Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1 Introduction

Phosphorus is one of the most common elements on earth. In cell it exists in its phosphate form (PO4 3−) and plays fundamental and diverse roles. Phosphate performs a structural role in the charged backbone of nucleic acids as well as regulating cell behaviour through its ability to post-translationally modify proteins in signalling processes. Furthermore, phosphate groups linked together by the ‘high-energy’ phosphoanhydride bounds, as found in ATP, represent the principal biological energy store.

In eukaryote cells there are two groups of molecules especially rich in phosphate, inorganic polyphosphate (polyP) (Kornberg et al. 1999) and the inositol pyrophosphates (PP-IPs) (Saiardi 2012a). The polymer polyP contains from few to hundreds of phosphate residues linked by phosphoanhydride bonds (Fig. 5.1a). Although polyP is termed ‘inorganic’ – it contains no carbons – it is ubiquitously present in every living organism from bacteria to human. The cellular free phosphate concentration is buffered by polyP that due to its chelating property also regulates cation homoeostasis (Kornberg et al. 1999; Rao et al. 2009). Besides these basic attributes, polyP has chaperone-like activity (Gray et al. 2014) and regulates stress response (Jahid et al. 2006), ion channels (Zakharian et al. 2009; Seidlmayer et al. 2012) and infectivity (Moreno and Docampo 2013). Furthermore polyP appears to play a role in blood coagulation (Muller et al. 2009; Morrissey et al. 2012), though this function is debated (Faxalv et al. 2013).

Fig. 5.1
figure 1

Structure of inorganic polyphosphate and of typical inositol phosphates. Graphic representation of the minimal polyP structure (a, left) where n can range from 1 to several hundreds. Elongated representation of polyP molecule (a, right) showing the bonds targeted by the endopolyphosphatase activity of Ppn1 and Ddp1 and the exopolyphosphatase activity of Ppx1. Structure of myo-inositol (b, left); the dashed line between carbons 2 and 5 define the axis of symmetry on this molecule. Structure of the calcium-release factor IP3 (b, centre) generated by the action of phospholipase C. The structure of the ‘typical’ IP7 (b, right), the isomer 5PP-IP5 (5-diphosphoinositol pentakisphosphate) generated by IP6Ks. Circled P indicates the phosphate group (PO3 2−)

Full size image

Amongst the ‘organic’ (carbon containing) molecules, the PP-IPs are extraordinarily rich in phosphate. The best characterised inositol pyrophosphate, the diphosphoinositol pentakisphosphate (hereafter called IP7), is notable for its arrangement of seven phosphates groups around the six-carbon inositol ring (Fig. 5.1b). Moreover, PP-IPs containing eight, nine or even more phosphates groups have been described (Losito et al. 2009; Pisani et al. 2014). Because several inositol pyrophosphates have the unique property of possessing more phosphates than carbon atoms, it is not unexpected that they too have been linked to phosphate homoeostasis. In fact the literature provides numerous examples of PP-IPs controlling cellular phosphate metabolism (Saiardi 2012a, b). The inositol phosphates are involved in some way in virtually every physiological process. In fact, several biological processes are specifically regulated by PP-IPs (for review see (Wundenberg and Mayr 2012; Wilson et al. 2013; Shears 2015; Thota and Bhandari 2015)). These include the ability to regulate cellular energetic status by controlling ATP levels (Szijgyartovc et al. 2011). Similarly, many specific biological functions have been attributed to polyP, and several chapters of this book are dedicated to these roles.

The objective of the current chapter is to highlight the metabolic and functional connection between PP-IPs and polyP, since in the yeast Saccharomyces cerevisiae these two classes of molecule share a strong metabolic relationship (Auesukaree et al. 2005; Lonetti et al. 2011). We mainly, but not exclusively, discuss the roles and interconnection of these two molecules in lower eukaryote the yeast and the amoeba Dictyostelium discoideum. Since the readership of book is primary polyP oriented, we should start by presenting some inositol phosphate background.

2 Introduction to Inositol Polyphosphates

The metabolism and physiological roles played by PP-IPs cannot be properly appreciated without introducing the inositol phosphates family of molecules. An extensive exploration of the inositol phosphates is not the objective of the current essay; thus we invite the interested reader to read the following review (Irvine and Schell 2001; Resnick and Saiardi 2008). Here we aim to give some historical background and the key facts about inositol phosphates.

