title: Functional and structural role of amino acid residues in the matrix alpha-helices, termini and cytosolic loops of the bovine mitochondrial oxoglutarate carrier.
abstract: The mitochondrial oxoglutarate carrier belongs to the mitochondrial carrier family and exchanges oxoglutarate for malate and other dicarboxylates across the mitochondrial inner membrane. Here, single-cysteine mutant carriers were engineered for every residue in the amino- and carboxy-terminus, cytoplasmic loops, and matrix alpha-helices and their transport activity was measured in the presence and absence of sulfhydryl reagents. The analysis of the cytoplasmic side of the oxoglutarate carrier showed that the conserved and symmetric residues of the mitochondrial carrier motif [DE]XX[RK] localized at the C-terminal end of the even-numbered transmembrane alpha-helices are important for the function of the carrier, but the non-conserved cytoplasmic loops and termini are not. On the mitochondrial matrix side of the carrier most residues of the three matrix alpha-helices that are in the interface with the transmembrane alpha-helical bundle are important for function. Among these are the residues of the symmetric [ED]G motif present at the C-terminus of the matrix alpha-helices; the tyrosines of the symmetric YK motif at the N-terminus of the matrix alpha-helices; and the hydrophobic residues M147, I171 and I247. The functional role of these residues was assessed in the structural context of the homology model of OGC. Furthermore, in this study no evidence was found for the presence of a specific homo-dimerisation interface on the surface of the carrier consisting of conserved, asymmetric and transport-critical residues.
authors.affiliation: Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy.
authors.forename: Daniela V
authors.initials: DV
authors.lastname: Miniero
authors.forename: Anna R
authors.initials: AR
authors.lastname: Cappello
authors.forename: Rosita
authors.initials: R
authors.lastname: Curcio
authors.forename: Anna
authors.initials: A
authors.lastname: Ludovico
authors.forename: Lucia
authors.initials: L
authors.lastname: Daddabbo
authors.forename: Italo
authors.initials: I
authors.lastname: Stipani
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Robinson
authors.forename: Edmund R S
authors.initials: ER
authors.lastname: Kunji
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Palmieri
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D003600:Cytosol / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013439:Sulfhydryl Reagents / Q000378:metabolism
chemical_list: D000596:Amino Acids; D002352:Carrier Proteins; D007656:Ketoglutaric Acids; D026901:Membrane Transport Proteins; D013439:Sulfhydryl Reagents; C021723:oxoglutarate translocator