PI 5 P and PI ( 3 , 5 ) P 2 : Minor , but Essential Phosphoinositides

In most eukaryotes, phosphoinositides (PIs) have crucial roles in multiple cellular functions. Although the cellular levels of phosphatidylinositol 5-phosphate (PI5P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) are extremely low relative to some other PIs, emerging evidence demonstrates that both lipids are crucial for the endocytic pathway, intracellular signaling, and adaptation to stress. Mutations that causes defects in the biosynthesis of PI5P and PI(3,5)P2 are linked to human diseases including neurodegenerative disorders. Here, we review recent findings on cellular roles of PI5P and PI(3,5)P2, as well as the pathophysiological importance of these lipids.


Introduction
Cellular organelles, including endosomes, lysosomes, and mitochondria, are membrane-bound compartments that maintain specialized sub-environments within the cell.Membranes are composed of proteins and lipids.Membrane lipids include cholesterol, glycerophospholipids, and sphingolipids that include sphingomyelin (van Meer et al., 2008), which function in part to maintain the structural integrity of organelles.In addition, some membrane lipids function as key effectors that regulate intracellular signaling as well as remodeling of the cytoskeleton (Eyster, 2007;Fernandis and Wenk, 2007).
Phosphorylated derivatives of phosphatidylinositol, called phosphoinositides (PIs), form a family of acidic phospholipids embedded in membranes.Despite their rarity compared to other phospholipids such as phosphatidylcholine and phosphatidylethanolamine (phosphatidylinositol makes up approximately 10-20% of total cellular phospholipids), PIs have an indispensable role in cellular processes.Seven PIs, differing in phosphorylation at the 3-, 4-, and 5hydroxyls of the inositol head group, have been detected in cells (Di Paolo and De Camilli, 2006;Sasaki et al., 2009;Mayinger, 2012;Balla, 2013;Schink et al., 2016).Production and turnover of PIs by phosphorylation and dephosphorylation is spatiotemporally regulated through the controlled action of PI kinases, phosphatases, and phospholipases (Fig. 1).PIs are localized at cytosolic leaflet of distinct membranes (Fig. 2) and can recruit effector proteins to membranes to modulate intracellular signaling as well as membrane dynamics (Balla, 2013;Tsujita and Itoh, 2015;Schink et al., 2016).
Determination of the functions of phosphatidylinositol 5phosphate (PI5P) or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ), has advanced slowly relative to other PIs, due to their low abundance and limitations in their detection including a lack of bioprobes specifically recognizing these lipids.Moreover, there are no methods currently available to solely deplete PI5P or PI(3,5)P 2 .Despite these limitations, hundreds of recent studies demonstrate that these minor PIs have important and intriguing cellular roles.This review summarizes and discusses current findings on the physiological and pathological roles of PI5P and PI(3,5)P 2 .

Phosphatidylinositol 5-phosphate (PI5P)
Regulation of PI5P synthesis and turnover PI5P is the most recently detected of the seven known PIs, and is present at levels similar to PI3P (approximately 0.5% of total PI) in mammalian cells (Zolov et al., 2012;McCartney et al., 2014b).PI5P was discovered in mammalian NIH-3T3 cells in 1997 (Rameh et al., 1997).The same report revealed that, in vitro, PI5P is a substrate of type II PIP kinase (PI5P 4-kinase), which generates PI(4,5)P 2 .Most cellular pools of PI5P depend on the activity of the lipid kinase PIKfyve.Direct phosphorylation of PI at the 5position by PIKfyve has been proposed to be the primary source of PI5P (Sbrissa et al., 1999(Sbrissa et al., , 2002).An alternative model proposes that most PI5P is generated by PIKfyve indirectly.According to the indirect model, most pools of PI5P are derived from 3-phosphatase mediated dephosphorylation of PI(3,5)P 2 , which is produced by PIKfyve catalyzed phosphorylation of PI3P.(Schaletzky et al., 2003;Tronchère et al., 2004;Vaccari et al., 2011;Zolov et al., 2012;Oppelt et al., 2013).Importantly, in either scenario, depletion or inhibition of PIKfyve simultaneously lowers the cellular levels of PI5P and PI(3,5)P 2 .