In living organisms the inositol sugar is almost exclusively in its myo-inositol isomeric arrangement, possessing one single axial hydroxyl group on positions two and five remaining equatorial hydroxyl groups, in the same plane as the sugar ring (Fig. 5.1b). The combinatorial substitution of phosphate moieties for the six hydroxyls imparts complexity and functional significance to the inositol phosphates. Over 40 different inositol polyphosphates have been observed in diverse eukaryotic organisms resulting in a fairly busy metabolic map (Irvine and Schell 2001; Resnick and Saiardi 2008). The best characterised inositol phosphate is the calcium-release factor I(1,4,5)P3 (IP3) (Fig. 5.1b). The paradigm of receptor activation with subsequent phospholipase C (PLC)-mediated release of IP3, followed by its binding to IP3 receptor and the release of calcium from intracellular stores, represents one of the most classical signal transduction pathways. In this cascade IP3 acts as the stereotypical ‘second messenger’ (Streb et al. 1983; Berridge et al. 2000; Irvine 2003).

After the establishment of IP3 signalling paradigm by the middle 1980s, an era of proliferation and discovery of novel inositol polyphosphates began. A simplified inositol phosphate metabolic pathway leading to the synthesis of PP-IPs is depicted in Fig. 5.2. Over the following years, it became clear that not all inositol polyphosphates are signalling molecules. In many cases the newly identified inositol phosphates simply represent transient metabolites towards the synthesis of more complex inositol phosphates (Saiardi and Cockcroft 2008). Nevertheless, some inositol phosphates do exhibit specific signalling roles, including Ins(3,4,5,6)P4, which regulates calcium-activated chloride (Cl−) channel (Shears 2009b). The inositol polyphosphate multikinase (IPMK in yeast known as Arg82, Ipk2) is the enzyme responsible for the synthesis of higher phosphorylated inositol phosphate, converting IP3 to the inositol pentakisphosphate isomer I(1,3,4,5,6)P5 (Saiardi et al. 1999, 2000b, 2001a; Odom et al. 2000). The formation of IP5 represents the penultimate step in the synthesis of the most abundant inositol phosphate on earth, Ins(1,2,3,4,5,6)P6 (thereafter called IP6) also known as phytic acid or inositol hexakisphosphate. Its synthesis is achieved via the phosphorylation of IP5’s lone remaining axial hydroxyl by an IP5 2-kinase (IPPK1 in yeast known as Ipk1) (York et al. 1999). The abundance of IP6 in nature is owed largely to its use by plants for phosphate storage in seeds. In fact this molecule was originally discovered in seeds (Posternak 1919), hence the name phytic acid (Raboy 2003).

Fig. 5.2
figure 2

Inositol pyrophosphate biosynthetic pathway. This simplified metabolic pathway illustrates the possible routes of inositol pyrophosphate biosynthesis starting from inositol. The yeast S. cerevisiae exclusively uses the lipid route to synthesise the PP-IPs derived from the fully phosphorylated ring of IP6 or IP7, or the inositol pyrophosphates derived from IP5 or PP-IP4. The lipid route requires the inositol to be converted into the lipid phosphoinositide phosphoinositol (PI), phosphoinositol phosphate (PIP) and phosphoinositol bisphosphate (PIP2). The social amoeba D. discoideum utilises a direct cytosolic route to synthesise higher phosphorylated inositol species, the enzymology of which is not fully elucidated (dashed line). The names of the kinases catalysing each enzymatic step are indicated in red (yeast) and blue (mammalian). Phospholipase (PLC1), inositol polyphosphate multikinase (IPMK, Arg82), inositol pentakisphosphate 2-kinase (PPK1. Ipk1), inositol hexakisphosphate kinase (IP6K, Kcs1), diphosphoinositol pentakisphosphate kinase (PPIP5K, Vip1)

Full size image

Even in non-plant eukaryotic cells, IP6 is present at an intracellular concentration ranging from 10 to 100 μM, making it the most abundant inositol phosphate species in these cells too, often exceeding most other inositol phosphate by an order of magnitude or more (Shears 2001). Higher concentrations have been observed in the slime mould D. discoideum where the IP6 concentrations can reach as high as 700 μM (Letcher et al. 2008; Pisani et al. 2014). Owing to its high charge density, IP6, as polyP, is a strong chelator that readily forms insoluble salts with polyvalent cations. The formation of these complexes can result in the precipitation of IP6 at higher concentrations. Under the cellular ionic conditions found in animal cells, soluble IP6 concentrations are limited to <50 μM (likely in the form of a neutral, stable pentamagnesium salt) (Torres et al. 2005; Veiga et al. 2006). Thus, it is likely that much of cellular IP6 is actually found in a ‘bound state’ or sequestered in vesicular compartments especially in slime mould.

