VTC- and 5-IP7-dependent polyP synthesis by isolated vacuoles

Vacuoles were isolated from logarithmic cultures of BY4742 wildtype cells (BY) or from isogenic vtc4τι cells strains. They were incubated in polyphosphate synthesis assays without (A) or (B) in presence of 50 µM 5-InsP7. At indicated times, aliquots were withdrawn, solubilized in Triton X-100 and polyphosphate was quantified through the polyP-dependent fluorescence of added DAPI. Means ± SEM (standard error of the mean) of at least three independent experiments are shown.

VTC-dependent accumulation of Pi in isolated vacuoles

Vacuoles were isolated from the indicated, logarithmically growing cells. PolyP overproduction was achieved either through (A, B) overexpressing VTC5 from the strong GPD promotor, or (C,D) by expressing the hyperactivating vtc3k126A and vtc4K129A alleles form their native promotors as the sole source of these two proteins. The vacuoles were incubated with an ATP-regenerating system and 50 µM 5-InsP7 under conditions allowing polyP synthesis. At indicated times, the vacuoles were solubilized with Triton X-100 and Pi was assayed through malachite green. Graphs represent the mean ± SEM of at least three independent experiments for each strain.

Effect of Pi on polyphosphatase activity.

Vacuoles were isolated from the indicated, logarithmically growing strains. The organelles were diluted in polyphosphatase assay reaction buffer, which contained 0.1% Triton X-100 and hence liberated the luminal polyphosphatases. This lysate was incubated with polyP300 as a substrate and with A) 1 mM MgCl2 or B) 1 mM ZnCl2. Where indicated, 30 mM K-Pi pH 6.8 had been added. After the indicated times of incubation, the remaining polyP was quantified through DAPI. The DAPI signal at the beginning of the incubation served as 100% reference. Graphs represent the means ± SEM of three independent experiments.

Effects of N- and C-terminal fluorescent protein tags on the localization of Pho91.

PHO91 was fused with a variety of N- or C-terminal protein tags and peptide spacers as indicated. They were expressed from the endogenous PHO91 promotor or, where indicated, from ADH1 or GPD1 promotors. Cells were logarithmically grown overnight in SC medium, harvested at OD600nm of 1-2, and analyzed by fluorescence microscopy.

Pho91 cannot replace other Pi transporters to support growth of yeast.

We generated a BY4741 strain with a quintuple deletion of the known Pi transporters Pho84, Pho87, Pho89, Pho90 and Pho91 (D5m). These cells were kept alive by expressing the plasma membrane Pi transporter Pho89 from a URA3-based centromeric (single copy) plasmid (pRS416). Pho91 was expressed from a HIS3-based centromeric plasmid (pRS315). Cells were plated in a dilution series on SC lacking histidine (SC-HIS) to verify that the cells had the HIS3-based PHO91 plasmid, or on SC with 5-fluoro-orotic acid (5-FOA), a drug that forces cells to lose the URA3-based pRS416 and thus to live without the Pho89 transporter. Pho91 as the sole Pi transporter (on SC + 5-FOA) does not allow cells to grow.

Accumulation of Pi in isolated vacuoles

VTC- and Pho91-dependence. Vacuoles were isolated from the indicated, logarithmically growing strains. The purified organelles were incubated as in Fig. 1, i.e. in a buffer with an ATP-regenerating system that allows the synthesis of polyP, and either without (w/o) or in the presence of 50 µM 5-IP7. After the indicated periods of incubation at 27°C, an 80 µl aliquot was withdrawn, the vacuoles were sedimented by centrifugation, washed and then lysed. Released vacuolar Pi was determined by malachite green assay. Graphs represent the mean ± SEM of at least three independent experiments for each strain.

PolyP accumulation in pho91 mutant vacuoles.

Vacuoles from wildtype (BY) and isogenic pho91τι cells were isolated and incubated under conditions supporting polyP synthesis and Pi accumulation as in Fig. 4, in the absence (w/o) or presence of 50 µM 5-IP7. At the indicated timepoints, aliquots were withdrawn, the vacuoles were lysed in detergent, and polyP was assayed through DAPI fluorescence.

Impact of the vacuolar polyP/Pi cycle on cytosolic Pi signalling

A) Illustration of nucleo-cytoplasmic relocation of Pho4-GFP in response to Pi availability. Wildtype yeast cells were grown in SC medium under Pi replete conditions. During exponential phase (OD600nm=1), cells were transferred for 30 min to synthetic complete media with 200 µM phosphate (-Pi) or 7.5 mM Pi (+Pi) and imaged by fluorescence microscopy. B) The indicated yeast strains were logarithmically grown over night in SC medium with 7.5 mM Pi, harvested at OD600nm=1 and immediately imaged by fluorescence microscopy as in A. The graph shows the means and SEM of the percentage of cells showing Pho4 predominantly in the nucleus. n=3.

Impact of Pho91 and vacuolar polyphosphatases on InsPP levels.

pho91τι and ppn1τιppn2τι cells, as well as their isogenic wildtypes, were logarithmically grown in Pi-replete SC medium as in Fig. 6. At OD600nm=1 the cells were extracted with perchloric acid as previously described (Wilson et al, 2015) and analyzed for the indicated InsPs through capillary electrophoresis coupled to mass spectrometry (CE-MS) as described (Qiu et al, 2023).

Working model of acidocalcisome-like vacuoles as Pi buffering systems.

ATP drives the conversion of Pi into polyP and its translocation into the organelle. Here, polyP is degraded by the vacuolar polyphosphatases Ppn1 and Ppn2 to establish a vacuolar pool of free Pi. Feedback inhibition of Pi gradually reduces polyP degradation, enabling the buildup of a vacuolar polyP stock. Cytosolic Pi scarcity decreases InsPP levels, which triggers two compensatory, SPX-controlled effects: The transfer of Pi from the cytosol into vacuoles through VTC ceases; and Pho91-dependent export of Pi from vacuoles is activated. Both measures synergize to stabilize cytosolic Pi.