Cellular levels of PI5P can be altered by pathogen infection.Some virulence factors of invasive bacterial pathogens change the host levels of PI5P through the type III secretion system (Norris et al., 1998;Marcus et al., 2001;Niebuhr et al., 2002).These factors facilitate invasion of host cells by manipulating host phospholipid metabolism in order to direct actin remodeling and modify their uptake machinery (Cossart and Sansonetti, 2004).The Shigella flexneri virulence factor IpgD converts PI(4,5)P 2 to PI5P in vitro and in vivo (Niebuhr et al., 2002).Interestingly, expression of IpgD in mammalian cells elevates intracellular levels of PI5P as measured by HPLC (Niebuhr et al., 2002;Pendaries et al., 2006;Viaud et al., 2014) or by an in vitro mass assay (Vaccari et al., 2011).Exogenous expression of IpgD to increase PI5P levels has been used to analyze the function of PI5P in mammalian cells.However, it is not known whether these increased pools of PI5P are produced at the proper intracellular location.The homologues of IpgD in Salmonella dublin and Salmonella typhimurium are SopB and SigD, respectively (Norris et al., 1998;Marcus et al., 2001).Each of these virulence factors contains the consensus sequence for 4-phosphatases (C-X-X-X-X-X-R; X is not an acidic residue), and prefer PI(4,5)P 2 as a substrate over other PIs, supporting the model that these virulence factors generate PI5P from PI(4,5)P 2 .A search for similar proteins in mammalian databases led to the identification of type I and type II PI(4,5)P 2 4-phosphatases in mammalian cells (Ungewickell et al., 2005).Both proteins dephosphorylate PI(4,5)P 2 to generate PI5P in vitro.These 4phosphatases localize to lysosomes and are involved in epidermal growth factor (EGF) receptor degradation, suggesting that these enzymes, and perhaps the lipid species they produce, are involved in degradation pathways.However, in vivo conversion of PI(4,5)P 2 to PI5P by these phosphatases has not yet been demonstrated and their mechanistic roles remain to be determined.
PI5P 4-kinases (PI5P4Ks) are conserved from flies and worms to humans.Mammals have three PI5P4K isoforms (α, β, and γ) (Lecompte et al., 2008;Clarke and Irvine, 2013).PI5P4Ks prefer PI5P as a substrate and can generate PI(4,5)P 2 in vitro and in vivo (Rameh et al., 1997;Roberts et al., 2005;Sarkes and Rameh, 2010).The contribution of these PI5P4Ks to total intracellular levels of PI(4,5)P 2 is reported to be minor and it has been proposed that their primary cellular function might be to reduce PI5P.

Changes in PI5P levels upon stress
Cellular levels of PI5P can be altered by extracellular stimulation.For instance, thrombin induced platelet aggregation at sites of vascular injury is associated with a 3-fold increase in the levels of PI5P as measured by an in vitro mass assay (Morris et al., 2000).Interestingly, the increase of PI5P is acute (peaking at 5 minutes after stimulation), suggesting that this lipid may stimulate platelet aggregation.Hyperosmotic stress has been shown to increase the PI5P levels in plants (Meijer et al., 2001) and hypo-osmotic shock induces a significant decrease of PI5P levels in mammalian cells (Sbrissa et al., 2002), as measured by an in vitro mass assay.Changes in PI5P levels resulting from osmotic stress are acute (within 10 minutes), implying that PI5P plays early roles in adaptation to osmotic shock.In response to oxidative stress, such as hydrogen peroxide (H 2 O 2 ), PI5P levels are transiently elevated, whereas the levels of PI(4,5)P 2 are decreased as measured by HPLC (Sarkes and Rameh, 2010;Jones et al., 2013), suggesting that the source of H 2 O 2 -induced PI5P is PI(4,5)P 2 .In addition, exposure to insulin causes a transient increase of PI5P in 3T3-L1 adipocytes by an in vitro mass assay (Sbrissa et al., 2004), CHO cells stably expressing insulin receptor by HPLC (Sarkes and Rameh, 2010), and skeletal muscle cells by an in vitro mass assay (Grainger et al., 2011).Together these data suggest that PI5P is an important second messenger regulating intracellular signaling pathways in response to stress.However, the mechanisms of the synthesis and turnover of PI5P during diverse stresses are not known.

