Vtc5 Is Localized to the Vacuole Membrane by the Conserved AP-3 Complex to Regulate Polyphosphate Synthesis in Budding Yeast.

Polyphosphates (polyP) are energy-rich polymers of inorganic phosphates assembled into chains ranging from 3 residues to thousands of residues in length. They are thought to exist in all cells on earth and play roles in an eclectic mix of functions ranging from phosphate homeostasis to cell signaling, infection control, and blood clotting. In the budding yeast Saccharomyces cerevisiae, polyP chains are synthesized by the vacuole-bound vacuolar transporter chaperone (VTC) complex, which synthesizes polyP while simultaneously translocating it into the vacuole lumen, where it is stored at high concentrations. VTC's activity is promoted by an accessory subunit called Vtc5. In this work, we found that the conserved AP-3 complex is required for proper Vtc5 localization to the vacuole membrane. In human cells, previous work has demonstrated that mutation of AP-3 subunits gives rise to Hermansky-Pudlak syndrome, a rare disease with molecular phenotypes that include decreased polyP accumulation in platelet dense granules. In yeast AP-3 mutants, we found that Vtc5 is rerouted to the vacuole lumen by the endosomal sorting complex required for transport (ESCRT), where it is degraded by the vacuolar protease Pep4. Cells lacking functional AP-3 have decreased levels of polyP, demonstrating that membrane localization of Vtc5 is required for its VTC stimulatory activity in vivo. Our work provides insight into the molecular trafficking of a critical regulator of polyP metabolism in yeast. We speculate that AP-3 may also be responsible for the delivery of polyP regulatory proteins to platelet dense granules in higher eukaryotes. IMPORTANCE Long polymers of inorganic phosphates called polyphosphates are ubiquitous across biological kingdoms. From bacteria to humans, they have diverse functions related to protein homeostasis, energy metabolism, and cell signaling. In this study, we provide new insights into the intracellular trafficking of the polyphosphate biosynthetic machinery in the budding yeast S. cerevisiae. The critical advances of the work are 2-fold. First, it provides an explanation for decreased polyphosphate levels observed in cells mutated for a conserved intracellular trafficking machine. Second, it defines critical pathways that are highly likely to serve as hubs for polyphosphate regulation in yeast and other species.