PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection.

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.

Phosphatidylinositol 3,5-bisphosphate and Fab1p/PIKfyve underPPIn endo-lysosome function.

PtdIns(3,5)P(2) is one of the seven regulatory PPIn (polyphosphoinositides) that are ubiquitous in eukaryotes. It controls membrane trafficking at multiple points in the endosomal/lysosomal system and consequently regulates the size, shape and acidity of at least one endo-lysosomal compartment. PtdIns(3,5)P(2) appears to exert this control via multiple effector proteins, with each effector specific for a subset of the various PtdIns(3,5)P(2)-dependent processes. Some putative PtdIns(3,5)P(2) effectors have been identified, including Atg18p-related PROPPIN [beta-propeller(s) that bind PPIn] proteins and the epsin-like proteins Ent3p and Ent5p, whereas others remain to be defined. One of the principal functions of PtdIns(3,5)P(2) is to regulate the fission/fragmentation of endo-lysosomal sub-compartments. PtdIns(3,5)P(2) is required for vesicle formation during protein trafficking between endo-lysosomes and also for fragmentation of endo-lysosomes into smaller compartments. In yeast, hyperosmotic stress accelerates the latter process. In the present review we highlight and discuss recent studies that reveal the role of the HOPS-CORVET complex and the vacuolar H(+)-ATPase in the process of endo-lysosome fission, and speculate on connections between these machineries and the Fab1p pathway. We also discuss new evidence linking PtdIns(3,5)P(2) and PtdIns5P to the regulation of exocytosis.

Characterization of phosphatidylinositol phosphate kinases from the moss Physcomitrella patens: PpPIPK1 and PpPIPK2.

Phosphoinositides (PIs) play a major role in eukaryotic cells, despite being a minor component of most membranes. This is the first report on PI metabolism in a bryophyte, the moss Physcomitrella patens. Moss PI composition is similar to that of other land plants growing under normal conditions. In contrast to the large number of PIPK genes present in flowering plants, the P. patens genome encodes only two type I/II PIPK genes, PpPIPK1 and PpPIPK2, which are very similar at both the nucleotide and protein product levels. However, the expression of the two genes is differentially regulated, and in vitro biochemical characterization shows that the resulting enzymes have different substrate specificities. PpPIPK1 uses PtdIns4P and PtdIns3P with similar preference and also metabolizes PtdIns(3,4)P(2) to produce PtdIns(3,4,5)P(3), a PI not yet detected in intact plant cells. PpPIPK2 prefers PtdIns as substrate and is much less active towards PtdIns4P and PtdIns3P. Thus, PpPIPK2 shows properties reminiscent of both PtdInsP-kinase and PtdIns-kinases. Moreover, a substitution of glutamic acid by alanine in the activation loop drastically reduced PpPIPK1 activity and altered the substrate specificity to PtdIns5P being the preferred substrate compared with PtdIns4P and PtdIns3P. These findings demonstrate that the substrate specificity of plant PIPKs is determined in a plant-specific manner, which provides new insights into the regulatory modes of PIPK activity in plants.

A protein complex that regulates PtdIns(3,5)P2 levels.

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is needed for retrograde membrane trafficking from lysosomal and late endosomal compartments and its synthesis is tightly regulated. But how cells regulate PtdIns(3,5)P2 synthesis--for example, in response to hyperosmotic shock--remains unexplained. A paper from the Weisman group gives the most complete picture so far of a multiprotein complex that controls PtdIns(3,5)P2 synthesis and explains how a VAC14 mutation functionally impairs the scaffold protein at the heart of the complex and causes a neurodegenerative condition in mice.

Inositol lipid-dependent functions in Saccharomyces cerevisiae: analysis of phosphatidylinositol phosphates.

Inositol phospholipids regulate many cellular processes, including cell survival, membrane trafficking, and actin polymerization. Quantification of inositol lipids is one of the essential techniques needed for studies that aim to decipher inositol lipid-dependent cellular functions. The study of phosphoinositides in most organisms is hampered by a lack of facile genetic tools. However, the essential elements of most inositol lipid signaling pathways appear to be conserved across eukaryote phylogeny. They can therefore readily be elucidated (both genetically and biochemically) in the budding yeast Saccharomyces cerevisiae. Because of the low abundance of polyphosphoinositides in cells, many analytical methods start by radioactively labeling intact cells and then extracting the lipids with chloroform/methanol/ water mixtures based on those first devised half a century ago. Yeast present special extraction problems because the cell wall must be broken in order to facilitate solvent access and maximize lipid yield. Once lipids have been extracted, fatty acids are removed and the resulting water-soluble glycerophosphoinositol phosphates are analysed by anion-exchange HPLC. This chapter describes how to extract and quantify the inositol lipids of S. cerevisiae cells that have been radiolabeled to isotopic equilibrium with [3H]myo-inositol.