Intracellular localization of PI5P
PI5P was initially discovered to have a signaling role in the nucleus (Clarke et al., 2001;Gozani et al., 2003;Jones et al., 2006).A plant homeodomain (PHD) finger of the chromatin-associated protein ING2 strongly binds to PI5P in vitro and in vivo.Importantly, the interaction of ING2 with PI5P regulates a p53-dependent apoptotic pathway, suggesting that ING2 functions as a nuclear receptor for PI5P (Gozani et al., 2003).Furthermore, PI5P4Kβ, which localizes to the nucleus (Ciruela et al., 2000), modulates nuclear levels of PI5P, regulating the association of ING2 and chromatin in response to cellular stress such as UV irradiation (Jones et al., 2006).Notably, nuclear levels of PI5P increase in cells during the G1 phase of the cell cycle (Clarke et al., 2001), indicating that this lipid might be involved in regulation of the cell cycle.
Biochemical subcellular fractionation, combined with HPLC detection, revealed that PI5P is also present at the plasma membrane as well as on non-nuclear intracellular membrane compartments including the endoplasmic reticulum and Golgi apparatus (Sarkes and Rameh, 2010).Consistent with this finding, several reports indicate relationships between PI5P and the plasma membrane as well as the endomembrane system.The localization of the PI5P binding peptide ING2 PHD indicates that PI5P is concentrated at host cell entry sites during early phases of infection by S. flexner, which involve injection of the virulence factor IpgD to remodel the actin cytoskeleton (Niebuhr et al., 2002;Pendaries et al., 2006).The localization of PI5P at the plasma membrane and early endosomes is also supported by recruitment of the PI5P binding protein Tiam1 to these compartments (Viaud et al., 2014).Upregulation of PI5P induced by introduction of the virulence factor IpgD enhances the internalization of the EGF receptor from the cell surface to early endosomes, but suppresses the maturation of early endosomes to late endosomes (Ramel et al., 2011;Boal et al., 2015).Moreover, elevation of PI5P alters actin and membrane dynamics at the plasma membrane to promote cell migration (Oppelt et al., 2013;Viaud et al., 2014).
PI5P has also been reported to be involved in autophagy.A recent study shows that PI5P localizes to autophagosomes and activates autophagy (Vicinanza et al., 2015).Moreover, this report shows that depletion of PI5P4Ks, especially PI5P4Kγ, which phosphorylates PI5P to produce PI(4,5)P 2 , enhances autophagy and reduces aggregates of mutant huntingtin associated with Huntington's disease.
Taken together, these data suggest PI5P is localized to the nucleus and to non-nuclear membranes including the plasma membrane and early endosomes.It is clear that, although cellular levels of this lipid are quite low compared to other PIs, PI5P plays crucial roles at each of these compartments including remodeling of the actin cytoskeleton, endocytosis, and nuclear signaling.

Physiological and pathological roles of PI5P
Insulin stimulation, which leads to increased cellular PI5P (Sbrissa et al., 2004;Sarkes and Rameh, 2010;Grainger et al., 2011), causes translocation of the glucose transporter GLUT4 from intracellular compartments to the plasma membrane enhancing glucose uptake from extracellular fluid (Leney and Tavaré, 2009).Intriguingly, the microinjection of PI5P, but not other PIs, into cells induces GLUT4 translocation to the plasma membrane (Sbrissa et al., 2004;Grainger et al., 2011).Moreover, insulin-induced GLUT4 localization to the cell surface is completely blocked in 3T3-L1 adipocytes expressing ING2 PHD (Sbrissa et al., 2004), a PI5P binding peptide predicted to interfere with PI5P binding to appropriate effectors.In addition, the increase in PI5P levels and glucose uptake induced by insulin stimulation are abolished in L6 myotubes by the expression of PI5P4Kα, which removes PI5P by phosphorylating it to generate PI(4,5)P 2 (Grainger et al., 2011).These studies demonstrate that PI5P is a key factor regulating insulininduced glucose uptake through the translocation of GLUT4 vesicles in both adipocytes and myotube cells.