In contrast to the rapid changes upon receptor activation of IP3, IP6 concentrations appear rather static, leading to the improper characterisation of IP6 as lethargic molecule. However, the identification of IP6 as a precursor to more phosphorylated inositol, the PP-IPs has revealed the true dynamic nature of cellular pool of IP6. Although it has been more than 30 years since the definition of IP3 as a second messenger, the PP-IPs continue to offer a different perspective to an already complex network of molecules (Burton et al. 2009; Shears 2009a).

3 Inositol Pyrophosphate Metabolism and Functions

The inositol pyrophosphates were identified in 1993 in D. discoideum and fluoride-treated pancreatoma cells (Menniti et al. 1993; Stephens et al. 1993). Subsequent studies confirmed the widespread evolutionary conservation of the PP-IPs. The best characterised of these ‘high-energy’ molecules are IP7 (or PP-IP5) and bis-diphosphoinositol tetrakisphosphate (IP8, PP2-IP4) species (Fig. 5.1b). In most cell types, these species are present in submicromolar concentrations, representing 1–5 % of the total IP6 levels. However, despite their low levels, the turnover of these molecules is remarkably rapid with up to 50 % of IP6 cycling through the PP-IPs every hour in mammalian cells (Glennon and Shears 1993; Menniti et al. 1993). Thus, the cell invests a considerable amount of energy to maintain the PP-IPs steady-state level; this is suggestive of a molecular switching property of the PP-IPs. Triphosphate-containing ‘IP8’ species (PPP-IP5) have also been observed (Draskovic et al. 2008). In addition to the inositol pyrophosphates derived from IP6, inositol pyrophosphates can also be derived from the pyrophosphorylation of IP5 and then called PP-IP4 and (PP)2-IP3 (Fig. 5.2). These IP5-derived PP-IPs are common constituents of mammalian cells where the cellular levels of IP5 and IP6 are similar (Wilson et al. 2013) and in the yeast ipk1Δ mutant that cannot synthesise IP6 and thus accumulate IP5 (Fig. 5.2).

The synthesis of PP-IPs has so far been attributed to two classes of enzymes, both conserved across the evolutionary spectrum. The IP6-kinases (IP6Ks) synthesise IP7 from IP6 pyrophosphorylating the inositol ring on position five (Saiardi et al. 1999). These enzymes can also utilise IP5 as substrate to generate PP-IP4. The yeast genome encodes a single IP6K, called Kcs1 (Saiardi et al. 2000a), while mammals possess three isoforms called IP6K1 to 3 (Saiardi et al. 2000b). The second class of enzymes able to synthesise PP-IPs is the PP-IP5Ks (Choi et al. 2007; Mulugu et al. 2007). In vivo, these enzymes mainly convert IP7 to IP8 by pyrophosphorylating the inositol ring on position one. In vitro, this enzyme can also metabolise IP6 although not IP5. Yeast possesses a single PP-IP5K called Vip1 (Mulugu et al. 2007), while mammals possess two isoforms. The amoeba D. discoideum possesses a well-defined IP6K (gene called I6KA) (Luo et al. 2003) but as well other inositol pyrophosphate synthesising enzymes not yet fully characterised (Tom Livermore, AS unpublished result 2015). Furthermore, the amoeba also possesses a single PP-IP5K type of enzyme (Tom Livermore, AS unpublished result 2015).

PP-IPs can be dephosphorylated by at least two phosphatase activities. Oddly, one of these activities is attributed to PP-IP5K, which besides its kinase domain possesses an acid phosphatase domain (Pohlmann et al. 2014; Wang et al. 2015). This domain is able to dephosphorylate PP-IPs in position one, removing the pyrophosphate moiety added by the kinase domain of the same protein. This remarkable ability to generate and degrade pyrophosphate moiety in position one is an area of intense research. The second phosphatases able to degrade PP-IPs belong to the Nudix family of enzymes. Once more, yeast has a single member of this family called Ddp1. At least three have been identified in mammalian genomes, denoted DIPP1 to 3 (Safrany et al. 1998; Kilari et al. 2013). These enzymes have a broad substrate specificity and are also able to degrade nucleotide dimers such A6pA and polyP (see below) (Lonetti et al. 2011).

Both yeast and amoeba are powerful genetic experimental models; the deletion by homologous recombination of Kcs1 and I6KA gene, respectively, generates organism depleted of PP-IPs (Saiardi et al. 2002; Luo et al. 2003). The analysis of these mutants revealed many processes regulated by PP-IPs. In D. discoideum PP-IPs regulate chemotaxis (Luo et al. 2003), while the better characterised kcs1Δ reveals a key role of PP-IPs in vesicular trafficking (Saiardi et al. 2002), telomere length regulation (Saiardi et al. 2005; York et al. 2005), epigenetic transcriptional control (Burton et al. 2013; Worley et al. 2013), ribosomal biogenesis (Horigome et al. 2009; Thota et al. 2015) and cell wall stability (Dubois et al. 2002). This large variety of functions is suggestive that PP-IPs are controlling a very basic cellular task alteration of which is manifested by this variety of phenotypes. The discovery that PP-IPs regulate yeast energetic metabolism, by repressing mitochondrial functionality and enhancing the glycolytic flux, gives an explanation to the diversity of phenotype attribute to PP-IPs (Wundenberg and Mayr 2012; Wilson et al. 2013). In fact the absence of PP-IPs in kcs1Δ yeast results in substantial elevated level of the central molecules of intermediate metabolism ATP (Szijgyartovc et al. 2011) that is virtually involved in any biochemical process taking place in living organism. It has been hypnotised that the ability of PP-IPs to regulate energy metabolism is manifested through their metabolic connection with polyP (Saiardi 2012b) as discussed below.

Intense debate surrounds the PP-IPs’ molecular mechanism of action. As is the case for other inositol polyphosphates, binding/allosteric mechanisms for inositol pyrophosphate functionality have been proposed (Luo et al. 2003; Chakraborty et al. 2010). However, the high energetic potential of the β-phosphates in inositol pyrophosphates suggests a unique mechanism for this class of inositol phosphates. Indeed, early characterisation of IP6-kinase demonstrated its capacity for ATP formation in the reverse by transferring a β-phosphate from IP7 to ADP (Voglmaier et al. 1996). More importantly IP7 appears to physiologically phosphorylate a variety of eukaryote protein targets. This ability allows IP7 to transfer its β-phosphate to a prephosphorylated serine generating pyrophosphorylated-serine residue (Saiardi et al. 2004; Bhandari et al. 2007; Azevedo et al. 2009). Studying protein pyrophosphorylation has led to the discovery of a new protein post-translational modification directly mediated by polyP protein polyphosphorylation (Azevedo et al. 2015) described below.

4 Inorganic Polyphosphate Eukaryote Metabolism

Bacteria were the organisms where polyP metabolism was originally explored with the identification of the polyP biosynthetic enzyme in the protein polyphosphate kinase (PPK) (Ahn and Kornberg 1990; Zhang et al. 2002). Uniquely in eukaryotes D. discoideum possesses a polyP biosynthetic route of bacterial origin (Zhang et al. 2005, 2007). The amoeba genome contains a PPK enzyme homologous to the bacterial gene, called DdPPK1, acquired by horizontal gene transfer facilitated by the amoeba’s bacterial feeding habit. This eukaryote PPK gene is present in a few related amoeba genomes belonging to the order of the Dictyosteliida; however it is unidentifiable by homology search in any other eukaryotic genome sequenced. For this reason there was great excitement when Kornberg’s laboratory biochemically purified and identified a second amoeba polyphosphate kinase activity called DdPPK2 in an actin and actin-related protein complex (Gomez-Garcia and Kornberg 2004; Hooley et al. 2008). While homologues of these proteins are present in any eukaryote genome, the identification of the exact peptides responsible for DdPPK2 activity by characterisation of the recombinant protein has not been accomplished, casting serious doubt on the real nature of the original purified DdPPK2 activity. In fact, recent characterisation of D. discoideum DdPPK1 null mutant revealed that the enzyme of bacterial origin is the only protein responsible for polyP synthesis in this amoeba (Livermore et al. 2016).

The second class of eukaryote where polyP synthesis has been elucidated is the yeast S. cerevisiae. In serendipitous fashion, the presence of polyP in the crystal structure of the cytosolic domain of subunit 4 of the vacuolar transporter chaperone complex (VTC) Vtc4 (or Phm3), which is important in regulating vacuolar membrane fusion (Muller et al. 2002), led to the discovery of an intrinsic polyphosphate polymerase activity (Hothorn et al. 2009). This vacuolar transporter chaperone complex consists of four subunits integrated into the vacuolar membrane; interestingly while the catalytic domain is located on the cytoplasmic side, polyP accumulates in the vacuolar lumen. Thus it has been proposed that the synthesis of polyP is coupled to its transport through a channel consisting of the VTC complex itself (Gerasimaite et al. 2014). The polyP translocation is dependent by the proton gradient generated by vacuolar membrane ATPase (V-ATPase or Vma2) since the vma2Δ mutant accumulates a reduce amount of polyP (Gerasimaite et al. 2014). Vtc4 homologous proteins, although absent in metazoa, can be identified in other protozoa such as in the order of Trypanosomatida (Lander et al. 2013). However, trypanosomes must possess a second enzyme able to synthesise polyP since the knockout of the Vtc4 homologous gene led to just a 20 % reduction in polyP levels (Lander et al. 2013). The genome of metazoa lacks Vtc4 or PPK1 homologous counterparts; thus the synthesis of polyP in higher eukaryotes is an area of active investigation.

