How Does a Membrane Protein Know What is In and What is Out? Lipids as Topological Determinants

Abstract

Primary functions of lipids are to define barrier properties of membranes and provide a scaffold within which membrane proteins are organized. Using a genetic approach to alter the phospholipid composition of the Escherichia coli coupled with biochemical approaches to monitor topological organization of membrane proteins, dependence of lactose permease (LacY) on phosphatidylethanolamine (PE) for proper orientation with respect to the plane of the membrane was determined. Assembly of LacY in the absence of PE results in topological inversion of its N-terminal half, which is largely reversed by post-assembly synthesis of PE. Replacement of PE by the foreign lipids phosphatidylcholine, monoglucosyl diacylglycerol, or diglucosyl diacylglycerol, which exhibit similar properties to PE, restores proper topology thereby supporting common functions for lipids with diverse structures. Topology of LacY in membranes lacking PE is dependent on a connecting flexible hinge region in order for the two halves of LacY to independently respond to the lipid environment. Final topology is determined after LacY exits the translocon by long-range and short-range interactions between the net charge of extra-membrane domains and the net charge density of the phospholipid bilayer surface. PE appears to dampen the translocation potential of acidic residues in normally cytoplasmic domains in favor of the cytoplasmic retention potential of basic residues. Thus a primary role for PE is to allow the presence of acidic residues in the cytoplasmic domains of membrane proteins for functional purposes without affecting protein topological. The topologies of two amino acid permeases (PheP and GabP) unrelated to LacY are also topologically sensitive to membrane lipid composition strongly indicating that lipid environment is a significant determinant of final topological organization of multiple membrane proteins. Supported in part by NIGMS R37-GM20478.