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• W2104414280 abstract "Group IVA cytosolic phospholipase A2 (cPLA2α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA2α binds zwitterionic lipids such as phosphatidylcholine in a Ca2+-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA2α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA2α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA2α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA2α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA2α. Group IVA cytosolic phospholipase A2 (cPLA2α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA2α binds zwitterionic lipids such as phosphatidylcholine in a Ca2+-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA2α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA2α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA2α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA2α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA2α. arachidonic acid Amot coiled-coil homology domain bin-amphiphysin-Rvs167 calcium binding loop ceramide-1-phosphate circular dichroism group IVA cytosolic phospholipase A2 electron microscopy epsin N-terminal homology Fc receptor large unilamellar vesicle Pleckstrin homology 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine surface plasmon resonance transmission electron microscopy wild type Group IVA cytosolic phospholipase A2 (cPLA2α) is an 85 kDa enzyme consisting of a N-terminal lipid-binding C2 domain (∼120 residues) and a C-terminal catalytic or lipase domain (∼600 residues) that is separated by a flexible linker (1Clark J.D. Schievella A.R. Nalefski E.A. Lin L.L. Cytosolic phospholipase A2.J. Lipid Mediat. Cell Signal. 1995; 12: 83-117Crossref PubMed Scopus (425) Google Scholar, 2Leslie C.C. Gangelhoff T.A. Gelb M.H. Localization and function of cytosolic phospholipase A2alpha at the Golgi.Biochimie. 2010; 92: 620-626Crossref PubMed Scopus (37) Google Scholar). cPLA2α regulates arachidonic acid (AA) release from the sn-2 positions of membrane phospholipids; AA is used in the synthesis of leukotrienes and prostaglandins in response to inflammatory agonists (3Shimizu T. Ohto T. Kita Y. Cytosolic phospholipase A2: biochemical properties and physiological roles.IUBMB Life. 2006; 58: 328-333Crossref PubMed Scopus (70) Google Scholar). cPLA2α has also been implicated in a number of pathological conditions, including asthma (4Hewson C.A. Patel S. Calzetta L. Campwala H. Havard S. Luscombe E. Clarke P.A. Peachell P.T. Matera M.G. Cazzola M. et al.Preclinical evaluation of an inhibitor of cytosolic phospholipase A2alpha for the treatment of asthma.J. Pharmacol. Exp. Ther. 2012; 340: 656-665Crossref PubMed Scopus (27) Google Scholar), cancers (5Sundarraj S. Kannan S. Thangam R. Gunasekaran P. Effects of the inhibition of cytosolic phospholipase A(2)alpha in non-small cell lung cancer cells.J. Cancer Res. Clin. Oncol. 2012; 138: 827-835Crossref PubMed Scopus (16) Google Scholar), arthritis (6Tai N. Kuwabara K. Kobayashi M. Yamada K. Ono T. Seno K. Gahara Y. Ishizaki J. Hori Y. Cytosolic phospholipase A2 alpha inhibitor, pyrroxyphene, displays anti-arthritic and anti-bone destructive action in a murine arthritis model.Inflamm. Res. 2010; 59: 53-62Crossref PubMed Scopus (42) Google Scholar), cerebral ischemia (7Kishimoto K. Li R.C. Zhang J. Klaus J.A. Kibler K.K. Dore S. Koehler R.C. Sapirstein A. Cytosolic phospholipase A2 alpha amplifies early cyclooxygenase-2 expression, oxidative stress and MAP kinase phosphorylation after cerebral ischemia in mice.J. Neuroinflammation. 2010; 7: 42Crossref PubMed Scopus (45) Google Scholar), and heart disease (8Kerkela R. Boucher M. Zaka R. Gao E. Harris D. Piuhola J. Song J. Serpi R. Woulfe K.C. Cheung J.Y. et al.Cytosolic phospholipase A(2)alpha protects against ischemia/reperfusion injury in the heart.Clin. Transl. Sci. 2011; 4: 236-242Crossref PubMed Scopus (15) Google Scholar). The general principles governing cPLA2α in vitro membrane binding (9Bittova L. Sumandea M. Cho W. A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues.J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 10Corbin J.A. Evans J.H. Landgraf K.E. Falke J.J. Mechanism of specific membrane targeting by C2 domains: localized pools of target lipids enhance Ca2+ affinity.Biochemistry. 