In Saccharomyces cerevisiae grown in synthetic minimal medium supplemented with non-fermentable carbon sources glutamate is synthesized within mitochondria

Abstract

In Saccharomyces cerevisiae the export of 2-oxoglutarate from the mitochondria, catalyzed by Yhm2p, Odc1p and Odc2p or by at least one of these transporters, has recently been shown to be essential for glutamate biosynthesis in glucose-supplemented minimal synthetic (SM) medium without glutamate, because the triple mutant yhm2∆odc1∆odc2∆ displays a growth defect under these conditions. Surprisingly, in this study it was found that yhm2∆odc1∆odc2∆ cells grow like wild-type (WT) cells in the same medium supplemented with non-fermentable carbon sources. Direct transport assays of 2-oxoglutarate/2-oxoglutarate homoexchange activity in mitochondria from WT and yhm2∆odc1∆odc2∆ cells (solubilized and reconstituted into liposomes) showed that the mitochondrial extract from yhm2∆odc1∆odc2∆ was completely inactive at variance with that from WT cells, showing that S. cerevisiae mitochondria do not contain additional proteins capable of catalyzing 2-oxoglutarate transport efficiently besides Yhm2p, Odc1p and Odc2p. Furthermore, quantitative real-time PCR experiments showed that in both WT and yhm2∆odc1∆odc2∆ cells the expression of GDH1 is low on lactate and high on glucose and, vice versa, the expression of GDH3 is high on lactate and low on glucose. These results may be interpreted to indicate that in S. cerevisiae, grown in glucose-supplemented SM medium, glutamate is synthesized by Gdh1p in the cytosol, whereas in lactate-supplemented SM medium glutamate is synthesized by Gdh3p in the mitochondria; therefore, the pathway of ammonia assimilation under fermentative conditions requires export of 2-oxoglutarate from the mitochondria, whereas the alternative pathway under respiratory conditions does not.