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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.

The free energy of nucleotide hydrolysis depends on phosphate concentration. Cells regulate cytosolic phosphate levels by orchestrating phosphate acquisition and storage through inositol pyrophosphates (PP-InsP) and SPX domains. Here, we report the synthesis of the novel 5-PPP-InsP containing a triphosphate subunit. Using this and a series of synthetic PP-InsP, we examined the ligand specificity of the SPX domain in the PP-InsP-controlled yeast polyphosphate polymerase VTC. SPX decodes the relative positioning of the phosphoric anhydrides, their structure (diphosphate vs triphosphate), and the presence of other phosphates on the inositol ring. Despite the higher potency of 1,5-(PP)-InsP, 5-PP-InsP is the primary activator of VTC in cells, indicating that its higher concentration compensates for its lower potency. 1,5-(PP)-InsP levels rise and could become relevant under stress conditions. Thus, SPX domains may integrate PP-InsP dependent signaling to adapt cytosolic phosphate concentrations to different metabolic situations.

SPX domains control phosphate homeostasis in eukaryotes.Ten genes in yeast encode SPX-containing proteins, among which YDR089W is the only one of unknown function. Here, we show that YDR089W encodes a novel subunit of the vacuole transporter chaperone (VTC) complex that produces inorganic polyphosphate (polyP). The polyP synthesis transfers inorganic phosphate (P i ) from the cytosol into the acidocalcisome-and lysosome-related vacuoles of yeast, where it can be released again. It was therefore proposed for buffer changes in cytosolic P i concentration (Thomas, M. R., and O'Shea, E. K. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 9565-9570). Vtc5 physically interacts with the VTC complex and accelerates the accumulation of polyP synthesized by it. Deletion of VTC5 reduces polyP accumulation in vivo and in vitro. Its overexpression hyperactivates polyP production and triggers the phosphate starvation response via the PHO pathway. Because this Vtc5-induced starvation response can be reverted by shutting down polyP synthesis genetically or pharmacologically, we propose that polyP synthesis rather than Vtc5 itself is a regulator of the PHO pathway. Our observations suggest that polyP synthesis not only serves to establish a buffer for transient drops in cytosolic P i levels but that it can actively decrease or increase the steady state of cytosolic P i .Phosphate is a limiting factor for the growth of living organisms. It is mainly taken up by the cells as P i and incorporated in biological molecules, including ATP, nucleic acids, and phospholipids. P i plays a major role in the regulation of biochemical pathways through phosphorylation, pyrophosphorylation, and polyphosphorylation. Its concentration affects the free energy liberated by the hydrolysis of ATP and other nucleoside di-and triphosphates. Therefore, P i homeostasis (import, usage, storage, and export) is regulated (1, 2), and P i can be accumulated and stored as a polymer of P i units called polyphosphate (polyP).3 One mode of regulation operates on the transcriptional level. The PHO pathway, which is a P i -dependent transcriptional program in yeast, has been extensively characterized (2). Transcription of the PHO genes is regulated by the transcription factors Pho4 and Pho2. Phosphorylated Pho2 interacts with Pho4 to induce expression of the PHO genes. Pho2 is a target of Cdc28 kinase in vitro suggesting a coordination between cell cycle progression and nutrient availability (3). Pho4 is itself regulated by the cyclin-dependent kinase Pho85 and its cyclin Pho80. Under P i -rich conditions, the Pho80-Pho85 complex phosphorylates Pho4, thereby restricting the localization of the transcription factor to the cytosol (4, 5). Under P i limitation, Pho80/Pho85 is inhibited, and the nonphosphorylated Pho4 is imported into the nucleus, allowing transcription of PHO genes. Pho80/85 is regulated by the cyclin-dependent kinase inhibitor Pho81. Inhibition of Pho80/85 by Pho81 is facilitated by the inositol pyrophosphate 1-IP 7 , which is produced by Vip1 (6). However,...

Hoechst dyes are among the most popular fluorophores used to stain DNA in living and fixed cells. Moreover, their high affinity and specificity towards DNA make Hoechst dyes excellent targeting moieties, which can be conjugated to various other molecules in order to tether them to DNA. The recent developments in the fields of microscopy and flow cytometry have sparked interest in such composite molecules, whose applications range from investigating nucleus microenvironment to drug delivery into tumours. Here we provide an overview of the properties of Hoechst dyes and discuss recent developments in Hoechst-based composite probes.