Cellular metabolism can be viewed as an economic system, managing metabolite supply and demand. While global metabolic programs regulate growth in response to nutrient environments, how cells prioritize restoration of specific metabolic resources after transient supply disruptions remains poorly understood. Amino acids are central to this cellular economy, essential for protein synthesis and metabolism. Cells possess mechanisms to sense amino acid sufficiency and restore supply (e.g., TORC1, Gcn4/ATF4). However, prioritization strategies for restoring different amino acids after supply disruptions are unclear. Most studies use complete amino acid starvation, failing to address the distinct metabolic origins, synthesis routes, chemical properties, and intracellular concentrations of individual amino acids. This study uses yeast to systematically investigate these prioritization strategies by transiently disrupting the supply of amino acid groups and assessing the restoration responses using Gcn4 activity as a reporter of supply-demand mismatches.

Existing literature highlights the role of global metabolic programs in regulating cell growth under varying nutrient conditions. However, there's limited understanding of how cells prioritize restoring individual metabolic resources after transient supply disruptions. The role of amino acids in cellular metabolism is well-established, but the relative contributions of protein synthesis versus metabolic processes are not well-quantified. Studies on amino acid sensing and restoration mechanisms often focus on complete starvation, overlooking the unique properties of individual amino acids. The Gcn4/ATF4 transcription factor is known as a key regulator of amino acid biosynthesis, functioning during starvation to restore amino acid supply, but its role in differential restoration of distinct amino acids is not well-explored.

Prototrophic yeast cells were used in a defined glucose and nitrogen-replete environment. Amino acids were grouped based on metabolic origins and chemical properties. Transient supply disruptions were achieved by removing each group from the medium while supplementing the others. Gcn4-luciferase reporter activity was used to assess supply-demand mismatches. The unit costs of biosynthesis for each amino acid were estimated by calculating the high-energy phosphate bonds (ATP) and reducing equivalents (NAD(P)H) required. Total demand was estimated from ribosome profiling data for protein synthesis and qualitative estimates for metabolic processes. The effects of individual amino acid dropouts on short-term growth were also analyzed using Gcn4-deficient cells. Finally, experiments using *arg1Δ* cells examined the contribution of metabolic demand (specifically polyamine synthesis) to the arginine response. TORC1 activity was assessed via Sch9 phosphorylation and ribosomal transcript levels.

The study revealed a hierarchical prioritization of amino acid restoration responses. The strongest response was to the disruption of glutamate-derived amino acids (Arg, Pro, Lys), followed by sulfur-containing, glycolytic/TCA, BCAA, and PPP, aromatic amino acids. The biosynthetic cost analysis showed that glutamate-derived amino acids had the lowest supply costs. The strongest Gcn4 response correlated with low supply costs and high total demand. Arginine, within the glutamate-derived group, showed the lowest individual supply cost and the highest demand, primarily due to its role in polyamine synthesis. Disrupting arginine supply elicited the strongest restoration response, consistent with the law of demand (inverse correlation between price and quantity demanded). TORC1 activity also showed the greatest reduction upon arginine limitation, complementing the Gcn4 response. Experiments with *arg1Δ* cells indicated that approximately half of arginine demand is attributed to metabolic requirements (polyamine synthesis).

The findings support a demand-driven framework for amino acid allocation in cells. Amino acids with low supply costs and high demand elicit the strongest restoration responses. This framework helps predict prioritization responses under different nutrient conditions and estimate amino acid reserve requirements. Treating amino acids as distinct entities rather than homogenous goods based on their metabolic origins and demand reveals potential roles for amino acid-specific sensing and restoration machinery. Although readily applicable to autonomous cells, extending this model to complex systems like tissues and organs requires addressing multiple supply sources and estimating demand elasticity.

This study demonstrates that amino acid restoration prioritization in yeast cells follows a demand-driven economic principle. High demand coupled with low supply costs drives the restoration response, exemplified by arginine. This framework provides insights into cellular resource allocation and has implications for metabolic engineering. Future research should focus on predicting responses to changing nutrient environments, estimating amino acid reserves, and identifying novel amino acid-specific sensing mechanisms.

The study primarily focuses on yeast cells in a defined nutrient environment. Extending these findings to other organisms or more complex conditions requires further investigation. The demand estimations involve some qualitative aspects, particularly for metabolic demand, due to limitations in available data. The model might not be directly generalizable to multicellular organisms with more complex regulatory networks.