Lipid-Protein Interactions Drive Membrane Protein Topogenesis in Accordance with the Positive Inside Rule

Abstract

Transmembrane domain orientation within some membrane proteins is dependent on membrane lipid composition. Initial orientation occurs within the translocon, but final orientation is determined after membrane insertion by interactions within the protein and between lipid headgroups and protein extramembrane domains. Positively and negatively charged amino acids in extramembrane domains represent cytoplasmic retention and membrane translocation forces, respectively, which are determinants of protein orientation. Lipids with no net charge dampen the translocation potential of negative residues working in opposition to cytoplasmic retention of positive residues, thus allowing the functional presence of negative residues in cytoplasmic domains without affecting protein topology. Membrane Protein AssemblyMembrane protein topogenesis, the process by which TM 4 orientation of a membrane protein is determined, is directed by a combination of several factors, leading to a predictable final organization for many but not all membrane proteins (1, 2). Initial TM topology is determined by topogenic signals residing within the protein sequence (1, 3) and protein interaction with the ribosomal channel (4) and the translocon machinery (5). Final topology is determined after the completed polypeptide chain exits the translocon by internal protein-protein interactions and protein-membrane lipid interactions during folding into the final compact protein (6 -8). Although TM organization of many membrane proteins is established, the process by which these domains achieve their final orientation with respect to the plane of the membrane bilayer is not fully understood.The biogenesis of polytopic membrane proteins has been extensively reviewed (9, 10). The signal recognition particle binds to the N-terminal signal sequence of a nascent polypeptide as it exits the ribosome and directs the docking of the complex onto the membrane-integrated translocon via interaction with the signal recognition particle receptor. Translation proceeds with the N-terminal extramembrane domain exiting the translocon on either the cytoplasmic (cis) or opposite (trans) side of the membrane. The first TM translates into the translocon pore and then exits laterally into the hydrophobic core of the membrane bilayer. The next extramembrane domain generally exits the translocon on the opposite side of the membrane relative to the preceding extramembrane domain, followed by a repeat of the above cycle until the initial insertion and orientation of TMs are completed. Therefore, the side of the membrane through which the flanking extramembrane domains exit the translocon initially determines the orientation of each TM. This initial orientation is governed by interaction of the nascent polypeptide with the ribosomal and translocon channels and is largely determined by the "positive inside rule," which is based on the statistically and biochemically verified observation that the overwhelming majority of extramembrane domains facing the cytoplasm carry a net positive ch...

Lipids and Topological Rules of Membrane Protein Assembly

“…Testing the Lipid-dependent Charge Balance Hypothesis with PheP-We suggested that polytopic membrane proteins containing competing opposite charges on one side of the membrane might share a common mechanism for topogenesis (1,14,19). To further test this hypothesis, we extended our studies to PheP as another model protein with little homology to the sugar permeases.…”

“…Therefore, it was concluded that interaction between the collective charge properties of the membrane surface and the protein cytoplasmically exposed extramembrane domains directs the final orientation of TMs (i.e. the effects of changes in net charge were the same whether resulting from the lipid or the protein) (14,19). A role for lipids with no net charge appears to be a dampening of the translocation potential of negative residues that work in opposition to the positive inside rule.…”

“…This dampening allows the functional presence of negative residues in cytoplasmic domains without affecting protein organization (1). Therefore, according to the net charge balance hypothesis of membrane protein assembly (1,14,19), the proper charge balance between translocation signals and retention signals required to achieve proper TM topology is maintained by the appropriate charge density of the membrane surface. In Escherichia coli, membrane surface charge is determined by the mol % of anionic (phosphatidylglycerol and cardiolipin) and zwitterionic (PE) phospholipids.…”

“…Although a net positive charge dictates cytoplasmic disposition, negatively charged residues can be topogenic and attenuate the effect of positively charged residues, thus favoring periplasmic location if they lie within a critical distance (up to 6 residues) from a TM (18). Acidic residues located within the normally cytoplasmic domain of the above permeases were postulated to act as lipidsensitive topogenic signals (1,14,19). An increase in the net positive charge of the cytoplasmic surface of LacY, either by eliminating negatively charged residues or introducing positively charged residues in a position-independent manner, resulted in the N-terminal half of LacY adopting its native orientation even in PE-lacking membranes (1).…”

Annular Anionic Lipids Stabilize the Integrin αIIbβ3 Transmembrane Complex

“…Membrane proteins are arranged such that positively charged residues localize to the intracellular membrane face (4). The structural basis of this phenomenon is the interaction of anionic lipids, which are concentrated to the intracellular membrane leaflet, with cationic protein residues (5,6). This fundamental mechanism implies that anionic lipids also compete with electrostatic interactions between transmembrane (TM) 3 helices within the intracellular lipid headgroup region.…”