title: Identification of amino acid residues underlying the antiport mechanism of the mitochondrial carnitine/acylcarnitine carrier by site-directed mutagenesis and chemical labeling.
abstract: The mitochondrial carnitine/acylcarnitine carrier catalyzes the transport of carnitine and acylcarnitines by antiport as well as by uniport with a rate slower than the rate of antiport. The mechanism of antiport resulting from coupling of two opposed uniport reactions was investigated by site-directed mutagenesis. The transport reaction was followed as [(3)H]carnitine uptake in or efflux from proteoliposomes reconstituted with the wild type or mutants, in the presence or absence of a countersubstrate. The ratio between the antiport and uniport rates for the wild type was 3.0 or 2.5, using the uptake or efflux procedure, respectively. This ratio did not vary substantially in mutants H29A, K35R, G121A, E132A, K135A, R178A, D179E, E191A, K194A, K234A, and E288A. A ratio of 1.0 was measured for mutant K35A, indicating a loss of antiport function by this mutant. Ratios of >1.0 but significantly lower than that of the wild type were measured for mutants D32A, K97A, and D231A, indicating the involvement of these residues in the antiport mechanism. To investigate the role of the countersubstrate in the conformational changes underlying the transport reaction, the m-state of the transporter (opened toward the matrix side) was specifically labeled with N-ethylmaleimide while the c-state of the carrier (opened toward the cytosolic side) was labeled with fluorescein maleimide. The labeling results indicated that the addition of an external substrate, on one hand, reduced the amount of protein in the m-state and, on the other, increased the protein fraction in the c-state. This substrate-induced conformational change was abolished in the protein lacking K35, pointing to the role of this residue as a sensor in the mechanism of the antiport reaction.
authors.affiliation: CNR Institute of Biomembranes and Bioenergetics , via Amendola 165/A, 70126 Bari, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Giangregorio
authors.forename: Lara
authors.initials: L
authors.lastname: Console
authors.forename: Annamaria
authors.initials: A
authors.lastname: Tonazzi
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Palmieri
authors.forename: Cesare
authors.initials: C
authors.lastname: Indiveri
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D002331:Carnitine / Q000737:chemistry; D002417:Cattle; D017124:Conserved Sequence; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
chemical_list: D008081:Liposomes; D026901:Membrane Transport Proteins; C120109:SLC25A20 protein, human; D002331:Carnitine