A principal role for the proteasome in endoplasmic reticulum-associated degradation of misfolded intracellular cystic fibrosis transmembrane conductance regulator.
Endoplasmic reticulum-associated degradation of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein is known to involve the ubiquitin-proteasome system. In addition, an ATP-independent proteolytic system has been suggested to operate in parallel with this pathway and become up-regulated when proteasomes are inhibited (Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Cell 83, 129-135). In this study, we use two independent techniques, pulse-chase labeling and a noninvasive fluorescence cell-based assay, to investigate the proteolytic pathways underlying the degradation of misfolded CFTR. Here we report that only inhibitors of the proteasome have a significant effect on preventing the degradation of CFTR, whereas cell-permeable inhibitors of lysosomal degradation, autophagy, and several classes of protease had no measurable effect on CFTR degradation, when used either alone or in combination with the specific proteasome inhibitor carbobenzoxy-l-leucyl-leucyl-l-leucinal (MG132). Our results suggest that ubiquitin-proteasome-mediated degradation is the dominant pathway for disposal of misfolded CFTR in mammalian cells and provide new mechanistic insight into endoplasmic reticulum-associated degradation.
Gelman
Marina S
MS
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
Kannegaard
Elisa S
ES
Kopito
Ron R
RR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D020134:Catalytic Domain; D002460:Cell Line; D002469:Cell Separation; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism; D015854:Up-Regulation
D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C078846:epoxomicin