Evidence That Endoplasmic Reticulum (ER)-associated Degradation of Cystic Fibrosis Transmembrane Conductance Regulator Is Linked to Retrograde Translocation from the ER Membrane*
- From the Department of Molecular and Cellular Engineering and Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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 β-lactone, ALLN, andN α-tosyl-l-phenylalanine chloromethyl ketone and was relatively unaffected by lactacystin andN-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.
Footnotes
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↵* This work was supported by Grants GM53457 and DK51818 from the National Institutes of Health and by the North American Cystic Fibrosis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed. Present address: Division of Molecular Medicine, 3181 SW Sam Jackson Park Rd., NRC-3, Oregon Health Sciences University, Portland, OR 97201. Tel.: 503-494-7322; Fax: 503-494-7368; E-mail: skachw{at}ohsu.edu.
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↵2 E. Chong and W. R. Skach, unpublished observations.
- Abbreviations:
- ER
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endoplasmic reticulum
- ERAD
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ER associated degradation
- CFTR
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cystic fibrosis transmembrane conductance regulator
- DTT
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dithiothreitol
- HMW
-
high molecular weight
- RRL
-
rabbit reticulocyte lysate
- ALLN
-
N-acetyl-l-leucyl-l-leucinyl-norleucinal
- TPCK
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N α-tosyl-l-phenylalanine chloromethyl ketone
- TLCK
-
N-tosyl lysyl chloromethyl ketone
- TM
-
transmembrane
- PAGE
-
polyacrylamide gel electrophoresis.
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- Received July 24, 1998.
- Revision received October 23, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
