Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity

Abstract

Eukaryotes contain inorganic polyphosphate (polyP) and acidocalcisomes, which sequester polyP and store amino acids and divalent cations. Why polyP is sequestered in dedicated organelles is not known. We show that polyP produced in the cytosol of yeast becomes toxic. Reconstitution of polyP translocation with purified vacuoles, the acidocalcisomes of yeast, shows that cytosolic polyP cannot be imported, whereas polyP produced by the vacuolar transporter chaperone (VTC) complex, an endogenous vacuolar polyP polymerase, is efficiently imported and does not interfere with growth. PolyP synthesis and import require an electrochemical gradient, probably as a driving force for polyP translocation. VTC exposes its catalytic domain to the cytosol and carries nine vacuolar transmembrane domains. Mutations in the VTC transmembrane regions, which are likely to constitute the translocation channel, block not only polyP translocation but also synthesis. Given that they are far from the cytosolic catalytic domain of VTC, this suggests that the VTC complex obligatorily couples synthesis of polyP to its import in order to avoid toxic intermediates in the cytosol. Sequestration of otherwise toxic polyP might be one reason for the existence of acidocalcisomes in eukaryotes.

Eukaryotic Phosphate Homeostasis: The Inositol Pyrophosphate Perspective

“…In these proteins the SPX domain is associated with other functional regions, defining six diverse groups. The SPX domain is present in four components of the vacuolar transport complex (VTC) that is responsible for synthesis of inorganic polyphosphate (polyP; see Glossary) in yeast [8][9][10], polyP is a linear polymer of phosphate groups [11,12] whose main function is to buffer free phosphate (see below). Other proteins containing SPX domains are the low-affinity phosphate transporters Pho87 and Pho90, in which SPX deletion revealed an inhibitory function: the truncated transporters allowed increased phosphate uptake [13].…”

Organelle size control – increasing vacuole content activates SNAREs to augment organelle volume through homotypic fusion

“…Because VTC has since been identified as the major polyP polymerase of yeast (Hothorn et al, 2009), we have reconsidered the role of VTC in vacuole fusion and have now tested whether it could stimulate vacuole fusion by changing the abundance of vacuole content. We exploited the availability of structural information on the catalytic center of VTC and of methods to reliably measure synthesis and translocation of polyP by yeast vacuoles (Gerasimaite et al, 2014) in order to explore a possible link between the vacuolar fusion machinery and polyP accumulation in the vacuolar lumen.…”

Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

“…The intact VTC complex is required for transport of polyP into the vacuole lumen (15), leading to the accumulation of polyP in the vacuole. Both exo-and endo polyphosphatases have also been identified in yeast (16,17).…”