Two different classes of phosphatase degrade polyP: the exopolyphosphatases removing the phosphate from the polyP terminus, shortening the polyP polymer by one unit (Fig. 5.1a), and the endopolyphosphatases cleaving internal phosphoanhydride linkages, generating two smaller polyP molecules (Fig. 5.1b). A yeast exopolyphosphatase Ppx1 is a very active protein that progressively hydrolyses linear polyP chains longer than three phosphates, releasing free phosphate indispensable for metabolic processes and therefore regulating cellular phosphate homoeostasis. Ppx1 does not metabolise pyrophosphate, ATP, the cyclic form of tripolyphosphate or PP-IPs. Ppx1 is a member of the so-called DHH phosphoesterase family characterised by the aspartic acid (D) followed by two histidine (H) signatures in domain three of the protein (Aravind and Koonin 1998). Phosphatases possessing this DHH signature represent a group of functionally related enzymes that include pyrophosphatases. Yeast Ppx1 is usually localised in the cytoplasm; however mitochondrial matrix localisation has been also observed (Andreeva et al. 2008). In humans the Ppx1 homologue is called h-prune or simply Prune. The characterisation of its biochemical activity revealed that Prune is a short-chain exopolyphosphatase. Interestingly, Prune has been associated with metastatic cancer progression since it is a binding partner of the metastasis suppressor nm23-H1; thus it has been proposed that Prune’s ability to metabolise polyP plays some kind of role in cancer (Tammenkoski et al. 2008).

The yeast endopolyphosphatase Ppn1 (also known as Phm5) cleaves long chains of polyP to generate shorter chains (Sethuraman et al. 2001). Purified Ppn1 is a homotetramer of a 35-kDa subunit that derives from the proteolytic cleavage of a 78-kDa polypeptide (674 amino acids). Although originally characterised as endopolyphosphatase, Ppn1 possesses both endo- and exopolyphosphatase activities. In the presence of magnesium, Ppn1 generates shorter chain polyP, while Co2+ stimulates free-phosphate release (Andreeva et al. 2015). The maturation by proteolytic cleavage and N-glycosylation is essential to obtain catalytically active Ppn1 proteins (Shi and Kornberg 2005). Thus the production of bacterially expressed recombinant Ppn1 has not been achieved. The need for an active and easy to express endopolyphosphatase is very important since there are pools of polyP that are not degraded by the single action of an exopolyphosphatase as Ppx1 (Wurst et al. 1995). Some polyPs appear to be protected possibly by the attachment of a protecting group at the polyP terminus (Kornberg et al. 1999).

A new yeast endopolyphosphatase, capable of being recombinantly expressed in bacteria, has been recently discovered in the gene named Ddp1 (Lonetti et al. 2011). This discovery is not surprising because endopolyphosphatase activity has been detected in the cytosol of the double mutant of ppn1Δppx1Δ (Lichko et al. 2008). Ddp1 stands for diadenosine and diphosphoinositol polyphosphate phosphohydrolase since this enzyme was originally characterised to degrade nucleotide analogues, such as diadenosine hexaphosphate (A6pA) and PP-IPs (Safrany et al. 1999). Thus Ddp1 is a multi-substrate phosphatase able to attack the phosphoanhydride bond of A6pA, PP-IPs and polyP (Lonetti et al. 2011). Ddp1 belongs to the Nudix (nucleoside diphosphate-linked moiety X) hydrolase family possessing a 23-amino acid catalytic domain denominated the MutT motif (McLennan 2006). In mammals, four putative Ddp1/DIPPs have been characterised: DIPP1, DIPP2, and DIPP3a/b. All four mammalian proteins present a robust polyP endopolyphosphohydrolase activity (Lonetti et al. 2011).

