Eisosomes Regulate Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Cortical Clusters and Mitogen-activated Protein (MAP) Kinase Signaling upon Osmotic Stress

Eisosomes are multiprotein structures that generate linear invaginations at the plasma membrane of yeast cells. The core component of eisosomes, the BAR domain protein Pil1, generates these invaginations through direct binding to lipids including phosphoinositides. Eisosomes promote hydrolysis of phosphatidylinositol 4,5 bisphosphate (PI(4,5)P₂) by functioning with synaptojanin, but the cellular processes regulated by this pathway have been unknown. Here, we found that PI(4,5)P₂ regulation by eisosomes inhibits the cell integrity pathway, a conserved MAPK signal transduction cascade. This pathway is activated by multiple environmental conditions including osmotic stress in the fission yeast Schizosaccharomyces pombe. Activation of the MAPK Pmk1 was impaired by mutations in the phosphatidylinositol (PI) 5-kinase Its3, but this defect was suppressed by removal of eisosomes. Using fluorescent biosensors, we found that osmotic stress induced the formation of PI(4,5)P₂ clusters that were spatially organized by eisosomes in both fission yeast and budding yeast cells. These cortical clusters contained the PI 5-kinase Its3 and did not assemble in the its3-1 mutant. The GTPase Rho2, an upstream activator of Pmk1, also co-localized with PI(4,5)P₂ clusters under osmotic stress, providing a molecular link between these novel clusters and MAPK activation. Our findings have revealed that eisosomes regulate activation of MAPK signal transduction through the organization of cortical lipid-based microdomains.     

Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress

During hyperosmotic shock, Saccharomyces cerevisiae adjusts to physiological challenges, including large plasma membrane invaginations generated by rapid cell shrinkage. Calcineurin, the Ca²⁺/calmodulin–dependent phosphatase, is normally cytosolic but concentrates in puncta and at sites of polarized growth during intense osmotic stress; inhibition of calcineurin-activated gene expression suggests that restricting its access to substrates tunes calcineurin signaling specificity. Hyperosmotic shock promotes calcineurin binding to and dephosphorylation of the PI(4,5)P₂ phosphatase synaptojanin/Inp53/Sjl3 and causes dramatic calcineurin-dependent reorganization of PI(4,5)P₂-enriched membrane domains. Inp53 normally promotes sorting at the trans-Golgi network but localizes to cortical actin patches in osmotically stressed cells. By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells. In response to hyperosmotic shock and calcineurin-dependent regulation, Inp53 shifts from associating predominantly with clathrin to interacting with endocytic proteins Sla1, Bzz1, and Bsp1, suggesting that Inp53 mediates stress-specific endocytic events. This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion. We propose that activation of Ca²⁺/calcineurin and PI(4,5)P₂ signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.     

Characterization of the S. cerevisiae inp51 mutant links phosphatidylinositol 4,5-bisphosphate levels with lipid content,membrane fluidity and cold growth

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and its derivatives diphosphoinositol phosphates (DPIPs) play key signaling and regulatory roles. However, a direct function of these molecules in lipid and membrane homeostasis remains obscure. Here, we have studied the cold tolerance phenotype of yeast cells lacking the Inp51-mediated phosphoinositide-5-phosphatase. Genetic and biochemical approaches showed that increased metabolism of PI(4,5)P₂ reduces the activity of the Pho85 kinase by increasing the levels of the DPIP isomer 1-IP₇. This effect was key in the cold tolerance phenotype. Indeed, pho85 mutant cells grew better than the wild-type at 15 °C, and lack of this kinase abolished the inp51-mediated cold phenotype. Remarkably, reduced Pho85 function by loss of Inp51 affected the activity of the Pho85-regulated target Pah1, the yeast phosphatidate phosphatase. Cells lacking Inp51 showed reduced Pah1 abundance, derepression of an INO1-lacZ reporter, decreased content of triacylglycerides and elevated levels of phosphatidate, hallmarks of the pah1 mutant. However, the inp51 phenotype was not associated to low Pah1 activity since deletion of PAH1 caused cold sensitivity. In addition, the inp51 mutant exhibited features not shared by pah1, including a 40%-reduction in total lipid content and decreased membrane fluidity. These changes may influence the activity of membrane-anchored and/or associated proteins since deletion of INP51 slows down the transit to the vacuole of the fluorescent dye FM4-64. In conclusion, our work supports a model in which changes in the PI(4,5)P₂ pool affect the 1-IP₇ levels modulating the activity of Pho85, Pah1 and likely additional Pho85-controlled targets, and regulate lipid composition and membrane properties.     

