Developmental Accumulation of Inorganic Polyphosphate Affects Germination and Energetic Metabolism in Dictyostelium Discoideum

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Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

Thomas Miles Livermorea, Jonathan Robert Chubba,b, and Adolfo Saiardia,1

aMedical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom; and bDepartment of Cell and Developmental Biology, University College London, London WC1E 6BT, United Kingdom

Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved December 11, 2015 (received for review October 2, 2015)

Inorganic polyphosphate (polyP) is composed of linear chains of The intact VTC complex is required for transport of polyP into the phosphate groups linked by high-energy phosphoanhydride bonds. vacuole lumen (15), leading to the accumulation of polyP in the However, this simple, ubiquitous molecule remains poorly under- vacuole. Both exo- and endo polyphosphatases have also been stood. The use of nonstandardized analytical methods has contrib- identified in yeast (16, 17). Furthermore, the yeast model has uted to this lack of clarity. By using improved polyacrylamide gel revealed a metabolic link between polyP and the phosphate-rich electrophoresis we were able to visualize polyP extracted from Dic- inositol pyrophosphates. In yeast, the levels of inositol pyrophos- tyostelium discoideum. We established that polyP is undetectable in phates closely correlate with the level of polyP, and yeast strains cells lacking the polyphosphate kinase (DdPpk1). Generation of this lacking inositol pyrophosphates possess almost no polyP (17). PolyP has also been documented in the social amoeba Dictyos- ppk1 null strain revealed that polyP is important for the general fit- telium discoideum. Its presence in amoebae was first recognized by ness of the amoebae with the mutant strain displaying a substantial biochemical extraction in the early 1970s (18). Subsequently, in the growth defect. We discovered an unprecedented accumulation of 31 late 1980s, the use of P-NMR analysis revealed that both InsP6 polyP during the developmental program, with polyP increasing and polyP are present in Dictyostelium spores (19). Besides more than 100-fold. The failure of ppk1 spores to accumulate polyP

