The mitochondrial oxoglutarate carrier: cysteine-scanning mutagenesis of transmembrane domain IV and sensitivity of Cys mutants to sulfhydryl reagents.
Using a functional mitochondrial oxoglutarate carrier mutant devoid of Cys residues (C-less carrier), each amino acid residue in transmembrane domain IV and flanking hydrophilic loops (from T179 to S205) was replaced individually with Cys. The great majority of the 27 mutants exhibited significant oxoglutarate transport in reconstituted liposomes as compared to the activity of the C-less carrier. In contrast, Cys substitution for G183, R190, Q198, and Y202, in either C-less or wild-type carriers, yielded molecules with complete loss of oxoglutarate transport activity. G183 and R190 could be partially replaced only by Ala and Lys, respectively, whereas Q198 and Y202 were irreplaceable with respect to oxoglutarate transport. Of the single-Cys mutants tested, only T187C, A191C, V194C, and N195C were strongly inactivated by N-ethylmaleimide and by low concentrations of methanethiosulfonate derivatives. Oxoglutarate protects Cys residues at positions 187, 191, and 194 against reaction with N-ethylmaleimide. These positions as well as the residues found to be essential for the carrier activity, except Y202 which is located in the extramembrane loop IV-V, reside on the same face of transmembrane helix IV, probably lining part of a water-accessible crevice or channel between helices of the oxoglutarate carrier.
Stipani
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Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy.
Cappello
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Daddabbo
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Natuzzi
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Miniero
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Stipani
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Palmieri
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D003545:Cysteine / Q000235:genetics*; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007656:Ketoglutaric Acids / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008698:Mesylates / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary / Q000187:drug effects / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D002352:Carrier Proteins; D007656:Ketoglutaric Acids; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D011510:Proteolipids; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; C093171:methanethiosulfonate ethylammonium; C021723:oxoglutarate translocator; C020485:proteoliposomes; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D005020:Ethyl Methanesulfonate; D003545:Cysteine; D005033:Ethylmaleimide