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

Deletion of Phe-508 (ΔF508) in cystic fibrosis transmembrane conductance regulator causes cystic fibrosis because of misfolding of the protein. P-glycoprotein (P-gp) containing the equivalent mutation (ΔY490) 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 ΔY490 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 ΔY490 P-gp by 6-fold. Most other changes to Ile-306 or Phe-343 did not enhance maturation of ΔY490 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.