Acidocalcisome-like vacuoles constitute a feedback-controlled phosphate buffering system for the cytosol
- Département d'immunobiologie, Université de Lausanne, Switzerland
- Institute of Organic Chemistry University of Freiburg and CIBSS – Centre for Integrative Biological Signalling Studies, University of Freiburg, Germany
Figures
Figure 1
VTC- and 5-IP7-dependent polyphosphate (polyP) synthesis by isolated vacuoles.
Vacuoles were isolated from logarithmic cultures of BY4742 wildtype cells (WT) or from isogenic vtc4Δ cell strains. They were incubated in polyphosphate synthesis assays without (A) or (B) in the presence of 50 µM 5-InsP7. At indicated times, aliquots were withdrawn, solubilised in Triton X-100, and polyphosphate was quantified through the polyP-dependent fluorescence of added DAPI. Means ± SD (standard deviation) of three independent experiments are shown. *p<0.05, **p<0.005 for a paired t-test comparing WT with and without 5-InsP7 at each time point.
Figure 2 with 1 supplement
VTC-dependent accumulation of Pi in isolated vacuoles.
Vacuoles were isolated from the indicated logarithmically growing BY4742 (WT) cells. Polyphosphates (polyP) overproduction was achieved (A, B) either through overexpressing VTC5 from the strong GPD promoter, or (C, D) by expressing the hyperactivating vtc3k126A and vtc4K129A alleles from their native promoters 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 solubilised with Triton X-100 and Pi was assayed through malachite green. Graphs represent the mean ± SEM of three independent experiments for each strain. *p<0.05, **p<0.005, ***p<0.001 from a paired t-test comparing each time WT vs VTC5 overexpression or vtc3k126A vtc4K129A.
Figure 2—figure supplement 1
Isolated vacuoles during polyphosphate (polyP) synthesis.
BJ 4742 (WT) vacuoles were incubated in vitro as in Figure 1B. After 10 min, 0.1 μM of the fluorophore FM4-64 was added to stain the membranes. The suspension was immediately imaged on a Yokogawa spinning disc microscope with a Gataca LiveSR unit and a 100× 1.45 NA objective. Stacks were taken at 0.2 µM z-distance. The image shows an optical section out of such a stack. To determine vacuole diameters, the stacks were subjected to maximum intensity projection in ImageJ. The largest visible diameter for 100 organelles was measured manually using ImageJ, yielding an average diameter of 0.8 µm with an SD of 0.2 µm. Scale bar: 1 µm.
Figure 3
Effect of Pi on polyphosphatase activity.
(A) Pi titration. Vacuoles were isolated from logarithmically growing wildtype cells (BY4742). 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 30 μM polyP300 as a substrate and supplemented with 1 mM ZnCl2 and the indicated concentrations of K-Pi pH 6.8. 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. (B, C) Differentiation of Pi effects on Ppn1 and Ppn2. Vacuoles were isolated from the indicated logarithmically growing strains, lysed and used in polyP degradation assays with 300 µM polyP300 as in (A). These assays were performed in the presence or absence of 30 mM K-Pi pH 6.8 and 1 mM MgCl2 (B) or, instead of this, with 1 mM ZnCl2 (C) as cation supporting catalytic activity.
Figure 4 with 1 supplement
Pho91 cannot replace other Pi transporters to support growth of yeast.
We generated a BY4741 strain with a quintuple deletion of the genes for the known Pi transporters, PHO84, PHO87, PHO89, PHO90, and PHO91 (D5m). These cells were kept alive by expressing the gene for 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 synthetic complete (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 PHO89. PHO91 as the sole Pi transporter gene (on SC + 5-FOA) does not allow cells to grow.
Figure 4—figure supplement 1
Effects of N- and C-terminal fluorescent protein tags on the localisation 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 promoter or, where indicated, from the overexpressing ADH1 or GPD1 promoters. Cells were logarithmically grown overnight in synthetic complete (SC) medium, harvested at OD600nm of 1–2, and analysed by fluorescence microscopy. Scale bar: 1 µm.
Figure 5
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 Figure 1, i.e., in a buffer with an ATP-regenerating system that allows the synthesis of polyphosphates (polyP), and either (A) without (w/o) or (B) 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 lysed. Released vacuolar Pi was determined by malachite green assay. Graphs represent the mean ± SEM of three independent experiments. p<0.05 from a paired t-test comparing each time WT vs pho91Δ. (B) The indicated yeast strains were logarithmically grown overnight in synthetic complete (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 independent experiments with 200 cells quantified per sample. **p<0.005, ***p<0.001, ****p<0.0001 from a paired t-test comparing WT with the mutants.
Figure 6
Polyphosphate (PolyP) accumulation in pho91 mutant vacuoles.
