Abstract
Amino acid (AA) detection is fundamental for cellular function, balancing translation demands, biochemical pathways, and signaling networks. Although the GCN2 and mTORC1 pathways are known to regulate AA sensing, the global cellular response to AA deprivation remains poorly understood, particularly in non-transformed cells, which may exhibit distinct adaptive strategies compared with cancer cells. Here, we employed murine pluripotent embryonic stem (ES) cells as a model system to dissect responses to AA stress. Using multi-omics analyses over an extended time course, we examined the effects of arginine (Arg) and leucine (Leu) deprivation. We uncovered a broad array of proteomic, phosphoproteomic, transcriptomic, and metabolomic adaptations, including an increase in lysosome production, all occurring without lethality. We found that Arg or Leu starvation induces reversible cell cycle exit, promoting a quiescent state that enhances resistance to cytotoxic stressors. In contrast, cysteine (Cys) and threonine (Thr) deprivation led to cell death via distinct pathways: ferroptosis for Cys starvation, whereas Thr deprivation triggered a previously uncharacterized form of cell death, which could be entirely suppressed by methionine (Met) co-starvation, and mTOR or translational inhibition. These findings suggest that ES cells implement specialized survival strategies in response to different AA limitations, highlighting their ability to reprogram cellular biochemistry under nutrient stress.