The expanding roles of PI4P and PI(4,5)P2 at the plasma membrane: Role of phosphatidylinositol transfer proteins

Phosphoinositides are phosphorylated derivatives of phosphatidylinositol, a phospholipid that is synthesised at the endoplasmic reticulum. The plasma membrane contains the enzymes to phosphorylate phosphatidylinositol and is therefore rich in the phosphorylated derivatives, PI4P and PI(4,5)P2. PI(4,5)P2 is a substrate for phospholipase C and during cell signaling, PI(4,5)P2 levels are reduced. Here I discuss a family of proteins, phosphatidylinositol transfer proteins (PITPs) that can restore PI(4,5)P2 levels

Undifferentiated HL60 cells respond to extracellular ATP and UTP by stimulating phospholipase C activation and exocytosis

AbstractWe have recently characterised the presence of a Ca2+-mobilising receptor for ATP which stimulates exocytosis in differentiated HL60 cells. Here we demonstrate that the Undifferentiated HL60 cells also respond to extracellular ATP by stimulating an increase in inositol phosphates and exocytosis. Of the nucleotides (ATP, UTP, ITP, ATPyS, AppNHp, XTP, CTP, GTP, 8-Br-ATP and GTPγS) that were active in stimulating inositol phosphate formation, only UTP, ATP, ITP, ATPγS and AppNHp were active in stimulating secretion. On differentiation, the extent of secretion due to the purinergic agonists ATP, ITP, ATPγS and AppNHp remained unchanged whilst secretion due to UTP, a pyrimidine, was substantially increased. These results indicate that the effect of ATP and UTP may be mediated via separate purinergic and pyrimidinergic receptors, respectively

ARF1(2–17) does not specifically interact with ARF1-dependent pathways Inhibition by peptide of phospholipases Cβ, D and exocytosis in HL60 cells

AbstractThe small GTP-binding protein ARF has been shown recently to regulate phospholipase D (PLD). In order to investigate the role of ARF proteins in regulated exocytosis, we have used the N-terminal peptide ARF1(2–17) of the ARF1 protein. ARF1 reconstituted PLD activity in cytosol-depleted HL60 cells was inhibited by ARF1(2–17). In the presence of endogenous cytosol, ARF1(2–17) also inhibited GTP-γ-S-stimulated PLD activity and exocytosis. Mastoparan Politses jadwagae and mastoparan Vespula lewisii which exhibit similar structural properties to ARF1(2–17) also inhibited GTP-γ-S-stimulated PLD and exocytosis. GTP-γ-S-stimulated phospholipase C-β (PLC-β) was also inhibited by ARF(2–17) and mastoparan. In cytosol-depleted HL60 cells, the ARF(2–17) inhibited the reconstitution of GTP-γ-S-stimulated PLC-β activity with exogenously-added PLC-β1 and phosphatidylinositol transfer protein. We conclude that the widely-used ARF1(2–17) peptide inhibits both ARF-independent (i.e. PLC-β) and ARF-dependent pathways (i.e. PLD) and therefore cannot be regarded as a specific inhibitor of ARF function

Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2

AbstractBackground: Phosphatidylinositol transfer protein (PI-TP), which has the ability to transfer phosphatidylinositol (PI) from one membrane compartment to another, is required in the inositol lipid signalling pathway through phospholipase C–β (PLC–β) that is regulated by GTP-binding protein(s) in response to extracellular signals. Here, we test the hypothesis that the principal role of PI-TP is to couple sites of lipid hydrolysis to sites of synthesis, and so to replenish depleted substrate for PLC–β.Results We have designed an experimental protocol that takes advantage of the different rates of release of endogenous PI-TP and PLC-β from HL60 cells permeabilized with streptolysin O. We have examined the kinetics of stimulated inositol lipid hydrolysis in cells depleted of PI-TP, but not of endogenous PLC-β, in the presence and absence of exogenous PI-TP. Linear time-courses were observed in the absence of any added protein, and the rate was accelerated by PI-TP using either guanosine 5′[γ-thio]-triphosphate (GTPγS) or the receptor-directed agonist fMetLeuPhe as activators. In addition, depletion from the cells of both PI-TP and PLC-β isoforms by extended permeabilization (40 minutes) allowed us to control the levels of PLC–β present in the cells. Once again, PI-TP increased the rates of reactions. To identify whether the role of PI-TP was to make available the substrate phosphatidylinositol bisphosphate (PIP2) for the PLC, we examined the synthesis of PIP2 in cells depleted of PI-TP. We found that PI-TP was essential for the synthesis of PIP2.Conclusion The predicted function of PI-TP in inositol lipid signalling is the provision of substrate for PLC–β from intracellular sites where PI is synthesized. We propose that PI-TP is in fact a co-factor in inositol lipid signalling and acts by interacting with the inositol lipid kinases. We hypothesize that the preferred substrate for PLC–β is not the lipid that is resident in the membrane but that provided through PI-TP

Identification of a Plasmodium falciparum phospholipid transfer protein.

Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte

Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid

Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPβ (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBβ binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBβ when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBβ was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBβ. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBβ was greater at the expense of PI binding. We propose that RdgBβ, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments

Individual phosphatidylinositol transfer proteins have distinct functions that do not involve lipid transfer activity

Platelets utilize signal transduction pathways facilitated by Class I phosphatidylinositol transfer proteins (PITPs). The two mammalian Class I PITPs, PITPα and PITPβ, are single PITP domain soluble proteins that are encoded by different genes and have 77% sequence identity, though their individual roles in mammalian biology remain uncharacterized. These proteins are believed to shuttle phosphatidylinositol and phosphatidylcholine between separate intracellular membrane compartments, thereby regulating phosphoinositide synthesis and second messenger formation. Previously, we observed that platelet-specific deletion of PITPα, the predominant expressed murine PITP isoform, had no effect on hemostasis, but had impaired tumor metastasis formation and disrupted phosphoinositide signaling. Here, we find that mice lacking the lesser expressed PITPβ in their platelets exhibit a similar phenotype. However, in contrast to PITPα-null platelet lysates that have impaired lipid transfer activity, PITPβ-null platelet lysates have essentially normal lipid transfer activity, although both isoforms contribute to phosphoinositide synthesis in vitro. Moreover, we found that platelet-specific deletion of both PITPs leads to ex vivo platelet aggregation/secretion and spreading defects, impaired tail bleeding, and profound tumor dissemination. Our studies also demonstrate that PITP isoforms are required for maintaining endogenous phosphoinositide PI(4,5)P2 levels and agonist stimulated second messenger formation. The data shown here demonstrate that both class I PITP isoforms contribute to phosphoinositide signaling in platelets, likely through distinct biochemical mechanisms or in different subcellular domains. They are functionally overlapping and either single isoform is able to maintain the homeostasis of platelets