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J. Biol. Chem., Vol. 280, Issue 6, 4968-4974, February 11, 2005

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Destabilization of the Transmembrane Domain Induces Misfolding in a Phenotypic Mutant of Cystic Fibrosis Transmembrane Conductance Regulator^*

Mei Y. Choi¶, Anthony W. Partridge||, Craig Daniels**, Kai Du**, Gergely L. Lukacs**, and Charles M. Deber

From the Division of Structural Biology and Biochemistry and ^**Program in Cell and Lung Biology, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8 and the Departments of Biochemistry and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Two phenotypic missense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel pore (L346P and R347P in transmembrane (TM) segment 6) involve gain of a proline residue, but only L346P represents a significant loss of segment hydropathy. We show here that, for synthetic peptides corresponding to sequences of CFTR TM6 segments, circular dichroism spectra of wild type and R347P TM6 in membrane mimetic environments are virtually identical, but L346P loses 50% helicity, implying a membrane insertion defect in the latter mutant. A similar defect was observed in the corresponding double-spanning ("hairpin") TM5/6-L346P synthetic peptide. Examination of the biogenesis of CFTR revealed that the full-length protein harboring the L346P mutation is rapidly degraded at the endoplasmic reticulum (ER), whereas the wild type and the R347P protein process normally. Furthermore, a second site mutation (R347I) that restores in vitro membrane insertion and folding of the TM5/6-L346P peptide also rescues the folding and cell surface chloride channel function of full-length L346P CFTR. The correlated in vitro/in vivo results demonstrate that destabilizing local hydrophobic character represents a sufficient signal for marking CFTR as a non-native protein by the ER quality control, with accompanying deleterious consequences to global protein folding events.

Received for publication, September 1, 2004 , and in revised form, November 8, 2004.

^* This work was supported, in part, by grants (to G. L. L. and C. M. D.) from the Canadian Cystic Fibrosis Foundation, the Canadian Institutes of Health Research (CIHR), and the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by an award from the Hospital for Sick Children Research Training Committee.

^|| Held a CIHR doctoral fellowship award.

To whom correspondence should be addressed. Tel.: 416-813-5924; Fax: 416-813-5005; E-mail: deber{at}sickkids.ca (to C. M. D.) or glukacs{at}sickkids.ca (to G. L. L.).

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