2007; 46: 4322-4336Crossref PubMed Scopus (84) Google Scholar) and activation (11Tucker D.E. Ghosh M. Ghomashchi F. Loper R. Suram S. John B.S. Girotti M. Bollinger J.G. Gelb M.H. Leslie C.C. Role of phosphorylation and basic residues in the catalytic domain of cytosolic phospholipase A2alpha in regulating interfacial kinetics and binding and cellular function.J. Biol. Chem. 2009; 284: 9596-9611Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) as well as cellular translocation (12Evans J.H. Leslie C.C. The cytosolic phospholipase A2 catalytic domain modulates association and residence time at Golgi membranes.J. Biol. Chem. 2004; 279: 6005-6016Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Evans J.H. Spencer D.M. Zweifach A. Leslie C.C. Intracellular calcium signals regulating cytosolic phospholipase A2 translocation to internal membranes.J. Biol. Chem. 2001; 276: 30150-30160Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) are well established, where the C2 domain binds with high affinity to zwitterionic membranes in a Ca2+-dependent manner (9Bittova L. Sumandea M. Cho W. A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues.J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), whereas the catalytic domain binds to membranes independent of Ca2+ weakly (14Das S. Cho W. Roles of catalytic domain residues in interfacial binding and activation of group IV cytosolic phospholipase A2.J. Biol. Chem. 2002; 277: 23838-23846Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). This functionality allows the C2 domain to act as a Ca2+ sensor in cells, which drives the cellular localization to the Golgi, ER, and nuclear membranes (2Leslie C.C. Gangelhoff T.A. Gelb M.H. Localization and function of cytosolic phospholipase A2alpha at the Golgi.Biochimie. 2010; 92: 620-626Crossref PubMed Scopus (37) Google Scholar, 12Evans J.H. Leslie C.C. The cytosolic phospholipase A2 catalytic domain modulates association and residence time at Golgi membranes.J. Biol. Chem. 2004; 279: 6005-6016Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Evans J.H. Spencer D.M. Zweifach A. Leslie C.C. Intracellular calcium signals regulating cytosolic phospholipase A2 translocation to internal membranes.J. Biol. Chem. 2001; 276: 30150-30160Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). The C2 domain (∼120 amino acids) contains three calcium-binding loops (CBLs), two of which (CBL1 and -3) harbor hydrophobic and aromatic amino acids (Fig. 1) that promote binding to zwitterionic membranes (9Bittova L. Sumandea M. Cho W. A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues.J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Ca2+ binding induces a dramatic change in electrostatic potential, lowering the desolvation penalty associated with membrane insertion (15Murray D. Honig B. Electrostatic control of the membrane targeting of C2 domains.Mol. Cell. 2002; 9: 145-154Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) and promoting the docking of the C2 domain to the membrane bilayer. This penetration into the membrane has been shown to be significant, with a depth of ∼15 Å (16Frazier A.A. Wisner M.A. Malmberg N.J. Victor K.G. Fanucci G.E. Nalefski E.A. Falke J.J. Cafiso D.S. Membrane orientation and position of the C2 domain from cPLA2 by site-directed spin labeling.Biochemistry. 2002; 41: 6282-6292Crossref PubMed Scopus (100) Google Scholar), which extends extensively into the hydrocarbon region of the membrane. The significant membrane penetration of cPLA2α is important for its membrane targeting to zwitterionic membranes and also to its ability to produce arachidonic acid. Recently, two anionic lipids—ceramide-1-phosphate (C1P) (17Pettus B.J. Bielawska A. Subramanian P. Wijesinghe D.S. Maceyka M. Leslie C.C. Evans J.H. Freiberg J. Roddy P. Hannun Y.A. et al.Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2.J. Biol. Chem. 2004; 279: 11320-11326Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 18Subramanian P. Stahelin R.V. Szulc Z. Bielawska A. Cho W. Chalfant C.E. Ceramide 1-phosphate acts as a positive allosteric activator of group IVA cytosolic phospholipase A2 alpha and enhances the interaction of the enzyme with phosphatidylcholine.J. Biol. Chem. 2005; 280: 17601-17607Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and PtdIns(4,5)P2 (19Balsinde J. Balboa M.A. Li W.H. Llopis J. Dennis E.A. Cellular regulation of cytosolic group IV phospholipase A2 by phosphatidylinositol bisphosphate levels.J. Immunol. 2000; 164: 5398-5402Crossref PubMed Scopus (66) Google Scholar, 20Mosior M. Six D.A. Dennis E.A. Group IV cytosolic phospholipase A2 binds with high affinity and specificity to phosphatidylinositol 4,5-bisphosphate resulting in dramatic increases in activity.J. Biol. Chem. 1998; 273: 2184-2191Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar)—have been demonstrated to bind and activate cPLA2α with emerging roles in the cellular translocation of the enzyme (21Stahelin R.V. Subramanian P. Vora M. Cho W. Chalfant C.E. Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain.J. Biol. Chem. 2007; 282: 20467-20474Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 22Casas J. Gijon M.A. Vigo A.G. Crespo M.S. Balsinde J. Balboa M.A. Phosphatidylinositol 4,5-bisphosphate anchors cytosolic group IVA phospholipase A2 to perinuclear membranes and decreases its calcium requirement for translocation in live cells.Mol. Biol. Cell. 2006; 17: 155-162Crossref PubMed Scopus (62) Google Scholar). Besides its role in eicosanoid biosynthesis, cPLA2α is selectively activated upon Fc receptor (FcR)-mediated phagocytosis in macrophages, where it rapidly translocates to the nascent phagosome (23Zizza P. Iurisci C. Bonazzi M. Cossart P. Leslie C.C. Corda D. Mariggio S. Phospholipase A2IValpha regulates phagocytosis independent of its enzymatic activity.J. Biol. Chem. 2012; 287: 16849-16859Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Unexpectedly, however, it was shown that membrane binding by the isolated C2 domain of cPLA2α was sufficient to induce phagosome formation (23Zizza P. Iurisci C. Bonazzi M. Cossart P. Leslie C.C. Corda D. Mariggio S. Phospholipase A2IValpha regulates phagocytosis independent of its enzymatic activity.J. Biol. Chem. 2012; 287: 16849-16859Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), suggesting that the C2 domain alone has membrane binding activity that regulates phagocytosis. This was further verified with a mutation in the C2 domain, D43N, which abrogates Ca2+-binding and could not rescue phagocytosis (23Zizza P. Iurisci C. Bonazzi M. Cossart P. Leslie C.C. Corda D. Mariggio S. Phospholipase A2IValpha regulates phagocytosis independent of its enzymatic activity.J. Biol. Chem. 2012; 287: 16849-16859Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). cPLA2α also plays a role in membrane curvature generation through regulation of aberrant Golgi vesiculation (24Grimmer S. Ying M. Walchli S. van Deurs B. Sandvig K. Golgi vesiculation induced by cholesterol occurs by a dynamin- and cPLA2-dependent mechanism.Traffic. 2005; 6: 144-156Crossref PubMed Scopus (53) Google Scholar), Golgi tubulation (25San Pietro E. Capestrano M. Polishchuk E.V. DiPentima A. Trucco A. Zizza P. Mariggio S. Pulvirenti T. Sallese M. Tete S. et al.Group IV phospholipase A(2)alpha controls the formation of inter-cisternal continuities involved in intra-Golgi transport.PLoS Biol. 2009; 7: e1000194Crossref PubMed Scopus (80) Google Scholar), and vesiculation of cholesterol-rich, GPI-anchored, protein-containing endosomes (26Cai B. Caplan S. Naslavsky N. cPLA2alpha and EHD1 interact and regulate the vesiculation of cholesterol-rich, GPI-anchored, protein-containing endosomes.Mol. Biol. Cell. 2012; 23: 1874-1888Crossref PubMed Google Scholar). Although it is speculated the C2 domain may induce changes to membrane structure (23Zizza P. Iurisci C. Bonazzi M. Cossart P. Leslie C.C. Corda D. Mariggio S. Phospholipase A2IValpha regulates phagocytosis independent of its enzymatic activity.J. Biol. Chem. 2012; 287: 16849-16859Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), direct evidence is lacking. These recent studies suggest that cPLA2α and its C2 domain have membrane remodeling activity that is critical to biological signaling pathways. Recently, a number of peripheral proteins, mainly attributed to their modular lipid binding domains, have been found to induce membrane curvature changes (27Campelo F. McMahon H.T. Kozlov M.M. The hydrophobic insertion mechanism of membrane curvature generation by proteins.Biophys. J. 2008; 95: 2325-2339Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), including the epsin N-terminal homology (ENTH) (28Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Curvature of clathrin-coated pits driven by epsin.Nature. 2002; 419: 361-366Crossref PubMed Scopus (793) Google Scholar), bin-amphiphysin-Rvs167 (BAR) (29Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. McMahon H.T. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure.Science. 2004; 303: 495-499Crossref PubMed Scopus (1348) Google Scholar), Pleckstrin homology (PH) (30Cao X. Coskun U. Rossle M. Buschhorn S.B. Grzybek M. Dafforn T.R. Lenoir M. Overduin M. Simons K. Golgi protein FAPP2 tubulates membranes.Proc. Natl. Acad. Sci. USA. 2009; 106: 21121-21125Crossref PubMed Scopus (53) Google Scholar), Amot coiled-coil homology domain (ACCH) (31Heller B. Adu-Gyamfi E. Smith-Kinnaman W. Babbey C. Vora M. Xue Y. Bittman R. Stahelin R.V. Wells C.D. Amot recognizes a juxtanuclear endocytic recycling compartment via a novel lipid binding domain.J. Biol. Chem. 2010; 285: 12308-12320Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), and C2 domains (32Martens, S., Kozlov, M. M., McMahon, H. T., . 2007. How synaptotagmin promotes membrane fusion. Science 316: 1205–1208.Google Scholar). Here, we investigate the ability of the isolated C2 domain of cPLA2α to induce changes to lipid structure. A number of imaging assays are used, including electron microscopy (EM) of large unilamellar vesicles (LUVs), imaging of giant unilamellar vesicles (GUVs), and imaging of membrane sheets. In addition, biophysical assays, including monolayer penetration analysis and surface plasmon resonance (SPR), were used to correlate membrane penetration and affinity with membrane remodeling activity. Results provide evidence that Ca2+-dependent membrane insertion of CBL1 and -3 of the C2 domain drive membrane curvature changes. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Octyl glucoside, (3-[3-cholamidopropyl) dimethylammonio]1-propane-sulfonate, Nunc Lab-Tek I Chambered Cover Glasses (8-well), and a bicinchoninic acid protein assay kit were from Thermo Fisher Scientific (Waltham, MA). L1 sensor chips were from GE Healthcare (Piscataway, NJ). N-(3-(triethylammoniumpropyl)-4-(4-(diethylamino)styryl)pyridinium dibromide (FM® 2-10) lipophilic dye was from Life Technologies (Grand Island, NY). Restriction endonucleases and enzymes for molecular biology were obtained from New England Biolabs (Beverly, MA). The QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) was used to introduce mutations into the pET28a vector with a His6 tag engineered into the N-terminus of the cPLA2α C2 domain gene (9Bittova L. Sumandea M. Cho W. A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues.J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). All mutated constructs were sequenced to ensure the presence of the desired mutation. The C2 domain and respective mutations were expressed and purified from Escherichia coli BL21(DE3) cells as previously described (9Bittova L. Sumandea M. Cho W. A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues.J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Protein concentrations were determined by the bicinchoninic acid method, and all protein aliquots were stored in 20 mM HEPES (pH 7.4) containing 160 mM KCl. Two hundred microliters of 1 mg/ml POPC LUVs were prepared as previously described (32Martens, S., Kozlov, M. M., McMahon, H. T., . 2007. How synaptotagmin promotes membrane fusion. Science 316: 1205–1208.Google Scholar). Briefly, the lipids were dried under nitrogen gas and resuspended in 20 mM HEPES (pH 7.4) containing 160 mM KCl and either 100 μM CaCl2 or 100 μM EGTA. The vesicles were incubated at 37°C and extruded through an 800 nm filter (Avanti Polar Lipids, Alabaster, AL). The respective C2 domain and mutations were incubated at a concentration of 10 μM with the POPC vesicles for 30 min at 25°C. Samples were then applied to a carbon-formvar-coated copper grid and stained with 2% uranyl acetate. Liposome morphologies were then imaged at 80 kV on a FEI 80-300 D3203 electron microscope at 6,300× magnification. An aliquot of lipids containing POPC, POPE, and POPS suspended in chloroform were prepared in a 60:20:20 molar ratio. The suspension was dried under nitrogen gas and resuspended in chloroform to a final concentration of 0.4 mg/ml. The lipid suspension was dried onto an indium tin oxide-coated slide and dehydrated under a vacuum for 1 h. The GUV apparatus was assembled, and a 350 mM D-sucrose solution was placed into the reservoir containing the dried lipids. Another glass plate was placed on top to eliminate air from the system, and then a sin wave generator was applied at 3V and 20 Hz for 5 h at 25°C. The GUV solution was collected and stored at 25°C until use. The GUV solution was diluted 20-fold in 20 mM HEPES (pH 7.4) containing 160 mM KCl and 10 μM FM® 2-10 lipophilic dye. Samples were prepared with 100 μM EGTA, 10 μM CaCl2, or 500 nM CaCl2 as necessary for experimental conditions. GUV vesiculation was assessed after a 5 min incubation with 2 μM or 500 nM cPLA2α-C2, 500 nM full-length cPLA2α, or 2 μM of respective mutants and was imaged via confocal microscopy (Zeiss LSM 710) on Nunc Lab-Tek I Chambered Cover Glasses (8 well) using a 63× 1.4 NA oil objective. Three replicates for each control or sample were quantified by counting 60–100 GUVs per replicate. The number of vesiculated GUVs was determined separately and compared with the total number of GUVs in each replicate. The degree of vesiculation was then expressed as a percentage, compared with the control, and quantified using an unpaired Student t-test. Two microliters of 10 mM POPC was prepared in chloroform, spotted onto Nunc Lab Tek I (8 well) Chambered Cover Glasses, and dried under a vacuum. The lipid was rehydrated with 20 mM HEPES (pH 7.4) containing 160 mM KCl and 20 μM FM® 2-10 and allowed to rehydrate for 15 min. Samples were prepared with 100 μM EGTA or 100 μM CaCl2 as necessary for experimental conditions. The experiments contained 2 μM cPLA2α-C2 or respective mutants and were subsequently imaged after a 15 min incubation with a confocal microscope (Nikon A1R-MP with a 100× 1.4 NA oil objective). Surface pressure (π) of solution in a circular Teflon trough (2 ml) was measured using a wire probe connected to a Kibron MicroTrough X (Kibron, Inc., Helsinki, Finland) as previously described (33Hom R.A. Vora M. Regner M. Subach O.M. Cho W. Verkhusha V.V. Stahelin R.V. Kutateladze T.G. pH-dependent binding of the Epsin ENTH domain and the AP180 ANTH domain to PI(4,5)P2-containing bilayers.J. Mol. Biol. 2007; 373: 412-423Crossref PubMed Scopus (36) Google Scholar). Phospholipid solution (2–8 μl) in hexane/ethanol (9:1 v/v) was spread onto 2 ml of subphase to form a monolayer with a given initial surface pressure (π0). The subphase was stirred continuously at 30 revolutions/min with a small magnetic stir bar. After stabilization of the surface pressure of the monolayer (∼5 min), the protein solution (typically 10 μl) was injected into the subphase, and the change in surface pressure (Δπ) was measured as a function of time. Generally, the Δπ value reached a maximum after 20 min. The maximal Δπ value depends upon the protein concentration and reached saturation at cPLA2α-C2 > 1 μg/ml, as previously reported (34Stahelin R.V. Cho W. Roles of calcium ions in the membrane binding of C2 domains.Biochem. J. 2001; 359: 679-685Crossref PubMed Scopus (56) Google Scholar). Protein concentrations in the subphase are maintained above such values to ensure that the Δπ represents a maximal value. The Δπ versus π0 plots were constructed from these measurements to obtain the x-intercept or critical pressure (πc) defined as the value to which the protein penetrates (35Cho W. Bittova L. Stahelin R.V. Membrane binding assays for peripheral proteins.Anal. Biochem. 2001; 296: 153-161Crossref PubMed Scopus (114) Google Scholar). All SPR measurements were performed at 25°C. A detailed protocol for coating the L1 sensor chip has been described elsewhere (34Stahelin R.V. Cho W. Roles of calcium ions in the membrane binding of C2 domains.Biochem. J. 2001; 359: 679-685Crossref PubMed Scopus (56) Google Scholar). Briefly, after washing the sensor chip surface, 90 μl of POPC vesicles were injected at 5 μl/min to give a response of 6,500 resonance units. An uncoated flow channel was used as a control surface. Under our experimental conditions, no binding was detected to this control surface beyond the refractive index change for the C2 domain of cPLA2α as previously reported (18Subramanian P. Stahelin R.V. Szulc Z. Bielawska A. Cho W. Chalfant C.E. Ceramide 1-phosphate acts as a positive allosteric activator of group IVA cytosolic phospholipase A2 alpha and enhances the interaction of the enzyme with phosphatidylcholine.J. Biol. Chem. 2005; 280: 17601-17607Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 34Stahelin R.V. Cho W. Roles of calcium ions in the membrane binding of C2 domains.Biochem. J. 2001; 359: 679-685Crossref PubMed Scopus (56) Google Scholar). Each lipid layer was stabilized by injecting 10 μl of 50 mM NaOH three times at 100 μl/min. SPR measurements were done at the flow rate of 5 μl/min. To give an association time to reach saturation of binding signal (Req), 50–90 μl of protein in 10 mM HEPES (pH 7.4) containing 160 mM KCl, and 50 μM Ca2+ was injected (see Fig. 5C). The saturation responses for wild type (WT) and mutations were normalized where maximum WT saturation response was set to 1 to compare the binding capacity of WT versus mutations. The lipid surface was regenerated using 10 μl of 50 mM NaOH. Each of the sensorgrams was corrected for refractive index change by subtracting the control surface response from the binding curve. A minimum of three data sets was collected for each protein at a minimum of five different concentrations for each protein within a 10-fold range of Kd. Req values were then plotted versus protein concentration and the Kd value was determined by a nonlinear least-squares analysis of the binding isotherm using the equation Req= Rmax/(1 + Kd/C) (36Stahelin R.V. Karathanassis D. Murray D. Williams R.L. Cho W. Structural and membrane binding analysis of the PX domain of Bem1p: basis of phosphatidylinositol-4-phosphate specificity.J. Biol. Chem. 2007; 282: 25737-25747Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Each data set was repeated three times to calculate a standard deviation. To ensure the WT and mutant proteins retained a stable structure, circular dichroism (CD) was used to assess the secondary structure of each recombinant protein used in the study. The spectra were taken on a JASCO 815 CD spectrometer scanned from 195 to 250 nm in a 1 mm quartz spectrophotometer cell (Starna Cells Inc., Atascadero, CA) at 20°C in 10 mM Hepes and 80 mM KCl (pH 7.4). Each measurement was performed in triplicate and averaged to yield the representative scans shown in supplementary Fig. IA. Molar ellipticity was defined according to the JASCO software and was subtracted from a control buffer scan. WT and mutations displayed overlapping spectra consistent with β-sheet structure. To measure the calcium-binding capacity of WT cPLA2α-C2 and the point mutants, the calcium detector Bis-Fura-2 (Life Technologies, Carlsbad, CA) was used according to the manufacture's protocol. Briefly, 2 μM protein was incubated for 30 min with control or 10 μM Ca2+ standard in a black fluorescent plate with a clear bottom (Costar Life Science, Tewksbury, MA). The difference in the unknown Ca2+ concentration was determined in relation to a standard curve by measuring the ratio of the emission at 510 nm at excitation wavelengths of 350 nm and 380 nm, respectively. Percent binding was normalized to the average WT binding capacity. Measurements were performed in triplicate for WT and mutations to determine the standard deviation (supplementary Fig. IB). EM has been used to effectively characterize changes in liposome morphologies induced by the ENTH (28Ford M.G. Mills I.G. Peter B.J. Vallis Y. Praefcke G.J. Evans P.R. McMahon H.T. Curvature of clathrin-coated pits driven by epsin.Nature. 2002; 419: 361-366Crossref PubMed Scopus (793) Google Scholar), BAR (29Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. McMahon H.T. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure.Science. 2004; 303: 495-499Crossref PubMed Scopus (1348) Google Scholar), ACCH (31Heller B. Adu-Gyamfi E. Smith-Kinnaman W. Babbey C. Vora M. Xue Y. Bittman R. Stahelin R.V. Wells C.D. Amot recognizes a juxtanuclear endocytic recycling compartment via a novel lipid binding domain.J. Biol. Chem. 2010; 285: 12308-12320Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), and C2 domains (32Martens, S., Kozlov, M. M., McMahon, H. T., . 2007. How synaptotagmin promotes membrane fusion. Science 316: 1205–1208.Google Scholar). To assess the ability of the C2 domain of cPLA2α to induce changes to liposome morphologies, we used transmission EM (TEM) with negative staining to visualize liposomes before and after incubation with the C2 domain. The C2 domain induced dramatic changes in POPC liposome structures as long tubules were extensively visualized through the grids (Fig. 2). Moreover, the tubulation of liposomes induced by the C2 domain was Ca2+ dependent; experiments performed in the presence of 100 μM EGTA in place of CaCl2 did not display detectable changes in liposome morphology (Fig. 2). The ENTH, BAR, and ACCH domains insert into the hydrocarbon region of the membrane bilayer, which is a prerequisite for their abi" @default.
• W2104414280 created "2016-06-24" @default.
• W2104414280 creator A5000603569 @default.
• W2104414280 creator A5006866639 @default.
• W2104414280 creator A5020924158 @default.
• W2104414280 creator A5078195240 @default.
• W2104414280 date "2012-12-01" @default.
• W2104414280 modified "2023-10-16" @default.
• W2104414280 title "C2 domain membrane penetration by group IVA cytosolic phospholipase A2 induces membrane curvature changes" @default.
• W2104414280 cites W1522134183 @default.
• W2104414280 cites W1550274293 @default.
• W2104414280 cites W1964027175 @default.
• W2104414280 cites W1971212998 @default.
• W2104414280 cites W1971962926 @default.
• W2104414280 cites W1987835269 @default.
• W2104414280 cites W1988088765 @default.
• W2104414280 cites W1991131490 @default.
• W2104414280 cites W1995865956 @default.
• W2104414280 cites W1996870265 @default.
• W2104414280 cites W1999045066 @default.
• W2104414280 cites W2001033348 @default.
• W2104414280 cites W2002012958 @default.
• W2104414280 cites W2002189009 @default.
• W2104414280 cites W2002339878 @default.
• W2104414280 cites W2004344365 @default.
• W2104414280 cites W2010104107 @default.
• W2104414280 cites W2020052689 @default.
• W2104414280 cites W2021285837 @default.
• W2104414280 cites W2021717885 @default.
• W2104414280 cites W2023863818 @default.
• W2104414280 cites W2026886026 @default.
• W2104414280 cites W2031143643 @default.
• W2104414280 cites W2040470620 @default.
• W2104414280 cites W2040566502 @default.
• W2104414280 cites W2041622964 @default.
• W2104414280 cites W2043634254 @default.
• W2104414280 cites W2052424724 @default.
• W2104414280 cites W2053132501 @default.
• W2104414280 cites W2055761513 @default.
• W2104414280 cites W2058070463 @default.
• W2104414280 cites W2058563514 @default.
• W2104414280 cites W2072023677 @default.
• W2104414280 cites W2072606718 @default.
• W2104414280 cites W2072637321 @default.
• W2104414280 cites W2076132445 @default.
• W2104414280 cites W2081166773 @default.
• W2104414280 cites W2082412503 @default.
• W2104414280 cites W2106333373 @default.
• W2104414280 cites W2108586733 @default.
• W2104414280 cites W2110913125 @default.
• W2104414280 cites W2112827571 @default.
• W2104414280 cites W2114628856 @default.
• W2104414280 cites W2117649547 @default.
• W2104414280 cites W2119253842 @default.
• W2104414280 cites W2120151870 @default.
• W2104414280 cites W2121822849 @default.
• W2104414280 cites W2125215711 @default.
• W2104414280 cites W2128386610 @default.
• W2104414280 cites W2133011371 @default.
• W2104414280 cites W2144996654 @default.
• W2104414280 cites W2145945997 @default.
• W2104414280 cites W2156827480 @default.
• W2104414280 cites W2158620418 @default.
• W2104414280 cites W2159301814 @default.
• W2104414280 cites W2165914953 @default.
• W2104414280 cites W2166335706 @default.
• W2104414280 cites W2168185723 @default.
• W2104414280 cites W2169082359 @default.
• W2104414280 cites W2169857435 @default.
• W2104414280 cites W2171502794 @default.
• W2104414280 cites W4253476580 @default.
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