5 Inositol Pyrophosphate and Inorganic Polyphosphate Metabolic Connected

The fact that a common phosphatase Ddp1 (DIPPs in mammals) can metabolise both PP-IPs and polyP is indicative of a metabolic connection between these two classes of molecules. More compelling evidence of a metabolic link between polyP and PP-IPs came from genetic screening. Microarray analysis performed on arg82Δ and kcs1Δ strains revealed the constitutive expression of the secreted acid phosphatase Pho5 (El Alami et al. 2003). Accordingly, the kcs1Δ yeast strain was also shown to exhibit a reduced uptake of phosphate from the culture medium (Saiardi et al. 2004). Subsequently, a functional genome-wide screen, designed to identify secreted acid phosphatase activity, identified Plc1, Arg82 and Kcs1 as genes responsible for the constitutive expression of Pho5 (Auesukaree et al. 2005). By using 31P-NMR to detect polyP, this work also identified the pathway of PP-IPs synthesis as essential for the accumulation of polyP (Auesukaree et al. 2005).

In apparent conflict, the yeast ipk1Δ strain possesses normal levels of polyP, and although it lacks IP7, it does possess PP-IPs derived from IP5 (Fig. 5.2); thus the authors concluded that these molecules, PP-IP4 and/or (PP)2-IP3, regulate polyP metabolism, not IP7. However, systematic biochemical analysis of polyP by gel electrophoresis in a panel of PP-IP pathway mutants has revealed that any species of PP-IP is able to control the level of polyP (Lonetti et al. 2011). Furthermore, the dynamic turnover of PP-IPs is linked to polyP biosynthesis, with their levels fluctuating in tandem in response to manipulation of phosphate. The phosphate overplus assay, in which phosphorus is withdrawn and then resupplied to the growing medium of yeast, induces the reduction and subsequent resynthesis of both polyP and PP-IPs (Lonetti et al. 2011). Understanding how PP-IPs regulate polyP metabolism is an area of intense research (Saiardi 2012b), and since polyP is buffering cellular phosphates, this connection might have far-reaching implication.

In D. discoideum, preliminary analysis revealed that, to the contrary of yeast, the single deletion of IP6K does not result in a dramatic alteration of polyP metabolism (Tom Livermore, AS unpublished result 2015). However, while wild-type (WT) D. discoideum undergoes a dramatic developmental accumulation of polyP, the analysis of ppk1 null amoeba revealed a failure to accumulate polyP (Livermore et al. 2016). This strain revealed a compensatory increase in PP-IPs, demonstrating a reciprocal metabolic connection between PP-IPs and polyP (Livermore et al. 2016).

Nevertheless, the ability of PP-IPs to regulate polyP metabolism appears to be conserved in mammals since the analysis of the knockout ip6k1−/− mouse revealed a decrease in polyP abundance in blood platelet (Ghosh et al. 2013). Interestingly, regulation of mammalian phosphate homoeostasis, probably through polyP metabolism, has been associated to all the three mammalian IP6Ks. In fact, the IP6K2 was initially cloned while searching for a novel plasma membrane phosphate transporter and initially called Phosphate inorganic Uptake Stimulator (PiUS) (Norbis et al. 1997; Schell et al. 1999). Transfection of PiUS into Xenopus oocytes stimulated the uptake of radioactive phosphate, a result reminiscent of the reduced phosphate uptake of kcs1Δ yeast (Saiardi et al. 2004). Furthermore, single-nucleotide polymorphisms in the human IP6K3 promoter were identified as important for regulation of serum phosphate levels in humans (Kestenbaum et al. 2010). While the exact nature of the metabolic connection between PP-IPs and polyP/phosphate is still unclear, it appears obvious that deciphering this important link will help to clarify many PP-IPs and polyP attributes.

6 Inorganic Polyphosphate Function in Saccharomyces cerevisiae

The baker’s yeast S. cerevisiae is a free-living organism, exposing it to an ever-changing phosphate environment. Thus, yeast has developed a sophisticated phosphate regulation pathway, known as the PHO regulon. This pathway controls the phosphate needs of the cell from membrane transport to transcriptional response (Ogawa et al. 2000; Korber and Barbaric 2014). In phosphate-rich media (environment), yeast accumulates as much phosphate as possible for use in the event of entering a low-phosphate environment. However, the cellular concentration of free phosphate must remain fixed. Consequently, in phosphate-rich condition, phosphate is converted to the osmotically inert form of polyP.