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55^Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4⁺ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P₂, therefore controlling Pr55^Gag membrane association. Rab27a also controls PI(4,5)P₂ levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55^Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P₂ production and, consequently, HIV-1 replication.     

Retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides

Retroviruses acquire a lipid envelope during budding from the membrane of their hosts. Therefore, the composition of this envelope can provide important information about the budding process and its location. Here, we present mass spectrometry analysis of the lipid content of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). The results of this comprehensive survey found that the overall lipid content of these viruses mostly matched that of the plasma membrane, which was considerably different from the total lipid content of the cells. However, several lipids are enriched in comparison to the composition of the plasma membrane: (i) cholesterol, ceramide, and GM3; and (ii) phosphoinositides, phosphorylated derivatives of phosphatidylinositol. Interestingly, microvesicles, which are similar in size to viruses and are also released from the cell periphery, lack phosphoinositides, suggesting a different budding mechanism/location for these particles than for retroviruses. One phosphoinositide, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂], has been implicated in membrane binding by HIV Gag. Consistent with this observation, we found that PI(4,5)P₂ was enriched in HIV-1 and that depleting this molecule in cells reduced HIV-1 budding. Analysis of mutant virions mapped the enrichment of PI(4,5)P₂ to the matrix domain of HIV Gag. Overall, these results suggest that HIV-1 and other retroviruses bud from cholesterol-rich regions of the plasma membrane and exploit matrix/PI(4,5)P₂ interactions for particle release from cells.     

Proteome of Acidic Phospholipid-binding Proteins: SPATIAL AND TEMPORAL REGULATION OF CORONIN 1A BY PHOSPHOINOSITIDES*

Reversible interactions between acidic phospholipids in the cellular membrane and proteins in the cytosol play fundamental roles in a wide variety of physiological events. Here, we present a novel approach to the identification of acidic phospholipid-binding proteins using nano-liquid chromatography-tandem mass spectrometry. We found more than 400 proteins, including proteins with previously known acidic phospholipid-binding properties, and confirmed that several candidates, such as Coronin 1A, mDia1 (Diaphanous-related formin-1), PIR121/CYFIP2, EB2 (end plus binding protein-2), KIF21A (kinesin family member 21A), eEF1A1 (translation elongation factor 1α1), and TRIM2, directly bind to acidic phospholipids. Among such novel proteins, we provide evidence that Coronin 1A activity, which disassembles Arp2/3-containing actin filament branches, is spatially and temporally regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Whereas Coronin 1A co-localizes with PI(4,5)P₂ at the plasma membrane in resting cells, it is dissociated from the plasma membrane during lamellipodia formation where the PI(4,5)P₂ signal is significantly reduced. Our in vitro experiments show that Coronin 1A preferentially binds to PI(4,5)P₂-containing liposomes and that PI(4,5)P₂ antagonizes the ability of Coronin 1A to disassemble actin filament branches, indicating a spatiotemporal regulation of Coronin 1A via a direct interaction with the plasma membrane lipid. Collectively, our proteomics data provide a list of potential acidic phospholipid-binding protein candidates ranging from the actin regulatory proteins to translational regulators.     

