Functional and structural role of amino acid residues in the matrix alpha-helices, termini and cytosolic loops of the bovine mitochondrial oxoglutarate carrier.

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.

Structural-dynamical properties of the transmembrane segment VI of the mitochondrial oxoglutarate carrier studied by site directed spin-labeling.

Site directed spin-labeling (SDSL) has been used to probe the structural and dynamic features of residues comprising the sixth transmembrane segment of the mitochondrial oxoglutarate carrier. Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe. The labeled proteins, reconstituted into liposomes, have been assayed for their transport activity and analyzed with continuous-wave electron paramagnetic resonance.

Functional and structural role of amino acid residues in the odd-numbered transmembrane alpha-helices of the bovine mitochondrial oxoglutarate carrier.

The mitochondrial oxoglutarate carrier (OGC) plays an important role in the malate-aspartate shuttle, the oxoglutarate-isocitrate shuttle and gluconeogenesis. To establish amino acid residues that are important for function, each residue in the transmembrane alpha-helices H1, H3 and H5 was replaced systematically by a cysteine in a fully functional mutant carrier that was devoid of cysteine residues. The transport activity of the mutant carriers was measured in the presence and absence of sulfhydryl reagents.

Functional and structural role of amino acid residues in the even-numbered transmembrane alpha-helices of the bovine mitochondrial oxoglutarate carrier.

The mitochondrial oxoglutarate carrier exchanges cytosolic malate for 2-oxoglutarate from the mitochondrial matrix. Orthologs of the carrier have a high degree of amino acid sequence conservation, meaning that it is impossible to identify residues important for function on the basis of this criterion alone. Therefore, each amino acid residue in the transmembrane alpha-helices H2 and H6 was replaced by a cysteine in a functional mitochondrial oxoglutarate carrier that was otherwise devoid of cysteine residues.

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study.

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp.