Three mitochondrial transporters of Saccharomyces cerevisiae are essential for ammonium fixation and lysine biosynthesis in synthetic minimal medium

摘要

Abstract The nuclear genes of Saccharomyces cerevisiae YHM2 , ODC1 and ODC2 encode three transporters that are localized in the inner mitochondrial membrane. In this study, the roles of YHM2 , ODC1 and ODC2 in the assimilation of nitrogen and in the biosynthesis of lysine have been investigated. Both the odc1 Δ odc2 Δ double knockout and the yhm2 Δ mutant grew similarly as the YPH499 wild-type strain on synthetic minimal medium (SM) containing 2% glucose and ammonia as the main nitrogen source. In contrast, the yhm2 Δ odc1 Δ odc2 Δ 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. Furthermore, the lack of growth of yhm2 Δ odc1 Δ odc2 Δ on 2% glucose SM was rescued by the addition of glutamate, but not glutamine, to the medium. Using lysine-prototroph YPH499-derived strains, the yhm2 Δ odc1 Δ odc2 Δ knockout (but not the odc1 Δ odc2 Δ and yhm2 Δ mutants) also displayed a growth defect in lysine biosynthesis on 2% glucose SM, which was rescued by the addition of lysine and, to a lesser extent, by the addition of 2-aminoadipate. Additional analysis of the triple mutant showed that it is not respiratory-deficient and does not display mitochondrial DNA instability. These results provide evidence that only the simultaneous absence of YHM2 , ODC1 and ODC2 impairs the export from the mitochondrial matrix of i) 2-oxoglutarate which is necessary for the synthesis of glutamate and ammonium fixation in the cytosol and ii) 2-oxoadipate which is required for lysine biosynthesis in the cytosol. Finally, the data presented allow one to suggest that the yhm2 Δ odc1 Δ odc2 Δ triple knockout is suitable in complementation studies aimed at assessing the pathogenic potential of human SLC25A21 (ODC) mutations. Highlights ? Investigating the role of mitochondrial carriers in vivo is crucial for metabolism and pathophysiology ? Simultaneous deletion of S. cerevisiae YHM2 , ODC1 and ODC2 is required to impair ammonium fixation on SM medium ? Similarly YHM2 , ODC1 and ODC2 exhibit redundancy in lysine biosynthesis ? The triple knockout yhm2 Δ odc1 Δ odc2 Δ is not respiratory-deficient and is not involved in mtDNA stability ? Complementation of yhm2 Δ odc1 Δ odc2 Δ cells is suitable for assessing the pathogenic potential of SLC25A21 (ODC) mutations