In Saccharomyces cerevisiae, the rapamycin-sensitive TOR signaling pathway plays an essential role in up-regulating translation initiation and cell cycle progression in response to nutrient availability. One of the mechanisms by which TOR regulates cell proliferation is by excluding the GLN3 transcriptional activator from the nucleus and, in consequence, preventing its transcriptional activation therein. We examined the possibility that the TOR cascade could also control the transcriptional activity of Gcn4p, which is known to respond to amino acid availability. The results presented in this paper indicate that GCN4 plays a role in the rapamycinsensitive signaling pathway, regulating the expression of genes involved in the utilization of poor nitrogen sources, a previously unrecognized role for Gcn4p, and that the TOR pathway controls GCN4 activity by regulating the translation of GCN4 mRNA. This constitutes an additional TOR-dependent mechanism which modulates the action of transcriptional activators.

It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP ؉ -glutamate dehydrogenase (NADP ؉ -GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP ؉ -GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis.Two pathways for ammonium assimilation and glutamate biosynthesis have been found in a variety of organisms. The first one, described by Holzer and Schneider in 1957 (12), is mediated by NADP ϩ -glutamate dehydrogenase (NADP ϩ -GDH; EC 1.4.1.4), which catalyzes the reductive amination of 2-oxoglutarate to form glutamate. In an alternative pathway demonstrated by Tempest et al. (25), glutamate is aminated to form glutamine by glutamine synthetase (GS; EC 6.3.1.2), the amide group of which is then transferred reductively to 2-oxoglutarate by glutamate synthase (GOGAT; EC 1.4.1.13), resulting in the net conversion of ammonium and 2-oxoglutarate to glutamate. The GS-GOGAT pathway has been found in several microorganisms (2,13,16,23) and in higher plants (18).In Saccharomyces cerevisiae, both pathways for glutamate biosynthesis are present (7,19). Mutants altered in NADP ϩ -GDH have been isolated (6); these show a higher doubling time than that of the wild type when both strains are grown on minimal medium supplemented with ammonia as the sole nitrogen source. Mutants impaired in GOGAT activity were selected from NADP ϩ -GDH-less mutants as glutamate auxotrophs (7,19). Genetic analysis of one of these mutants showed that the lack of GOGAT activity was due to the presence of two mutations (gus1 and gus2), which suggested the existence of two GOGAT enzymes in S. cerevisiae (7). Cloning of the GOGAT structural gene (GLT1) and construction of null GOGAT mutants definitively established that this yeast possesses a single NADH-GOGAT enzyme (4) and that GOGATless mutants (7) which cannot be complemented with GLT1 (unpublished results) are probably impaired in GLT1 regulation. In this paper we report the characterization of strains impaired in either GDH1, GLT1, or both. Our results show that there is a third pathway for glutamate biosynthesis, mediated by an N...

In the yeast Saccharomyces cerevisiae, two NADP؉ -dependent glutamate dehydrogenases (NADP-GDHs) encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and ␣-ketoglutarate. The GDH2-encoded NAD ؉ -dependent glutamate dehydrogenase degrades glutamate producing ammonium and ␣-ketoglutarate. Until very recently, it was considered that only one biosynthetic NADP-GDH was present in S. cerevisiae. This fact hindered understanding the physiological role of each isoenzyme and the mechanisms involved in ␣-ketoglutarate channeling for glutamate biosynthesis. In this study, we purified and characterized the GDH1-and GDH3-encoded NADP-GDHs; they showed different allosteric properties and rates of ␣-ketoglutarate utilization. Analysis of the relative levels of these proteins revealed that the expression of GDH1 and GDH3 is differentially regulated and depends on the nature of the carbon source. Moreover, the physiological study of mutants lacking or overexpressing GDH1 or GDH3 suggested that these genes play nonredundant physiological roles. Our results indicate that the coordinated regulation of GDH1-, GDH3-, and GDH2-encoded enzymes results in glutamate biosynthesis and balanced utilization of ␣-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed.

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