Lipid-Assisted Membrane Protein Folding and Topogenesis (original) (raw)
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Lipids and topological rules governing membrane protein assembly
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2013
Membrane protein folding and topogenesis are tuned to a given lipid profile since lipids and proteins have coevolved to follow a set of interdependent rules governing final protein topological organization. Transmembrane domain (TMD) topology is determined via a dynamic process in which topogenic signals in the nascent protein are recognized and interpreted initially by the translocon followed by a given lipid profile in accordance with the Positive Inside Rule. The net zero charged phospholipid phosphatidylethanolamine and other neutral lipids dampen the translocation potential of negatively charged residues in favor of the cytoplasmic retention potential of positively charged residues (Charge Balance Rule). This explains why positively charged residues are more potent topological signals than negatively charged residues. Dynamic changes in orientation of TMDs during or after membrane insertion are attributed to non-sequential cooperative and collective lipid-protein charge interactions as well as long-term interactions within a protein. The proportion of dual topological conformers of a membrane protein varies in a dose responsive manner with changes in the membrane lipid composition not only in vivo but also in vitro and therefore is determined by the membrane lipid composition. Switching between two opposite TMD topologies can occur in either direction in vivo and also in liposomes (designated as fliposomes) independent of any other cellular factors. Such lipid-dependent post-insertional reversibility of TMD orientation indicates a thermodynamically driven process that can occur at any time and in any cell membrane driven by changes in the lipid composition. This dynamic view of protein topological organization influenced by the lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
Molecular genetic and biochemical approaches for defining lipid-dependent membrane protein folding
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012
We provide an overview of lipid-dependent polytopic membrane protein folding and topogenesis. Lipid dependence of this process was determined by employing Escherichia coli cells in which specific lipids can be eliminated, substituted, tightly titrated or controlled temporally during membrane protein synthesis and assembly. The secondary transport protein lactose permease (LacY) was used to establish general principles underlying the molecular basis of lipid-dependent effects on protein domain folding, protein transmembrane domain (TM) orientation, and function. These principles were then extended to several other secondary transport proteins of E. coli. The methods used to follow proper conformational organization of protein domains and the topological organization of protein TMs in whole cells and membranes are described. The proper folding of an extramembrane domain of LacY that is crucial for energy dependent uphill transport function depends on specific lipids acting as non-protein molecular chaperones. Correct TM topogenesis is dependent on charge interactions between the cytoplasmic surface of membrane proteins and a proper balance of the membrane surface net charge defined by the lipid head groups. Short-range interactions between the nascent protein chain and the translocon are necessary but not sufficient for establishment of final topology. After release from the translocon short-range interactions between lipid head groups and the nascent protein chain, partitioning of protein hydrophobic domains into the membrane bilayer, and long-range interactions within the protein thermodynamically drive final membrane protein organization. Given the diversity of membrane lipid compositions throughout nature, it is tempting to speculate that during the course of evolution the physical and chemical properties of proteins and lipids have co-evolved in the context of the lipid environment of membrane systems in which both are mutually dependent on each other for functional organization of proteins. This article is part of a Special Issue entitled: Protein Folding in Membranes
Proceedings of the National Academy of Sciences, 2013
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.
Lipid-dependent Generation of Dual Topology for a Membrane Protein
Journal of Biological Chemistry, 2012
Background: Determinants of membrane protein dual topological orientation are not known. Results: Dual topological conformers of lactose permease co-exist in proportion to membrane phospholipid composition. Post-assembly changes in phospholipid composition alter the proportional amounts. Conclusion: Dual conformations of membrane proteins are determined during and post-assembly by lipid-protein interactions. Significance: A thermodynamic model explains the existence of multiple conformers of membrane proteins in eukaryotic cells. The mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is unknown. We posit that such duality is determined by both the protein sequence and the membrane lipid composition wherein a spatial or temporal change in the latter can result in a post-assembly change in protein structure and function. To investigate whether coexistence of multiple topological conformers is dependent on the membrane lipid composition, we determined the topological organization of lactose permease in an Escherichia coli model cell system in which phosphatidylethanolamine membrane content can be systematically varied. At intermediate levels of phosphatidylethanolamine a mixture of native and topologically mis-oriented conformers co-existed. There was no threshold level of phosphatidylethanolamine determining a sharp transition from one conformer to the other. Co-existing conformers were not in rapid equilibrium at a static lipid composition indicating that duality of topology is established during an early folding step. Depletion of intermediate levels of phosphatidylethanolamine after final protein assembly resulted in complete mis-orientation of the native conformer. Combined with previous results, such topological dynamics are reversible in both directions. We propose a thermodynamically based model for how lipid-protein interactions can result in a mixed topological organization and how changes in lipid composition can result in changes in the ratio of topologically distinct conformers of proteins. These observations demonstrate a potential lipid-dependent biological switch for generating dynamic structural and functional heterogeneity for a protein within the same membrane or between different membranes in more complex eukaryotic cells.
