Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein

Palmieri, Luigi; Palmieri, Ferdinando; Runswick, Michael J.; Walker, John E.

doi:10.1016/s0014-5793(96)01350-6

Cited by 109 publications

(118 citation statements)

References 39 publications

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“…In yeast, the existence and importance of the malate-aspartate shuttle has been debatable until the transporters required for the malate-aspartate shuttle to operate were identified (Palmieri et al 1996(Palmieri et al , 1999(Palmieri et al , 2001Cavero et al 2003). Here our data showed that the malate-aspartate shuttle played a major role in mediating life span extension under CR as well as in Multiple CR pathways act to affect longevity in yeast.…”

Section: Discussionmentioning

confidence: 72%

The malate–aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast

Easlon¹,

Tsang²,

Skinner³

et al. 2008

Genes Dev.

138

118

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Recent studies suggest that increased mitochondrial metabolism and the concomitant decrease in NADH levels mediate calorie restriction (CR)-induced life span extension. The mitochondrial inner membrane is impermeable to NAD (nicotinamide adenine dinucleotide, oxidized form) and NADH, and it is unclear how CR relays increased mitochondrial metabolism to multiple cellular pathways that reside in spatially distinct compartments. Here we show that the mitochondrial components of the malate-aspartate NADH shuttle (Mdh1 [malate dehydrogenase] and Aat1 [aspartate amino transferase]) and the glycerol-3-phosphate shuttle (Gut2, glycerol-3-phosphate dehydrogenase) are novel longevity factors in the CR pathway in yeast. Overexpressing Mdh1, Aat1, and Gut2 extend life span and do not synergize with CR. Mdh1 and Aat1 overexpressions require both respiration and the Sir2 family to extend life span. The mdh1⌬aat1⌬ double mutation blocks CR-mediated life span extension and also prevents the characteristic decrease in the NADH levels in the cytosolic/nuclear pool, suggesting that the malate-aspartate shuttle plays a major role in the activation of the downstream targets of CR such as Sir2. Overexpression of the NADH shuttles may also extend life span by increasing the metabolic fitness of the cells. Together, these data suggest that CR may extend life span and ameliorate age-associated metabolic diseases by activating components of the NADH shuttles.[Keywords: Calorie restriction (CR); Sir2; aging; NADH shuttles; respiration; metabolism] Supplemental material is available at http://www.genesdev.org.

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Section: Discussionmentioning

confidence: 72%

The malate–aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast

Easlon¹,

Tsang²,

Skinner³

et al. 2008

Genes Dev.

138

118

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“…In addition, Yhm2p shares only 21 and 22% identical amino acids with Ctp1p and CTP, respectively. Yhm2p also differs from the dicarboxylate (38,39) and oxaloacetate (16,40) 1 from Plasmodium falciparum (31%) and XP_001238657.1 from Eimeria tenella (31%)), Euglenozoa (e.g. XP_811279.1 from Trypanosoma cruzi (32%)), and Stramenopiles (e.g.…”

Section: Discussionmentioning

confidence: 99%

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

Castegna

Scarcia

Agrimi

et al. 2010

Journal of Biological Chemistry

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117

103

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Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydrylblocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP ؉ and GSH/GSSG ratios in the cytosol of ⌬YHM2 cells as well as an increase in the NADPH/ NADP ؉ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ⌬YHM2 strain and more so by the ⌬YHM2⌬ZWF1 strain upon H 2 O 2 exposure, implying that Yhm2p has an antioxidant function.Yhm2p is a protein of unknown function that has been found to be present in the inner mitochondrial membrane of Saccharomyces cerevisiae (1). This protein was shown to complement the growth defect displayed at 37°C by S. cerevisiae cells lacking ABF2, which encodes a mitochondrial DNA-binding protein involved in mitochondrial DNA replication and recombination (1, 2). In addition, the primary structure of Yhm2p exhibits all the characteristic features of the mitochondrial carrier (MC) 3 protein family, i.e. a tripartite structure consisting of three tandemly repeated homologous domains of about 100 amino acids in length, each of which contains a distinct signature motif and two hydrophobic stretches that span the membrane as ␣-helices (for reviews see Refs. 3-5). S. cerevisiae possesses 35 members of this family, one of which is localized in peroxisomes and not in mitochondria. The majority of yeast MCs has been functionally characterized and shown to transport specific metabolites, nucleotides, and coenzymes across the mitochondrial membrane (for review see Ref. 6). By contrast, the substrate(s) transported by Yhm2p have not yet been discovered, and its physiological function is yet unknown.In this study, we provide evidence that the gene product of YMR241w, named Coc1p and known as Yhm2p, is a citrateoxoglutarate carrier in S. cerevisiae. Yhm2p was overexpressed in S....

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“…Inclusion bodies were purified on a sucrose density gradient (15) and washed at 4°C first with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0), then twice with a buffer containing Triton X-114 (3%, w/v), 1 mM EDTA, and 10 mM PIPES, pH 7.0, and once again with TE buffer. Proteins were solubilized in sarkosyl (w/v), as described (17,18). Small residues were removed by centrifugation (258,000 ϫ g for 20 min at 4°C).…”

Section: Methodsmentioning

confidence: 99%

α-Isopropylmalate, a Leucine Biosynthesis Intermediate in Yeast, Is Transported by the Mitochondrial Oxalacetate Carrier

Marobbio

Giannuzzi

Paradies

et al. 2008

Journal of Biological Chemistry

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In Saccharomyces cerevisiae, ␣-isopropylmalate (␣-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported ␣-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di-and tricarboxylate transporters as well as the previously proposed ␣-IPM transporter. Transport was saturable with a half-saturation constant of 75 ؎ 4 M for ␣-IPM and 0.31 ؎ 0.04 mM for ␤-IPM and was inhibited by the substrates of Oac1p. Though not transported, ␣-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, ␣-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an ␣-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with ␣-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant ⌬OAC1⌬LEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of ⌬OAC1⌬LEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates ␣-ketoisocaproate and ␣-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of ␣-IPM, probably in exchange for oxalacetate.

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Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein

Cited by 109 publications

References 39 publications

The malate–aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast

The malate–aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

α-Isopropylmalate, a Leucine Biosynthesis Intermediate in Yeast, Is Transported by the Mitochondrial Oxalacetate Carrier

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