Substrate Specificity of the Two Mitochondrial Ornithine Carriers Can Be Swapped by Single Mutation in Substrate Binding Site

Monné, Magnus; Miniero, Daniela Valeria; Daddabbo, Lucia; Robinson, Alan J.; Kunji, Edmund R. S.; Palmieri, Ferdinando

doi:10.1074/jbc.m111.324855

Cited by 52 publications

(53 citation statements)

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“…The yeast ADP/ATP carriers have one brace and three salt bridges in the extended matrix network, providing a significant energy barrier that prevents conversion to the m-state in the absence of substrate. A single substrate-binding site has been identified in the central cavity, which corresponds to the middle of the membrane (25,29). There is consensus on the residues involved in ADP binding to the ADP/ATP carrier (25)(26)(27)(28).…”

Section: Discussionmentioning

confidence: 99%

“…Previous proposals for the mechanism were based on a functional dimer (19,21,22), but subsequent work has shown that carriers are functionally monomeric (23,24). The first step toward a mechanism consistent with a monomeric carrier was the identification of a single substrate-binding site in the central cavity, as shown by distance and chemical constraints (25), molecular dynamics simulations (26,27), sequence analysis (28), and mutagenesis (29). Sequence analysis of the three homologous repeats found in members of the mitochondrial carrier family highlighted a conserved motif [YF][DE]xx[KR] on the cytoplasmic side, which has been suggested to form a salt-bridge network when the carrier is in the m-state (28).…”

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Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism

Ruprecht

Hellawell

Harding

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

199

416

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The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix. The carrier cycles by an unresolved mechanism between the cytoplasmic state, in which the carrier accepts ADP from the cytoplasm, and the matrix state, in which it accepts ATP from the mitochondrial matrix. Here we present the structures of the yeast ADP/ATP carriers Aac2p and Aac3p in the cytoplasmic state. The carriers have three domains and are closed at the matrix side by three interdomain salt-bridge interactions, one of which is braced by a glutamine residue. Glutamine braces are conserved in mitochondrial carriers and contribute to an energy barrier, preventing the conversion to the matrix state unless substrate binding occurs. At the cytoplasmic side a second salt-bridge network forms during the transport cycle, as demonstrated by functional analysis of mutants with charge-reversed networks. Analyses of the domain structures and properties of the interdomain interfaces indicate that interconversion between states involves movement of the even-numbered α-helices across the surfaces of the odd-numbered α-helices by rotation of the domains. The odd-numbered α-helices have an L-shape, with proline or serine residues at the kinks, which functions as a lever-arm, coupling the substrate-induced disruption of the matrix network to the formation of the cytoplasmic network. The simultaneous movement of three domains around a central translocation pathway constitutes a unique mechanism among transport proteins. These findings provide a structural description of transport by mitochondrial carrier proteins, consistent with an alternating-access mechanism.membrane protein | cardiolipin binding | X-ray crystallography | serine kinks | adenine nucleotide translocase M itochondrial carriers are a family of proteins that transport a diverse range of nucleotides, amino acids, inorganic ions, fatty acids, keto acids, and cofactors across the inner mitochondrial membrane (1). The carriers link the biochemical pathways in the cytoplasm with those in the mitochondrial matrix, thereby playing key roles in many aspects of cell physiology. There are many rare, but severe, human diseases associated with defective mitochondrial carriers (2).The ADP/ATP carriers are archetypal members of the mitochondrial carrier family (3). ADP/ATP carriers play the essential role of importing ADP into the mitochondrial matrix, where it can be phosphorylated by ATP synthase, and of exporting newly synthesized ATP into the cytosol, replenishing the cell with metabolic energy. ADP/ATP carriers have been intensively studied, because of their high natural abundance and the availability of specific inhibitors, which lock the carrier in two distinct states. The atractylosides, such as carboxyatractyloside (CATR) (4, 5), lock the carrier in the cytosolic state (c-state) with the substrate-binding site accessible to the intermembrane space, which is confluent with the cytosol. Bongkrekic acid (6) locks the carrier in the matrix state (m-state), wit...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism

