Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events.
Many aberrant or unassembled proteins synthesized in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. To investigate how soluble glycoproteins destined for degradation are retrotranslocated across the ER membrane, we analyzed the fate of two IgM subunits, mu and J, retained in the ER by myeloma cells that do not synthesize light chains. Degradation of mu and J is prevented by proteasome inhibitors, suggesting that both chains are retrotranslocated to be disposed of by proteasomes. Indeed, when proteasomes are inhibited, some deglycosylated J chains that no longer contain intrachain disulfide bonds accumulate in the cytosol. However, abundant glycosylated J chains are still present in the ER at time points in which degradation would have been almost complete in the absence of proteasome inhibitors, suggesting that retrotranslocation and degradation are coupled events. This was confirmed by protease protection and cell fractionation assays, which revealed that virtually all mu chains are retained in the ER lumen in a glycosylated state when proteasomes are inhibited. Association with calnexin correlated with the failure of mu chains to dislocate to the cytosol. Taken together, these results suggest that active proteasomes are required for the extraction of Ig subunits from the ER, though the requirements for retrotranslocation may differ among individual substrates.
Mancini
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Department of Molecular Pathology and Medicine, DIBIT-San Raffaele Scientific Institute, 20132 Milan, Italy.
Fagioli
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Maggioni
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Sitia
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D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037281:Calnexin; D037282:Calreticulin; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology / Q000276:immunology; D004721:Endoplasmic Reticulum / Q000276:immunology; D006031:Glycosylation; D007144:Immunoglobulin J-Chains / Q000737:chemistry / Q000378:metabolism*; D007148:Immunoglobulin mu-Chains / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D046988:Proteasome Endopeptidase Complex; D012261:Ribonucleoproteins / Q000378:metabolism; D012995:Solubility; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D015853:Cysteine Proteinase Inhibitors; D007144:Immunoglobulin J-Chains; D007148:Immunoglobulin mu-Chains; D009097:Multienzyme Complexes; D012261:Ribonucleoproteins; D037281:Calnexin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex