title: In vitro reconstitution of lipid-dependent dual topology and postassembly topological switching of a membrane protein.
abstract: Phospholipids could exert their effect on membrane protein topology either directly by interacting with topogenic signals of newly inserted proteins or indirectly by influencing the protein assembly machinery. In vivo lactose permease (LacY) of Escherichia coli displays a mixture of topological conformations ranging from complete inversion of the N-terminal helical bundle to mixed topology and then to completely native topology as phosphatidylethanolamine (PE) is increased from 0% to 70% of membrane phospholipids. These topological conformers are interconvertible by postassembly synthesis or dilution of PE in vivo. To investigate whether coexistence of multiple topological conformers is dependent solely on the membrane lipid composition, we determined the topological organization of LacY in an in vitro proteoliposome system in which lipid composition can be systematically controlled before (liposomes) and after (fliposomes) reconstitution using a lipid exchange technique. Purified LacY reconstituted into preformed liposomes of increasing PE content displayed inverted topology at low PE and then a mixture of inverted and proper topologies with the latter increasing with increasing PE until all LacY adopted its native topology. Interconversion between topological conformers of LacY was observed in a PE dose-dependent manner by either increasing or decreasing PE levels in proteoliposomes postreconstitution of LacY, clearly demonstrating that membrane protein topology can be changed simply by changing membrane lipid composition independent of other cellular factors. The results provide a thermodynamic-based lipid-dependent model for shifting the equilibrium between different conformational states of a membrane protein.
authors.affiliation: Department of Biochemistry and Molecular Biology and the Center for Membrane Biology, University of Texas Medical School, Houston, TX 77030, USA.
authors.forename: Heidi
authors.initials: H
authors.lastname: Vitrac
authors.forename: Mikhail
authors.initials: M
authors.lastname: Bogdanov
authors.forename: William
authors.initials: W
authors.lastname: Dowhan
mesh_terms: D015153:Blotting, Western; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D047468:Immunoprecipitation; D008081:Liposomes / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D010743:Phospholipids / Q000378:metabolism; D011487:Protein Conformation; D027981:Symporters / Q000737:chemistry* / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008081:Liposomes; D009004:Monosaccharide Transport Proteins; D010743:Phospholipids; D027981:Symporters