In vitro Reconstitution of a Membrane Switch Mechanism for the Polarity Protein LGL. Issue 24 (4th December 2016)

• Record Type: Journal Article
• Authors: Visco, Ilaria; Hoege, Carsten; Hyman, Anthony A.; Schwille, Petra
• Is Part Of: Journal of molecular biology. Volume 428:Issue 24:Part A(2016:Dec. 04)
• Page Start: 4828
• Page End: 4842
• Publication Date: 2016-12-04
• Subjects: aPKC atypical protein kinase C -- bPI(4, 5)P2 L-α-phosphatidylinositol-4, 5-bisphosphate -- BSA bovine serum albumin -- DGS-NTA(Ni) 1, 2-di-(9Z-octadecenoyl)-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] -- DLS dynamic light scattering -- DOPC 1, 2-dioleoyl-sn-glycero-3-phosphocholine -- DOPS 1, 2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine -- eGFP enhanced green fluorescent protein -- GFP green fluorescent protein -- GUV giant unilamellar vesicle -- His6 hexahistidine tag -- LUVs large unilamellar vesicles -- LGL-1 lethal giant larvae 1 -- MBS membrane binding sequence -- PAR partitioning-defective proteins -- PI(4, 5)P2 1, 2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′, 5′-bisphosphate) -- PKC protein kinase C -- Pt platinum -- RNAi RNA interference -- RT room temperature -- SUVs small unilamellar vesicles -- SLBs supported lipid bilayers -- TFE trifluoroethanol -- WT wild type; cell polarity -- lethal giant larvae (LGL) -- synthetic biology -- giant unilamellar vesicles -- C. elegans; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2016.10.003
• Languages: English
• ISSNs: 0022-2836

Abstract

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 . Graphical Abstract: Highlights: Lethal giant larvae 1 (LGL-1) binds to negatively charged phospholipids through specific basic residues of a membrane binding sequence. LGL-1 membrane binding induces the formation of an alpha-helix in the membrane binding sequence. Reconstitution of LGL-1 phosphorylation in giant unilamellar vesicles by atypical protein kinase C LGL-1 residues required for membrane binding are required for localization and cell polarity in vivo .