title: Membrane targeting of the Spir·formin actin nucleator complex requires a sequential handshake of polar interactions.
abstract: Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by fluorescence cross-correlation spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes.
authors.affiliation: From the Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany, Biotechnology Center (BIOTEC), Biophysics Research Group and.
authors.forename: Janine
authors.initials: J
authors.lastname: Tittel
authors.affiliation: Molecular Cell Biology Laboratory, Department of Neurology, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany, and.
authors.forename: Tobias
authors.initials: T
authors.lastname: Welz
authors.affiliation: Paul Langerhans Institute, Technische Universität (TU) Dresden, Tatzberg 47-51, D-01307 Dresden, Germany, German Center for Diabetes Research (Deutsches Zentrum für Diabetesforschung), 85764 Neuherberg, Germany.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Czogalla
authors.affiliation: Molecular Cell Biology Laboratory, Department of Neurology, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany, and.
authors.forename: Susanne
authors.initials: S
authors.lastname: Dietrich
authors.affiliation: Molecular Cell Biology Laboratory, Department of Neurology, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany, and.
authors.forename: Annette
authors.initials: A
authors.lastname: Samol-Wolf
authors.affiliation: Molecular Cell Biology Laboratory, Department of Neurology, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany, and.
authors.forename: Markos
authors.initials: M
authors.lastname: Schulte
authors.affiliation: From the Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany, Biotechnology Center (BIOTEC), Biophysics Research Group and.
authors.forename: Petra
authors.initials: P
authors.lastname: Schwille
authors.affiliation: From the Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany, Biotechnology Center (BIOTEC), Biophysics Research Group and.
authors.forename: Thomas
authors.initials: T
authors.lastname: Weidemann
authors.affiliation: Molecular Cell Biology Laboratory, Department of Neurology, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany, and eugen.kerkhoff@klinik.uni-regensburg.de.
authors.forename: Eugen
authors.initials: E
authors.lastname: Kerkhoff
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000080865:Formins; D057809:HEK293 Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D060066:Protein Interaction Maps / Q000235:genetics
chemical_list: D000199:Actins; C587953:Fmn2 protein, human; D000080865:Formins; D026901:Membrane Transport Proteins; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; C556674:Spir-2 protein, mouse
keywords: Actin; FYVE; Formin; Membrane Trafficking; Protein Complex; Spir; Vesicles