Yeast primarily stores polyP inside the vacuolar lumen. This organelle possesses analogous characteristics of an acidocalcisome, being acidic and rich in calcium (Docampo et al. 2005; Gerasimaite et al. 2014). Besides in the vacuole polyP has been detected in the yeast cytosol, mitochondria and nucleus (Lichko et al. 2006a, b). The vacuolar accumulation of polyP is counterbalanced by the simultaneous accumulation of basic amino acid such as arginine and lysine as demonstrated in Neurospora crassa (Cramer and Davis 1984); however, the negative polyP charge has also been shown to be neutralised by bivalent cations, mainly calcium and spermidine (Kornberg et al. 1999). The compartmentalisation of polyP in the vacuole and the appropriate cytosolic neutralisation of its charge are essential to yeast well-being. In fact, the overexpression of Escherichia coli PPK enzyme in the yeast cytosol results in a marked toxicity (Gerasimaite et al. 2014).

While the discovery of Vtc4 as the yeast polyP synthesising enzymes is relatively recent (Ogawa et al. 2000; Hothorn et al. 2009), the VTC complex has been extensively studied. This complex has been shown to regulate vacuolar physiology, protein transport, membrane fusion and even microautophagy suggesting that some of these functions might be mediated by polyP. The null mutant of the endopolyphosphatase, ppn1Δ, accumulates long-chain polyP (Ogawa et al. 2000; Lonetti et al. 2011) and is defective in growth in minimal media. Meanwhile the double mutant of ppn1Δppx1Δ rapidly loses viability in stationary phase (Sethuraman et al. 2001), underlining a basic and generic role of polyP to maintain cell fitness.

7 Inorganic Polyphosphate Function in Dictyostelium discoideum

The experimental model organism D. discoideum was originally selected to study the development of multicellularity. This amoeba grows and replicates as a unicellular organism in favourable conditions. However, in stress conditions, it undergoes a developmental transition into a multicellular form. Upon starvation thousands of unicellular amoebas aggregate to form multicellular complexes, culminating in the formation of ‘fruiting body’, which is comprised of spore cells and stalk cells. The amoeba’s simplicity and its remarkable developmental behaviour have led the study of biological processes from transcription to signalling in this organism (Franca-Koh et al. 2006; Muramoto et al. 2012; Pisani et al. 2014).

In addition, the polyP research field is amongst the many to have taken advantage of this organism. In the early 1970s, the presence of polyP in D. discoideum was recorded by the pioneering work of Gezelius (1974). By the 1980s, the use of 31P-NMR allowed the direct detection of polyP in the amoeba spore extract (Klein et al. 1988). Twenty years later, the Kornberg laboratory biochemically characterised the two enzymes, DdPPK1 and DdPPK2, described above. The analysis of a ppk1 null strain generated in the Kornberg laboratory revealed that polyP regulates amoeba development, predation and the late stages of cytokinesis (Zhang et al. 2005, 2007). However, only about 3 % of PPK1 gene was deleted in this strain, and it cannot be excluded that a partially active DdPPK1 protein was still present in these cells.

Recently, a new D. discoideum ppk1 mutant line was generated, this time deleting about 69 % of the Ppk1 gene. This new mutant revealed a defect in vegetative growth, development and spore germination (Livermore et al. 2016). The defective spore germination observed in the new ppk1 mutant can be explained by the previously mentioned dramatic increase (more than 100-fold) in the level of polyP during development of D. discoideum. This accumulation of polyP occurs predominantly in the spore and plays a fundamental role during amoeba spore germination, as demonstrated by the reduction in germination efficiency in ppk1 cells.

In dormant cells, polyP can be primarily considered as a phosphate storage and/or cation-sequestering molecule. Therefore, it seems likely that catabolism of polyP in the amoeba spore plays a key role in releasing these essential molecules in germinating spores. In fact, polyP accumulation in the amoeba spore is reminiscent of role played by IP6 in plant seeds (Raboy 2003), where IP6 accumulates and acts as both a phosphate and cation store during seed germination.

However, in vegetative D. discoideum cells, polyP must play a more dynamic role, since the general fitness of ppk1 amoebae is substantially reduced. The analysis of ppk1 demonstrated the dependence of cellular energetic metabolism on polyP, since a 2.5-fold decrease in cellular ATP level was recorded in vegetative ppk1 amoeba. The unexpected increase in the level of polyP after treating cells with mitochondrial poisons (Livermore et al. 2016) revealed in amoeba a not-yet-understood connection between polyP and energetic metabolism confirming mammalian observation (Abramov et al. 2007). The observed compensatory increase of inositol pyrophosphates and ATP during ppk1 development supports a model in which there is a functional interplay between inositol pyrophosphates, ATP and polyP (Livermore et al. 2016).