yeast, the social amoeba is the only other eukaryote where the CELL BIOLOGY results in a germination defect. These phenotypes are underpinned enzymology of polyP synthesis has been studied. Kornberg’s by the ability of polyP to regulate basic energetic metabolism, dem- laboratory described two enzymes able to synthesize polyP in onstrated by a 2.5-fold decrease in the level of ATP in vegetative D. discoideum. The first, DdPPK1 (hereafter refer as Ppk1), was ppk1. Finally, the lack of polyP during the development of ppk1 identified by homology to the bacterial polyphosphate kinase mutant cells is partially offset by an increase of both ATP and ino- (PPK) sequence (20). Dictyostelids represent the only eukaryote sitol pyrophosphates, evidence for a model in which there is a func- class possessing a bacterial-like PPK, suggesting the gene was tional interplay between inositol pyrophosphates, ATP, and polyP. obtained by a horizontal gene transfer event (20). The second protein complex, named DdPPK2, was identified after biochemical purifi- inorganic polyphosphate | phosphate | metabolism | cation of polyphosphate kinase activity and was found to comprise inositol pyrophosphate | mitochondria one actin-related protein and two actin-associated proteins (21). Whereas the in vitro biochemical properties of these two enzymes have been studied (20, 22), little is known about D. discoideum polyP he simplest biological polymer is inorganic polyphosphate metabolism and the contribution of these proteins to in vivo polyP T(polyP), which consists of a linear chain of phosphates linked synthesis, or the physiological function of polyP. Therefore, we have by high-energy phosphoanhydride bonds (1). Regardless of its used a recently refined biochemical procedure to study polyP me- origin, polyP is ubiquitously present in today’s living organisms. tabolism in D. discoideum. Our analysis failed to detect the presence The synthesis, enzymology, and role of polyP are best character- of polyP in cells lacking the bacterial-like Ppk1. Importantly, we ized in the bacteria Escherichia coli (2). However, polyP is also observed a dramatic increase of polyP during amoebae development present in archaea (3), eukaryotes (4), and human cells (5). Whilst polyP is present in virtually all cellular compartments (6), it ac- Significance cumulates in acidocalcisomes, organelles common to bacteria and mammalian cells (7). The most basic biological polymer is a linear chain of linked PolyP has been implicated in a range of cellular processes over phosphate groups, simply called inorganic polyphosphate (polyP). recent years, but its most basic proposed function is to act as a By ablating polyP synthesis in the amoebae, Dictyostelium dis- buffer for the level of free phosphate (8). Phosphate buffering is coideum essential for general metabolism, and the synthesis or degradation , we discovered that polyP regulates basic metabolism, of polyP is able to safeguard the level of free phosphate in cells. In general fitness, and spore germination. We also discovered a addition, the polyanionic nature of polyP makes it a strong chelator massive increase in the level of polyP during developmental pro- of bivalent cations; thus polyP plays a key role in cation homeo- gression. Interestingly this accumulation is linked to the accumu- stasis. The chelation of calcium by polyP might even play a role as a lation of another family of phosphate-rich molecules, the inositol calcium sink, regulating calcium signaling (8). Besides these com- pyrophosphates. Thus, by using a genetic model, we reveal a link mon roles, many specific functions have been attributed to polyP in between polyP, ATP production, and inositol polyphosphates. eukaryotes (see reviews in refs. 9–11). Recently its ability to function as a protein-folding chaperone (12) and to drive a newly discovered Author contributions: T.M.L., J.R.C., and A.S. designed research; T.M.L. performed research; posttranslational protein modification, protein polyphosphorylation J.R.C. contributed new reagents/analytic tools; T.M.L., J.R.C., and A.S. analyzed data; and T.M.L., J.R.C., and A.S. wrote the paper. (13), have provided further insight into the mechanism of action of this simple polymer. The authors declare no conflict of interest. Little is known about the synthesis or metabolism of polyP in This article is a PNAS Direct Submission. eukaryotes. The yeast Saccharomyces cerevisiae is the only eukary- Freely available online through the PNAS open access option. otic model where polyP physiology has been properly characterized 1To whom correspondence should be addressed. Email: [email protected]. by a genetic approach. In yeast, polyP synthesis is carried out by a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. subunit of the vacuolar transporter chaperone (VTC), Vtc4 (14). 1073/pnas.1519440113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1519440113 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 with an accumulation in the spore ultimately affecting germination. endopolyphosphatase Ddp1 (17) and the exopolyphosphatase Ppx1 These defects are due to the influence of polyP on the energetic (16). This enzymatic treatment removed the negative smear gen- status of the cell and inositol pyrophosphate metabolism. erated by polyP standards as well as that present in cell extracts (Fig. 1). We further confirmed the smear to be due to polyP by treating Results with acid at high temperature (acid boiled), because phos- D. discoideum Possesses polyP. Several polyP detection methods lack phoanhydride bonds are rapidly degraded by these conditions. These specificity (23). Furthermore quantification of polyP using tradi- analyses demonstrated the presence of polyP in vegetative D. dis- tional biochemical assays, such as the PPK assay (8) or measure- coideum, not only from the standard laboratory strain AX2, but also ment of free phosphate (Pi) after polyP hydrolysis, are greatly in the natural isolate, NC4 (Fig. 1). By comparing the migration of D. discoideum impaired by the inability to extract polyP without copurifying other polyP with polyP standards, we estimate that amoe- ∼ phosphate rich molecules (see below). Therefore, the only approach bae possess polyP with an average length of 50 phosphate residues. to unambiguously establish the presence of polyP in cells is its visual detection once resolved by polyacrylamide gel electrophoresis Ppk1 the Only Enzyme Responsible for polyP Synthesis Control General D. discoideum (PAGE) (24). Furthermore the ability of the phosphoanhydride Fitness and ATP Levels. The characterization of two bonds of polyP to induce photobleaching of DAPI has improved the enzymes Ppk1 (20) and DdPPK2 (22) able to synthesize polyP in vitro sensitivity of this method over traditional Toluidine staining (25). suggested that both enzymes might contribute to the synthesis of We extracted polyP from vegetative growing D. discoideum AX2 polyP in vivo. DdPPK2 has been identified as an actin-related protein complex but the specific gene was not cloned. Given that the cells using phenol/chloroform, which also extracts RNA and inositol D. discoideum pyrophosphates, and resolved 70 μg of RNA by 20% PAGE. DAPI genome encodes 41 actins and actin-related pro- staining revealed the negatively stained smear typical of polyP- teins (26) it was impractical to systematically delete all of these genes. Because only one bacterial-like Ppk1 is present in the Dic- DAPI photobleaching in cell extracts (Fig. 1). tyostelium Our extraction procedure copurifies polyP and nucleic acids, genome, we deleted, by homologous recombination, 69% of the ORF of this gene (ID: DDB_G0293524) as confirmed mainly RNA; thus to be confident of the nature of the polyP negative by Southern blots (Fig. 2 A and B). We referred to the Ppk1 null smear, we subjected the extract to DNase and RNase treatment. strain as ppk1. Two independent clones were generated and used in Whereas RNase treatment removed the positive fluorescence visible our experiments. The homology between the 688 aa of E. coli at the top of the gel (Fig. 1), neither DNase nor RNase affected PPK1 and the 1,050 aa of D. discoideum Ppk1 is restricted to the C the negative staining. To test whetherpolyPwastheconstituent terminus; therefore, our knockout approach deleted >95% of the of this negative staining, we treated the samples with the Ppk1 catalytic domain (Fig. 2A). We first analyzed the general fitness of ppk1 cells by performing a growth assay in rich HL5 medium (Fig. 2C). We observed that ppk1 mutant cells have a growth defect, with a mean generation time of 15 h, compared with 11 h for wild type (WT), demonstrating the importance of Ppk1 for D. discoideum physiology. We next analyzed polyP content by resolving 90 μg of RNA/polyP extract from WT and ppk1 cells by PAGE, revealing the complete loss of the polyP negative stain in ppk1 (Fig. 2D). This analysis demonstrates that Ppk1 is in fact the only enzyme responsible for polyP synthesis in D. discoideum cells. Previous analysis of the level of polyP in ppk1 cells reported that polyP was merely reduced to 20–50% of the WT level (27). There are two explanations for this discrepancy: The first is the use of indirect assays to determine polyP levels. These assays developed using synthetic polyP fail to account for the effect of copurifying interfering molecules. PolyP extraction not only purifies polyP and RNA but also inositol phosphates (Fig. 2D), nucleotides (28), and likely free phosphate. The abundance of these phosphate-containing molecules influences these polyP quantification methods (8). The second explanation is that the previously generated ppk1 line deleted only 114 nt of 3,153 nt, just ∼3% of the Ppk1 ORF (27) and that this mutant retains active Ppk1 enzyme. This hypothesis is supported by the identification of PPK activity in this mutant (27). Because polyP has been associated with primary metabolism (29) and that any change to Pi buffering seems likely to affect basic metabolism, we decided to investigate whether the level of ATP was affected. In fact, ppk1 cells displayed a 2.5-fold reduction in ATP, offering a simple explanation for the overall fitness defect of these cells (Fig. 2E). We next analyzed the level of polyP after inhibiting both the cytochrome mediated respiration (with KCN) and the al- Fig. 1. D. discoideum polyP detected by PAGE analysis. Synthetic polyP stan- ternative oxidase (AOX) pathway with benzohydroxamate (BHAM) dards with an average chain length of 25 (T25) and 65 (T65) were loaded, either (30). Sublethal doses of these drugs induced a ∼40% decrease in the untreated or after incubation with the exo- and endopolyphosphatase Ppx1 and level of ATP in WT amoebae (Fig. 2G), but did not kill the cells Ddp1. Equal amounts of vegetative AX2 D. discoideum phenol chloroform ex- during the 2-h time course. During this time course, we also observed tract, as normalized RNA (70 μg) were treated with DNase, RNase, exo-, and F ppk1 endopolyphosphatase (Ppx1 and Ddp1) or treated in acid at high temperature an unexpected increase in polyP level (Fig. 2 ). Conversely, (acid boiled). Samples were analyzed by 20% PAGE and stained with DAPI. cells did not display any accumulation of polyP nor change in ATP levels (Fig. 2 H and I). Meanwhile, the levels of inositol pyrophos- Phenol extract from wild-isolate NC4 cells (Right lane) revealed the same type of F H polyP. The negative stained smears depend on the polyP property to induce DAPI phatewereunaffectedbythesetreatments(Fig.2 and ). photobleaching (25). The signal disappears after Ppx1 and Ddp1 incubation or acid treatment, whereas the white fluorescence reveals RNA species degraded by polyP Levels Increase During D. discoideum Development. D. discoideum RNase treatment. The results shown are of a representative experiment that was was originally selected as an experimental model to study the repeated more than three times. transition to multicellularity. Single cells undergo a defined