Our FABulous VACation: a decade of phosphatidylinositol 3,5-bisphosphate.

PtdIns(3,5)P2 was discovered about a decade ago and much of the machinery that makes, degrades and senses it has been uncovered. Despite this, we still lack a complete understanding of how the pieces fit together but some patterns are beginning to emerge. Molecular functions for PtdIns(3,5)P2 are also elusive, but the identification of effectors offers a way into some of these processes. An examination of the defects associated with loss of synthesis of PtdIns(3,5)P2 in lower and higher eukaryotes begins to suggest a unifying theme; this lipid regulates membrane retrieval via retrograde trafficking from distal compartments to organelles that are more proximal in the endocytic/lysosomal system. Another unifying theme is stress signalling to organelles, possibly both to change their morphology in response to external insults and to maintain the lumenal pH or membrane potential of organelles. The next few years seem likely to uncover details of the molecular mechanisms underlying the biology of this fascinating lipid. This review also highlights some areas where further research is needed.

Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells.

The inositol lipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] is involved in a myriad of cellular processes, including the regulation of exocytosis and endocytosis. In this paper, we address the role of PtdIns(4,5)P2 in compound exocytosis from rat peritoneal mast cells. This process involves granule-plasma membrane fusion as well as homotypic granule membrane fusion and occurs without any immediate compensatory endocytosis. Using a novel quantitative immunofluorescence technique, we report that plasma membrane PtdIns(4,5)P2 becomes transiently depleted upon activation of exocytosis, and is not detected on the membranes of fusing granules. Depletion is caused by phospholipase C activity, and is mandatory for exocytosis. Although phospholipase C is required for Ca2+ release from internal stores, the majority of the requirement for PtdIns(4,5)P2 hydrolysis occurs downstream of Ca2+ signalling - as shown in permeabilised cells, where the inositol (1,4,5)-trisphosphate-Ca2+ pathway is bypassed. Neither generation of the PtdIns(4,5)P2 metabolite, diacylglycerol (DAG) or simple removal and/or sequestration of PtdIns(4,5)P2 are sufficient for exocytosis to occur. However, treatment of permeabilised cells with DAG induces a small potentiation of exocytosis, indicating that it may be required. We propose that a cycle of PtdIns(4,5)P2 synthesis and breakdown is crucial for exocytosis to occur in mast cells, and may have a more general role in all professional secretory cells.

Analysis of intact phosphoinositides in biological samples.

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P(3)], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [(32)P]inorganic phosphate or [(3)H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS(3). Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.

Phosphatidylinositol 3,5-bisphosphate: metabolism and cellular functions.

Polyphosphoinositides (PPIn) are low-abundance membrane phospholipids that each bind to a distinctive set of effector proteins and, thereby, regulate a characteristic suite of cellular processes. Major functions of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)] are in membrane and protein trafficking, and in pH control in the endosome-lysosome axis. Recently identified PtdIns(3,5)P(2) effectors include a family of novel beta-propeller proteins, for which we propose the name PROPPINs [for beta-propeller(s) that binds PPIn], and possibly proteins of the epsin and CHMP (charged multi-vesicular body proteins) families. All eukaryotes, with the exception of some pathogenic protists and microsporidians, possess proteins needed for the formation, metabolism and functions of PtdIns(3,5)P(2). The importance of PtdIns(3,5)P(2) for normal cell function is underscored by recent evidence for its involvement in mammalian cell responses to insulin and for PtdIns(3,5)P(2) dysfunction in the human genetic conditions X-linked myotubular myopathy, Type-4B Charcot-Marie-Tooth disease and fleck corneal dystrophy.

Phospholipase D activity is essential for actin localization and actin-based motility in Dictyostelium.

PLD (phospholipase D) activity catalyses the generation of the lipid messenger phosphatidic acid, which has been implicated in a number of cellular processes, particularly the regulation of membrane traffic. In the present study, we report that disruption of PLD signalling causes unexpectedly profound effects on the actin-based motility of Dictyostelium. Cells in which PLD activity is inhibited by butan-1-ol show a complete loss of actin-based structures, accompanied by relocalization of F-actin into small clusters, and eventually the nucleus, without a visible fall in levels of F-actin. Addition of exogenous phosphatidic acid reverses the effects of butan-1-ol, confirming that these effects are caused by inhibition of PLD. Loss of motility correlates with complete inhibition of endocytosis and a reduction in phagocytosis. Inhibition of PLD caused a major decrease in the synthesis of PtdIns(4,5)P2, which could again be reversed by exogenously applied phosphatidic acid. Thus the essential role of PLD signalling in both motility and endocytosis appears to be mediated directly via regulation of PtdIns(4)P kinase activity. This implies that localized PLD-regulated synthesis of PtdIns(4,5)P2 is essential for Dictyostelium actin function.

Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors.

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies. To isolate PtdIns(3,5)P2 effectors, we identified Saccharomyces cerevisiae mutants that display fab1delta-like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p displays PtdIns(3,5)P2 binding of exquisite specificity, GFP-Svp1p localises to the vacuole membrane in a Fab1p-dependent manner, and svp1delta cells fail to recycle a marker protein from the vacuole to the Golgi. Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P2. These observations identify Svp1p as a PtdIns(3,5)P2 effector required for PtdIns(3,5)P2-dependent membrane recycling from the vacuole. Other Svp1p-related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P2 similarly. Svp1p and related proteins almost certainly fold as beta-propellers, and the PtdIns(3,5)P2-binding site is on the beta-propeller. It is likely that many of the Svp1p-related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P2 effectors. Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P2 to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P2 effectors must exist.

I-proteins - a proposed switch in myotubularin function.

In this article, we consider the functions of the myotubularins - a large family of phosphoinositide 3-phosphatases. By analogy with the phosphatidylinositol 3-phosphatase PTEN (phosphatase and tensin homolog deleted in chromosome ten) and many protein phosphatases, it has been proposed that the primary function of this protein family is to regulate substrate levels, in this case phosphatidylinositol (3)-phosphate. We propose an alternate, or additional, function that is analogous to the G-protein family of phosphatases, which use nucleotide-dependent conformational changes to transduce signals or do mechanical work.

PtdIns-specific MPR pathway association of a novel WD40 repeat protein, WIPI49.

WIPI49 is a member of a previously undescribed family of WD40-repeat proteins that we demonstrate binds 3-phosphorylated phosphoinositides. Immunofluorescent imaging indicates that WIPI49 is localized to both trans-Golgi and endosomal membranes, organelles between which it traffics in a microtubule-dependent manner. Live cell imaging establishes that WIPI49 traffics through the same set of endosomal membranes as that followed by the mannose-6-phosphate receptor (MPR), and consistent with this, WIPI49 is enriched in clathrin-coated vesicles. Ectopic expression of wild-type WIPI49 disrupts the proper functioning of this MPR pathway, whereas expression of a double point mutant (R221,222AWIPI49) unable to bind phosphoinositides does not disrupt this pathway. Finally, suppression of WIPI49 expression through RNAi, demonstrates that its presence is required for normal endosomal organization and distribution of the CI-MPR. We conclude that WIPI49 is a novel regulatory component of the endosomal and MPR pathway and that this role is dependent upon the PI-binding properties of its WD40 domain.

Hypo-osmotic stress activates Plc1p-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol Hexakisphosphate accumulation in yeast.

Polyphosphoinositide-specific phospholipases (PICs) of the delta-subfamily are ubiquitous in eukaryotes, but an inability to control these enzymes physiologically has been a major obstacle to understanding their cellular function(s). Plc1p is similar to metazoan delta-PICs and is the only PIC in Saccharomyces cerevisiae. Genetic studies have implicated Plc1p in several cell functions, both nuclear and cytoplasmic. Here we show that a brief hypo-osmotic episode provokes rapid Plc1p-catalyzed hydrolysis of PtdIns(4,5)P2 in intact yeast by a mechanism independent of extracellular Ca2+. Much of this PtdIns(4,5)P2 hydrolysis occurs at the plasma membrane. The hydrolyzed PtdIns(4,5)P2 is mainly derived from PtdIns4P made by the PtdIns 4-kinase Stt4p. PtdIns(4,5)P2 hydrolysis occurs normally in mutants lacking Arg82p or Ipk1p, but they accumulate no InsP6, showing that these enzymes normally convert the liberated Ins(1,4,5)P3 rapidly and quantitatively to InsP6. We conclude that hypo-osmotic stress activates Plc1p-catalyzed PtdIns(4,5)P2 at the yeast plasma membrane and the liberated Ins(1,4,5)P3 is speedily converted to InsP6. This ability routinely to activate Plc1p-catalyzed PtdIns(4,5)P2 hydrolysis in vivo opens up new opportunities for molecular and genetic scrutiny of the regulation and functions of phosphoinositidases C of the delta-subfamily.

Phosphatidylinositol-5-phosphate activation and conserved substrate specificity of the myotubularin phosphatidylinositol 3-phosphatases.

