Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane - PubMed (original) (raw)
. 1999 Jan 29;274(5):2616-24.
doi: 10.1074/jbc.274.5.2616.
Affiliations
- PMID: 9915789
- DOI: 10.1074/jbc.274.5.2616
Free article
Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane
X Xiong et al. J Biol Chem. 1999.
Free article
Abstract
The ubiquitin-proteasome pathway has been implicated in the degradation of newly synthesized, misfolded 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 hemin and MG132. Degradation was inhibited to a lesser extent by clasto-lactacystin beta-lactone, ALLN, and Nalpha-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 from the lipid bilayer.
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