A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.Glutamate biosynthesis can be achieved through the reductive amination of 2-oxoglutarate catalyzed by NADP+-dependent glutamate dehydrogenase (NADP+-GDH) (2). In 1970, Tempest et al. (24) demonstrated the existence of an alternative pathway for glutamate biosynthesis in Klebsiella aerogenes. This pathway comprised glutamate synthase (GOGAT) and glutamine synthetase. The function of this pathway has been demonstrated in several microorganisms (3,12,23) and in higher plants (18). Physiological studies indicate that the main role of the glutamine synthetase-GOGAT pathway is in ammonium assimilation and glutamate biosynthesis under ammonium-limited conditions (23). In Neurospora crassa, GOGAT also appears to play an important role in glutamine degradation (4). N. crassa (21), Salmonella typhimurium (16), and Escherichia coli (5, 19) mutants lacking GOGAT activity have been obtained, and their characterization has contributed to the understanding of the role of GOGAT in glutamate biosynthesis.Although GOGAT activity has been previously detected in Saccharomyces cerevisiae (22), the role of GOGAT in glutamate biosynthesis and its regulation are poorly understood. Mutants altered in NADP+-GDH have been isolated from S. cerevisiae (7). The isolation of GOGAT-less mutants has been reported elsewhere (26); however, their genetic and physiological characterization has not been published, so the physiological function of GOGAT still remains obscure. Our results indicate that GOGAT plays a role in glutamate biosynthesis under conditions of ammonium excess and that under ammonium-limited conditions, both NADP+-GDH and GOGAT can participate in the synthesis of glutamate. We also present evidence suggesting that S. cerevisiae has two GOGAT activities.MATERIALS AND METHODS Strains. Table 1 describes the characteristics of the different strains used in this study.Growth conditions. Strains were routinely grown on minimal medium (MM) containing salts, trace elements, and vitamins following the formula of yeast nitrogen base (Difco * Corresponding author.Laboratories, Detroit, Mich.). Glucose (2% [wt/vol]) was used as the carbon source, and 40 mM (NH4)2SO4 was used as the nitrogen source. Amino acids needed to satisfy auxotrophic requirements were added at 0.01% (wt/vol). Cells wer...

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The apocytochrome b genes from two strains of the yeast Kluyveromyces lactis, have been isolated and sequenced. The coding sequences in strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140) were identical but the upstream noncoding regions were slightly different. The sequences demonstrated the presence of a continuous open reading frame with no introns. The amino acid sequence, derived from the coding strand, showed 82% homology to the apocytochrome b of Saccharomyces cerevisiae strain D273-10B and only 58% homology to the protein from Schizosaccharomyces pombe strain 50. CUN and CGN codon families were absent from the K. lactis gene. Codon usage was very similar to that of other mitochondrial genomes with mostly U or A in the third position. There were two unusual features. All threonines were coded by ACA(U) and all arginines by AGA.