PtdIns4P synthesis by PI4KIIIα at the plasma membrane and its impact on plasma membrane identity

Plasma membrane phosphatidylinositol (PI) 4-phosphate (PtdIns4P) has critical functions via both direct interactions and metabolic conversion to PI 4,5-bisphosphate (PtdIns(4,5)P₂) and other downstream metabolites. However, mechanisms that control this PtdIns4P pool in cells of higher eukaryotes remain elusive. PI4KIIIα, the enzyme thought to synthesize this PtdIns4P pool, is reported to localize in the ER, contrary to the plasma membrane localization of its yeast homologue, Stt4. In this paper, we show that PI4KIIIα was targeted to the plasma membrane as part of an evolutionarily conserved complex containing Efr3/rolling blackout, which we found was a palmitoylated peripheral membrane protein. PI4KIIIα knockout cells exhibited a profound reduction of plasma membrane PtdIns4P but surprisingly only a modest reduction of PtdIns(4,5)P₂ because of robust up-regulation of PtdIns4P 5-kinases. In these cells, however, much of the PtdIns(4,5)P₂ was localized intracellularly, rather than at the plasma membrane as in control cells, along with proteins typically restricted to this membrane, revealing a major contribution of PI4KIIIα to the definition of plasma membrane identity.     

Molecular Determinants of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly

Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag–PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag–PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]; however, previous studies suggested that HTLV-1 Gag–PM interactions and therefore virus assembly are less dependent on PI(4,5)P₂. We have recently shown that PI(4,5)P₂ binds directly to HTLV-1 unmyristoylated MA [myr(–)MA] and that myr(–)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P₂. Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA–membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P₂–binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P₂. Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly.     

Rous sarcoma virus gag has no specific requirement for phosphatidylinositol-(4,5)-bisphosphate for plasma membrane association in vivo or for liposome interaction in vitro

The MA domain of the retroviral Gag protein mediates interactions with the plasma membrane, which is the site of productive virus release. HIV-1 MA has a phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] binding pocket; depletion of this phospholipid from the plasma membrane compromises Gag membrane association and virus budding. We used multiple methods to examine the possible role of PI(4,5)P₂ in Gag-membrane interaction of the alpharetrovirus Rous sarcoma virus (RSV). In contrast to HIV-1, which was tested in parallel, neither membrane localization of RSV Gag-GFP nor release of virus-like particles was affected by phosphatase-mediated depletion of PI(4,5)P₂ in transfected avian cells. In liposome flotation experiments, RSV Gag required acidic lipids for binding but showed no specificity for PI(4,5)P₂. Mono-, di-, and triphosphorylated phosphatidylinositol phosphate (PIP) species as well as high concentrations of phosphatidylserine (PS) supported similar levels of flotation. A mutation that increases the overall charge of RSV MA also enhanced Gag membrane binding. Contrary to previous reports, we found that high concentrations of PS, in the absence of PIPs, also strongly promoted HIV-1 Gag flotation. Taken together, we interpret these results to mean that RSV Gag membrane association is driven by electrostatic interactions and not by any specific association with PI(4,5)P₂.     

Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca²⁺ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca²⁺ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], and Ca²⁺ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P₂ levels in ³²P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P₂ resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca²⁺ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.     

Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis

Cell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non‐plasma membrane PI(4,5)P₂, and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P₂ phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over‐activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P₂ phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable.     

Regulation of TRPV1 Ion Channel by Phosphoinositide (4,5)-Bisphosphate: THE ROLE OF MEMBRANE ASYMMETRY*

Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P₂) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P₂ in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P₂ was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P₂ in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P₂ in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P₂ whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P₂ must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P₂ may resolve the long standing controversy about whether PI(4,5)P₂ is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.     

Seg1 controls eisosome assembly and shape

Eisosomes are stable domains at the plasma membrane of the budding yeast Saccharomyces cerevisiae and have been proposed to function in endocytosis. Eisosomes are composed of two main cytoplasmic proteins, Pil1 and Lsp1, that form a scaffold around furrow-like plasma membrane invaginations. We show here that the poorly characterized eisosome protein Seg1/Ymr086w is important for eisosome biogenesis and architecture. Seg1 was required for efficient incorporation of Pil1 into eisosomes and the generation of normal plasma membrane furrows. Seg1 preceded Pil1 during eisosome formation and established a platform for the assembly of other eisosome components. This platform was further shaped and stabilized upon the arrival of Pil1 and Lsp1. Moreover, Seg1 abundance controlled the shape of eisosomes by determining their length. Similarly, the Schizosaccharomyces pombe Seg1-like protein Sle1 was necessary to generate the filamentous eisosomes present in fission yeast. The function of Seg1 in the stepwise biogenesis of eisosomes reveals striking architectural similarities between eisosomes in yeast and caveolae in mammals.     

Insights into the inhibitory mechanism of triazole-based small molecules on phosphatidylinositol-4,5-bisphosphate binding pleckstrin homology domain

BackgroundPhosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is an important regulator of several cellular processes and a precursor for other second messengers which are involved in cell signaling pathways. Signaling proteins preferably interact with PI(4,5)P₂ through its pleckstrin homology (PH) domain. Efforts are underway to design small molecule-based antagonist, which can specifically inhibit the PI(4,5)P₂/PH-domain interaction to establish an alternate strategy for the development of drug(s) for phosphoinositide signaling pathways.MethodsSurface plasmon resonance, molecular docking, circular dichroism, competitive Förster resonance energy transfer, isothermal titration calorimetric analyses and liposome pull down assay were used.ResultsIn this study, we employed 1,2,3-triazol-4-yl methanol containing small molecule (CIPs) as antagonists for PI(4,5)P₂/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance analysis (IC₅₀ ranges from 53 to 159 nM for PI(4,5)P₂/PLCδ1-PH domain binding assay). We also used phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂], PI(4,5)P₂ specific PH-domains to determine binding selectivity of the compounds. Various physicochemical analyses showed that the compounds have weak affect on fluidity of the model membrane but, strongly interact with the phospholipase C δ1 (PLCδ1)-PH domains. The 1,2,3-triazol-4-yl methanol moiety and nitro group of the compounds are essential for their exothermic interaction with the PH-domains. Potent compound can efficiently displace PLCδ1-PH domain from plasma membrane to cytosol in A549 cells.ConclusionsOverall, our studies demonstrate that these compounds interact with the PIP-binding PH-domains and inhibit their membrane recruitment.General significanceThese results suggest specific but differential binding of these compounds to the PLCδ1-PH domain and emphasize the role of their structural differences in binding parameters. These triazole-based compounds could be directly used/further developed as potential inhibitor for PH domain-dependent enzyme activity.     

Signal-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate without activation of phospholipase C: implications on gating of Drosophila TRPL (transient receptor potential-like) channel

In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P₂ in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P₂ was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P₂ is not an inhibitor of TRPL channel activation. PI(4,5)P₂ hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P₂ levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P₂ is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.     

Courier service for phosphatidylinositol: PITPs deliver on demand

Phosphatidylinositol is the parent lipid for the synthesis of seven phosphorylated inositol lipids and each of them play specific roles in numerous processes including receptor-mediated signalling, actin cytoskeleton dynamics and membrane trafficking. PI synthesis is localised to the endoplasmic reticulum (ER) whilst its phosphorylated derivatives are found in other organelles where the lipid kinases also reside. Phosphorylation of PI to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂) at the plasma membrane and to phosphatidylinositol 4-phosphate (PI4P) at the Golgi are key events in lipid signalling and Golgi function respectively. Here we review a family of proteins, phosphatidylinositol transfer proteins (PITPs), that can mobilise PI from the ER to provide the substrate to the resident kinases for phosphorylation. Recent studies identify specific and overlapping functions for the three soluble PITPs (PITPα, PITPβ and PITPNC1) in phospholipase C signalling, neuronal function, membrane trafficking, viral replication and in cancer metastases.     

PLCζ causes Ca(2+) oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P(2)

Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca(2+) oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate-mediated Ca(2+) release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P(2) targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P(2) with a green fluorescent protein-tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P(2) from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca(2+) oscillations. Specific depletion of this PI(4,5)P(2) pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca(2+) oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P(2), PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P(2) after PLCζ injection. Targeted depletion of vesicular PI(4,5)P(2) by expression of ciPLCζ-fused Inp54p inhibited the Ca(2+) oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca(2+) mobilization by hydrolyzing internal PI(4,5)P(2) stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.     

A Novel Phosphatidylinositol 4,5-Bisphosphate Binding Domain Mediates Plasma Membrane Localization of ExoU and Other Patatin-like Phospholipases

Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A₂ activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P₂, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P₂ for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P₂ and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P₂. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P₂ binding domains.