Pseudophosphatases Enhance Siw14, an Inositol Pyrophosphate Phosphatase that Negatively Regulates the Environmental Stress Response through Msn2

Abstract

Inositol pyrophosphates are high energy signaling molecules found across eukaryotes that are important for diverse cellular processes like phosphate homeostasis, the immune response, fungal pathogenicity, endocytosis, energy homeostasis and stress responses. Although inositol pyrophosphates have been implicated in many processes, the mechanism(s) by which inositol pyrophosphates influence them is largely unknown. Siw14 is an inositol pyrophosphate phosphatase in Saccharomyces cerevisiae that specifically cleaves the 5-position β-phosphate of inositol pyrophosphates 5PP-InsP5 and 1,5PP2-InsP4. We found that Oca1 and Oca2, Siw14 homologs in yeast which are catalytically inactive, enhance the activity of Siw14 with its preferred substrate 5PP-InsP5. Our data support a model in which Oca1 and Oca2 are novel pseudophosphatases that employ a modulator mechanism of action to affect their active canonical phosphatase, Siw14. The loss of Siw14 in S. cerevisiae leads to a significant increase in inositol pyrophosphates. Unsurprisingly, the loss of either Oca1 or Oca2 in yeast does not strongly increase inositol pyrophosphate levels, however the siw14Δoca1Δ and siw14Δoca2Δ mutants have levels of PP-InsPs similar to siw14Δ. Interestingly, we found that the siw14Δ mutant is resistant to multiple stresses including oxidative stress, osmotic stress, heat stress, and nutrient deprivation. Under normal growth conditions, the siw14Δ mutant has 444 genes that are misregulated, which largely overlap genes induced in the environmental stress response. Notably, 75% of the genes misregulated in the siw14Δ mutant are known to be regulated by the master cell regulator Target of Rapamycin Complex 1 (TORC1). We show that Msn2, a general stress transcription factor and a major target of TORC1, exhibits increased nuclear localization in the siw14Δ mutant. Our data suggest that inositol pyrophosphates affect the ESR in part by increasing the nuclear localization and transcription of Msn2 targets. Additionally, our data support a model in which inositol pyrophosphates negatively regulate TORC1 signaling.