title: In vitro Reconstitution of a Membrane Switch Mechanism for the Polarity Protein LGL.
abstract: Cell polarity arises from a combination of interactions between biological molecules, such as activation, inhibition, and positive or negative feedback between specific polarity units. Activation and inhibition often take place in the form of a membrane binding switch. Lethal giant larvae (LGL), a conserved regulator of cell polarity in animals, was suggested to function as such a switch. LGL localizes to both the cytoplasm and, asymmetrically, the membrane. However, the spatial regulation mechanism of LGL membrane localization has remained unclear. For systematic elucidation, we set out to reconstitute a minimal polarity unit using a model membrane, Caenorhabditis elegans LGL (LGL-1), and atypical protein kinase C (aPKC) supposed to activate the membrane switch. We identified a membrane binding sequence (MBS) in LGL-1 by a screen in vivo, reconstituted LGL-1 membrane binding in vitro, and successfully implemented the membrane switch by aPKC phosphorylation activity, detaching LGL from membranes. Upon membrane binding, LGL-1 MBS folds into an alpha-helix in which three regions can be identified: a positively charged patch, a switch area containing the three aPKC phosphorylation sites, and a hydrophobic area probably buried in the membrane. Phosphorylation by aPKC dramatically reduces the binding affinity of the LGL-1 MBS to negatively charged model membranes, inducing its detachment. Specific residues in the MBS are critical for LGL-1 function in C. elegans.
authors.affiliation: Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: ilaria.visco@mpi-dortmund.mpg.de.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Visco
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Hoege
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Hyman
authors.affiliation: Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: schwille@biochem.mpg.de.
authors.forename: Petra
authors.initials: P
authors.lastname: Schwille
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology*; D016764:Cell Polarity; D004252:DNA Mutational Analysis; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational
chemical_list: D029742:Caenorhabditis elegans Proteins; C553666:LGL-1 protein, C elegans; C115015:PKC-3 protein; D011493:Protein Kinase C
keywords: C. elegans; cell polarity; giant unilamellar vesicles; lethal giant larvae (LGL); synthetic biology