Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81
- Département d'immunobiologie, Université de Lausanne, Switzerland
- Institute of Organic Chemistry, Centre for Integrative Biological Signalling Studies, University of Freiburg, Germany
Figures
Figure 1
Pathways of inositol pyrophosphate metabolism in S. cerevisiae.
Figure 2 with 3 supplements
Cytosolic concentrations of 5-IP7, 1-IP7, and 1,5-IP8.
(A) Inositol pyrophosphate concentrations in the cytosol. The indicated strains were grown logarithmically in synthetic complete (SC) medium containing 7.5 mM of inorganic phosphate (Pi) (30°C, 150 …
Figure 2—figure supplement 1
Recovery of inositol pyrophosphates extracted from S. cerevisiae cells.
(A) Illustration of the procedure of inositol pyrophosphate extraction using TiO2 beads. 0.1 µM [13C6]1,5-IP8, 0.5 µM [13C6]5-IP7, and 0.5 µM [13C6]1-IP7 were spiked into the yeast culture …
Figure 2—figure supplement 2
Determination of cell dimensions from wildtype cells stained with trypan blue.
Cells were grown in synthetic complete (SC) medium over night until they reached an OD600nm of 1. 10 µg/mL of trypan blue was added, and the cells were analyzed on a fluorescence microscope. Cells …
Figure 2—figure supplement 3
Loss of inositol pyrophosphates from siw14Δ cells upon inorganic phosphate (Pi) starvation.
The indicated strains were grown logarithmically in synthetic complete (SC) medium containing 7.5 mM of Pi (30°C, 150 rpm, overnight). The cells were spun down, washed twice with Pi starvation …
Figure 3
Inositol pyrophosphate analysis in C. neoformans and S. pombe.
Inositol pyrophosphates were measured in C. neoformans (A) and S. pombe (B). Both fungi were logarithmically grown in synthetic complete (SC) medium for 17 h up to an OD600nm of 1. Cells were …
Figure 4 with 1 supplement
Inhibition of the PHO pathway by excessive 5-IP7.
The indicated cells producing Pho4yEGFP and the histone Hta2mCherry as a nuclear marker were logarithmically grown in inorganic phosphate (Pi)-replete synthetic complete (SC) medium, washed, and …
Figure 4—figure supplement 1
Segmentation of fluorescence microscopy time-lapse experiments.
The subcellular localization of Pho4-yEGFP has been quantified from time-lapse fluorescence microscopy experiments. This quantification required the segmentation of microscopy images to discriminate …
Figure 5
Effect of Vip1, Kcs1, and Pho81 on the time course of Pho4 translocation and PHO-gene activation.
The indicated cells were logarithmically grown in inorganic phosphate (Pi)-replete synthetic complete (SC) medium, washed, and transferred to Pi starvation medium as in Figure 2A. At the indicated …
Figure 6 with 1 supplement
SPX-dependent activation of the PHO pathway.
Cells were logarithmically grown in inorganic phosphate (Pi)-replete synthetic complete (SC) medium as in Figure 2A, washed, and incubated for further 4 hr in medium with 7.5 mM of Pi (+Pi), or in …
Figure 6—figure supplement 1
Myo-inositol polyphosphate-binding pocket of yeast SPX domains.
(A) Crystal structure close-up view of Chaetomium thermophilum Vtc4 SPX domain complexing IP6 (5IJP). (B) Structure prediction (generated with SWISS-MODEL) of the S. cerevisiae Pho81 SPX domain …
Figure 7
Pho81 residues leading to constitutive activation of the PHO pathway.
The image shows an Alphafold prediction of the Pho81 SPX domain (amino acids 1–215), taken from Alphafold database model AF-P17442-F1 (Varadi et al., 2022), in yellow. Basic residues of the putative …
Figure 8
Structure predictions of the minimum domain in complex with Pho80.
Alphafold multimer v3 was used to generate the following structure predictions. (A) Surface representation of Pho80 in complex with a peptide corresponding to the minimum domain of Pho81 (residues …
Figure 9
Localization of Pho81yEGFP in pho80R121K and pho80E154V loss-of-affinity mutants.
Cells were logarithmically grown in inorganic phosphate (Pi)-replete synthetic complete (SC) medium as in Figure 2A, washed, and incubated for further 4 hr in medium with 7.5 mM of Pi (+Pi), or in …
Figure 10
Recruitment of Pho81SPXyEGFP to the nucleus by Pho85-Pho80.
The indicated wildtype or isogenic mutant cells expressing the SPX domain of Pho81 as a yEGFP fusion (Pho81SPXyEGFP) from a centromeric plasmid under the ADH promotor were logarithmically grown in …
Figure 11
Working model on the control of the PHO pathway through 1,5-IP8 and Pho81.
At high Pi concentrations, inositol pyrophosphates accumulate. 1,5-IP8 binds the SPX domain of Pho81, which labilizes the interaction of Pho81 with Pho80 and prevents Pho81 from inhibiting …
Tables
Table 1
Parameters for MRM transitions.
| Compound | Precursor ion | Product ion | dwell | CE (V) | Cell acc (V) | Polarity |
|---|---|---|---|---|---|---|
| [13C6]IP8 | 411.9 | 362.9 | 80 | 10 | 1 | Negative |
| IP8 | 408.9 | 359.9 | 80 | 10 | 1 | Negative |
| [13C6]IP7 | 371.9 | 322.9 | 80 | 10 | 1 | Negative |
| IP7 | 368.9 | 319.9 | 80 | 10 | 1 | Negative |
| [13C6]IP6 | 331.9 | 486.9 | 80 | 17 | 1 | Negative |
| IP6 | 328.9 | 480.9 | 80 | 17 | 1 | Negative |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/87956/elife-87956-mdarchecklist1-v1.docx
-
Supplementary file 1
This supplementary file contains information on the genetic constitution of the strains used in this study, their origin, and on the plasmids and primers used to generate them.
(a) List of strains. (b) List of plasmids. (c) List of primers used for genetic manipulations.
- https://cdn.elifesciences.org/articles/87956/elife-87956-supp1-v1.docx
