title: Uncoupling proteasome peptidase and ATPase activities results in cytosolic release of an ER polytopic protein.
abstract: The 26S proteasome is the primary protease responsible for degrading misfolded membrane proteins in the endoplasmic reticulum. Here we examine the specific role of beta subunit function on polypeptide cleavage and membrane release of CFTR, a prototypical ER-associated degradation substrate with 12 transmembrane segments. In the presence of ATP, cytosol and fully active proteasomes, CFTR was rapidly degraded and released into the cytosol solely in the form of trichloroacetic acid (TCA)-soluble peptide fragments. Inhibition of proteasome beta subunits markedly decreased CFTR degradation but surprisingly, had relatively minor effects on membrane extraction and release. As a result, large TCA-insoluble degradation intermediates derived from multiple CFTR domains accumulated in the cytosol where they remained stably bound to inhibited proteasomes. Production of TCA-insoluble fragments varied for different proteasome inhibitors and correlated inversely with the cumulative proteolytic activities of beta1, beta2 and beta5 subunits. By contrast, ATPase inhibition decreased CFTR release but had no effect on the TCA solubility of the released fragments. Our results indicate that the physiologic balance between membrane extraction and peptide cleavage is maintained by excess proteolytic capacity of the 20S subunit. Active site inhibitors reduce this capacity, uncouple ATPase and peptidase activities, and generate cytosolic degradation intermediates by allowing the rate of unfolding to exceed the rate of polypeptide cleavage.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA.
authors.forename: Jon
authors.initials: J
authors.lastname: Oberdorf
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Carlson
authors.forename: William R
authors.initials: WR
authors.lastname: Skach
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism*; D061988:Proteasome Inhibitors; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D047992:TNF Receptor-Associated Factor 2; D014238:Trichloroacetic Acid / Q000737:chemistry; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: C101338:PSMD2 protein, human; D010446:Peptide Fragments; D061988:Proteasome Inhibitors; D021122:Protein Subunits; D047992:TNF Receptor-Associated Factor 2; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014238:Trichloroacetic Acid; D006427:Hemin; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000251:Adenosine Triphosphatases