Inositol pyrophosphate dynamics reveals control of the yeast PHO starvation program through 1,5-IP₈ and the SPX domain of Pho81

Reviewer #1 (Public Review):

Recent studies in plants and human cell lines argued for a central role of 1,5-InsP8 as the central nutrient messenger in eukaryotic cells, but previous studies concluded that this function is performed by 1-InsP7 in baker's yeast. Chabert et al now performed an elegant set of capillary electrophoresis coupled to mass spectrometry time course experiments to define the cellular concentrations of different inositol pyrophosphosphates (PP-InsPs) in wild-type yeast cells under normal and phosphate (Pi) starvation growth conditions. These experiments, in my opinion, form the center of the present study and clearly highlight that the levels of all major PP-InsPs drop under Pi starvation, with the 1,5-InsP8 isomer showing the most rapid changes.

The analysis of known mutants in the PP-InsP biosynthetic pathways furthermore demonstrate that loss-of-function of the PPIP5K enzymes Kcs1 and Vip1 result in a loss of 1,5-InsP8 and a hyperaccumulation of 5-InsP7, respectively. In line with this, loss-of-function of known PP-InsP phosphatases Ddp1 and Swi14 result in hyperaccumulation of either 1- or 5-InsP7, as anticipated from their in vitro substrate specificities. These experiments are of high technical quality and add to our understanding of the kinetics of PP-InsP metabolism/catabolism in yeast.

Next, the authors use changes in subcellular localisation of the central transcription factor Pho4 to assay at which time point after onset of Pi starvation the PHO pathway becomes activated. The early onset of the response, the behavior of the kcs1D mutant and of the ksc1D/vip1D all strongly argue for 1,5-InsP8 as the central nutrient messenger. I find this part of the manuscript well argued, nicely correlating PP-InsP levels, dynamics and the different mutant phenotypes.

The third part of the manuscript is a structure-function study of the CDK inhibitor Pho81, basically using a reverse genetics approach. This analysis demonstrates at the genetic level that the Pho81 SPX domain is required for activation of the PHO pathway. Next, the authors design point mutations that should block either interaction of Pho81-SPX with 1,5-InsP8 or interaction of Pho81 with the Pho80/Pho85 complex. In my opinion, these data can only provide limited insight into the molecular mechanism, as no complementary in vitro binding assays / in vivo co-IP experiments with the wild-type and mutant forms of Pho81 are presented.

The discussion section of the manuscript contains additional data such as PP-InsP levels for C. neoformans and complex structure predictions of Pho80 - Pho81. This, in my opinion, renders the discussion section of the work overly speculative. Perhaps, these results should be presented in the results section, and ideally (in the case of the complex structure predictions), be complemented by quantitative in vitro and/or qualitative in vivo binding assays.

Taken together, the work by Chabert et al, reinvestigates and clarifies the activation of the yeast PHO pathway by PP-InsP nutrient messengers and their cellular SPX receptors. From this work, a more unified eukaryotic mechanism emerges, in which 1,5-InsP8 represents the central signaling molecule in different species, with conserved SPX receptors sensing this signaling molecule.

Reviewer #2 (Public Review):

The manuscript by Chambert et al. describes a thorough and careful characterization of inositol pyrophosphate isomers and the PHO pathways in different genetic backgrounds in S. cerevisiae. The paper ultimately arrives at a proposed model in which the inositol pyrophosphate 1,5-IP8 signals phosphate abundance to SPX-domain containing proteins. To arrive at their conclusion, the authors rely heavily on CE-MS analysis of inositol pyrophosphates in different yeast strains, and monitoring inositol pyrophosphate depletion over time in response to phosphate starvation. This analysis is complemented by different reporter systems of PHO pathway activation, such as Pho4 translocation and Pho81 expression.

The experiments are well-designed and the results interpreted with care. With their findings, the authors demonstrate convincingly, that a previous study by O'Shea and co-workers (reference 15 and 16) had been misleading. Lee et al. claimed that the PHO pathway in S. cerevisiae is triggered by an increase in 1-IP7. This claim has been debated heavily in the community, and several groups were not able to reproduce this putative increase of inositol pyrophosphates (references 6, 11, 18). The confusion regarding these discrepancies has been resolved by the current study and is of significant importance to the community.

Reviewer #3 (Public Review):

Summary. This study sought to clarify the connection between inositol pyrophosphates (PP-IPs) and their regulation of phosphate homeostasis in the yeast Saccharomyces cerevisiae to answer the question of whether any of the PP-IPs (1-IP7, 5-IP7, and IP8) or only particular PP-IPs are involved in regulation. PP-IPs bind to SPX domains in proteins to affect their activity, and there are several key proteins in the PHO pathway that have an SPX domain, including Pho81. The authors use the latest methodology, capillary electrophoresis and mass spectrometry (CE-MS), to examine the cytosolic concentrations of PP-IPs in wild-type and strains carrying mutations in the enzymes that metabolize these compounds in rich medium and during a phosphate starvation time-course for the wild-type.

Major strengths and weaknesses. The authors have strong premises for performing these experiments: clarifying the regulatory molecule(s) in yeast and providing a unifying mechanism across eukaryotes. They use the latest methodologies and a variety of approaches including genetics, biochemistry, cell biology and protein structure to examine phosphate regulation. Their experiments are rigorous and well controlled, and the story is clearly told. The consideration of physiological levels of PP-IPs throughout the study was critical to the interpretation of the data and the strength of the manuscript.

There were a few places in which a deeper discussion of the data was warranted: not discussed was an explanation for the decrease in the levels of all of the PP-IPs upon phosphate starvation, nor of the phosphate regulation of two target genes of Pho4 when Pho4 is constitutively nuclear.

Appraisal. The authors achieved their goal of determining the mechanistic details for phosphate regulation, revising the prior model with new insights. Additionally, they provided strong support for the idea that IP8 regulates phosphate metabolism across eukaryotes - including animals and plants in addition to fungi.

Impact. This study is likely to have a broad impact because it addresses prior findings that are inconsistent with current understanding, and they provide good reasoning as to how older methods were inadequate.