Functional characterization of residues within the carnitine/acylcarnitine translocase RX2PANAAXF distinct motif.
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.
De Lucas
J Ramon
JR
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Campus Universitario, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain.
Indiveri
Cesare
C
Tonazzi
Annamaria
A
Perez
Patricia
P
Giangregorio
Nicola
N
Iacobazzi
Vito
V
Palmieri
Ferdinando
F
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001233:Aspergillus nidulans / Q000201:enzymology / Q000254:growth & development; D001692:Biological Transport; D002151:Calorimetry; D002331:Carnitine / Q000378:metabolism; D002333:Carnitine Acyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013997:Time Factors; D014170:Transformation, Genetic; D014316:Tritium / Q000378:metabolism
D000596:Amino Acids; D008081:Liposomes; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014316:Tritium; D002333:Carnitine Acyltransferases; D002331:Carnitine