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

被引:27
作者
De Lucas, J. Ramon [2 ]
Indiveri, Cesare [1 ,3 ]
Tonazzi, Annamaria [1 ,4 ]
Perez, Patricia [2 ]
Giangregorio, Nicola [1 ,4 ]
Iacobazzi, Vito [1 ,4 ]
Palmieri, Ferdinando [1 ,4 ]
机构
[1] Univ Bari, Dipartimento Farm Biol, Biochem & Mol Biol Lab, I-70125 Bari, Italy
[2] Univ Alcala de Henares, Campus Univ, Fac Farm, Dept Microbiol & Parasitol, Madrid, Spain
[3] Univ Calabria, Dept Cellular Biol, Arcavacata Di Rende, Rende, Italy
[4] Inst Biomembranes & Bioengn, CNR, Bari, Italy
关键词
Aspergillus nidulans; carnitine/acylcarnitine translocase; liposomes; mitochondrial carriers; site-directed mutagenesis;
D O I
10.1080/09687680701697476
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane -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.
引用
收藏
页码:152 / 163
页数:12
相关论文
共 40 条
[1]  
[Anonymous], 1989, Molecular Cloning
[2]  
AQUILA H, 1982, H-S Z PHYSIOL CHEM, V363, P345
[3]   ANALYSIS OF ACETATE NON-UTILIZING (ACU) MUTANTS IN ASPERGILLUS-NIDULANS [J].
ARMITT, S ;
MCCULLOUGH, W ;
ROBERTS, CF .
JOURNAL OF GENERAL MICROBIOLOGY, 1976, 92 (FEB) :263-282
[4]   TRANSFORMATION OF ASPERGILLUS-NIDULANS BY THE OROTIDINE-5'-PHOSPHATE DECARBOXYLASE GENE OF NEUROSPORA-CRASSA [J].
BALLANCE, DJ ;
BUXTON, FP ;
TURNER, G .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1983, 112 (01) :284-289
[5]   Replacements of basic and hydroxyl amino acids identify structurally and functionally sensitive regions of the mitochondrial phosphate transport protein [J].
Briggs, C ;
Mincone, L ;
Wohlrab, H .
BIOCHEMISTRY, 1999, 38 (16) :5096-5102
[6]   Functional and structural role of amino acid residues in the odd-numbered transmembrane α-helices of the bovine mitochondrial oxoglutarate carrier [J].
Cappello, Anna R. ;
Miniero, Daniela V. ;
Curcio, Rosita ;
Ludovico, Anna ;
Daddabbo, Lucia ;
Stipani, Italo ;
Robinson, Alan J. ;
Kunji, Edmund R. S. ;
Palmieri, Ferdinando .
JOURNAL OF MOLECULAR BIOLOGY, 2007, 369 (02) :400-412
[7]   Functional and structural role of amino acid residues in the even-numbered transmembrane α-helices of the bovine mitochondrial oxoglutarate carrier [J].
Cappello, Anna Rita ;
Curcio, Rosita ;
Miniero, Daniela Valeria ;
Stipani, Italo ;
Robinson, Alan J. ;
Kunji, Edmund R. S. ;
Palmieri, Ferdinando .
JOURNAL OF MOLECULAR BIOLOGY, 2006, 363 (01) :51-62
[8]   The acuH gene of Aspergillus nidulans, required for growth on acetate and long-chain fatty acids, encodes a putative homologue of the mammalian carnitine acylcarnitine carrier [J].
De Lucas, JR ;
Dominguez, AI ;
Valenciano, S ;
Turner, G ;
Laborda, F .
ARCHIVES OF MICROBIOLOGY, 1999, 171 (06) :386-396
[9]  
De Lucas JR, 1997, ARCH MICROBIOL, V168, P504
[10]   Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study [J].
Della Rocca, BM ;
Miniero, DV ;
Tasco, G ;
Dolce, V ;
Falconi, M ;
Ludovico, A ;
Cappello, AR ;
Sanchez, P ;
Stipani, I ;
Casadio, R ;
Desideri, A ;
Palmieri, F .
MOLECULAR MEMBRANE BIOLOGY, 2005, 22 (05) :443-452