Title
In vitro reconstitution of a membrane switch mechanism for the polarity protein LGL
Author
Ilaria Visco, Carsten Hoege, Anthony A. Hyman, Petra Schwille
Year
2016
Journal
Journal of Molecular Biology
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 both to the cytoplasm and, asymmetrically, to 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, C. elegans LGL (LGL-1) and atypical PKC (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.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Vesicle interactions, Ligand binding, Biochemistry