title: Cystic fibrosis transmembrane conductance regulator has an altered structure when its maturation is inhibited.
abstract: Inefficient maturation and trafficking to the cell surface of the cystic fibrosis transmembrane conductance regulator (CFTR) is the primary cause of cystic fibrosis. CFTR protein that fails to mature accumulates as an immature core-glycosylated protein and is rapidly degraded. To determine how the structures of mature and immature CFTR are different, we compared the properties of CFTR that had been expressed in the presence or absence of the proteasome inhibitor, MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal). Transient expression of wild-type CFTR in the presence of submicromolar concentrations of MG-132 blocks maturation of the protein. We found that expression of CFTR in the presence of MG-132 trapped the protein in a trypsin-sensitive conformation. In addition, the structure of the carboxyl-terminus of immature and mature CFTR differed as histidine-tagged mature CFTR was preferentially recovered by metal-chelate chromatography. No chloride channel activity was detected when membranes containing immature CFTR were fused with planar lipid bilayers. These results show that expression of CFTR in the presence of MG-132 traps the protein in an altered conformation that may be inactive.
authors.affiliation: Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Ontario, Canada M5S 1A8.
authors.forename: E Y
authors.initials: EY
authors.lastname: Chen
authors.forename: M C
authors.initials: MC
authors.lastname: Bartlett
authors.forename: D M
authors.initials: DM
authors.lastname: Clarke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics; D014357:Trypsin / Q000378:metabolism
chemical_list: C505032:CFTR protein, human; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde