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 sulfhydryl-blocking 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 DeltaYHM2 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 DeltaYHM2 strain and more so by the DeltaYHM2DeltaZWF1 strain upon H(2)O(2) exposure, implying that Yhm2p has an antioxidant function.

Highlights

  • Ricerca, the Center of Excellence in Genomics, Apulia Region, the University of Bari, and the Italian Human ProteomeNet No RBRN07BMCT_009

  • A strong immunoreactive band was detected in mitochondria isolated from YHM2-pYES2 cells and a faint band in mitochondria from wild-type cells, and no reaction was observed in mitochondria from the ⌬YHM2 mutant

  • Overexpression in S. cerevisiae of a previously unidentified mitochondrial carrier (MC) and reconstitution of the recombinant protein in liposomes as well as phenotype analysis of yeast knock-out cells have been employed to investigate the function of Yhm2p

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Summary

Introduction

The Center of Excellence in Genomics, Apulia Region, the University of Bari, and the Italian Human ProteomeNet No RBRN07BMCT_009. Yhm2p was overexpressed in S. cerevisiae, purified, reconstituted into phospholipid vesicles, and identified for its transport properties as a novel carrier for citrate and oxoglutarate. Besides these substrates, oxaloacetate, succinate, and fumarate are transported, to a lesser extent, by the carrier via a counter-exchange mechanism. ⌬YHM2 cells (devoid of YHM2) and more so ⌬YHM2⌬ZWF1 cells (lacking YHM2 and ZWF1 which encodes glucose-6-phosphate dehydrogenase, an important source of NADPH) exhibit a growth defect upon exposure to H2O2 This phenotype correlates with a decrease in the cytosolic NADPH/NADPϩ and GSH/GSSG ratios. NADPH citrate-oxoglutarate redox shuttle that catalyzes a net transfer of NADPH reducing equivalents from the mitochondria to the cytosol

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