In Saccharomyces cerevisiae there are 35 putative transport proteins which belong to the mitochondrial carriers family. The identified members of this family shuttle metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. We have functionally defined and characterized the mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae and its product was purified and reconstituted into liposomes. Its transport properties and kinetic parameters showed that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. It also transported oxaloacetate, succinate and fumarate to a lesser extent, but not malate and isocitrate. Yhm2p catalyzed a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate. By mass spectrometry analysis we observed a decrease in the NADPH/NADP+ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/ NADP+ ratio in their mitochondria compared to wild-type cells. Probably, Yhm2p acts as a key component of a citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol, and its physiological role is to increase the NADPH reducing power in the cytosol. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H2O2 exposure, implying that Yhm2p has an antioxidant function.
Identification of a novel mitochondrial carrier involved in a citrate-oxoglutarate NADPH redox shuttle in Saccharomyces cerevisiae.
SCARCIA, PASQUALE;CASTEGNA, Alessandra;AGRIMI, GENNARO;Palmieri L.;Spera I.;
2011-01-01
Abstract
In Saccharomyces cerevisiae there are 35 putative transport proteins which belong to the mitochondrial carriers family. The identified members of this family shuttle metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. We have functionally defined and characterized the mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae and its product was purified and reconstituted into liposomes. Its transport properties and kinetic parameters showed that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. It also transported oxaloacetate, succinate and fumarate to a lesser extent, but not malate and isocitrate. Yhm2p catalyzed a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate. By mass spectrometry analysis we observed a decrease in the NADPH/NADP+ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/ NADP+ ratio in their mitochondria compared to wild-type cells. Probably, Yhm2p acts as a key component of a citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol, and its physiological role is to increase the NADPH reducing power in the cytosol. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H2O2 exposure, implying that Yhm2p has an antioxidant function.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
