Multilayered regulation of TORC1 signaling by Ait1, Gcn2, and SEAC/GATOR during nitrogen limitation and starvation

The Target of Rapamycin kinase Complex I (TORC1) is a central hub in the cell growth and metabolic control network of eukaryotes. How its upstream regulators cooperate to tune signaling across environmental conditions remains unclear. Here, we combine phosphoproteomics, TORC1 activity assays, and targeted genetic perturbations to dissect TORC1 regulation in Saccharomyces cerevisiae during transitions from a high-quality nitrogen source (glutamine) to a low-quality nitrogen source (proline), and on to complete nitrogen starvation. In proline medium, Ait1 and Gcn2 attenuate TORC1 activity, establishing a partially inhibited "Low Nitrogen Adaptive" state marked by extensive metabolic reprogramming without growth arrest. In contrast, during nitrogen starvation, SEAC, Ait1, and Gcn2 cooperate to drive TORC1 into a fully inhibited state, triggering widespread dephosphorylation of its downstream targets and entry into quiescence. Our results define a multilayered regulatory circuit that governs graded TORC1 control-a design likely conserved across eukaryotes.