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One of the functions of inorganic polyphosphates (polyP) as compounds with phosphoester bonds is participation in energy metabolism. While yeast mitochondria contain their own pool of polyphosphates, the ways in which these polymers are involved in the functioning of mitochondria in these microorganisms are not well understood. The goal of the present work was to identify the effect of knockout mutations of the VTC4 gene of polyphosphate synthetase and the PPN1 gene of one of the polyphosphatases on the content of polyphosphates and polyphosphatase activity in mitochondria of S. cerevisiae and on the growth characteristics of the mutant strains on ethanol. Knockout of the VTC4 gene was shown to result in a significant decrease in the polyP content in the mitochondria. Knockout of the PPN1 gene led to the disappearance of polyphosphatase activity, but only to a slight increase in the content of mitochondrial polyphosphates during growth on glucose. When grown on ethanol, the polyP content in the mitochondria of this strain was similar to that of the parental strain, and in both strains it was approximately two times less than when grown on glucose. Both mutants were able to grow with ethanol as the carbon source; however, they exhibited a longer lag phase upon transition from glucose consumption to ethanol consumption. It was suggested that mitochondrial polyphosphates may represent the energy reserve of these organelles, which is necessary for the formation of full-fledged mitochondria during transition from glycolysis to oxidative phosphorylation. [ABSTRACT FROM AUTHOR]

Endocytosis and autophagy are the main pathways to deliver cargoes in vesicles and autophagosomes, respectively, to vacuoles/lysosomes in eukaryotes. Multiple positive regulators but few negative ones are reported to regulate the entry of vesicles and autophagosomes into vacuoles/lysosomes. In yeast, the Rab5 GTPase Vps21 and the ESCRT (endosomal sorting complex required for transport) are positive regulators in endocytosis and autophagy. During autophagy, Vps21 regulates the ESCRT to phagophores (unclosed autophagosomes) to close them. Phagophores accumulate on vacuolar membranes in both vps21∆ and ESCRT mutant cells under a short duration of nitrogen starvation. The vacuolar transport chaperon (VTC) complex proteins are recently found to be negative regulators in endocytosis and autophagy. Phagophores in vps21∆ cells are promoted to enter vacuoles when the VTC complex proteins are absent. Phagophores are easily observed inside vacuoles when any of these VTC complex proteins (Vtc1, 2, 4, 5) are removed. However, it is unknown whether the removal of VTC complex proteins will also promote the entry of phagophores into vacuoles in ESCRT mutant cells under the same conditions. Snf7 is a core subunit of ESCRT subcomplex III (ESCRT-III), and phagophores accumulate in snf7∆ cells under a short duration of nitrogen starvation. We used green fluorescence protein (GFP) labeled Atg8 to display phagophores and FM4-64-stained or Vph1-GFP-labeled membrane structures to show vacuoles, then examined fluorescence localization and GFP-Atg8 degradation in snf7∆ and snf7∆vtc4∆ cells. Results showed that Vtc4 depletion promoted the entry of phagophores in snf7∆ cells into vacuoles as it did for vps21∆ cells, although the promotion level was more obvious in vps21∆ cells. This observation indicates that the VTC complex proteins may have a widespread role in negatively regulating cargos to enter vacuoles in yeast. [ABSTRACT FROM AUTHOR]

Polyphosphates (polyP) are polymers of inorganic phosphates joined by high-energy bonds to form long chains. These chains are present in all forms of life but were once disregarded as 'molecular fossils'. PolyP has gained attention in recent years following new links to diverse biological roles ranging from energy storage to cell signaling. PolyP research in humans and other higher eukaryotes is limited by a lack of suitable tools and awaits the identification of enzymatic players that would enable more comprehensive studies. Therefore, many of the most important insights have come from single-cell model systems. Here, we review determinants of polyP metabolism, regulation, and function in major microbial systems, including bacteria, fungi, protozoa, and algae. We highlight key similarities and differences that may aid in our understanding of how polyP impacts cell physiology at a molecular level. [ABSTRACT FROM AUTHOR]