8 Protein Polyphosphorylation

The novel protein post-translational modification (PTM) lysine polyphosphorylation offers a new perspective on polyP research, representing an ideal molecular mechanism by which polyP might regulate protein activities. Protein polyphosphorylation was discovered while studying protein pyrophosphorylation (Saiardi et al. 2004; Bhandari et al. 2007; Azevedo et al. 2009). This latter PTM consists of the transfer of the β-phosphate of IP7 to a prephosphorylated serine, generating a pyrophosphoserine residue. Therefore protein pyrophosphorylation must be directly dependent on PP-IPs’ cellular level. Accordingly, it was noticed that an in vitro target of IP7, Nsr1 (Saiardi et al. 2004) migrated on SDS-PAGE differently depending on the amount of IP7 present (Azevedo et al. 2009). In kcs1Δ extracts, which have no detectable amounts of IP7, Nsr1 migrated as a sharp band of ~67 kDa, while in WT extracts, Nsr1 showed a huge mobility shift with a smeary appearance migrating around ~130 kDa (Azevedo et al. 2015). Since, as discussed above, in yeast there is a direct correlation between the cellular levels of PP-IPs and polyP, it was thought that the dramatic mobility shift of Nsr1 might correlate with the presence of both PP-IPs and polyP in WT yeast, and conversely with their absence in kcs1Δ. To test the possibility that polyP was mediating Nsr1 mobility shift, vtc4Δ extracts were analysed; these extracts are lacking polyP but possess normal level of IP7. The observation that Nsr1 mobility on SDS-PAGE in vtc4Δ was similar to that in kcs1Δ indicated that the mobility shift of Nsr1 was directly dependent on the level of polyP and not on IP7 levels (Azevedo et al. 2015). Interestingly, polyphosphorylation was shown to be non-enzymatic, resulting from a nucleophilic attack on an internal polyP phosphate ester linkage. Non-enzymatic PTMs are very common, for example, cysteine nitrosylation, and are often dependent on certain features: metabolic status, specific structural determinants and/or the local environment. Indeed, polyphosphorylation occurs at lysine residues embedded in an acidic, serine-rich domain called the PASK (for polyacidic serine and lysine rich) cluster and depends on the polyP cellular level. Interestingly lysine is the amino acid subjected to the highest variety of PTMs due to its chemical properties (Azevedo and Saiardi 2014). Another way to control non-enzymatic PTMs is by regulating their removal. The exopolyphosphatase Ppx1 has been shown to depolyphosphorylate Nsr1. Thus, regulating Ppx1 expression or subcellular localization will regulate protein polyphosphorylation signalling.

Besides Nsr1, topoisomerase Top1 was also shown to be polyphosphorylated. Polyphosphorylation regulates the ability of Nsr1 and Top1 to interact; when both proteins are polyphosphorylated, in WT yeast, they fail to interact. On the contrary in kcs1Δ or vtc4Δ yeast when both proteins are non-polyphosphorylated, they interact. Mechanistically, topoisomerase binds and relaxes supercoiled DNA. Like polyP DNA is a highly negatively charged molecule. We can envisage a mechanism in which polyphosphorylated Top1 mimics Top1 binding to DNA. Thus it has been proposed that polyphosphorylation is acting as a molecular switch preventing Top1 association with DNA (Azevedo and Saiardi 2014). Upon requirement, Top1 would be depolyphosphorylated and thus become available to associate with nucleic acids. Since the nucleus and nucleolus in particular appear to possess a well-defined set of acidic proteins (Kuehl et al. 1986), protein polyphosphorylation might represent an additional layer of regulation of nuclear signalling. In this model polyphosphorylation mimics nucleic acid association, keeping the enzyme away from DNA/RNA, and once depolyphosphorylated, the proteins can then associate with DNA/RNA and become active.

9 Concluding Remarks

The polymer polyP has for many years been considered a molecular fossil with minor if any real functions. Similarly, until recently PP-IPs have been considered an interesting curiosity and nothing more. Research into these molecules is gaining momentum, and both polyP and PP-IPs have now been established as important fields of research. Elucidating polyPs’ and PP-IPs’ physiological roles may not only lead to the uncovering of fundamental processes, such as the molecular basis of phosphate homoeostasis, but could help combat human diseases. Both molecules have in fact been associated with cancer and metabolic disorders. To reach these goals, research using the yeast and the amoeba will be of foremost importance; many major polyP and PP-IPs discoveries are likely still to come from working with these simple eukaryotes.