Journal of Cell Biology, 2008
The molecular details of how lipids influence final topological organization of membrane proteins are not well understood. Here, we present evidence that final topology is influenced by lipid–protein interactions most likely outside of the translocon. The N-terminal half of Escherichia coli lactose permease (LacY) is inverted with respect to the C-terminal half and the membrane bilayer when assembled in mutants lacking phosphatidylethanolamine and containing only negatively charged phospholipids. We demonstrate that inversion is dependent on interactions between the net charge of the cytoplasmic surface of the N-terminal bundle and the negative charge density of the membrane bilayer surface. A transmembrane domain, acting as a molecular hinge between the two halves of the protein, must also exit from the membrane for inversion to occur. Phosphatidylethanolamine dampens the translocation potential of negative residues in favor of the cytoplasmic retention potential of positive residu...
Methods in molecular biology (Clifton, N.J.), 2010
A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system.
Dynamic membrane protein topological switching upon changes in phospholipid environment
Proceedings of the National Academy of Sciences of the United States of America, 2015
A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, r...
Phospholipids As Determinants of Membrane Protein Topology
Journal of Biological …, 2005
Evidence is accumulating that the topological organization and hence function of some membrane proteins are not solely determined by the amino acid sequence of the protein but are also influenced by the lipid composition of the membrane. The ␥-aminobutyric acid (GABA) permease (GabP) of Escherichia coli has been found in this study to be affected both topologically and kinetically by membrane lipids. Using single cysteine accessibility methods with viable E. coli strains of natural lipid composition and those lacking phosphatidylethanolamine (PE), we have shown that the N-terminal hairpin of GabP is inverted relative to the membrane in PE-lacking cells, with a hinge point in transmembrane domain III. The rate of GABA transport is reduced by more than 99% in PE-lacking cells. The Michaelis constant for GABA transport is not greatly affected nor is the dependence of transport on energy. However, "transport specificity ratio" analysis demonstrated a clear transition state stability difference for GABA and nipecotic acid between the protein in PEcontaining and PE-lacking cells. The patterns of observed effects are similar to those seen with the phenylalanine transporter of E. coli (Zhang, W., Bogdanov, M.
Biochemistry, 2013
Folding of β-barrel membrane proteins, either from a urea-unfolded form or from chaperone-bound aqueous forms, has been characterized for pure lipid bilayers. The impact of preinserted integral proteins from biomembranes has not been examined in biophysical comparisons, but this knowledge is important for the characterization of protein assembly machinery in membranes to distinguish specific effects from unspecific effects. Here, folding was studied for a β-barrel membrane protein, outer membrane protein A (OmpA) from Escherichia coli, in the absence and presence of two other preinserted integral proteins, BamA of the β-barrel assembly machinery complex (BAM) from E. coli and FomA from Fusobacterium nucleatum. Three different preformed lipid membranes of phosphatidylcholine were prepared to compare the folding kinetics of OmpA, namely, proteoliposomes containing either BamA or FomA and pure liposomes. Urea-unfolded OmpA folded faster into phosphatidylcholine bilayers containing FomA than into pure lipid bilayers, but the kinetics of OmpA folding and insertion were fastest for bilayers containing BamA. Incorporation of BamA into lipid bilayers composed of phosphatidylcholine and phosphatidylethanolamine greatly weakened the inhibiting effect of phosphatidylethanolamine on the folding of OmpA. Folding of OmpA from its complex with the periplasmic chaperone Skp into bilayers composed of phosphatidylethanolamine and phosphatidylcholine was inhibited in the absence of BamA but facilitated when BamA was present, indicating an interaction of Skp−OmpA complexes with BamA.