As described above, virulence factors secreted by some pathogens alter specific host pathways through elevation of PI5P.IpgD from S. flexneri generates PI5P from PI(4,5)P 2 inducing membrane blebbing and actin remodeling (Niebuhr et al., 2002).Moreover, ectopic expression of IpgD or the addition of short-chain penetrating PI5P induces the activation of the PI 3-kinase/Akt pathway, which promotes long-term survival of the host cells, potentially facilitating efficient replication of the bacteria (Pendaries et al., 2006).It was also recently reported that S. flexneri infection accelerates the internalization and degradation of the intercellular adhesion molecule-1 (ICAM-1), an essential factor in immune cell recruitment, in an IpgDdependent manner (Boal et al., 2016).Importantly, the reduction of cell surface ICAM-1 by IpgD decreases neutrophil recruitment to the apical side of the epithelial cells, suggesting that pathogen induced increases in PI5P in host cells may serve to protect infected cells from the immune system.Thus, PI5P production (or decreased PI(4,5)P 2 ) in the host cell is beneficial for invasive bacteria on multiple levels.PI5P and the factors responsible for its production including IpgD are therefore potential drug targets for the treatment and prevention of bacteria-associated diseases.T cells, a major cell type in the immune system, are strongly activated through T cell receptor (TCR) stimulation by antigen presenting cells, such as dendritic cells and macrophages, promoting T cell maturation.The stimulation of TCR on T cells induces the activation of protein kinases including Akt and mTORC1, which control cell adhesion and gene expression (Acuto et al., 2008).Downstream of tyrosine kinase (Dok) proteins Dok-1 and Dok-2 also regulate T cell signaling (Acuto et al., 2008).It has been reported that the PH domains of Dok-1/Dok-2 are essential for them to be phosphorylated and for T cell signaling (Guittard et al., 2009).Interestingly, both PH domains bind to PI5P in vitro and elevation of PI5P through expression of IpgD results in an increase in tyrosine phosphorylation on Dok-1/Dok-2.In addition, IpgD-induced increases in PI5P cause the activation of Src kinase and Akt, and stimulate interleukin II promoter activity in T cells (Guittard et al., 2010).These studies indicate that PI5P is required to maintain T cell homeostasis through modification of T cell signaling.
Several pathogen components, such as lipopolysaccharides (LPS; a major component of the outer cell wall of gram-negative bacteria) and dsRNA from the genomes of dsRNA viruses, are detected by innate immune receptors including Toll-like receptors (TLRs) (Kawai and Akira, 2011;Kondo et al., 2012).Stimulation by either LPS or poly I:C (a synthetic analog of dsRNA), to mimic infection, results in increased cellular PI5P (Kawasaki et al., 2013).The suppression of PIKfyve, which is either directly or indirectly responsible for PI5P generation, impairs intracellular signaling triggered by TLRs, such as activation of interferon regulatory factor and NF-κB transcription factor, both of which control expression of the cytokine genes (Kawasaki et al., 2013).In addition, exogenous administration of synthetic PI5P induces the production of interferon β.These data suggest that PI5P is a critical signal in the innate immune response.

PI5P-binding proteins and their potential use as PI5P probes
Several proteins/domains bind to PI5P in vitro (Table I).The PHD finger of ING2 (ING2 PHD) is a potent binder of PI5P both in vitro and in vivo (Gozani et al., 2003).ING2 PHD is commonly used as a probe to investigate PI5P localization and function.However, this peptide also weakly binds PI3P and PI4P in vitro, which raises questions of whether ING2 PHD is an ideal PI5P probe.Intriguingly, the PHD finger of ATP-dependent chromatin remodeling factor (ACF) strongly and specifically interacts with PI5P in a protein-lipid overlay (PLO) assay (Gozani et al., 2003), suggesting that it could have potential for use as an in vivo probe for PI5P.The PH domains of Dok proteins bind mono-phosphoinositides including PI5P (Guittard et al., 2009(Guittard et al., , 2010) ) and, among these, the PH domain of Dok-5 displays the highest binding affinity for PI5P, as measured by PLO assay and surface plasmon resonance.The PH domain of Tiam1, a GEF of Rac1, also has high affinity for PI5P (Viaud et al., 2014), however, it also has a high affinity for PI3P (Baumeister et al., 2003;Viaud et al., 2014) indicating that this domain would be unsuitable for a PI5P specific probe.TOM1 has recently been reported to bind PI5P with an affinity that is two-fold higher than that observed with other mono-phosphoinositides (Boal et al., 2015).Thus, there is potential for the use of the VHS (Vps27-Hrs-STAM) domain of TOM1 as a probe for PI5P.
