Identification by site-directed mutagenesis and chemical modification of three vicinal cysteine residues in rat mitochondrial carnitine/acylcarnitine transporter

The proximity of the Cys residues present in the mitochondrial rat carnitine/acylcarnitine carrier (CAC) primary structure was studied by using site-directed mutagenesis in combination with chemical modification. CAC mutants, in which one or more Cys residues had been replaced with Ser, were overexpressed in E.coli and reconstituted into liposomes. The effect of SH oxidizing, cross-linking and coordinating reagents was evaluated on the carnitine/carnitine exchange catalyzed by the recombinant reconstituted CAC proteins. All the reagents tested efficiently inhibited the wild-type CAC. The inhibitory effect of diamide, Cu2+-phenanthroline or phenylarsine oxide was largely reduced or abolished by the double substitutions C136/155S, C58/136S and C58/155S. The decrease in sensitivity to these reagents was much lower in double mutants in which Cys-23 was substituted with Cys-136 or Cys- 155. No decrease in inhibition was found when Cys-89 and/or Cys-283 were replaced with Ser. Sb3+, which coordinates three cysteines, inhibited only the Cys-replacement mutants containing all three cysteines 58, 136 and 155 out of the six native cysteines. In addition, the mutant C23/89/155/283S, in which double tandem fXa recognition sites were inserted in position 65-72, i.e. between Cys-58 and Cys-136, was not cleaved into two fragments by fXa protease after treatment with diamide. These results are interpreted in the light of the homology model of CAC based on the available X-ray structure of the ADP/ATP carrier. They indicate that Cys-58, Cys-136 and Cys-155 become close in the tertiary structure of the CAC during its catalytic cycle.