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

Abstract

The proximity of the Cys residues present in the mitochondrial rat carnitine/acylcamitine 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 Escherichia coli and reconstituted into liposomes. The effect of SH oxidizing, cross-linking, and coordinating reagents was evaluated on the carnitine/camitine exchange catalyzed by the recombinant reconstituted CAC proteins. All the tested reagents 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 C136S/C155S, C58S/C136S, and C58S/C155S. The decrease in sensitivity to these reagents was much lower in double mutants in which Cys23 was substituted with Cys136 or Cys155. No decrease in inhibition was found when Cys89 and/or Cys283 were replaced with Ser. Sb 3+, which coordinates three cysteines, inhibited only the Cys replacement mutants containing cysteines 58, 136, and 155 of the six native cysteines. In addition, the mutant C23S/C89S/C155S/C283S, in which double tandem fXa recognition sites were inserted in positions 65-72, i.e. between Cys 58 and Cys136, was not cleaved into two fragments by fXa protease after treatment with diamide. These results are interpreted in light of the homology model of CAC based on the available x-ray structure of the ADP/ATP carrier. They indicate that Cys58, Cys136, and Cys 155 become close in the tertiary structure of the CAC during its catalytic cycle. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.