Vac7 in yeast plays an essential but poorly understood role in activation of Fab1 (Gary et al., 2002;Duex et al., 2006aDuex et al., , 2006b)).Vac7 is the only protein containing a transmembrane region among the regulatory proteins of Fab1 complex, but has no conserved motifs, and no known metazoan homologues (Gary et al., 2002).
In summary, Fab1 forms a complex with Fig4, Vac14, Vac7, and Atg18 in yeast, and PIKfyve forms a complex with Fig4 and Vac14 in mammals, to tightly control the cellular levels of PI(3,5)P 2 .Although some regulators present in the yeast complex have not been identified in mammals, the fundamental mechanism underlying the maintenance of PI(3,5)P 2 levels are highly conserved between these organisms.
It was recently proposed that the cytosolic N-terminal region of mucolipin transient receptor potential 1 (TRPML1) (ML1N) may be useful as a PI(3,5)P 2 probe (Li et al., 2013).ML1N colocalizes with LAMP1 and its localization is disrupted by treatment with YM-201636, a PIKfyve inhibitor (Jefferies et al., 2008), suggesting that the PI(3,5)P 2 is primarily localized to late endosomes and lysosomes.
Takatori et al. developed a method to detect PI(3,5)P 2 combining freeze-fracture electron microscopy with the use of tagged Atg18 protein as a PI(3,5)P 2 binding probe (Takatori et al., 2016).Atg18 binds to both PI(3,5)P 2 and PI3P.In order to improve specificity of Atg18 for PI(3,5)P 2 , a PI3P binding p40phox PX domain was used to compete with Atg18 binding to PI3P.Using this approach, PI(3,5)P 2 was found to be concentrated at specific microdomains of the vacuole membrane in yeast.In addition, in mammalian cells, PI(3,5)P 2 was detected at lysosomes, but not other organelles such as mitochondria and the Golgi apparatus.Together, these studies suggest that PI(3,5)P 2 is primarily localized to early and late endosomes and lysosomes/ vacuoles (Fig. 2).
Studies of several mouse mutants indicate that the PIKfyve complex has critical functions in both the central and peripheral nervous systems.Vac14 -/-mice display massive brain defects, including a spongiform-like appearance and increased apoptosis (Zhang et al., 2007).Fig4 -/-and Vac14 L156R/L156R (ingls) mice, a mutant that prevents the interaction of Vac14 with PIKfyve, as well as PIKfyve hypomorphic mice, which have decreased but not complete loss of PI(3,5)P 2 and PI5P, exhibit similar neurodegeneration (Chow et al., 2007;Jin et al., 2008;Ferguson et al., 2009;Lenk et al., 2011;Zolov et al., 2012).These observations demonstrate that nervous system defects are a prominent phenotype resulting from disruption of the PIKfyve complex.Additional tissues are also affected.Intestinespecific depletion of PIKfyve results in increased vacuolation as well as inflammation and fibrosis in the intestine resulting in malnutrition and a lean phenotype (Takasuga et al., 2013).Moreover, PIKfyve hypomorphic mice display large vacuoles and spongiform degeneration in the heart and reduction of airspace in the lung (Zolov et al., 2012).The spleen from Fig4 -/-mice or the PIKfyve hypomorph is small and has reduced density compared to wild-type (Chow et al., 2007;Zolov et al., 2012).Interestingly, pigment-containing hair follicles and melanosomes in hair are significantly reduced in Fig4 -/-mice (Chow et al., 2007), which suggests that the PIKfyve complex might be involved in the biogenesis of melanosomes.Plateletspecific deletion of PIKfyve induces inflammation and thrombosis, which are caused by impaired release of lysosomal enzymes in platelets (Min et al., 2014).Thus, PI(3,5)P 2 plays important roles in multiple tissue types.