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Fig. 2. Ppk1 is the only polyphosphate kinase in D. discoideum affecting growth and basic metabolism. (A) Diagram showing the homologous recombination knockout strategy to generate ppk1 mutant cells. (B) Southern blot of WT and ppk1 genomic DNA. Genomic DNA was digested with BsrGI and probed for the 5′ region, as indicated in A.(C) Cell growth curves in HL5 media. WT, black line; ppk1, dashed line. Average ± SD of three experiments were run in duplicate (*P < 0.05). (D) Phenol chloroform extracts, normalized by loading 90 μg of copurified RNA, from vegetative growing WT and ppk1 cells were analyzed by 20% PAGE and stained with DAPI. The figure shows the result of representative experiments that were repeated more than three times. (E) Quantification of ATP levels in vegetative growing WT and ppk1 amoebae. Average ± SD of three experiments were run in duplicate (**P < 0.01). Phenol chloroform extracts, normalized by loading 65 μg of copurified RNA, from WT (F)andppk1 (H) cells treated with KCN and BHAM for the indicated times were analyzed by 20% PAGE and stained with DAPI. The results shown are of representative experiments that were repeated more than three times. Parallel ATP level determination, normalized to untreated control (time 0), of KCN- and BHAM- treated WT (G) and ppk1 (I) culture. The data represent the average ± SD of three experiments run in duplicate (*P < 0.05, **P < 0.01).

