The nuclear genes of Saccharomyces cerevisiae YHM2, ODC1 and ODC2 encode three mitochondrial 2-oxoglutarate transporters. Mitochondrial extract from yhm2∆odc1∆odc2∆ cells reconstituted into liposomes showed that 2-oxoglutarate/2-oxoglutarate homo-exchange activity was completely inactive, showing that in S. cerevisiae Yhm2p, Odc1p and Odc2p are the sole 2-oxoglutarate transporters. Both the odc1Δodc2Δ double knockout and the yhm2Δ mutants showed a growth like that of the wild-type strain on a synthetic minimal medium (SM) containing 2% glucose and ammonia as the main nitrogen source. In contrast, the yhm2Δodc1Δodc2Δ triple knockout exhibited a marked growth defect under the same conditions. This defect was fully restored by the individual expression of YHM2, ODC1 or ODC2 in the triple deletion strain and by the addition of glutamate, but not glutamine, to the medium. Surprisingly, it was found that yhm2Δodc1Δodc2Δ cells grew like wild-type cells in the lactate-supplemented SM medium without glutamate. In S. cerevisiae, glutamate is synthesized by glutamate dehydrogenase isoforms (GDH). GDH1 is localized in the cytosol and expressed when cells grow on glucose, while GDH3 is localized in the mitochondria and expressed when cells grow on non-fermentable carbon sources. By quantitative real-time PCR experiments we showed that in both WT and yhm2Δodc1Δodc2Δ cells the expression of cytosolic GDH1 is low on lactate and high on glucose and, vice versa, the expression of mitochondrial GDH3 is high on lactate and low on glucose, suggesting that in lactate-supplemented SM medium glutamate biosynthesis is synthesized in the mitochondria by Gdh3p. In conclusion, the simultaneous absence of YHM2, ODC1 and ODC2 impairs the export from the mitochondrial matrix of 2-oxoglutarate required in the cytosol for glutamate when cells grew in glucose-supplemented medium.
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|Titolo:||Role of mitochondrial 2-oxoglutarate transporters in the assimilation of nitrogen in S. cerevisiae.|
PALMIERI, Luigi (Corresponding)
|Data di pubblicazione:||2018|
|Appare nelle tipologie:||4.3 Poster|