Publications

• Functional characterization of residues within the carnitine/acylcarnitine translocase RX2PANAAXF distinct motif.

  De Lucas J.R., Indiveri C., Tonazzi A., Perez P., Giangregorio N., Iacobazzi V., Palmieri F.

  The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane alpha-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by usi ... >> More

  The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane alpha-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by using two complementary approaches. First, we performed functional analysis in the model fungus Aspergillus nidulans of selected mutations with structural and functional relevance. Second, similar mutant human CACs were biochemically characterized after their reconstitution into liposomes. Both analyses have provided relevant information on the importance and role of the CAC motif residues in the activity and metabolic function of CAC. Only the two adjacent alanines, Ala281 and Ala282 in the human CAC, have been found not to be crucial for transport activity and in vivo function. Results obtained from amino acid substitutions of residues Arg275, Asn280 and Phe284 of human CAC together with structural analysis using molecular modelling of the carrier suggest that R275, N280 and F284 are involved in substrate binding during acylcarnitine/carnitine translocation. Furthermore, functional analysis of mutations of residues Pro278 and Ala279 in A. nidulans, together with kinetic data in reconstituted liposomes, suggest a predominant structural role for these amino acids. << Less

  Mol. Membr. Biol. 25:152-163(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Bacterial overexpression, purification, and reconstitution of the carnitine/acylcarnitine carrier from rat liver mitochondria.

  Indiveri C., Iacobazzi V., Giangregorio N., Palmieri F.

  The carnitine/acylcarnitine carrier from rat liver mitochondria was overexpressed in Escherichia coli. The expressed protein, recovered as inclusion bodies, was solubilized with sarkosyl and purified by Sephadex G-200 and celite chromatography. A yield of 15 mg of purified transport protein per li ... >> More

  The carnitine/acylcarnitine carrier from rat liver mitochondria was overexpressed in Escherichia coli. The expressed protein, recovered as inclusion bodies, was solubilized with sarkosyl and purified by Sephadex G-200 and celite chromatography. A yield of 15 mg of purified transport protein per liter of cell culture was obtained. Upon reconstitution into liposomes, the purified carrier catalyzed a [3H]carnitine/carnitine exchange inhibited by maleimides, mercurials, and sulfobetaines. Carnitine esters of various lengths were also transported. The Km for carnitine uptake was 0.47 +/-0.11 mM, the Vmax of the exchange was 0.78 +/-0.24 mmol/min per gram of protein, and the Ki for octanoylcarnitine was 13.5 +/-4.3 microM. The transport properties of the recombinant carrier were virtually identical to those of the native transporter. These studies represent the first overexpression of the functionally active mitochondrial carnitine/acylcarnitine carrier, thus enabling structure/function analysis of this protein by site-directed mutagenesis. << Less

  Biochem. Biophys. Res. Commun. 249:589-594(1998) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.