title: Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.
abstract: Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins.
authors.affiliation: Biological Sciences, Kent State University, PO Box 5190, 44242 Kent, OH, USA.
authors.forename: Priya
authors.initials: P
authors.lastname: Putta
authors.affiliation: Biological Sciences, Kent State University, PO Box 5190, 44242 Kent, OH, USA.
authors.forename: Johanna
authors.initials: J
authors.lastname: Rankenberg
authors.affiliation: University of Amsterdam, Swammerdam Institute for Life Sciences, Section of Plant Physiology, Postbus 94215, 1090, GE, Amsterdam, The Netherlands; University of Amsterdam, Swammerdam Institute for Life Sciences, Section of Plant Cell Biology, Postbus 94215, 1090 GE Amsterdam, The Netherlands.
authors.forename: Ruud A
authors.initials: RA
authors.lastname: Korver
authors.affiliation: University of Amsterdam, Swammerdam Institute for Life Sciences, Section of Plant Physiology, Postbus 94215, 1090, GE, Amsterdam, The Netherlands; University of Amsterdam, Swammerdam Institute for Life Sciences, Section of Plant Cell Biology, Postbus 94215, 1090 GE Amsterdam, The Netherlands.
authors.forename: Ringo
authors.initials: R
authors.lastname: van Wijk
authors.affiliation: University of Amsterdam, Swammerdam Institute for Life Sciences, Section of Plant Physiology, Postbus 94215, 1090, GE, Amsterdam, The Netherlands; University of Amsterdam, Swammerdam Institute for Life Sciences, Section of Plant Cell Biology, Postbus 94215, 1090 GE Amsterdam, The Netherlands.
authors.forename: Teun
authors.initials: T
authors.lastname: Munnik
authors.affiliation: University of Amsterdam, Swammerdam Institute for Life Sciences, Section of Plant Physiology, Postbus 94215, 1090, GE, Amsterdam, The Netherlands; University of Amsterdam, Swammerdam Institute for Life Sciences, Section of Plant Cell Biology, Postbus 94215, 1090 GE Amsterdam, The Netherlands.
authors.forename: Christa
authors.initials: C
authors.lastname: Testerink
authors.affiliation: Biological Sciences, Kent State University, PO Box 5190, 44242 Kent, OH, USA. Electronic address: ekooijma@kent.edu.
authors.forename: Edgar E
authors.initials: EE
authors.lastname: Kooijman
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D001681:Biological Assay; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D028044:Phosphate-Binding Proteins; D010712:Phosphatidic Acids / Q000737:chemistry* / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism; D050766:Qb-SNARE Proteins / Q000737:chemistry / Q000378:metabolism; D050767:Qc-SNARE Proteins / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D029681:Arabidopsis Proteins; C112303:CAVIN2 protein, human; D002352:Carrier Proteins; C575856:ECA1 protein, Arabidopsis; D008081:Liposomes; D008244:Lysophosphatidylcholines; C096105:OPI1 protein, S cerevisiae; D028044:Phosphate-Binding Proteins; D010712:Phosphatidic Acids; D010714:Phosphatidylethanolamines; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C497084:Spo20 protein, S cerevisiae; C483858:phosphatidylethanolamine; D064413:3-Phosphoinositide-Dependent Protein Kinases; D019908:Proto-Oncogene Proteins c-raf
keywords: Epsin-like clathrin adaptor (ECA); Liposome binding assays; Membrane curvature stress; PA target proteins; PA-binding; Phosphatidic acid; Type I and type II lipids