Exploring Polyphosphate Biology Regulation in Saccharomyces cerevisiae

Abstract

Polyphosphate (polyP) is a polymer of inorganic phosphate residues that can range from three residues to thousands in length. These polymers are conserved across most organisms where they participate in a multitude of cellular functions. In yeast, polyP is synthesized and stored within the vacuole where it is involved in phosphate, ion, and pH homeostasis. Despite growing interest in polyP’s role in a variety of diseases there are still many questions relating to its fundamental biology that remain unanswered. We first set out to expand the scope of polyP-binding proteins in yeast. We performed an expanded screen of 200 yeast proteins that were not screened in our groups previous screen for polyP-binding proteins published in 2018. We identify 8 new polyP-binding proteins that interact genetically and physically with previous targets involved in ribosome biogenesis. These include two proteins involved in vacuolar function – the Prb1 protease and the Apl5 subunit of the AP-3 complex. The ability of polyP to bind to these proteins suggests a model for feedback regulation of polyP synthesis in the vacuole. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In budding yeast, polyP synthesis by the membrane-bound vacuolar transporter chaperone (VTC) complex is coupled to its translocation into the lumen of the vacuole, a lysosomelike organelle, where it is stored at high concentrations. In contrast, the ectopic expression of the bacterial polyphosphate kinase (PPK) results in the toxic accumulation of polyP outside the vacuole. We used label-free mass spectrometry to investigate the mechanisms underlying this toxicity. We find that PPK expression results in the activation of a stress response mediated in part by the Hog1 and Yak1 kinases and the Msn2/Msn4 transcription factors as well as changes in protein kinase A (PKA) activity. This response is countered by the combined action of the Ddp1 and Ppx1 polyphosphatases that function together to counter polyP accumulation and downstream toxicity. Finally, we performed a protein mutagenesis screen to explore the structure and function of the Ddp1 endopolyphosphatase. We used our ectopic PPK expression system (described above) to compare the ability of Ddp1 mutants and wild-type Ddp1 to rescue the PPK-induced toxicity. Our screen confirms many phosphate recognition residues important for Ddp1’s polyphosphatase activity in vivo. We also developed a plasmid shuffle system in a pyrophosphatase mutant strain that presumably accumulates toxic levels of 1,5-bisdiphosphoinositol tetrakisphosphate (1,5-IP8) to expand our Ddp1 mutagenesis screen and study the regulation of its pyrophosphatase activity in vivo. Moreover, DIPP1, the mammalian homologue of Ddp1, can complement the loss of the yeast protein, highlighting that this system could be used to perform mutagenesis screens on other pyrophosphatases in the future. Together this work expands on potential functions of polyP in yeast and provides insight into the importance of polyP compartmentalization in eukaryotes. Moreover, it provides insight into the multiple functions of Ddp1 and how they might be regulated in vivo.