developmental process through a multicellular “slug,” culminating (Fig. 3 A and B). Analysis of ppk1 amoebae confirmed that Ppk1 is in a spore-containing fruiting body. We decided to investigate polyP the enzyme responsible for this accumulation of polyP (Fig. 3A). metabolism during the substantial physiological changes associated Analysis of the wild-isolate NC4 strain confirmed the striking accu- with D. discoideum development. Because the starvation response mulation of polyP during development (Fig. 3C). This accumulation induces D. discoideum development, we shifted a vegetative growing of polyP during the developmental progression is coherent with the culture from rich HL5 medium to nonnutrient (phosphate buffer) increased expression of the Ppk1 gene during the late stages of agar and collected cells at different time points corresponding to the development as reported by the DictyExpress database (https:// diverse developmental stages. PAGE analysis of phenol extract dictyexpress.research.bcm.edu/landing/). from WT amoebae revealed an impressive accumulation of polyP during development (Fig. 3A). Densitometry scanning of Toluidine- Ppk1 Is Required for Spore Germination. We did not observe any stained gels (Fig. 3B), which although less sensitive, offers a better developmental phase delay between WT and ppk1 cells. The dynamic range (31), showed the increase in polyP between vegeta- number of fruiting bodies formed was unaffected in the ppk1 tive stage and fruiting body was more than 100-fold. The average size background. The fruiting bodies of the mutant were correctly of polymers did not substantially change during this accumulation formed with identifiable basal disks, stalks, and spore heads.

Livermore et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 Fig. 3. D. discoideum accumulate polyP during development. (A)WTandppk1 cells were developed on nutrient-free agar and harvested at five time points. PolyP was extracted and 15 μg of RNA was analyzed by 20% PAGE and stained with DAPI. (B) A total of 70 μg of RNA WT cell extracts from five developmental time points was analyzed by 20% PAGE stained with Toluidine blue and analyzed by densitometry using ImageJ. (C) PolyP was extracted from NC4 cells at three time points during development, analyzed by 20% PAGE, and stained with DAPI. The results shown are of representative experiments that were repeated more than three times.

