Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane

Ximing Xiong, Elaine Chong, William R. Skach

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    95 Scopus citations

    Abstract

    The ubiquitin-proteasome pathway has been implicated in the degradation of newly synthesized, mis-folded and unassembled proteins in the endoplasmic reticulum (ER). Using a cell-free reticulocyte lysate system we have examined the relationship between biosynthesis and ER-associated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR), a polytopic protein with 12 predicted transmembrane segments. Our results provide direct evidence that full-length, glycosylated and membrane-integrated CFTR is a substrate for degradation and that degradation involves polyubiquitination and cytosolic proteolytic activity. CFTR ubiquitination was both temperature- and ATP-dependent. Degradation was significantly inhibited by EDTA, apyrase, and the proteasome inhibitors heroin and MG132. Degradation was inhibited to a lesser extent by clasto-lactacystin β-lactone, ALLN, and N(α)-tosyl-L- phenylalanine chloromethyl ketone and was relatively unaffected by lactacystin and N-tosyl lysyl chloromethyl ketone. In the presence of hemin, polyubiquitinated CFTR remained tightly associated with ER microsomes. However, membrane-bound ubiquitinated CFTR could be subsequently degraded into trichloroacetic acid-soluble fragments upon incubation in hemin-free, ATP-containing lysate. Thus ER-associated degradation of CFTR occurs via a membrane-bound, rather than cytosolic, intermediate and likely involves recruitment of degradation machinery to the ER membrane. Our data suggest a model in which the degradation of polytopic proteins such as CFTR is coupled to retrograde translocation and removal of the polypeptide lipid bilayer.

    Original languageEnglish (US)
    Pages (from-to)2616-2624
    Number of pages9
    JournalJournal of Biological Chemistry
    Volume274
    Issue number5
    DOIs
    StatePublished - Jan 29 1999

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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