Immunoglobulin Light Chains Dictate Vesicular Transport-dependent and -independent Routes for IgM Degradation by the Ubiquitin-Proteasome Pathway

Elkabetz, Yechiel; Kerem, Anat; Tencer, Lilach; Winitz, Dorit; Kopito, Ron R.; Bar-Nun, Shoshana

doi:10.1074/jbc.m208730200

Cited by 20 publications

(25 citation statements)

References 51 publications

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“…Under these conditions, s or accumulate in the microsomal fractions rather than the cytosol, implicating the proteasome in dislocation (8,9). Nevertheless, we detected ubiquitination of the luminal s when the proteasome proteolytic activity was inhibited (20), indicating that the proteasome was not required for s to gain access to the cytosol-facing ubiquitination machinery.…”

mentioning

confidence: 67%

“…Interestingly, BiP pulled down s assembled with into 2 2 monomers but not unassembled s ( In agreement with the genetic data in yeast (32), our results provide the first biochemical evidence for involvement of BiP in ERAD of luminal substrates in mammalian cells. (20) prompted us to examine the distribution of ubiquitinated s in fractionated, proteasome-inhibited B cells. Heavily ubiquitinated s was observed mostly in microsomes, in correlation with the distribution of the s protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, syntaxin 5 and the p97/p47 complex participate in homotypic membrane fusion (53). Although it has been shown that vesicular trafficking, which may involve homotypic fusion, is essential for ERAD of luminal substrates, it is dispensable for ERAD of membrane substrates (20,(55)(56)(57). The p97/ Cdc48 Ufd1/Npl4 complex, on the other hand, is clearly essential for ERAD of both luminal and membrane ERAD substrates (16, 21, 26 -28).…”

Section: Discussionmentioning

confidence: 99%

“…3A; see also Ref. 20). Next, wild-type or mutant (K48R) myc-tagged ubiquitin was overexpressed in naïve or s-expressing COS-7 cells.…”

Section: Luminal S Crosses the Er Membrane And Accumulates At The Er mentioning

confidence: 99%

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Distinct Steps in Dislocation of Luminal Endoplasmic Reticulum-associated Degradation Substrates

Elkabetz

Shapira

Rabinovich

et al. 2004

Journal of Biological Chemistry

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104

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Dislocation of endoplasmic reticulum-associated degradation (ERAD) substrates from the endoplasmic reticulum (ER) lumen to cytosol is considered to occur in a single step that is tightly coupled to proteasomal degradation. Here we show that dislocation of luminal ERAD substrates occurs in two distinct consecutive steps. The first is passage across ER membrane to the ER cytosolic face, where substrates can accumulate as ubiquitin conjugates. In vivo, this step occurs despite proteasome inhibition but requires p97/Cdc48p because substrates remain entrapped in ER lumen and are prevented from ubiquitination in cdc48 yeast strain. The second dislocation step is the release of accumulated substrates to the cytosol. In vitro, this release requires active proteasome, consumes ATP, and relies on salt-removable ERbound components, among them the ER-bound p97 and ER-bound proteasome, which specifically interact with the cytosol-facing substrates. An additional role for Cdc48p subsequent to ubiquitination is revealed in the cdc48 strain at permissive temperature, consistent with our finding that p97 recognizes luminal ERAD substrates through multiubiquitin. BiP interacts exclusively with ERAD substrates, suggesting a role for this chaperone in ERAD. We propose a model that assigns the cytosolic face of the ER as a midpoint to which luminal ERAD substrates emerge and p97/Cdc48p and the proteasome are recruited. Although p97/Cdc48p plays a dual role in dislocation and is involved both in passage of the substrate across ER membrane and subsequent to its ubiquitination, the proteasome takes part in the release of the substrate from the ER face to the cytosol en route to degradation.The endoplasmic reticulum-associated degradation (ERAD) 1 is a quality control process that selectively eliminates malfolded proteins or unassembled subunits of oligomeric proteins in the secretory pathway (1, 2). ERAD substrates are dislocated from the endoplasmic reticulum (ER) back to the cytosol via the Sec61 complex (3-6). In the cytosol, ubiquitin is conjugated to the ERAD substrates that are degraded by the proteasome (7). The proteolytically active proteasome has been implicated in the dislocation of ERAD substrates by virtue of their scarcity in cytosol of proteasome-inhibited cells (8 -11). Although stabilization of proteins in the secretory pathway by proteasome inhibitors is generally accepted as an indication for ERAD, ubiquitination of such proteins is a direct evidence for the access of the substrate to the cytosol (7). In most cases, however, accumulation of the multiubiquitinated ERAD substrates to detectable levels requires inhibition of the proteasome. If the proteasome is indeed required for dislocation (8 -11), then ubiquitination of ERAD substrates would not be observed. This applies especially to luminal ERAD substrates, as membrane ERAD substrates inherently display cytosolic domains, which may be ubiquitinated irrespective of dislocation (11-15). Therefore, it is our view that luminal ERAD substrates, whose dislocation is an ...