Ruprecht

Hellawell

Harding

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

199

416

View full text Add to dashboard Cite

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“…The coding sequences for SLC25A2 (NM_031947), SLC-25A12 (NM_003705), SLC25A13 (NM_014251), SLC-25A15 (NM_014252), SLC25A18 (NM_031481), SLC-25A22 (NM_024698), and SLC25A29 (NM_001039355) were amplified as perviously described (Fiermonte et al 2002(Fiermonte et al , 2003Lasorsa et al 2003;Monné et al 2012;Porcelli et al 2014). The amplified products were cloned into the pRUN (SLC25A2, SLC25A15, SLC25A18, SLC25A22, and SLC25A29), the pMW7 (SLC25A12), and pET-15b (SLC25A13) expression vectors and transformed into E. coli DH5α cells.…”

Section: Construction Of Expression Plasmidsmentioning

confidence: 99%

Asymmetric dimethylarginine is transported by the mitochondrial carrier SLC25A2

et al. 2015

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Asymmetric dimethyl L-arginine (ADMA) is generated within cells and in mitochondria when proteins with dimethylated arginine residues are degraded. The aim of this study was to identify the carrier protein(s) that transport ADMA across the inner mitochondrial membrane. It was found that the recombinant, purified mitochondrial solute carrier SLC25A2 when reconstituted into liposomes efficiently transports ADMA in addition to its known substrates arginine, lysine, and ornithine and in contrast to the other known mitochondrial amino acid transporters SLC25A12, SLC25A13, SLC25A15, SLC25A18, SLC25A22, and SLC25A29. The widely expressed SLC25A2 transported ADMA across the liposomal membrane in both directions by both unidirectional transport and exchange against arginine or lysine. The SLC25A2-mediated ADMA transport followed first-order kinetics, was nearly as fast as the transport of the best SLC25A2 substrates known so far, and was highly specific as symmetric dimethylarginine (SDMA) was not transported at all. Furthermore, ADMA inhibited SLC25A2 activity with an inhibition constant of 0.38 ± 0.04 mM, whereas SDMA inhibited it poorly. We propose that a major function of SLC25A2 is to export ADMA from mitochondria missing the mitochondrial ADMA-metabolizing enzyme AGXT2. There is evidence that ADMA can also be imported into mitochondria, e.g., in kidney proximal tubulus cells, to be metabolized by AGXT2. SLC25A2 may also mediate this transport function.

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“…Molecular modeling of residue p. Arg179 showed from the lateral side (d) or from the top (e), with the side chain exposed to the inner part of the pocket. AAC1 protein was used as template (Monne et al 2012). The presence of a glutamine in position 179 impairs the ability of ORNT2 to complement DArg11 yeast cells, suggesting that ORNT2 functions primarily as a transporter of compounds other than ornithine.…”

Section: Discussionmentioning

confidence: 99%

Heterologous Expression in Yeast of Human Ornithine Carriers ORNT1 and ORNT2 and of ORNT1 Alleles Implicated in HHH Syndrome in Humans

et al. 2015

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Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive metabolic disorder usually presenting in the neonatal period with intermittent episodes of hyperammonemia, psychomotor delay, and progressive encephalopathy. Adult cases usually evolve into frank spastic paraparesis. The syndrome is caused by mutations in SLC25A15/ORNT1 encoding the mitochondrial ornithine transporter; a second ornithine transporter, ORNT2 of unknown function, is also present in most placental mammals. ORNT2 is believed to originate from an ancient retro-transposition event. In yeast Saccharomyces cerevisiae the major function of the transporter (encoded by Arg11) is to shuttle ornithine from the mitochondrial matrix to the cytosol. Its inactivation abolishes growth in the absence of arginine.In this work, we used functional complementation in S. cerevisiae to characterize the function of human ORNT2 and to test the pathogenicity of ORNT1 mutations found in HHH patients. Notably, we found that human ORNT1 but not ORNT2 complements the deletion of the yeast gene, despite their high level of homology. However, we identified some key residues in ORNT2, which may recover its functional competence when replaced with the corresponding residues of ORNT1, suggesting that roles of the two transporters are different. Moreover, we used this system to test a series of missense mutations of ORNT1 identified in patients with HHH syndrome. All mutations had a detrimental effect on the functionality of the human gene, without however clear genotype-phenotype correlations. Our data support yeast as a simple and effective model to validate missense mutations occurring in patients with HHH.

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Substrate Specificity of the Two Mitochondrial Ornithine Carriers Can Be Swapped by Single Mutation in Substrate Binding Site

Cited by 52 publications

References 54 publications

Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism

Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism

Asymmetric dimethylarginine is transported by the mitochondrial carrier SLC25A2

Heterologous Expression in Yeast of Human Ornithine Carriers ORNT1 and ORNT2 and of ORNT1 Alleles Implicated in HHH Syndrome in Humans

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