II-C p-6.Site-directed mutagenesis of the mitochondrial carnitine/acylcarnitine carrier: identification of four vicinal cysteine residues.

The mitochondrial carnitine/acylcarnitine carrier is responsible for the transfer of acylcarnitines into the mitochondrial matrix for the b-oxidation pathway. The structural features of the carnitine/acylcarnitine carrier demonstrate that it belongs to the mitochondrial carier protein family (1). We have previously investigated the role of the carnitine carrier Cys residues by site-directed mutagenesis. Single and multiple mutants of the carnitine carrier were constructed in which one or more of the six Cys residues were substituted with Ser. Inhibition analysis of single and multiple Cys-mutants with monofunctional SH reagents revealed that each of the six native Cys of the protein can be replaced without substantial variation of the activity and that Cys-136, the major target for SH reagents, is accessible from the cytosolic side of the carrier (2). In the present work we have studied the possibility that some Cys residues are in close proximity to each other in the protein structure. The effects of SH oxidizing, cross-linking and coordinating reagents were evaluated on single and multiple Cys-mutants of the carnitine/acylcarnitine carrier. All the reagents tested inhibited efficiently the wild-type protein. The inhibitory effect was reduced by the substitution C136S and abolished by the double substitution C136/155S. Reduction of inhibition was also observed in double mutants in which Cys-136 together with Cys-23 or Cys-58 were substituted with Ser or, alternatively, Cys-155 and Cys-58 were substituted with Ser. Among the four replacement mutants, i.e., proteins containing two of the six native Cys, only those containing the couples Cys-136/155, Cys-136/58, Cys-136/23 or Cys-155/58 were sensitive to the reagents. To confirm the formation of a S_S bridge in the four replacement mutants containing the couples Cys-136/58 and Cys-136/23, fXa recognition sites between Cys-58 (or 23) and Cys-136 were inserted. Pretreatment of the two proteins with diamide prevented their cleavage by the fXa protease. The result described, revealed that Cys-23, Cys-58, Cys-136 and Cys-155 form a cluster of four vicinal SH groups. From these data, the relationships among three hydrophobic intermembrane segments and two hydrophilic loops are defined. References [1] C. Indiveri, V. Iacobazzi, N. Giangregorio, F. Palmieri, Biochem J. 321 (1997) 713-719. [2] C. Indiveri, N. Giangregorio, V. Iacobazzi, F. Palmieri, Biochemistry 41 (2002) 8649-8656.