Protozoan parasites contain negatively charged polymers of a few up to several hundreds of phosphate residues. In other organisms, these polyphosphate (polyP) chains serve as an energy source and phosphate reservoir, and have been implicated in adaptation to stress and virulence of pathogenic organisms. In this study, we confirmed first that the polyP polymerase vacuolar transporter chaperone 4 (VTC4) is responsible for polyP synthesis in Leishmania parasites. During Leishmania in vitro culture, polyP is accumulated in logarithmic growth phase and subsequently consumed once stationary phase is reached. However, polyP is not essential since VTC4-deficient (vtc4–) Leishmania proliferated normally in culture and differentiated into infective metacyclic parasites and into intracellular and axenic amastigotes. In in vivo mouse infections, L. major VTC4 knockout showed a delay in lesion formation but ultimately gave rise to strong pathology, although we were unable to restore virulence by complementation to confirm this phenotype. Knockdown of VTC4 did not alter the course of L. guyanensis infections in mice, suggesting that polyP was not required for infection, or that very low levels of it suffice for lesion development. At higher temperatures, Leishmania promastigotes highly consumed polyP, and both knockdown or deletion of VTC4 diminished parasite survival. Thus, although polyP was not essential in the life cycle of the parasite, our data suggests a role for polyP in increasing parasite survival at higher temperatures, a situation faced by the parasite when transmitted to humans.

Inorganic polyphosphate (polyP) is an important factor of alkaline, heavy metal, and oxidative stress resistance in microbial cells. In yeast, polyP is synthesized by Vtc4, a subunit of the vacuole transporter chaperone complex. Here, we report reduced but reliably detectable amounts of acid-soluble and acid-insoluble polyPs in the Δvtc4 strain of Saccharomyces cerevisiae, reaching 10% and 20% of the respective levels of the wild-type strain. The Δvtc4 strain has decreased resistance to alkaline stress but, unexpectedly, increased resistance to oxidation and heavy metal excess. We suggest that increased resistance is achieved through elevated expression of DDR2, which is implicated in stress response, and reduced expression of PHO84 encoding a phosphate and divalent metal transporter. The decreased Mg2+-dependent phosphate accumulation in Δvtc4 cells is consistent with reduced expression of PHO84. We discuss a possible role that polyP level plays in cellular signaling of stress response mobilization in yeast.

Polyphosphorylation is a newly described non-enzymatic post-translational modification wherein long chains of inorganic phosphates are attached to lysine residues. The first targets of polyphosphorylation identified were S. cerevisiae proteins Nsr1 and Top1. Building on this theme, we recently exploited functional genomics tools in yeast to identify 15 new targets, including a conserved network of nucleolar proteins implicated in ribosome biogenesis. We also described the polyphosphorylation of six human proteins, suggesting that this unique post-translational modification could be conserved throughout eukaryotes. The study of polyphosphorylation seems poised to uncover novel modes of protein regulation in pathways spanning diverse biological processes. In this review, we establish a framework for future work by outlining critical questions related to the biochemistry of polyphosphorylation, its therapeutic potential, and everything in between. [ABSTRACT FROM AUTHOR]

To assess the ubiquity of the potential for inorganic polyphosphate (polyP) synthesis in microalgae, we searched databases for algal homologues to the polyP polymerase VTC4 of Chlamydomonas reinhardtii. Homologues of this protein were found within >40 species of microalgae known to inhabit marine, freshwater, and terrestrial environments. Phylogenetic analysis demonstrated that these proteins were organized into clades aligning with their taxonomic relationships. These similarities and evolutionary relationships suggest that polyP synthesis represents an ancient ability that has evolved with species as the microalgal lineage has spread out over time. Based on these results and prior knowledge on P metabolism, C. reinhardtii , Chlorella vulgaris , Desmodesmus cf. armatus , Gonium pectorale , and Microcystis aeruginosa were further tested in bioassays known to trigger the synthesis of polyP within dense granules, by addition of P following a period of P depletion. While the cellular P content of C. reinhardtii , G. pectorale , M. aeruginosa , and D. cf. armatus increased to similar maxima, ranging from 2.6 ± 0.5 % to 3.6 ± 1.3 % 24 h after P repletion, P content only reached 1.2 ± 0.2 % in C. vulgaris , suggesting a lesser ability to accumulate polyP than the strains of the other species. Models of predicted VTC4 proteins were generated from the four eukaryotic species tested and showed that the microalgae share the conserved VTC catalytic core and SPX phosphate-sensing domains found in the yeast VTC4 proteins. This confirms the role of microalgal VTC4 as polyP polymerase and suggests a similar regulation of VTC4 proteins to the one described in yeast. Further work is now needed to uncover the assembly of the microalgal VTC complex and its regulation. A deeper study of the microalgal VTC structure could also help to understand whether differences in VTC structures can explain observed differences in P accumulation kinetics. • Homologues of Chlamydomonas reinhardtii VTC4 protein were found in 48 algal species. • These proteins were organized into clades aligning with the species' taxonomic relationships. • Modelling of algal VTC4 structures shows conservation of the VTC and SPX domains. • For the first time, Gonium pectorale was shown to synthesize polyphosphate. [ABSTRACT FROM AUTHOR]

