Site-Directed Mutagenesis and Chemical Modification of the Six Native Cysteine Residues of the Rat Mitochondrial Carnitine Carrier: Implications for the Role of Cysteine-136 †

摘要: By use of site-directed mutagenesis in combination with chemical modification mutated proteins, the role six Cys residues transport function rat mitochondrial carnitine carrier (CAC) was studied. Several CAC mutants, which one or more had been replaced Ser, were overexpressed Escherichia coli, purified, and reconstituted liposomes. The efficiency incorporation into liposomes proteins lower for all constructs lacking Cys-23. Single, double, quadruple replacement mutants showed Vmax comparable to that wild type. On basis values internal external affinities ( Km) their comparison those measured mitochondria, recombinant is oriented unidirectionally liposomes, right side out compared mitochondria. Substitution Cys-136 Ser caused a nearly complete loss sensitivity N-ethylmaleimide, (2-aminoethyl)methanethiosulfonate hydrobromide (MTSES), other hydrophilic SH reagents but not very hydrophobic N-phenylmaleimide. wild-type containing substrate protection against NEM MTSES inhibition labeling. data show none native cysteines essential mechanism major target raise hypothesis accessible from medium located at, near, binding site. A model proposed matrix loop protrudes membrane between transmembrane domains protein.