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.
By use of site-directed mutagenesis in combination with chemical modification of mutated proteins, the role of the six Cys residues in the transport function of the rat mitochondrial carnitine carrier (CAC) was studied. Several CAC mutants, in which one or more Cys residues had been replaced with Ser, were overexpressed in Escherichia coli, purified, and reconstituted in liposomes. The efficiency of incorporation into liposomes of the reconstituted proteins was lower for all constructs lacking Cys-23. Single, double, and quadruple replacement mutants showed V(max) comparable to that of the wild type. On the basis of the values of internal and external transport affinities (K(m)) for carnitine and of their comparison with those measured in mitochondria, the recombinant CAC is oriented unidirectionally in the liposomes, right side out compared to mitochondria. Substitution of Cys-136 with Ser caused a nearly complete loss of sensitivity of the CAC to N-ethylmaleimide, (2-aminoethyl)methanethiosulfonate hydrobromide (MTSES), and other hydrophilic SH reagents but not to the very hydrophobic N-phenylmaleimide. The wild-type CAC and the mutants containing Cys-136 showed substrate protection against NEM and MTSES inhibition and against NEM labeling. The data show that none of the native cysteines is essential for the transport mechanism and that Cys-136 is the major target of SH reagents and raise the hypothesis that Cys-136 is accessible from the external medium and is located at, or near, the substrate binding site. A model of the CAC is proposed in which the matrix hydrophilic loop containing Cys-136 protrudes into the membrane between the transmembrane domains of the protein.
Indiveri
Cesare
C
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy.
Giangregorio
Nicola
N
Iacobazzi
Vito
V
Palmieri
Ferdinando
F
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D008698:Mesylates / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D027701:Organic Cation Transport Proteins; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000074058:Solute Carrier Family 22 Member 5; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002352:Carrier Proteins; D008698:Mesylates; D027701:Organic Cation Transport Proteins; D011994:Recombinant Proteins; C115441:Slc22a5 protein, rat; D000074058:Solute Carrier Family 22 Member 5; D013439:Sulfhydryl Reagents; C093171:methanethiosulfonate ethylammonium; C109153:(2-sulfonatoethyl)methanethiosulfonate; D005020:Ethyl Methanesulfonate; D003545:Cysteine; D005033:Ethylmaleimide