RESUMEN
Members of the SLC25 mitochondrial carrier family link cytosolic and mitochondrial metabolism and support cellular maintenance and growth by transporting compounds across the mitochondrial inner membrane. Their monomeric or dimeric state and kinetic mechanism have been a matter of long-standing debate. It is believed by some that they exist as homodimers and transport substrates with a sequential kinetic mechanism, forming a ternary complex where both exchanged substrates are bound simultaneously. Some studies, in contrast, have provided evidence indicating that the mitochondrial ADP/ATP carrier (SLC25A4) functions as a monomer, has a single substrate binding site, and operates with a ping-pong kinetic mechanism, whereby ADP is imported before ATP is exported. Here we reanalyze the oligomeric state and kinetic properties of the human mitochondrial citrate carrier (SLC25A1), dicarboxylate carrier (SLC25A10), oxoglutarate carrier (SLC25A11), and aspartate/glutamate carrier (SLC25A13), all previously reported to be dimers with a sequential kinetic mechanism. We demonstrate that they are monomers, except for dimeric SLC25A13, and operate with a ping-pong kinetic mechanism in which the substrate import and export steps occur consecutively. These observations are consistent with a common transport mechanism, based on a functional monomer, in which a single central substrate-binding site is alternately accessible.
Asunto(s)
Transportadores de Ácidos Dicarboxílicos , Humanos , Cinética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Mitocondrias/metabolismo , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Multimerización de Proteína , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Proteínas de Transporte de Anión/metabolismo , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/química , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Antiportadores/metabolismo , Antiportadores/genética , Antiportadores/química , Translocasas Mitocondriales de ADP y ATP/metabolismo , Translocasas Mitocondriales de ADP y ATP/genética , Transporte Biológico , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/química , Adenosina Trifosfato/metabolismo , Proteínas Portadoras , Proteínas de Transporte de MembranaRESUMEN
The mitochondrial ADP/ATP carrier (SLC25A4), also called the adenine nucleotide translocase, imports ADP into the mitochondrial matrix and exports ATP, which are key steps in oxidative phosphorylation. Historically, the carrier was thought to form a homodimer and to operate by a sequential kinetic mechanism, which involves the formation of a ternary complex with the two exchanged substrates bound simultaneously. However, recent structural and functional data have demonstrated that the mitochondrial ADP/ATP carrier works as a monomer and has a single substrate binding site, which cannot be reconciled with a sequential kinetic mechanism. Here, we study the kinetic properties of the human mitochondrial ADP/ATP carrier by using proteoliposomes and transport robotics. We show that the Km/Vmax ratio is constant for all of the measured internal concentrations. Thus, in contrast to earlier claims, we conclude that the carrier operates with a ping-pong kinetic mechanism in which substrate exchange across the membrane occurs consecutively rather than simultaneously. These data unite the kinetic and structural models, showing that the carrier operates with an alternating access mechanism.
Asunto(s)
Mitocondrias , Translocasas Mitocondriales de ADP y ATP , Humanos , Translocasas Mitocondriales de ADP y ATP/química , Translocasas Mitocondriales de ADP y ATP/metabolismo , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Difosfato/metabolismo , Cinética , Translocador 1 del Nucleótido Adenina/metabolismoRESUMEN
The mitochondrial pyruvate carrier (MPC) is critical for cellular homeostasis, as it is required in central metabolism for transporting pyruvate from the cytosol into the mitochondrial matrix. MPC has been implicated in many diseases and is being investigated as a drug target. A few years ago, small membrane proteins, called MPC1 and MPC2 in mammals and Mpc1, Mpc2 and Mpc3 in yeast, were proposed to form large protein complexes responsible for this function. However, the MPC complexes have never been isolated and their composition, oligomeric state and functional properties have not been defined. Here, we identify the functional unit of MPC from Saccharomyces cerevisiae In contrast to earlier hypotheses, we demonstrate that MPC is a hetero-dimer, not a multimeric complex. When not engaged in hetero-dimers, the yeast Mpc proteins can also form homo-dimers that are, however, inactive. We show that the earlier described substrate transport properties and inhibitor profiles are embodied by the hetero-dimer. This work provides a foundation for elucidating the structure of the functional complex and the mechanism of substrate transport and inhibition.
Asunto(s)
Proteínas de Transporte de Anión , Proteínas de Transporte de Membrana Mitocondrial , Transportadores de Ácidos Monocarboxílicos , Complejos Multiproteicos/fisiología , Multimerización de Proteína/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Transportadores de Ácidos Monocarboxílicos/química , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Organismos Modificados Genéticamente , Estructura Cuaternaria de Proteína/fisiología , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , TemperaturaRESUMEN
The mitochondrial ADP/ATP carrier, also called adenine nucleotide translocase, accomplishes one of the most important transport activities in eukaryotic cells, importing ADP into the mitochondrial matrix for ATP synthesis, and exporting ATP to fuel cellular activities. In the transport cycle, the carrier changes between a cytoplasmic and matrix state, in which the central substrate binding site is alternately accessible to these compartments. A structure of a cytoplasmic state was known, but recently, a structure of a matrix-state in complex with bongkrekic acid was solved. Comparison of the two states explains the function of highly conserved sequence features and reveals that the transport mechanism is unique, involving the coordinated movement of six dynamic elements around a central translocation pathway.
Asunto(s)
Translocasas Mitocondriales de ADP y ATP/química , Translocasas Mitocondriales de ADP y ATP/metabolismo , Transporte Biológico , Cristalografía por Rayos X , Humanos , Enlace de HidrógenoRESUMEN
Substrates of most transport proteins have not been identified, limiting our understanding of their role in physiology and disease. Traditional identification methods use transport assays with radioactive compounds, but they are technically challenging and many compounds are unavailable in radioactive form or are prohibitively expensive, precluding large-scale trials. Here, we present a high-throughput screening method that can identify candidate substrates from libraries of unlabeled compounds. The assay is based on the principle that transport proteins recognize substrates through specific interactions, which lead to enhanced stabilization of the transporter population in thermostability shift assays. Representatives of three different transporter (super)families were tested, which differ in structure as well as transport and ion coupling mechanisms. In each case, the substrates were identified correctly from a large set of chemically related compounds, including stereo-isoforms. In some cases, stabilization by substrate binding was enhanced further by ions, providing testable hypotheses on energy coupling mechanisms.