The Mitochondrial Oxoglutarate Carrier: Cysteine-Scanning Mutagenesis of Transmembrane Domain IV and Sensitivity of Cys Mutants to Sulfhydryl Reagents † Valentina Stipani, Anna Rita Cappello, Lucia Daddabbo, Dorotea Natuzzi, Daniela Valeria Miniero, Italo Stipani, and Ferdinando Palmieri* Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, UniVersity of Bari, 70125 Bari, Italy, and CNR Unit for the Study of Mitochondria and Bioenergetics, Bari, Italy ReceiVed August 6, 2001; ReVised Manuscript ReceiVed October 22, 2001 ABSTRACT: 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. The oxoglutarate transporter, also known as the oxoglu- tarate/malate carrier (OGC), 1 is a nuclear-encoded protein located in the inner mitochondrial membrane. This enzyme catalyzes the transport of 2-oxoglutarate in an electroneutral exchange for malate and plays an important role in several metabolic processes, including the malate-aspartate shuttle, the oxoglutarate-isocitrate shuttle, gluconeogenesis from lactate, and nitrogen metabolism (1). Encoded by the OGC gene that in man maps to chromo- some 17q25.3 (2), the carrier has been purified and kineti- cally characterized in reconstituted liposomes (for a review, see ref 3). Like other functionally characterized members of the mitochondrial carrier family, the protein has a tripartite structure, made up of 3 tandemly repeated sequences of about 100 amino acids in length. On the basis of the hydropathy profile of the primary amino acid sequence (4), a secondary structure was proposed in which the carrier has six R-helical transmembrane domains connected by hydrophilic loops. Evidence supporting the general features of the model and demonstrating that both the N and C termini of OGC are on the cytoplasmic face of the membrane has been obtained from limited proteolysis and immunological studies (5). By using cross-linking reagents, it has been shown that the OGC exists as a homodimer (6). In addition, the OGC has been expressed in E. coli and refolded in reconstitutively active form (7). Previous site-directed mutagenesis studies showed that the three cysteinyl residues present in the primary structure of OGC are not involved in the transport mechanism since the C-less mutant is fully active (8). In this study, Cys-scanning mutagenesis of the functional C-less mutant of OGC has been employed to examine the residues in transmembrane helix IV and in part of the flanking hydrophilic loops of OGC. The effects of individual Cys replacements for Thr179-Ser205 on the oxoglutarate transport activity are reported, as well as the sensitivity of the mutants to the alkylating agent N-ethylmaleimide (NEM). The results demonstrate that four of the residues (G183, R190, Q198, and Y202) are essential for the carrier activity, four Cys mutants (T187C, A191C, V194C, and N195C) are inactivated by NEM, and these mutants, except N195C, exhibit substrate protection against NEM inhibition. All these residues, except Y202 which is located in the extramembrane loop IV-V, appear to cluster on one face of helix IV, suggesting that this surface within the OGC protein may be important for substrate binding and/or translocation of oxoglutarate. MATERIALS AND METHODS Materials. 2-Keto[1- 14 C]glutaric acid and [ 14 C]NEM were purchased from Dupont De Nemours; Pipes, Triton X-114, Amberlite XAD-4, and egg yolk phospholipids (lecithin from † This work was supported by grants from MURST-PRIN, MURST L.488/92 CO3 and CO4, MURST-CNR L.95/95, CEGBA, and CNR target project on Biotechnology, and by the European Social Fund. * Address correspondence to this author at the Dipartimento Far- maco-Biologico, Universita ` di Bari,Via E. Orabona 4, 70125 Bari, Italy. Tel.: +39-80-5443374. Fax: +39-80-5442770. E-mail: fpalm@ farmbiol.uniba.it. 1 Abbreviations: DTE, dithioerythritol; MTS, methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate hydrobromide; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate; MTSET, [2-(trimethyl- ammonium)ethyl]methanethiosulfonate bromide; NEM, N-ethylmale- imide; OGC, oxoglutarate carrier; PCR, polymerase chain reaction; Pipes, 1,4-piperazinediethanesulfonic acid; SDS-PAGE, sodium dode- cyl sulfate-polyacrylamide gel electrophoresis. 15805 Biochemistry 2001, 40, 15805-15810 10.1021/bi011616j CCC: $20.00 © 2001 American Chemical Society Published on Web 11/22/2001