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

Although the yeast vacuole plays a crucial role in storing and mobilizing cationic amino acids (CAA), CAA transport at the vacuolar membrane remains only partially characterized. 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 when 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 is down-regulated 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.