A tRNA modification balances carbon and nitrogen metabolism by regulating phosphate homeostasis, to couple metabolism to cell cycle progression.

Abstract

10Cells must appropriately sense and integrate multiple metabolic resources to commit to 11 proliferation. Here, we report that cells regulate carbon and nitrogen metabolic homeostasis 12 through tRNA U 34 --thiolation. Despite amino acid sufficiency, tRNA--thiolation deficient cells appear 13 amino acid starved. In these cells, carbon flux towards nucleotide synthesis decreases, and trehalose 14 synthesis increases, resulting in a starvation--like metabolic signature. Thiolation mutants have only 15 minor translation defects. However, these cells exhibit strongly decreased expression of phosphate 16 homeostasis genes, resulting in an effectively phosphate--limited state. Reduced phosphate enforces 17 a metabolic switch, where glucose--6--phosphate is routed towards storage carbohydrates. Notably, 18 trehalose synthesis, which releases phosphate and thereby restores phosphate availability, is central 19 to this metabolic rewiring. Thus, cells use thiolated tRNAs to perceive amino acid sufficiency, and 20 balance carbon and amino acid metabolic flux to maintain metabolic homeostasis, by controlling 21 phosphate availability. These results further biochemically explain how phosphate availability 22 determines a switch to a 'starvation--state'. diverted away from the pentose--phosphate pathway/nucleotide synthesis axis, and towards storage 75 carbohydrates trehalose and glycogen. This thereby alters cellular commitments towards growth 76 and cell cycle progression. Counter--intuitively, we discover that this metabolic--state switch in cells 77 lacking tRNA thiolation is achieved by down--regulating a distant metabolic arm of phosphate 78 homeostasis. We biochemically elucidate how regulating phosphate balance can couple amino acid 79 and carbon utilization towards or away from nucleotide synthesis, and identify trehalose synthesis 80 as the pivotal control point for this metabolic switch. Through these findings we show how tRNA 81 thiol--modifications couple amino acid sensing with overall metabolic homeostasis. We further 82 present a general biochemical explanation for how inorganic phosphate homeostasis regulates 83 commitments to different arms of carbon and nitrogen metabolism, thereby determining how cells 84 commit to a 'growth' or 'starvation' state. 85 86 87 Results 88 89 Amino acid and nucleotide metabolism are decoupled in tRNA thiolation deficient cells 90Earlier studies observed an increased expression of amino acid biosynthetic genes, and an 91 activation of the amino acid starvation responsive transcription factor Gcn4, in cells lacking tRNA 92 thiolation (Laxman et al., 2013; Zinshteyn and Gilbert, 2013; Nedialkova and Leidel, 2015). These 93 studies therefore suggested that tRNA thiolation--deficient cells were amino--acid starved. We 94 investigated this surmise, by directly measuring free intracellular amino acids in wild--type (WT) and 95 tRNA thiolation mutant cells (uba4Δ and ncs2Δ). Note: these two independent pathway mutants 96 were chosen, to avoid misinterpretations coming from possible rol...