The Mitochondrial Ornithine Transporter

Fiermonte, Giuseppe; Dolce, Vincenza; David, Laura; Santorelli, Filippo M.; Dionisi‐Vici, Carlo; Palmieri, Ferdinando; Walker, John E.

doi:10.1074/jbc.m302317200

Cited by 118 publications

(51 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The mutation is at 183 BtAAC1 of contact point II and it changes the motif from R-E to R-K, which would disfavor ornithine from binding. An isoform of the ORNT (ORNT2) cannot replace fully the function of ORNT1 (22). It has the motif Q-E at contact point II, and so the amino acid group of ornithine would bind less well than to wild-type ORNT1, consistent with its reduced transport capability.…”

Section: Resultsmentioning

confidence: 99%

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

Robinson

Kunji

2006

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

237

289

View full text Add to dashboard Cite

Mitochondrial carriers link biochemical pathways in the cytosol and mitochondrial matrix by transporting substrates across the inner mitochondrial membrane. Substrate recognition is specific for each carrier, but sequence similarities suggest the carriers have similar structures and mechanisms of substrate translocation. By considering conservation of amino acids, distance and chemical constraints, and by modeling family members on the known structure of the ADP͞ATP translocase, we have identified a common substrate binding site. It explains substrate selectivity and proton coupling and provides a mechanistic link to carrier opening by substrate-induced perturbation of the salt bridges that seal the pathway to and from the mitochondrial matrix. It enables the substrate specificity of uncharacterized mitochondrial carriers to be predicted.comparative modeling ͉ membrane protein ͉ sequence alignment M embers of the mitochondrial carrier family are located in the inner mitochondrial membrane and allow exchange of substrates between the cytosol and the mitochondrial matrix (1). The family is exclusive to eukaryotes, and its members are unrelated to other transporter families, including the major facilitator superfamily and the ABC transporters. Their substrates include nucleotides, amino acids, cofactors, carboxylic acids, and inorganic anions required by the organelle for oxidative phosphorylation, gluconeogenesis, synthesis and degradation of amino and fatty acids, macromolecular synthesis of proteins and nucleic acids, sterol metabolism, and thermogenesis (1). Mutations of the carriers are associated with diseases, including progressive external opthalmoplegia, adult-and neonatal-onset type II citrullinaemia, Amish-type microcephaly, hyperornithinemia-hyperammonia-homocitrullinuria, carnitineacylcarnitine translocase deficiency, neonatal myoclonic epilepsy, severe obesity, and type II diabetes (1).The mitochondrial carriers have three tandem repeats of Ϸ100 aa, each containing two transmembrane ␣-helices linked by a large loop (2) and a conserved signature motif P-X-[DE]-X-X-[RK] (3). The carriers exist in two distinct conformational states: the cytoplasmic state (c-state) in which the carrier accepts its substrate from the cytoplasm, and the matrix state in which it accepts a substrate from the mitochondrial matrix. For the ADP͞ATP carrier, the inhibitor carboxyatractyloside (CATR) is known to bind to the carrier in the c-state only and prevent the binding of ADP. In the atomic model of the ADP͞ATP carrier 1 from Bos taurus (BtAAC1) in complex with CATR (4), the six-transmembrane ␣-helices form a barrel that has pseudo-3-fold symmetry. CATR is bound in the internal aqueous cavity (see Fig. 4, which is published as supporting information on the PNAS web site), which is open to the cytosol and closed to the mitochondrial matrix, consistent with the structure representing the c-state. The prolines of the signature motif kink the transmembrane helices, bringing their C-terminal ends together at the base of the cavit...

show abstract

Section: Resultsmentioning

confidence: 99%

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

Robinson

Kunji

2006

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

237

289

View full text Add to dashboard Cite

show abstract

“…For the closely related sequence of Ndt2p, which was also cloned and expressed in E. coli in this study, no biochemical data are available, because we were incapable of renaturing and/or reconstituting it functionally. However, because of the high degree of homology between Ndt1p and Ndt2p (70% homology and 55% identical amino acids), which is similar to that found for other mitochondrial carrier isoforms (33,39,45), it is likely that Ndt2p is an isoform of the NAD ϩ transporter in S. cerevisiae. The percentage of homology and identical amino acids is based on the sequence of Ndt1p without the first 54 residues of its long N-terminal extension.…”

Section: Discussionmentioning

confidence: 99%

Identification of the Mitochondrial NAD+ Transporter in Saccharomyces cerevisiae

Todisco

Agrimi

Castegna

et al. 2006

Journal of Biological Chemistry

Self Cite

203

218

View full text Add to dashboard Cite

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and cofactors across the inner mitochondrial membrane. In Saccharomyces cerevisiae, NAD ؉ is synthesized outside the mitochondria and must be imported across the permeability barrier of the inner mitochondrial membrane. However, no protein responsible for this transport activity has ever been isolated or identified. In this report, the identification and functional characterization of the mitochondrial NAD ؉ carrier protein (Ndt1p) is described. Mitochondria contain in their matrix the universal hydrogen transfer coenzyme NAD ϩ , which serves to transfer hydrogen from substrates to the respiratory chain by oxidative phosphorylation. In addition to its well known role as a coenzyme in redox reactions, NAD ϩ exerts other important functions in mitochondria. In Saccharomyces cerevisiae, mitochondrial NADH has been shown to participate in Fe/S protein biogenesis (1) and to be the source for NADPH (2), which is required in mitochondria for oxidative stress protection and for specific biogenesis reactions. NAD ϩ has been shown also to serve critical regulatory functions in gene transcription, enzyme activity, and other important processes through ADP-ribosylation and deacetylation reactions (3-5). In addition, the increase in the NAD ϩ /NADH ratio in mitochondria seems to be important for the extension of the life-span of yeast by calorie restriction (6). The enzymes of NAD ϩ biosynthesis are generally believed to be localized outside the mitochondria (Refs. 7-9, but see Refs. 10 and 11), therefore, NAD ϩ must be imported into these organelles. For a long time nicotinamide adenine dinucleotides were known to be unable to cross the inner membranes of mitochondria (12). However, NAD ϩ has been shown to be taken up by intact plant mitochondria, the uptake being concentration-and temperature-dependent and specifically inhibited by an azido derivative of NAD ϩ (13, 14). Moreover, using human cultured cells harvested under quiescent conditions (6 -8 days after medium change), Rustin et al. (15) observed a depletion of mitochondrial NAD ϩ and an influx of NAD ϩ into the mitochondrial matrix of these cells after adding external NAD ϩ to digitonin-permeabilized cells.These studies contradicted the notion of mitochondrial inner membrane impermeability to pyridine coenzymes and led to the hypothesis that NAD ϩ is transported into mitochondria by a carrier-mediated system. However, the one or more proteins responsible for the observed transport activities have not been hitherto isolated or identified. In this study we provide evidence that the gene products of YIL006W and YEL006W, named Ndt1p and Ndt2p, respectively, are two isoforms of the mitochondrial NAD ϩ transporter in S. cerevisiae. These proteins are 373 and 335 amino acids long, respectively, possess the characteristics of the MCF, 2 and display a high degree (70%) of homology. Ndt1p was overexpressed in Escherichia coli, purified, reconstituted in phospholipid vesicles,...

show abstract

“…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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

The Mitochondrial Ornithine Transporter

Cited by 118 publications

References 26 publications

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

Identification of the Mitochondrial NAD+ Transporter in Saccharomyces cerevisiae

Asymmetric dimethylarginine is transported by the mitochondrial carrier SLC25A2

Contact Info

Product

Resources

About