However, the overall size of the fruiting bodies was considerably level of inositol pyrophosphates (17). These molecules (InsP7 and reduced (Fig. 4A). Because fruiting bodies consist of two cell types, InsP8) are notable for their ability to control cellular ATP synthesis we decided to investigate whether polyP accumulates in spores or (32, 33). Because polyP influences the level of ATP in vegetatively stalk cells. We isolated pure preparations of spore cells and com- growing cells (Fig. 2E), we wondered if the massive absence of polyP pared polyP levels extracted from spores to those extracted from (and therefore phosphoanhydride bonds) during development of whole fruiting bodies. We normalized between these samples by ppk1 cells (Fig. 3A) might affect inositol pyrophosphate synthesis or spore number and extracted from 5 × 106 spores in each case. This ATP production. analysis revealed that a large proportion of polyP accumulates in The analysis of inositol phosphates extracted from WT and thespore(Fig.4B). ppk1 cells revealed that the ratio of InsP7 and InsP8 to their The storage of polyP in the spores suggested an important role precursor InsP6 is significantly altered (Fig. 5A). The quantifi- for this polymer during spore germination, potentially acting as a cation of several experiments revealed that ppk1 fruiting bodies store of phosphate, minerals, or energy. In fact, analysis of spore accumulated more than twice the level of inositol pyrophos- germination in rich HL5 medium revealed a germination delay in phates compared with WT (Fig. 5B). Although we were techni- the mutant strain (Fig. 4C). The polymeric nature of polyP also cally unable to quantify ATP in the later stages of D. discoideum offers clear osmotic benefit due to its ability to complex with development, we observed that during the initial phases of devel- counterions. Thus, polyP synthesis/degradation can also buffer opment ATP level in WT cells remained largely unchanged. osmotic pressure by capturing/realizing Pi and cations. We in- Meanwhile, the level of ATP in ppk1 cells was substantially in- vestigated the effect of osmotic stress on germination and found creased (Fig. 5C) and reached levels slightly exceeding those of WT that the defect observed between WT and ppk1 cells was ampli- amoebae. These results indicated that when polyP cannot be syn- fied in media supplemented with 0.2 M sorbitol (Fig. 4D). thesized, as in ppk1, cells accumulated inositol pyrophosphates InsP7 and InsP8. This perhaps represents a partial compensation, in Altered ATP and Accumulation of Inositol Pyrophosphates in ppk1 Mutant which energy is stored in phosphoanhydride bonds of inositol py- Spores. In yeast, the level of polyP is metabolically connected to the rophosphates rather than polyP. It is clear that the additional energy

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1519440113 Livermore et al. Downloaded by guest on September 28, 2021 PAGE analysis with the sensitivity and specificity of DAPI staining to reliably detect polyP in D. discoideum. Furthermore, we were unable to detect any polyP in cells lacking Ppk1. Earlier work described the involvement of Ppk1 in fruiting body development as well as an effect on the predation behavior of the amoebae (27). In this study, we generated a ppk1 mutant amoeba by deleting ∼70% of the Ppk1 ORF, revealing many previously unrecognized phenotypes. A modest developmental increase of polyP was previously noted (27); however, our analysis has revealed an unprecedented >100-fold increase in the level of polyP during D. discoideum development. The astonishing polyP increase and accumulation in the spore is reminiscent of InsP6 (phytic acid) accumulation in plant seeds (35). These phosphate-rich molecules share similar biophysical characteristics, including the ability to chelate di- valent cations. Therefore, as InsP6 is important to supply phosphate and cations during plant seed germination (35), polyP might play similar roles during spore germination as demonstrated by the reduction in germination efficiency in ppk1 cells. Because polyP can act as a Pi donor and buffer, it follows that polyP might regulate ATP metabolism. In yeast, a genome-wide screen revealed the interdependence of polyP with primary metabolism (29). In mammalian cells, alteration of mitochondrial metabolism affects polyP production (36). It has also been suggested that polyP is an activator or even a constituent of the CELL BIOLOGY

Fig. 4. ppk1 cells show impaired fruiting body formation and spore germination. (A) Representative images of fruiting bodies from WT and ppk1 cells. Images were taken at the same magnification. (B) Analysis by 20% PAGE of polyP extracted from whole fruiting bodies and pure spore preps, stained with DAPI. Time courses of spore germination in HL5 media (C)andHL5mediasupplemented with 0.2 M sorbitol (D)over24h.WTblackline;ppk1 dashed line. The date represent the average ± SD of three experiments run in duplicate (*P < 0.05).

stored in the phosphoanhydride bonds of InsP7 and InsP8 in ppk1 will not be equal to that accumulated in WT polyP. However, these results do confirm a metabolic interplay between these two families of phosphate-rich molecules and their ability to regulate or be regulated by cellular energy metabolism.

