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J. Biol. Chem., Vol. 281, Issue 40, 29436-29440, October 6, 2006

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Insertion of an Arginine Residue into the Transmembrane Segments Corrects Protein Misfolding^*

From the Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Deletion of Phe-508 (F508) in cystic fibrosis transmembrane conductance regulator causes cystic fibrosis because of misfolding of the protein. P-glycoprotein (P-gp) containing the equivalent mutation (Y490) is also misfolded but can be rescued with drug substrates. Whether rescue is due to direct binding of drug substrate to the transmembrane (TM) segments or to indirect effects on cellular protein folding pathways is still controversial. P-gp-drug substrate interactions likely involve hydrogen bonds. If the mechanism of drug rescue involves changes to TM packing then we should be able to identify suppressor mutations in the TM segments that can mimic the drug rescue effects. We predicted that an arginine residue in the TM segments predicted to line the drug-binding pocket of P-gp (I306(TM5) or F343(TM6)) might suppress Y490 P-gp protein misfolding because it has the highest propensity to form hydrogen bonds. We show that R306(TM5) or R343(TM6) increased the relative amount of mature Y490 P-gp by 6-fold. Most other changes to Ile-306 or Phe-343 did not enhance maturation of Y490 P-gp. The I306R mutant also promoted maturation of misprocessed mutants that had mutations in the second nucleotide-binding domain (L1260A), the cytoplasmic loops (G251V, F804A), the linker region (P709A), or in TM segments (G300V, G722A). These results show that arginine residues in the TM domains can mimic the drug rescue effects and are effective suppressor mutations for processing mutations located throughout the molecule.

Received for publication, August 4, 2006 , and in revised form, August 16, 2006.

^* This work was supported by grants from the National Cancer Institute of Canada through the Canadian Cancer Society and from the Canadian Institutes of Health Research. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Recipient of the Canada Research Chair in Membrane Biology. To whom correspondence should be addressed: Dept. of Medicine, University of Toronto, 1 King's College Circle, Rm. 7342, Medical Sciences Bldg., Toronto, Ontario M5S 1A8, Canada. Tel.:/Fax: 416-978-1105; E-mail: david.clarke{at}utoronto.ca.

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				T. W. Loo, M. C. Bartlett, and D. M. Clarke Arginines in the First Transmembrane Segment Promote Maturation of a P-glycoprotein Processing Mutant by Hydrogen Bond Interactions with Tyrosines in Transmembrane Segment 11 J. Biol. Chem., September 5, 2008; 283(36): 24860 - 24870. [Abstract] [Full Text] [PDF]

				T. W. Loo, M. C. Bartlett, and D. M. Clarke Suppressor Mutations in the Transmembrane Segments of P-glycoprotein Promote Maturation of Processing Mutants and Disrupt a Subset of Drug-binding Sites J. Biol. Chem., November 2, 2007; 282(44): 32043 - 32052. [Abstract] [Full Text] [PDF]