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1.
EMBO J ; 43(16): 3450-3465, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38937634

ABSTRACT

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.


Subject(s)
Dicarboxylic Acid Transporters , Humans , Kinetics , Dicarboxylic Acid Transporters/metabolism , Dicarboxylic Acid Transporters/genetics , Mitochondria/metabolism , Mitochondria/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Protein Multimerization , Amino Acid Transport Systems, Acidic/metabolism , Amino Acid Transport Systems, Acidic/genetics , Anion Transport Proteins/metabolism , Anion Transport Proteins/genetics , Anion Transport Proteins/chemistry , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Antiporters/metabolism , Antiporters/genetics , Antiporters/chemistry , Mitochondrial ADP, ATP Translocases/metabolism , Mitochondrial ADP, ATP Translocases/genetics , Biological Transport , Organic Anion Transporters/metabolism , Organic Anion Transporters/genetics , Organic Anion Transporters/chemistry , Adenosine Triphosphate/metabolism , Carrier Proteins , Membrane Transport Proteins
2.
EMBO Rep ; 24(8): e57127, 2023 08 03.
Article in English | MEDLINE | ID: mdl-37278158

ABSTRACT

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.


Subject(s)
Mitochondria , Mitochondrial ADP, ATP Translocases , Humans , Mitochondrial ADP, ATP Translocases/chemistry , Mitochondrial ADP, ATP Translocases/metabolism , Mitochondria/metabolism , Adenosine Triphosphate/metabolism , Adenosine Diphosphate/metabolism , Kinetics , Adenine Nucleotide Translocator 1/metabolism
3.
EMBO J ; 38(10)2019 05 15.
Article in English | MEDLINE | ID: mdl-30979775

ABSTRACT

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.


Subject(s)
Anion Transport Proteins , Mitochondrial Membrane Transport Proteins , Monocarboxylic Acid Transporters , Multiprotein Complexes/physiology , Protein Multimerization/physiology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Anion Transport Proteins/chemistry , Anion Transport Proteins/genetics , Anion Transport Proteins/metabolism , Gene Expression Regulation, Fungal , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Monocarboxylic Acid Transporters/chemistry , Monocarboxylic Acid Transporters/genetics , Monocarboxylic Acid Transporters/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Organisms, Genetically Modified , Protein Structure, Quaternary/physiology , Pyruvic Acid/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity Relationship , Temperature
4.
Curr Opin Struct Biol ; 57: 135-144, 2019 08.
Article in English | MEDLINE | ID: mdl-31039524

ABSTRACT

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.


Subject(s)
Mitochondrial ADP, ATP Translocases/chemistry , Mitochondrial ADP, ATP Translocases/metabolism , Biological Transport , Crystallography, X-Ray , Humans , Hydrogen Bonding
5.
Elife ; 72018 10 15.
Article in English | MEDLINE | ID: mdl-30320551

ABSTRACT

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.


Subject(s)
Biological Assay , Membrane Transport Proteins/metabolism , Temperature , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Humans , Ions , Ligands , Mitochondria/metabolism , Protein Stability , Reproducibility of Results , Substrate Specificity , Tetrahymena/metabolism
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