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

Abstract

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....

Characterization of Human and Yeast Mitochondrial Glycine Carriers with Implications for Heme Biosynthesis and Anemia

“…Furthermore, glycine uptake was strongly reduced in reconstituted hem25⌬ mitochondrial extract (0.95 Ϯ 0.06 nmol/15 min ϫ mg of protein) compared with wild-type (16.83 Ϯ 1.50 nmol/15 min ϫ mg of protein). This finding is not unrealistic, because many mitochondrial carriers display some overlap of substrate specificity, albeit with lower efficiency, and play different metabolic roles (38). Recently, it has been proposed that Ymc1p is a secondary mitochondrial glycine transporter, because the hem25⌬ymc1⌬ double mutant presented a lower level of heme content compared with the single hem25⌬ or ymc1⌬ mutant (14); however, data in the literature pointed out a role for YMC1 in carbon source utilization (39,40).…”

Citrate – new functions for an old metabolite

“…In the cytosol, citrate can be converted to isocitrate by cytosolic aconitase. Isocitrate is then transformed to α-KG by a cytosolic NADP-dependent isocitrate dehydrogenase and can be cycled back to pyruvate by mitochondrial and cytosolic pathways (Palmieri, 2004;Castegna et al, 2010). Inhibition of CIC activity by BTA in INS-1-derived 832/13 cells or primary rat islets and suppression of CIC expression via a recombinant adenovirus significantly inhibit GSIS in 832/13, whereas overexpression of CIC enhances GSIS and increases cytosolic citrate levels (Ronnebaum et al, 2006).…”

Saccharomyces cerevisiae Porin Pore Forms Complexes with Mitochondrial Outer Membrane Proteins Om14p and Om45p

“…Sfc1p, also known as Acr1p, is responsible for import of cytosolic succinate and export of fumarate (37). The antiporter Yhm2p (Coc1p) transports citrate out of and oxoglutarate into mt (7). A further detected IM protein, Phb2p, is a subunit of the conserved 1.0 -1.4-MDa prohibitin complex.…”

“…The genome of S. cerevisiae encodes for 34 putative members of MCF. Up to now, the transported molecules for 26 of those are known or are assumed (6,7).…”

“…However, expression of OM14 and OM45 is tightly co-regulated and increased after diauxic shift (14), suggesting a role in the transition to respiratory growth. Om45p has a molecular mass of 45-kDa and is anchored within the mt OM by an ␣-helical transmembrane domain at the N terminus (amino acids [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. Available data on the orientation of the protein in the membrane are conflicting (11-13, 15, 16).…”