normally, indicating further that import and export are distinct and that other cytosolic factors may pull polypeptides from the ER.

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Section: Discussionmentioning

confidence: 72%

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confidence: 99%

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The Requirement for Molecular Chaperones during Endoplasmic Reticulum-associated Protein Degradation Demonstrates That Protein Export and Import Are Mechanistically Distinct

Brodsky¹,

Werner²,

Dubas³

et al. 1999

Journal of Biological Chemistry

258

172

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“…In accordance with these hypotheses, several lines of evidence support a role for the lumenal Hsp70, BiP, in ERAD in both mammals and yeast. First, the proteolysis of ERAD substrates coincides with the rate at which they are released from BiP in the mammalian ER (Knittler et al, 1995;Beggah et al, 1996;Skowronek et al, 1998;Chillaron and Haas, 2000), and BiP associates preferentially with exposed regions of an unfolded ERAD substrate (Schmitz et al, 1995). Second, the degradation of an ERAD substrate is slowed when yeast contain a mutant allele in the gene encoding BiP, KAR2 (kar2-113;Plemper et al, 1997), and two ERAD substrates in yeast, CPY* and p␣F, aggregate in microsomes prepared from another kar2 mutant shifted to the nonpermissive temperature (kar2-203;Nishikawa et al, 2001).…”

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confidence: 99%

Dependence of Endoplasmic Reticulum-associated Degradation on the Peptide Binding Domain and Concentration of BiP

Kabani

Kelley

Morrow

et al. 2003

MBoC

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ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD. Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al. J. Biol. Chem. 274,(3453)(3454)(3455)(3456)(3457)(3458)(3459)(3460). The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate. Peptide-stimulated ATPase activity was also reduced, suggesting that the interdomain coupling in Kar2-1p is partially compromised. In contrast, Hsp40 cochaperone-activation of Kar2-1p's ATPase activity was unaffected. Consistent with UPR induction in kar2-1 yeast, an ERAD substrate aggregated in microsomes prepared from this strain but not from wild-type yeast. Overexpression of wild-type BiP increased substrate solubility in microsomes obtained from the mutant, but the ERAD defect was exacerbated, suggesting that simply retaining ERAD substrates in a soluble, retro-translocationcompetent conformation is insufficient to support polypeptide transit to the cytoplasm. INTRODUCTIONBefore being delivered to their ultimate locations, secreted proteins are monitored by a quality control "machine" associated with the endoplasmic reticulum (ER; reviewed by Ellgaard et al., 1999); aberrant polypeptides may be retrotranslocated from the ER to the cytoplasm and destroyed by the proteasome in a process termed ER-associated degradation (ERAD; McCracken and Brodsky, 1996). The importance of defining the molecular mechanism of ERAD is underscored by the fact that several human diseases arise from the accumulation or accelerated degradation of ERAD substrates and because some bacterial toxins and viruses coopt the ERAD pathway to exert their effects (reviewed in Thomas et al., 1995;Aridor and Hannan, 2000;Fewell et al., 2001).ERAD may result from inefficient protein folding, so it is not surprising that molecular chaperones are required for this process. Chaperones prevent the formation of off-pathway intermediates or directly catalyze folding and have been proposed to "judge" whether a nascent protein will ultimately fold or whether it should be targeted for degradation (reviewed by Hayes and Dice, 1996;Hartl, 1996;Horwich et al., 1999;Plemper and Wolf, 1999;Rö misch, 1999;Wickner et al., 1999;Fewell et al., 2001;Hö hfeld et al., 2001). Hsp70 (heat shock proteins with a molecular mass of ϳ70 kDa) molecular chaperones hydrolyze ATP concomitant with the binding of peptides with overall hydrophobic character (Flynn et al., 1991;Blond-Elguindi et al., 1993;Rü diger et al., 1997); therefore, Hsp70s might retain the solubility of unfolded, retro-translocating polypeptides during their voyage from the ER to the cytoplasm via the Sec61p translocation channel or might "gate" this c...

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“…This might be a consequence of an increased number of functional stress protein molecules available for the formation of a complex with the substrate protein. Such an interaction may limit the exposure of a determinant(s) on the surface of the protein required for its targeting to degradation, as suggested by Schmitz et al (1995), resulting in the prolonged retention and stability of the substrate protein in the ER. …”

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confidence: 99%

Degradation of a Short-lived Glycoprotein from the Lumen of the Endoplasmic Reticulum: The Role of N-linked Glycans and the Unfolded Protein Response

Virgilio

Kitzmüller

Schwaiger

et al. 1999

MBoC

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We are studying endoplasmic reticulum-associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI 332 , containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI 332 was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of ϳ45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI 332 in which the single used N-glycosylation consensus site had been removed (RI 332 -Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI 332 degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI 332 to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI 332 . After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI 332 and RI 332 -Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI 332 and RI 332 -Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI 332 was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives. INTRODUCTIONNewly synthesized proteins of the endomembrane system and most secreted proteins of eukaryotic cells enter the secretory pathway through the translocation channel at the membrane of the endoplasmic reticulum (ER). The lumen of the ER is the site where translocated proteins assume their secondary structure and where assembly of oligomeric complexes occurs. Additionally, newly synthesized proteins undergo cotranslational and posttranslational modifications in the lumen of the ER, some of which allow transient interactions of the folding polypeptide chains with a set of ER-resident proteins (Hammond and Helenius, 1995;Leitzgen and Haas, 1998). Only after acquiring a fully folded, native conformation can proteins complete their journey through the secretory pathPresent addresses:† Dipartimento Medicina Sperimentale e Diagnostica, Sezione di Patologia Generale, Universitá di Ferrara, Ferrara, Italy; ‡ Spiegelmayr K...

show abstract

In vivo iodination of a misfolded proinsulin reveals co-localized signals for Bip binding and for degradation in the ER.

Cited by 49 publications

References 46 publications

The Requirement for Molecular Chaperones during Endoplasmic Reticulum-associated Protein Degradation Demonstrates That Protein Export and Import Are Mechanistically Distinct

The Requirement for Molecular Chaperones during Endoplasmic Reticulum-associated Protein Degradation Demonstrates That Protein Export and Import Are Mechanistically Distinct

Dependence of Endoplasmic Reticulum-associated Degradation on the Peptide Binding Domain and Concentration of BiP

Degradation of a Short-lived Glycoprotein from the Lumen of the Endoplasmic Reticulum: The Role of N-linked Glycans and the Unfolded Protein Response

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