Abstract
Amino acid homeostasis is essential for cellular functions such as growth, metabolism, and signaling. In budding yeast Saccharomyces cerevisiae, the General Amino Acid Control (GAAC) and Target of Rapamycin Complex 1 (TORC1) pathways are utilized for intracellular amino acid sensing, while the Ssy1-Ptr3-Ssy5 (SPS) pathway is used for extracellular sensing. These pathways maintain homeostasis by responding to variations in amino acid levels to regulate amino acid biosynthesis and uptake. However, their interactions under various conditions and behavior at single-cell resolution remain insufficiently understood. We developed fluorescent transcriptional reporters to monitor amino acid biosynthesis and uptake pathways in single cells, revealing pathway engagement in response to different amino acid levels and types. Inhibition experiments demonstrated that the SPS pathway influences TORC1 and GAAC activities differently. Additionally, pathway engagement varied between liquid culture and colony environments. In colonies, some cells specialized in either amino acid synthesis or uptake. Disruption of the SPS pathway hindered this specialization and increased cell death rates in aging colonies, indicating a role for metabolic differentiation in maintaining colony viability. Collectively, this study introduces a new tool for exploring cellular amino acid homeostasis and highlights the importance of cellular differentiation in amino acid control for colony survival.