In diverse cells from bacterial to mammalian species, inorganic phosphate is stored in long chains called polyphosphate (polyP). These nearly universal polymers, ranging from three to thousands of phosphate moieties in length, are associated with molecular functions, including energy homeostasis, protein folding, and cell signaling. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In the budding yeast Saccharomyces cerevisiae, polyP synthesis by the membrane-bound v acuolar t ransporter c haperone (VTC) complex is coupled to its translocation into the lumen of the vacuole, a lysosome-like 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. In this study, 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 by 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. In contrast, the ectopic expression of previously proposed mammalian polyphosphatases did not impact PPK-mediated toxicity in this model, suggesting either that these enzymes do not function directly as polyphosphatases in vivo or that they require cofactors unique to higher eukaryotes. Our work provides insight into why polyP accumulation outside lysosome-like organelles is toxic. Furthermore, it serves as a resource for exploring how polyP may impact conserved biological processes at a molecular level. IMPORTANCE Cells from bacteria to humans have a molecule called polyphosphate (polyP) that functions in diverse processes. In many microbes, polyP is sequestered in granules or lysosome-related organelles such as vacuoles. In this study, we use an ectopic expression system to force budding yeast to accumulate polyP outside the vacuole. We use proteomics to demonstrate that this nonvacuolar polyP initiates a stress response mediated by a signaling cascade involving the Yak1 and Hog1 kinases and the Msn2 and Msn4 transcription factors. This response is countered by a pair of polyphosphatases with different enzymatic activities that function in concert to degrade polyP. Our results provide new insights into why polyP is confined to specific cell locations in many microbial cells.

Simple Summary: Inorganic polyphosphate, a linear polymer of orthophosphoric acid, plays an important role in microorganisms' stress resistance. Vtc4 protein synthetizes inorganic polyphosphate in yeast. Here we show that yeast lacking this protein exhibit very low polyphosphate level, decreased resistance to alkaline stress, but increased resistance to oxidative and heavy metal stresses. We suggest the increased stress resistance is achieved by constitutive up-regulation of oxidative stress-response genes and decreased expression of Pho84 that is responsible for manganese uptake. Inorganic polyphosphate (polyP) is an important factor of alkaline, heavy metal, and oxidative stress resistance in microbial cells. In yeast, polyP is synthesized by Vtc4, a subunit of the vacuole transporter chaperone complex. Here, we report reduced but reliably detectable amounts of acid-soluble and acid-insoluble polyPs in the Δvtc4 strain of Saccharomyces cerevisiae, reaching 10% and 20% of the respective levels of the wild-type strain. The Δvtc4 strain has decreased resistance to alkaline stress but, unexpectedly, increased resistance to oxidation and heavy metal excess. We suggest that increased resistance is achieved through elevated expression of DDR2, which is implicated in stress response, and reduced expression of PHO84 encoding a phosphate and divalent metal transporter. The decreased Mg2+-dependent phosphate accumulation in Δvtc4 cells is consistent with reduced expression of PHO84. We discuss a possible role that polyP level plays in cellular signaling of stress response mobilization in yeast. [ABSTRACT FROM AUTHOR]

Protozoan parasites contain negatively charged polymers of a few up to several hundreds of phosphate residues. In other organisms, these poly-phosphate (polyP) chains serve as an energy source and phosphate reservoir, and have been implicated in adaptation to stress and virulence of pathogenic organisms. In this study, we confirmed first that the polyP polymerase vacuolar transporter chaperone 4 ( VTC4 ) is responsible for polyP synthesis in Leishmania parasites. During Leishmania culture, polyP is accumulated in logarithmic growth phase and subsequently consumed once stationary phase is reached. However, polyP is not essential since VTC4-deficient ( in vitro culture, polyP is accumulated in logarithmic growth phase and subsequently consumed once stationary phase is reached. However, polyP is not essential since VTC4-deficient ( vtc4 - ) Leishmania proliferated normally in culture and differentiated into infective metacyclic parasites and into intracellular and axenic amastigotes. In in vivo mouse infections, L. major VTC4 knockout showed a delay in lesion formation but ultimately gave rise to strong pathology, although we were unable to restore virulence by complementation to confirm this phenotype. Knockdown of VTC4 did not alter the course of L. guyanensis infections in mice, suggesting that polyP was not required for infection, or that very low levels of it suffice for lesion development. At higher temperatures, Leishmania promastigotes highly consumed polyP, and both knockdown or deletion of VTC4 diminished parasite survival. Thus, although polyP was not essential in the life cycle of the parasite, our data suggests a role for polyP in increasing parasite survival at higher temperatures, a situation faced by the parasite when transmitted to humans., Competing Interests: Conflict of interest: The authors declare no conflict of interest.

Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called "polyphosphorylation" is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)