Fig. 1: Cells exhibit hierarchical responses to supply-disruptions of amino acid groups notably the glutamate-derived group.

A Amino acid supply sources, and demand requirements. Supply comes from de novo biosynthesis and uptake from the environment. Demand includes the use of amino acids for translation and metabolism. B A metabolic source and chemical-structure based grouping of amino acids: (i) PPP, aromatic (ii) sulfur containing (iii) Glycolytic/TCA, branched chain amino acids (BCAA) (iv) TCA, polar (v) Glycolytic, uncharged (vi) glutamate derived and (vii) PPP, polar, basic. The locations in the carbon network from which each amino acid is derived are highlighted. See Supplementary Fig. S2 for complete pathways. C Single-step transamination of α-ketoglutarate to glutamate (which forms glutamine). Glutamate and glutamine function as a nitrogen source for all amino acids. D How amino acid supply-demand mismatches in a batch culture experiment are sensed, managed and restored by Gcn4. E Temporal changes in Gcn4-luciferase reporter activity, estimating the restoration response to transient amino acid supply disruption. Upper panel: Gcn4-luciferase reporter. Data from n = 3 (biological replicates) displayed as mean ± SEM. Left to right p = 0.0324, 0.0322, 0.0015, 0.0043, <0.0001, 0.0072, 0.0087, 0.0284. F Relative Gcn4-luciferase reporter activity in the indicated dropout medium (at 75 min), compared to all amino acid dropout (-AA). Data displayed as means ± SEM, n ≥ 4 biological replicates. For E and F ****p < 0.0001, ns denotes non-significant difference, two-tailed student’s t-test. G Summary of relative levels of stabilized Gcn4 (sGcn4), in dropout medium (as in panel F) at 75 min (also see Supplementary Fig. S1B). H Native Gcn4 levels (detected using anti-HA antibody) in the indicated dropout medium, in conditions identical to panels (F and G). Image represents n = 3 biological replicates. I Summarized consolidated Gcn4 responses to the supply disruption of each amino acid group. This integrates results from the Gcn4-luciferase reporter, sGcn4, native Gcn4 protein, and Gcn4 dependant transcripts (see Supplementary Fig. S1D). The circles correspond to the intensity of the response. J Heat map showing relative cell growth for wild-type and gcn4∆ cells in the indicated dropout, after 6 h. Mean for n = 2, biological replicates. Data are provided in Source Data file.