The discovery of PIKfyve inhibitors such as YM-201636 (Jefferies et al., 2008), MF4 (de Lartigue et al., 2009), and apilimod (Cai et al., 2013) has greatly contributed to determining the cellular functions of PI(3,5)P 2 .The most notable phenotype of Fab1/PIKfyve suppression is enlarged vacuoles/lysosomes (Bonangelino et al., 2002;Duex et al., 2006b;Rutherford et al., 2006;Zhang et al., 2007).Overexpression of the TRPML1 calcium channel rescues the enlarged lysosome phenotype observed in PI(3,5)P 2deficient MEFs (Dong et al., 2010), indicating that the increased size of vacuoles/lysosomes in these cells is caused, at least in part, by defects in lysosomal ion homeostasis.Importantly, PI(3,5)P 2 has been shown to activate ion channels on endosomes and lysosomes.TRPML1 is activated by binding of PI(3,5)P 2 to its N-terminal region (Dong et al., 2010).Furthermore, PI(3,5)P 2 promotes the activity of two-pore channel proteins (TPC1, TPC2), which mediate calcium release from endolysosomes to the cytosol (Wang et al., 2012).Moreover, vacuole acidification is impaired in fab1Δ and vac7Δ yeast (Yamamoto et al., 1995;Bonangelino et al., 1997), although an independent study suggests that the pH of the vacuole is unaffected in fab1Δ yeast (Ho et al., 2015).These studies suggest that PI(3,5)P 2 is crucial for the regulation of specific ion channels on endosomes and lysosomes and possibly affects vacuole acidification as well.Effects on vacuolar pH may be due to direct regulation of the v-ATPase by PI(3,5)P 2 (Li et al., 2014).
The Fab1/PIKfyve complex is involved in a variety of intracellular membrane traffic pathways.In yeast, the depletion of Vac7 suppresses retrograde transport between vacuoles and Golgi (Bryant et al., 1998).In addition, fab1Δ yeast cells exhibit multivesicular bodies that contain fewer intraluminal vesicles compared to wild-type cells (Gary et al., 1998).Similarly, depletion of PIKfyve or Vac14 in mammalian cells perturbs retrieval of the cationindependent mannose 6-phosphate receptor (CI-MPR) and sortilin from endosomes to the trans-Golgi network (TGN) (Rutherford et al., 2006;Zhang et al., 2007).Furthermore, PIKfyve suppression using siRNA or PIKfyve inhibitors changes the cellular distribution of CI-MPR and TGN46 (de Lartigue et al., 2009).These reports also identify a minor effect of PIKfyve inhibition on EGF receptor degradation, indicating that the mammalian PIKfyve complex has an important role in retrograde traffic between endosomes and the TGN.However, whether there is direct involvement of PIKfyve in the degradation pathway remains an open question.
The mechanistic target of rapamycin (mTOR) signaling pathway regulates many major cellular processes and defects in mTOR signaling are implicated in a large number of diseases including cancer, diabetes, and neurodegeneration (Laplante and Sabatini, 2012).mTORC1 is localized to lysosomes and is activated in response to growth factor stimulation to control cell growth and autophagy through regulatory proteins including TSC1/2 and Raptor.It has been shown that PI(3,5)P 2 is required for TORC1 activity and the localization of TORC1 substrates in yeast (Jin et al., 2014).Moreover, PI(3,5)P 2 plays a role in the lysosomal localization and activation of mTORC1 in adipocytes (Bridges et al., 2012), suggesting that PI(3,5)P 2 is a regulator of mTORC1 in yeast and in at least some mammalian cell types.
Autophagy is an essential intracellular degradation system conserved from yeast to humans (Mizushima and Komatsu, 2011;Lamb et al., 2013;Shibutani and Yoshimori, 2014).Autophagosomes, which are generated under stress conditions including starvation, enclose cytosolic cargos such as proteins and organelles and facilitate their degradation through fusion with lysosomes.Inhibition of PIKfyve results in the accumulation of autophagosomes (de Lartigue et al., 2009;Martin et al., 2013).Brain tissue from Fig4 -/-mice display an accumulation of the autophagosome marker LC3-II and the autophagy substrate p62 (Ferguson et al., 2009), implying that PI(3,5)P 2 promotes autophagy.Given the role of PIKfyve in intracellular transport and lysosomal acidification, the phenotypes observed in these studies could be caused by a defect in autophagosome-lysosome fusion and/or lysosomal function.Interestingly, depletion of the PI 5-phosphatase INPP5E causes suppression of the fusion step between autophagosomes and lysosomes (Hasegawa et al., 2016).That INPP5E acts on PI(3,5)P 2 in vitro, suggests that lysosomal PI(3,5)P 2 is elevated during depletion of INPP5E.Notably, cortactin binds directly to PI(3,5)P 2 , which negatively regulates cortactin interactions with actin (Hong et al., 2015).Thus, elevation of PI(3,5)P 2 may underlie the defects observed during depletion of INPP5E, namely defects in cortactin-actin association with lysosomes, and inhibition of autophagosome-lysosome fusion (Hasegawa et al., 2016).These studies suggest that proper production and turnover of PI(3,5)P 2 are critical for the regulation of autophagy.
