title: The mechanism underlying cystic fibrosis transmembrane conductance regulator transport from the endoplasmic reticulum to the proteasome includes Sec61beta and a cytosolic, deglycosylated intermediary.
abstract: Endoplasmic reticulum (ER) degradation pathways can selectively route proteins away from folding and maturation. Both soluble and integral membrane proteins can be targeted from the ER to proteasomal degradation in this fashion. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral, multidomain membrane protein localized to the apical surface of epithelial cells that functions to facilitate Cl- transport. CFTR was among the first membrane proteins for which a role of the proteasome in ER-related degradation was described. However, the signals that route CFTR to ubiquitination and subsequent degradation are not known. Moreover, limited information is available concerning the subcellular localization of polyubiquitinated CFTR or mechanisms underlying retrograde dislocation of CFTR from the ER membrane to the proteasome either before or after ubiquitination. In the present study, we show that proteasome inhibition with clasto-lactacystin beta-lactone (4 microM, 1 h) stabilizes the presence of a deglycosylated CFTR intermediate for up to 5 h without increasing the core glycosylated (band B) form of CFTR. Deglycosylated CFTR is present under the same conditions that result in accumulation of polyubiquitinated CFTR. Moreover, the deglycosylated form of both wild type and DeltaF508 CFTR can be found in the cytosolic fraction. Both the level and stability of cytosolic, deglycosylated CFTR are increased by proteasome blockade. During retrograde translocation from the ER to the cytosol, CFTR associates with the Sec61 trimeric complex. Sec61 is the key component of the mammalian co-translational protein translocation system and has been proposed to function as a two way channel that transports proteins both into the ER and back to the cytosol for degradation. We show that the level of the Sec61.CFTR complexes are highest when CFTR degradation proceeds at the greatest rate (approximately 90 min after pulse labeling). Quantities of Sec61.CFTR complexes are also increased by inhibition of the proteasome. Based on these results, we propose a model in which complex membrane proteins such as CFTR are transported through the Sec61 trimeric complex back to the cytosol, escorted by the beta subunit of Sec61, and degraded by the proteasome or by other proteolytic systems.
authors.affiliation: Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Bebök
authors.forename: C
authors.initials: C
authors.lastname: Mazzochi
authors.forename: S A
authors.initials: SA
authors.lastname: King
authors.forename: J S
authors.initials: JS
authors.lastname: Hong
authors.forename: E J
authors.initials: EJ
authors.lastname: Sorscher
mesh_terms: D001692:Biological Transport; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000378:metabolism; D000069816:SEC Translocation Channels
chemical_list: C505032:CFTR protein, human; D015853:Cysteine Proteinase Inhibitors; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; C499388:SEC61B protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex