Abstract
In recent years, the functional roles of effectors from a wide variety of fungal and oomycete pathogens have begun to emerge. As a product of this work, the importance of effector-lipid interactions has been made apparent. Phospholipids are not only important signaling molecules, but they also play important roles in the trafficking of endosomes and the localization of proteins. Characterizing effector-lipid interactions can provide novel information regarding the functions of effectors relevant to their cellular and subcellular targeting and their potential effects on host signaling and vesicle trafficking. We present here two techniques that can be used to screen for and validate protein-lipid interactions without the need to access highly specialized machinery. We describe in detail how to perform lipid filter and liposome-binding assays and provide suggestions for troubleshooting potential problems with these assays.
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Kooijman EE et al (2003) Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid. Traffic 4: 162–174.
Athenstaedt K, Daum G (1999) Phosphatidic acid, a key intermediate in lipid metabolism. Eur J Biochem 266: 1–16.
Delon C et al (2004) Sphingosine kinase 1 is an intracellular effector of phosphatidic acid. J Biol Chem 279: 44763–44774.
Young BP et al (2010) Phosphatidic acid is a pH biosensor that links membrane biogenesis to metabolism. Science 329: 1085–1088.
Anthony RG et al (2006) The Arabidopsis proteinkinase PTI1-2 is activated by convergent phosphatidic acid and oxidative stress signaling pathways downstream of PDK1 and OXI1.
Park J et al (2004) Phosphatidic acid induces leaf cell death in Arabidopsis by activating the rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation. Plant Physiol 134: 129–136.
Daleke DL (2003) Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Research 44: 233–242.
An X et al (2004) Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability. Biochemistry 43: 310–315.
Sahu SK et al (2007) Phospholipid scramblases: an overview. Arch Biochem Biophys 462: 103–114.
Zwaal RF et al (2005) Surface exposure of phosphatidylserine in pathological cells. Cell Mol Life Sci 62: 971–988.
Vermeet JEM et al (2009) Mapping phosphatidylinositol 4-phosphate dynamics in living plant cells. Plant J 57: 356–372.
Garofalo RS et al (2003) Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta. J Clinic Invest 112: 197–208.
Franke TF et al (2003) PI3K/Akt and apoptosis: size matters. Oncogene, 22: 8983–8998.
Godi A et al (2004) FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P. Nat Cell Biol 6: 393–404.
D’Angelo et al (2008) The multiple roles of PtdIns(4)P- not just the precursor of PtdIns(4,5)P2. J Cell Sci 121: 1955–1963.
Wurmser AE, Emr SD (1998) Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires luminal vacuolar hydrolase activities. EMBO Journal 17: 4930–4942.
Zoncu R et al (2009) A Phosphoinositide switch controls the maturation and signaling properties of APPL endosomes. Cell 136: 1110–1121.
Kale SD et al (2010) External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells. Cell 142: 284–295.
Stahelin RV (2009) Lipid binding domains: more than simple lipid effectors. J Lipid Res 50: 299–304.
Colon-Gonzalez F et al (2006) C1 domains exposed: From diacylglycerol binding to protein-protein interactions. BBA- Mol Cell Biol L 1761: 827–837.
Bolsover SR et al (2003) Role of Ca2+/phospha-tidylserine binding region of the C2 domain in the translocation of protein kinase Cα to the plasma membrane. J Biol Chem 278: 10282–10290.
Craig KL, Harley CB (1996) Phosphorylation of human pleckstrin on Ser-113 and Ser-117 by protein kinase C. Biochem J 314: 937–942.
Dowler S et al (2000) Identification of pleckstrin-homology-domain containing proteins with novel phosphoinositide binding specificities. Biochem J 351: 19–31.
Gaullier JM et al (2004) FYVE finger bind PtdIns(3)p. Nat Cell Biol 5: 393–404.
Lee SA et al (2006) Molecular Mechanism of Membrane Docking by the Vam7p PX Domain J Biol Chem 281: 37091–37101.
McLaughlin S et al (2005) Reversible - through calmodulin - electrostatic interactions between basic residues on proteins and acidic lipids in the plasma membrane. Biochem Soc Symp 72: 189–198.
Ghosh S et al (1996) Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. J Biol Chem 271: 8472–8480.
Grange M et al (2000) The cAMP-specific phosphodiesterase PDE4D3 is regulated by phosphatidic acid binding. J Biol Chem: 275, 33379–33387.
Dou D et al (2008) RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen encoded machinery. Plant Cell 20: 1930–1947.
Rafiqi M et al (2010) Internalization of flax rust avirulence proteins into flax and tobacco cells can occur in the absence of the pathogen. Plant Cell 22: 2017–2032.
Whisson SC et al (2007) A translocation signal for delivery of oomycete effector proteins into host plant cells. Nature 450: 115–118.
Jiang RHY et al (2008) RXLR effector reservoir in two Phytophthora species is dominated by a single rapidly evolving superfamily with more than 700 members. Proc Natl Acad Sci USA 12: 4874–4879.
Dou D et al (2008) Conserved C-terminal motifs required for avirulence and suppression of cell death by Phytophthora sojae effector Avr1b. Plant Cell 20: 1118–1133.
Bos JI et al (2007) The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana. Plant Journal 48: 165–76.
Sohn KH et al (2007) The Downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana. Plant Cell 19: 4077–4090.
Lafont F et al (2004) Bacterial subversion of lipid rafts. Curr Opin Microbiol 7: 4–10.
Murata-Kamiya N et al (2010) Helicobacter pylori Exploits Host Membrane Phosphatidylserine for Delivery, Localization, and Pathophysiological Action of the CagA Oncoprotein. Cell Host Microbe 7: 338–339.
Rüter C et al (2010) A newly identified bacterial cell-penetrating peptide that reduces the transcription of pro-inflammatory cytokines. J Cell Sci 123: 2190–2198.
Schoebel, S et al (2010) High-affinity binding of phosphatidylinositol 4-phosphate by Legionella pneumophila DrrA. EMBO reports 11: 598–604.
Weber SS et al (2006) Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog 2(5): e46. doi:10.1371/journal.ppat.0020046.
Brombacher E et al (2009) Rab1 guanine nucleotide exchange factor SidM is a major PtdIns(4)P-binding effector protein of Legionella pneumophila. J Biol Chem 284: 4846–4856.
Tawk L et al (2010) Phosphatidylinositol 3-phosphate, an essential lipid in Plasmodium, localises to the food vacuole membrane and the apicoplast. Eukaryotic Cell doi:10.1128/EC.00124-10.
Lee SW et al (2005) PilT is required for PI(3,4,5)P3-mediated crosstalk between Neisseria gonorrhoeae and epithelial cells. Cell Microbiol 7: 1271–1284.
Narayan K, Lemmon MA (2006) Determining selectivity of phosphoinositide-binding domains. Methods 39: 122–133.
Echelon Biosciences Inc. (2005). Technical Data Sheet. Q&A P-6000 Rev: 1 (08/08/05). http://www.echelon-inc.com/corp/p-6000%-20qa.pdf.
Ju H et al (2009) Membrane insertion of the FYVE domain is modulated by pH. Proteins 76: 852–860.
Silvius JR (1982) Thermotropic phase transitions of pure lipids in model membranes and their modification by membrane proteins. In: Jost PC, Griffith OH (eds) Lipid-protein interactions, vol 2. Wiley, New York.
Diraviyam K et al (2003) Computer modeling of the membrane interaction of FYVE Domains. J Mol Biol 328: 721–736.
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Kale, S.D., Tyler, B.M. (2012). Identification of Lipid-Binding Effectors. In: Bolton, M., Thomma, B. (eds) Plant Fungal Pathogens. Methods in Molecular Biology, vol 835. Humana Press. https://doi.org/10.1007/978-1-61779-501-5_24
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DOI: https://doi.org/10.1007/978-1-61779-501-5_24
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