title: Characterization of PROPPIN-Phosphoinositide Binding and Role of Loop 6CD in PROPPIN-Membrane Binding.
abstract: PROPPINs (β-propellers that bind polyphosphoinositides) are a family of PtdIns3P- and PtdIns(3,5)P2-binding proteins that play an important role in autophagy. We analyzed PROPPIN-membrane binding through isothermal titration calorimetry (ITC), stopped-flow measurements, mutagenesis studies, and molecular dynamics (MD) simulations. ITC measurements showed that the yeast PROPPIN family members Atg18, Atg21, and Hsv2 bind PtdIns3P and PtdIns(3,5)P2 with high affinities in the nanomolar to low-micromolar range and have two phosphoinositide (PIP)-binding sites. Single PIP-binding site mutants have a 15- to 30-fold reduced affinity, which explains the requirement of two PIP-binding sites in PROPPINs. Hsv2 bound small unilamellar vesicles with a higher affinity than it bound large unilamellar vesicles in stopped-flow measurements. Thus, we conclude that PROPPIN membrane binding is curvature dependent. MD simulations revealed that loop 6CD is an anchor for membrane binding, as it is the region of the protein that inserts most deeply into the lipid bilayer. Mutagenesis studies showed that both hydrophobic and electrostatic interactions are required for membrane insertion of loop 6CD. We propose a model for PROPPIN-membrane binding in which PROPPINs are initially targeted to membranes through nonspecific electrostatic interactions and are then retained at the membrane through PIP binding.
authors.affiliation: Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Ricarda A
authors.initials: RA
authors.lastname: Busse
authors.affiliation: Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Andreea
authors.initials: A
authors.lastname: Scacioc
authors.affiliation: Institute of Cellular Biochemistry, University Medicine, Georg-August University, Göttingen, Germany.
authors.forename: Roswitha
authors.initials: R
authors.lastname: Krick
authors.affiliation: Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Pérez-Lara
authors.affiliation: Institute of Cellular Biochemistry, University Medicine, Georg-August University, Göttingen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Thumm
authors.affiliation: Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. Electronic address: kkuehne@gwdg.de.
authors.forename: Karin
authors.initials: K
authors.lastname: Kühnel
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D055672:Static Electricity; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C532949:HSV2 protein, S cerevisiae; D010716:Phosphatidylinositols; D029701:Saccharomyces cerevisiae Proteins; D053835:Unilamellar Liposomes; D010450:Endopeptidases