Vsb1, Ypq1, and Ypq2 control dynamic cationic amino acid storage in the yeast vacuole

Abstract Although the yeast vacuole plays a crucial role in storing and mobilizing cationic amino acids (CAA), CAA transport at the vacuolar membrane remains poorly understood. Here, by combining analysis of CAA pools, uptake and permeabilization assays, we establish Vsb1 as the principal vacuolar lysine transporter, enabling its strong accumulation in the vacuole while mitigating its toxicity. We further show that, although Ypq1 can mediate proton-independent vacuolar lysine import, it mainly functions as a lysine exporter necessary for lysine mobilization under conditions of lysine scarcity and downregulated as lysine stores are exhausted. Using quantitative models based on dynamic metabolic labeling, we further show that, surprisingly, in growing cells, CAA rapidly exchange between vacuolar and cytosolic compartments, a process involving the export activity of Ypq1 and its paralogue Ypq2, specific for lysine and arginine, respectively. Together, our findings reveal the unexpectedly complex function of Vsb1 and Ypq1/2 as the key transporters mediating dynamic vacuolar CAA storage. 40-word summary Zaremba et al . characterize Vsb1 as the main yeast vacuolar lysine importer and Ypq1/2 as bi- directional vacuolar transporters of lysine and arginine. Their study highlights the role of vacuolar transporters in regulating cationic amino acid homeostasis under fluctuating nutrient availability.