Discussion Fig. 5. Developmental failure to accumulate polyP affects inositol pyro- The ability of polyP to covalently modify proteins (13) and to phosphate and ATP metabolism. (A) Analysis by 35% PAGE of perchloric acid work as a chaperone (12) have recently boosted interest in this extract from WT and ppk1 cells during development, stained with Toluidine blue. (B) Quantification of the ratio between inositol pyrophosphates (InsP and polymer. Certainly polyP is no longer the “forgotten polymer” (34) 7 InsP8) and their precursor, InsP6 in fruiting bodies. Quantification by densitom- it was a few years ago. The study of polyP has long been inhibited by etry using ImageJ software, results represent the average ± SD of three in- the lack of reliable techniques, a fact not lost on Kornberg et al. who dependent experiments (***P < 0.0001). (C) Quantification of fold change in commented on “the inadequacy of methods to establish the au- ATP between vegetative cells and aggregating cells starved for 9 h. The data thenticity and size of polyP” (8). Here we have coupled high-quality represent the average ± SD of six experiments run in duplicate (*P < 0.05).

Livermore et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 mitochondrial permeability transition pore (37). Using our thus it is the ideal experimental model to investigate the metabolic clean genetic system, we demonstrated the interdependence between connection between these molecules. The yeast vtc4Δ strain and the polyP and cellular energetic metabolism, through the substantial ppk1 amoebae are the only two eukaryotic models in which a ge- decrease in cellular ATP in vegetative ppk1 cells. The unexpected netic approach has eliminated polyP. Whereas both experimental increase in the level of polyP after treatment with mitochondrial models offer distinctive advantages, the larger size of amoebae, poisons KCN and BHAM, revealed a further linkage between polyP absence of a cell wall, and a polyP level more similar to mammalian and energetic metabolism. Nevertheless, these results do appear cells, make D. discoideum an excellent model to dissect polyP counterintuitive as these drugs combine to decrease ATP levels. It is functions and the ideal model to develop more specific probes for possible that our treatment induced a starvation-like response and localizing polyP in the cell. thus an increase in polyP. However, we did not observe any signifi- cant change in ATP level during starvation, and mitochondrial Materials and Methods functionality actually increases during development (38). An alter- native explanation could be that an increase of polyP in cells with Detailed methods are provided in SI Materials and Methods. inhibited mitochondria might represent some compensatory mecha- Inorganic polyphosphate was extracted from cell pellets at indicated time nism. In bacteria, polyP complexes with β-hydroxybutyrate (PHB) to points. Cells were resuspended in one volume of LETS buffer (100 mM LiCl, 10 mM form calcium selective ion channel (39). This channel induces con- EDTA, 10 mM Tris·HCl, pH 8.0, 0.5% SDS), one volume of acidic phenol (pH 4.0) was ductance states similar to those recorded for the mitochondrial added, and samples were vortexed at 4 °C for 5 min. Samples were spun at permeability transition pore (37). Thus, it has been proposed that 17,000 × g for 5 min at 4 °C and aqueous phase was recovered. Two volumes polyP plays a key role in defining the mitochondrial permeability of chloroform were added and samples were again vortexed at 4 °C for transition pore (37). These counterintuitive results invite further work 5 min before spinning at 5,000 rpm for 5 min. The aqueous phase was col- with D. discoideum to define the precise role of polyP in mitochon- lected and precipitated by adding 2.5 volumes of EtOH and incubating at drial function and the mitochondrial permeability transition pore. −80 °C for 1–2h.Afterspinningat17,000× g for 10 min, pellets were resuspended The compensatory increase of inositol pyrophosphates and in 10 mM Tris·HCl, pH 7.4, 1 mM EDTA, and 0.1% SDS. RNA concentration was ATP observed during ppk1 development is supportive of a model measured by nanodrop and used to normalize samples before loading on in which there is a functional interplay between inositol pyro- PAGE. Inositol pyrophosphates were extracted as described before (31). phosphates, ATP, and polyP. This nascent hypothesis requires further work to be fully validated. However, our characterization ACKNOWLEDGMENTS. We acknowledge the contribution of members of of D. discoideum polyP metabolism and the development of ppk1 the A.S. laboratory for discussion and the core staff at the Laboratory for Molecular Cell Biology (LMCB) for facilitating our research. This work was cells, offer the unique platform to investigate the link between supported by the Medical Research Council (MRC) core support to the MRC/ polyP and ATP production. It is noteworthy to remember that University College London LMCB University Unit (MC_UU_1201814) (to A.S.). inositol pyrophosphates were discovered in D. discoideum (33); J.R.C. is supported by a Wellcome Trust Senior Fellowship (WT090904).

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