Vacuoles from BY4742 cells (WT) and isogenic pho91Δ cells were isolated and incubated under conditions supporting polyP synthesis and Pi accumulation as in Figure 5, in the absence (w/o) or presence of 50 µM 5-InsP7. At the indicated time points, aliquots were withdrawn, the vacuoles were lysed in detergent, and polyP was assayed through DAPI fluorescence. Graphs show the means and SEM from three independent experiments. p>0.05 from a paired t-test for all differences between WT and pho91Δ.
Figure 7
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 synthetic complete (SC) medium under Pi-replete conditions. During the exponential phase (OD600nm=1), cells were transferred for 30 min to SC media with 200 µM phosphate (-Pi) or 7.5 mM Pi (+Pi) and imaged by fluorescence microscopy. Scale bar: 1 µm. (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 independent experiments with 200 cells quantified per sample. ** p<0.005, *** p<0.001, ****p<0.0001 from a paired t-test comparing WT with the mutants.
Figure 8
Impact of Pho91 and vacuolar polyphosphatases on InsPP levels.
pho91Δ and ppn1Δppn2Δ cells, as well as their isogenic wildtypes, were logarithmically grown in Pi-replete synthetic complete (SC) medium as in Figure 7. At OD600nm=1, the cells were extracted with perchloric acid as previously described (Wilson et al., 2015) and analysed for the indicated InsPs through capillary electrophoresis coupled to mass spectrometry (CE-MS) as described (Qiu et al., 2023). n=3. *p<0.05 from a paired t-test comparing WT with the mutants.
Figure 9
Working model of acidocalcisome-like vacuoles as Pi buffering systems.
(A) Under Pi-replete conditions, ATP drives the conversion of Pi into polyphosphates (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. Red lines and SPX colouring indicate inhibitory action, green colouring stimulation. (B) 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 synergise to stabilise cytosolic Pi. The export of Pi from the vacuole lifts product inhibition on the polyphosphatases Ppn1 and Ppn2 and stimulates a compensatory degradation of polyP.
Tables
Table 1
Yeast strains used in this study.
| Background | Genotype | Source |
|---|---|---|
| BY4741 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | Euroscarf |
| BY4742 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 | Euroscarf |
| BY4742 | vtc4::kanMX | Gerasimaitė et al., 2017 |
| BY4741 | vtc4::kanMX | This study |
| BY4742 | ppn1::natNT2 | Gerasimaitė et al., 2017 |
| BY4742 | ppn2::URA3 | Gerasimaitė et al., 2017 |
| BY4742 | ppn1::natNT2 ppn2::URA3 | Gerasimaitė et al., 2017 |
| BY4742 | ppn1::natNT2 ppn2::URA3 vtc4::URA3 | Gerasimaitė et al., 2017 |
| BY4741 | PGPD1-VTC5 natNT2 | Desfougères et al., 2016a |
| BY4742 | VTC3K126A VTC4K129A | Wild et al., 2016 |
| BY4741 | pho91::kanMX | This study |
| BY4741 | pho91::kanMX PGPD1-VTC5 natNT2 | This study |
| BY4741 | pho91::kanMX pRS303-PPHO91-PHO91 (K237A) | This study |
| BY4741 | pho91::hyg vtc4::kanMX | This study |
| BY4741 | pRS415-PPHO4-PHO4-GFP | This study |
| BY4741 | vtc4::kanMX pRS415-PPHO4-PHO4-GFP | This study |
| BY4741 | pho91::kanMX pRS415-PPHO4-PHO4-GFP | This study |
| BY4741 | pho91::hyg vtc4::kanMX pRS415-PPHO4-PHO4-GFP | This study |
| BY4741 | PGPD1-VTC5 natNT2 pho91::hyg pRS415-PPHO4-PHO4-GFP | This study |
| BY4741 | PGPD1-VTC5 natNT2 pRS415-PPHO4-PHO4-GFP | This study |
| BY4742 | ppn1::natNT2 ppn2::URA3 pRS415-PPHO4-PHO4-GFP | This study |
| BY4741 | pho91::kanMX pRS303-PPHO91-PHO91 (K237A) pRS415-PPHO4-PHO4-GFP | This study |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/108181/elife-108181-mdarchecklist1-v1.docx
-
Supplementary file 1
Enrichment factors of marker proteins in the vacuolar fraction of BY4742 cells over the total extract of spheroblasts.
- https://cdn.elifesciences.org/articles/108181/elife-108181-supp1-v1.xlsx
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Acidocalcisome-like vacuoles constitute a feedback-controlled phosphate buffering system for the cytosol
eLife 14:RP108181.
https://doi.org/10.7554/eLife.108181.3
