Glutamine synthesis is a regulatory signal controlling glucose catabolism in Saccharomyces cerevisiae

Abstract

The effect of glutamine biosynthesis and degradation on glucose catabolism in Saccharomyces cerevisiae was studied. A wild-type strain and mutants altered in glutamine biosynthesis and degradation were analyzed. Cells having low levels of glutamine synthetase activity showed high ATP/ADP ratios and a diminished rate of glucose metabolism. It is proposed that glutamine biosynthesis plays a role in the regulation of glucose catabolism.The biosynthesis of glutamine proceeds through the action of glutamine synthetase (GS), and in this reaction ATP is consumed. In Neurospora crassa, decreased GS activity results in ATP accumulation (2, 12), and a functional cycle of glutamine synthesis and degradation, dissipating energy, has been reported (2, 12). Glucose catabolism in Bacillus subtilis is diminished in GS mutants (5), and in tumor cells glutamine reduces glycolytic flow (10) and can be a preferred carbon source (13). This report addresses such matters for Saccharomyces cerevisiae.In S. cerevisiae, glutamine is known to decrease GS activity (8,11). Table 1 shows that when the wild-type strain S288C (AATot mal gai2) was grown on glucose with glutamine as the sole nitrogen source, the ATP/ADP ratio was 2.5 times higher than when ammonium sulfate was used as nitrogen source, and in the former situation GS activity was severalfold lower. The ATP/ADP ratio found when the wild type was grown on ammonium is similar to that previously reported by Entian and Zimmermann (3). In mutant CN1 (MA4Ta inol-13 ino4-8 canl gln), which is impaired in GS activity (6), the ATP/ADP ratio was even higher than in the wild type grown with glutamine, while in a strain with the GS structural gene on a multicopy plasmid, CN6/pGDC1 (MA4Ta Iys2 leu2 gln), that shows high GS activity even when grown with glutamine (6), the ATP/ ADP ratio on glutamine or ammonium was similar to that found when the wild type was grown on ammonium. These results show that GS activity correlates with a low ATP/ADP ratio.For determination of levels of ATP, ADP, and GS, cells were grown at 30°C with agitation (250 rpm) to an optical density of 0.7, with the indicated nitrogen sources, on minimal medium containing the same salts, trace elements, and vitamins used in yeast nitrogen base medium (Difco Laboratories, Detroit, Mich.). Glucose (2% [wt/vol]) was used as the carbon source. Amino acids needed to satisfy auxotrophic requirements were added at 0.01% (wt/vol). Cells were collected by filtration with 1.2-,um-pore-size Millipore membranes. ATP and ADP were extracted by disintegrating cells in 6% perchloric acid with glass beads as previously described (6). ATP and ADP levels were determined as previously described (1, 7). For GS determination, soluble extracts were prepared by suspending whole cells in 5 mM K2HPO4 buffer containing 0.5 mM * Corresponding author.EDTA and 50 mM K2HPO4 (pH 7.2) and grinding them with glass beads in a Vortex mixer. Transferase activity was measured as previously described (4). Protein was measured by the method of Lowry et al. (9), with ...

A Sensing Role of the Glutamine Synthetase in the Nitrogen Regulation Network in Fusarium fujikuroi

“…The growth defect of the wild type under these conditions was attributed to the highly active ATP-consuming glutamine/glutamate cycling (GS-GOGAT) [ 91 ] contributing to an ATP-drain that would drive the ATP-producing glycolysis in the presence of glucose [ 90 , 92 ]. Similar observations have been reported in S. cervisiae [ 93 ]. The data obtained in this work make a comparable mechanism in F. fujikuroi highly conceivable.…”

The glutamate synthase (GOGAT) of plays an important role in central nitrogen metabolism

“…The concerted action of GOGAT and NAD-GDH could then cycle glutamate while producing NADH in the cytosol, maintaining a correct redox balance. A similar cycling reaction (GS and GOGAT) in the CNM has also been reported for controlling the ATP/ADP ratio [33]. This shuttle mechanism is similar to other shuttles between cytosol and mitochondria, such as the glycerol 3-phosphate shuttle [34] and the glutamatemalate shuttle.…”

The glutamate synthase (GOGAT) ofSaccharomyces cerevisiaeplays an important role in central nitrogen metabolism

“…The concerted action of GOGAT and NAD‐GDH could then cycle glutamate while producing NADH in the cytosol, maintaining a correct redox balance. A similar cycling reaction (GS and GOGAT) in the CNM has also been reported for controlling the ATP/ADP ratio [33]. This shuttle mechanism is similar to other shuttles between cytosol and mitochondria, such as the glycerol 3‐phosphate shuttle [34] and the glutamate‐malate shuttle.…”