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mentioning

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…3A; see also Ref. 20). Next, wild-type or mutant (K48R) myc-tagged ubiquitin was overexpressed in naïve or s-expressing COS-7 cells.…”

Section: Luminal S Crosses the Er Membrane And Accumulates At The Er mentioning

confidence: 99%

See 2 more Smart Citations

Distinct Steps in Dislocation of Luminal Endoplasmic Reticulum-associated Degradation Substrates

Elkabetz

Shapira

Rabinovich

et al. 2004

Journal of Biological Chemistry

Self Cite

104

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“…This mechanism protects against proteotoxicity by eliminating aberrant proteins from the ER (19,20). ERAD also rids the cell of normal ER proteins when they are no longer needed under specific developmental or metabolic circumstances (21)(22)(23) …”

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confidence: 99%

Metabolically Regulated Endoplasmic Reticulum-associated Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase

Leichner

Avner

Harats

et al. 2011

Journal of Biological Chemistry

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In mammalian cells, the enzyme 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR), which catalyzes the rate-limiting step in the mevalonate pathway, is ubiquitylated and degraded by the 26 S proteasome when mevalonate-derived metabolites accumulate, representing a case of metabolically regulated endoplasmic reticulum-associated degradation (ERAD). Here, we studied which mevalonate-derived metabolites signal for HMGR degradation and the ERAD step(s) in which these metabolites are required. In HMGR-deficient UT-2 cells that stably express HMGal, a chimeric protein between ␤-galactosidase and the membrane region of HMGR, which is necessary and sufficient for the regulated ERAD, we tested inhibitors specific to different steps in the mevalonate pathway. We found that metabolites downstream of farnesyl pyrophosphate but upstream to lanosterol were highly effective in initiating ubiquitylation, dislocation, and degradation of HMGal. Similar results were observed for endogenous HMGR in cells that express this protein. Ubiquitylation, dislocation, and proteasomal degradation of HMGal were severely hampered when production of geranylgeranyl pyrophosphate was inhibited. Importantly, inhibition of protein geranylgeranylation markedly attenuated ubiquitylation and dislocation, implicating for the first time a geranylgeranylated protein(s) in the metabolically regulated ERAD of HMGR.Mammalian cells satisfy most of their cholesterol requirements through receptor-mediated endocytosis of cholesterolrich plasma lipoproteins. However, when exogenous cholesterol supply is short, or when there is an increased demand for particular nonsterol isoprenoid(s), cells up-regulate the activity of the mevalonate (MVA) 3 pathway ( Fig. 1), in which sterols and essential MVA-derived nonsterol isoprenoids are produced (1, 2). Intracellular MVA levels are finely tuned and tightly regulated by a feedback mechanism that governs the rate of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reduction into MVA, the major rate-limiting step for the entire pathway (3, 4). This reaction is catalyzed by HMG-CoA reductase (HMGR), an integral glycoprotein resident to the endoplasmic reticulum (ER). Each of the 97-kDa subunits of HMGR is embedded in the ER membrane via a ϳ350 residue N-terminal noncatalytic region that spans the membrane 8 times (5-7), whereas the rest of the polypeptide faces the cytosol where it tetramerizes to form the active sites of the enzyme (5, 8, 9). To allow sufficient production of isoprenoids yet avoid overaccumulation of potentially toxic products, the activity of HMGR is stringently controlled and varies according to the cellular needs for sterol and the nonsterol products. HMGR activity increases when cells are starved for cholesterol or MVA. Conversely, abundant cholesterol and/or MVA acutely suppress the enzyme (4, 10). At the post-translational level, regulation of HMGR mainly involves modulation of its stability. Thus, in cells deprived of sterols and/or MVA, HMGR is a rather stable protein that turns over with a half-l...

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An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant monoclonal antibody production process titer

McLeod

O'Callaghan

Pybus

et al. 2011

Biotech & Bioengineering

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In this study we have combined empirically derived mathematical models of intracellular Mab synthesis to quantitatively compare the degree to which individual cellular processes limit recombinant IgG(4) monoclonal antibody production by GS-CHO cells throughout a state-of-the-art industrial fed-batch culture process. Based on the calculation of a production process control coefficient for each stage of the intracellular Mab synthesis and secretion pathway, we identified the major cellular restrictions on Mab production throughout the entire culture process to be recombinant heavy chain gene transcription and heavy chain mRNA translation. Surprisingly, despite a substantial decline in the rate of cellular biomass synthesis during culture, with a concomitant decline in the calculated rate constants for energy-intensive Mab synthetic processes (Mab folding/assembly and secretion), these did not exert significant control of Mab synthesis at any stage of production. Instead, cell-specific Mab production was maintained by increased Mab gene transcription which offset the decline in cellular biosynthetic rates. Importantly, this study shows that application of this whole-process predictive modeling strategy should rationally precede and inform cell engineering approaches to increase production of a recombinant protein by a mammalian host cell--where control of productivity is inherently protein product and cell line specific.

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Immunoglobulin Light Chains Dictate Vesicular Transport-dependent and -independent Routes for IgM Degradation by the Ubiquitin-Proteasome Pathway

Cited by 20 publications

References 51 publications

Distinct Steps in Dislocation of Luminal Endoplasmic Reticulum-associated Degradation Substrates

Distinct Steps in Dislocation of Luminal Endoplasmic Reticulum-associated Degradation Substrates

Metabolically Regulated Endoplasmic Reticulum-associated Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase

An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant monoclonal antibody production process titer

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