Phosphoinositides control many different processes required for normal cellular function. Myotubularins are a family of Phosphatidylinositol 3-phosphate (PtdIns3P) phosphatases identified by the positional cloning of the MTM1 gene in patients suffering from X-linked myotubular myopathy and the MTMR2 gene in patients suffering from the demyelinating neuropathy Charcot-Marie-Tooth disease type 4B. MTM1 is a phosphatidylinositol phosphatase with reported specificity toward PtdIns3P, while the related proteins MTMR2 and MTMR3 hydrolyze both PtdIns3P and PtdIns(3,5)P2. We have investigated MTM1 and MTMR6 and find that they use PtdIns(3,5)P2 in addition to PtdIns3P as a substrate in vitro. The product of PtdIns(3,5)P2 hydrolysis, PtdIns5P, causes MTM1 to form a heptameric ring that is 12.5 nm in diameter, and it is a specific allosteric activator of MTM1, MTMR3, and MTMR6. A disease-causing mutation at arginine 69 of MTM1 falling within a putative pleckstrin homology domain reduces the ability of the enzyme to respond to PtdIns5P. We propose that the myotubularin family of enzymes utilize both PtdIns3P and PtdIns(3,5)P2 as substrates, and that PtdIns5P functions in a positive feedback loop controlling their activity. These findings highlight the importance of regulated phosphatase activity for the control of phosphoinositide metabolism.

Activation of the G(i) heterotrimeric G protein by ANCA IgG F(ab')2 fragments is necessary but not sufficient to stimulate the recruitment of those downstream mediators used by intact ANCA IgG.

Anti-neutrophil cytoplasm autoantibodies (ANCA) are implicated in the pathogenesis of systemic vasculitis. Intact ANCA IgG activate superoxide generation in cytokine-primed neutrophils after binding their antigens and co-engaging Fcgamma receptors (FcgammaR). The contribution of antigen binding via ANCA F(ab')(2) fragments to signaling has been unclear. This study shows that both ANCA IgG and F(ab')(2) fragments of ANCA IgG induce significant GTPase activity, which could be blocked with pertussis toxin and anti-G(i) protein antibodies. Pertussis toxin inhibited ANCA IgG-induced superoxide generation but was without effect on superoxide production after conventional FcgammaR ligation. ANCA F(ab')(2) fragments did not induce superoxide generation. ANCA IgG activated PI 3-kinase-generating PIP(3), activated protein kinase B (PKB), and p21(ras); activation of each mediator was inhibited with pertussis toxin, but PI3K and PKB were not activated by ANCA IgG F(ab')(2) fragments. Intact ANCA IgG induced tyrosine phosphorylation, whereas F(ab')(2) fragments did not, and ANCA IgG-mediated superoxide generation was inhibited with genistein. Both genistein and pertussis toxin together completely abrogated the ANCA-induced oxidative burst. Genistein also inhibited ANCA IgG-induced PIP(3) generation and p21(ras) activation. These data implicate a novel ANCA IgG stimulated signaling pathway that involves both F(ab')(2)-mediated antigen binding and Fc-mediated FcgammaR ligation in cooperative interactions between G(i) proteins and tyrosine kinases that facilitates activation of downstream mediators.

Vac14 controls PtdIns(3,5)P(2) synthesis and Fab1-dependent protein trafficking to the multivesicular body.

BACKGROUND: The PtdIns3P 5-kinase Fab1 makes PtdIns(3,5)P(2), a phosphoinositide essential for retrograde trafficking between the vacuole/lysosome and the late endosome and also for trafficking of some proteins into the vacuole via multivesicular bodies (MVB). No regulators of Fab1 were identified until recently. RESULTS: Visual screening of the Eurofan II panel of S. cerevisiae deletion mutants identified YLR386w as a novel regulator of vacuolar function. Others recently identified this ORF as encoding the vacuolar inheritance gene VAC14. Like fab1 mutants, yeast lacking Vac14 have enlarged vacuoles that do not acidify correctly. FAB1 overexpression corrects these defects. vac14Delta cells make very little PtdIns(3,5)P(2), and hyperosmotic shock does not stimulate PtdIns(3,5)P(2) synthesis in the normal manner, implicating Vac14 in Fab1 regulation. We also show that, like fab1Delta mutants, vac14Delta cells fail to sort GFP-Phm5 to the MVB and thence to the vacuole: irreversible ubiquitination of GFP-Phm5 overcomes this defect. In the BY4742 genetic background, loss of Vac14 causes much more penetrant effects on phosphoinositide metabolism and vacuolar trafficking than does loss of Vac7, another regulator of Fab1. Vac14 contains motifs suggestive of a role in protein trafficking and interacts with several proteins involved in clathrin-mediated membrane sorting and phosphoinositide metabolism. CONCLUSIONS: Vac14 and Vac7 are both upstream activators of Fab1-catalysed PtdIns(3,5)P(2) synthesis, with Vac14 the dominant contributor to the hierarchy of control. Vac14 is essential for the regulated synthesis of PtdIns(3,5)P(2), for control of trafficking of some proteins to the vacuole lumen via the MVB, and for maintenance of vacuole size and acidity.