PIKfyve suppression impedes phagosome maturation but does not impair lysosomal acidification in macrophages (Kim et al., 2014).Additionally, PIKfyve facilitates the export of nutrients from vacuoles following engulfment of macromolecules (Krishna et al., 2016), indicating that PI(3,5)P 2 plays an important role in the maturation of phagosomes as well as reuse of phagocytosed nutrients in macrophages.
Glutamate stimulation of neurons increases PI(3,5)P 2 levels (McCartney et al., 2014b).Moreover, PIKfyve promotes the internalization and degradation of voltage-gated calcium channels in response to glutamate, leading to protection from excitotoxic cell death (Tsuruta et al., 2009).Vac14 is present at the synapse in hippocampal neurons and loss of Vac14 or Fig4 enhances excitatory synaptic function.In addition, surface levels of AMPA-type glutamate receptors are increased by the depletion of Vac14 due to reduced receptor endocytosis or an increase in receptor recycling (Zhang et al., 2012).Similarly, surface expression of AMPA-type glutamate receptors is reduced after treatment with PIKfyve inhibitors (Seebohm et al., 2012).Therefore, the PIKfyve complex and/or PI(3,5)P 2 play important roles in regulating postsynaptic function through regulating the surface levels of glutamate receptors and channels.
Several mutations of Fig4 have been found in CMT4J, a specific type of Charcot-Marie-Tooth disease (Chow et al., 2007;Zhang et al., 2008).CMT4J causes damage to myelin sheaths and nerve fibers resulting in neuropathy (Nicholson and Myers, 2006).Patients with the common CMT4J mutation Fig4 isoleucine 41 to threonine (I41T), have a delay in motor development and reduced nerve conduction velocity (Chow et al., 2007).The corresponding yeast mutation, Fig4 I59T, results in reduced cellular PI(3,5)P 2 levels (Chow et al., 2007).In addition, mutations in Vac14 have been associated with a progressive neurological disorder (Lenk et al., 2016).The PIKfyve-Vac14-Fig4 complex is therefore linked to human neurological disorders.While mutations in PIKfyve that are linked to neurological disorders have yet to be uncovered, mutations in PIKfyve have been linked to fleck corneal dystrophy, a syndrome with punctate corneal stromal opacities (Gee et al., 2015) .

Conclusions
This review covers some intracellular and physiological functions of PI5P and PI(3,5)P 2 .In metazoans, since the PIKfyve complex is implicated in the generation of both of these lipids, phenotypes observed upon depletion of the complex cannot yet be attributed to loss of PI(3,5)P 2 , PI5P, or both lipids.However, it is clear that both of these lipids play critical roles in physiology and pathology and a deeper understanding of metabolic pathways controlling production and turnover of PI5P and PI(3,5)P 2 is imperative.The success of these studies will require the development of sensitive and specific cellular probes for both of these lipids, as well as methods that specifically raise or lower PI5P alone, as well as PI(3,5)P 2 alone.

Fig. 1 .
Fig. 1.Interconversion pathways of seven phosphoinositide lipids.The action of lipid kinases is indicated as red arrows, and the action of phosphatases is indicated as blue arrows.The action of phospholipase C (PLC) is indicated by a green arrow.

Fig. 2 .
Fig. 2. Phosphoinositides are localized at distinct sites within the intracellular membrane system.The reported localization of each phosphoinositide is shown as the color indicated in the key.

Table I .
BINDING PROTEINS/DOMAINS TO PI5P