Polyphosphates (polyP) are long chains of phosphates residues linked via high-energy phosphoanhydride bonds. PolyP’s regulation and its functions have been studied in bacteria and in yeast, but the synthesis and degradation pathways of polyP in mammals are still unknown. This makes it challenging to study polyP biology in mammalian cells. In this thesis, the E. coli gene Polyphosphate kinase 1 (PPK1), which encodes for an enzyme that synthesizes polyP, was used as a tool to study polyP biology in both yeast and mammalian cells. Using yeast as a model organism, expression of EcPPK1 led to a decrease in alpha-synuclein toxicity. Additional evidence shows the usefulness of using yeast as a model organism to study polyP’s protective effect on alpha-synuclein toxicity. In mammalian cells, transfection of EcPPK1 allowed for the modulation of polyP concentration in the cell. This led to the identification of six human proteins that are able to be polyphosphorylated. Preliminary data from RNA sequencing also shows that polyP in HEK293T cells regulates gene expression. Overall, this work demonstrated that EcPPK1 is a useful tool to study polyP biology in yeast and mammals.

Polyphosphates (PolyP) are linear chains of inorganic phosphates joined together by phosphoanhydride bonds. This polyanionic molecule has been found in all organisms and has been implicated in diverse biological processes such as cell growth and blood coagulation. Nonetheless, the mechanism of polyP’s involvement in these processes and its impact at a molecular level is still unknown. In 2015, the addition of polyP chains to lysine residues was found to be a novel post-translational modification. However, a hurdle in studying the impact of polyP in the mammalian system is its low endogenous levels. In this study, I applied an ectopic expression system using E. coli-derived polyphosphate kinase 1 (PPK) in HEK293T cells to induce excess production of polyP. In mammalian cells, increased intracellular polyP levels lead to increased activation of both Erk1/2 and p70s6k. I also expanded our system by utilizing the yeast exopolyphosphatase, Ppx1, to deplete polyP. Overall, this work presents a novel role for polyP in mediating key signalling pathways and will help probe the function of polyP in mammalian cells.

The first report of polyphosphates (polyP) was in 1890 by L. Liberman and since then, polyP’s role in biology has been explored. PolyPs are chains of phosphoanhydride-linked inorganic phosphates ranging from 3-1000s of units in length. These chains are implicated in many cellular pathways including blood clotting, bacterial virulence, and neuroproteotoxic disease. Given the diversity of polyP, they make an excellent candidate in the development of novel therapeutics. In yeast, polyP is synthesized by the vacuolar transporter chaperone (VTC) complex as a translocation event into the vacuole lumen. In 2015, polyP chains were found to act as a post-translational modification termed polyphosphorylation on yeast proteins (Nsr1 and Top1). This modification occurs non-enzymatically on lysine residues within poly-acidic, serine, and lysine (PASK) motifs and can only be detected via electrophoretic mobility shift on NuPAGE gels. We have since expanded the pool of yeast polyphosphorylated substrates to 25, with an enrichment of proteins with roles related to RNA biology. Additionally, we were the first group to demonstrate polyphosphorylation of 6 human proteins by expressing E. coli PPK1 in HEK293T cells. We next focused on elaborating how polyP is being regulated via the VTC complex by assessing which protein trafficking pathways are critical for VTC localization at the vacuole membrane. We found the adaptor protein 3 (AP-3) complex is responsible for localizing Vtc5 subunit to the vacuole membrane and in AP-3 mutants, Vtc5 becomes mislocalized to the vacuole lumen and degraded. Vtc5 degradation, upon AP-3 mutation, is mediated by the endosomal sorting complex required for transport (ESCRT) complex. The loss of polyP in AP-3 mutants is imparted by Vtc5 mislocalization. In humans, mutations in AP-3 cause a rare genetic disorder termed Hermansky-Pudlak Syndrome (HPS) which has a wide range of symptoms. These include defects in polyP accumulation in platelets, likely related to a loss of polyP. We expect that our work using yeast will provide a framework for understanding fundamental aspects of polyP biology related to HPS and other health conditions.