A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition

To address the role of phospholipids in the topological organization of polytopic membrane proteins, the function and assembly of lactose permease (LacY) was studied in mutants of Escherichia coli lacking phosphatidylethanolamine (PE). PE is required for the proper conformation and active transport function of LacY. The N-terminal half of LacY assembled in PE-lacking cells adopts an inverted topology in which normally non-translocated domains are translocated and vice versa. Post-assembly synthesis of PE triggers a conformational change, resulting in a lipid-dependent recovery of normal conformation and topology of at least one LacY subdomain accompanied by restoration of active transport. These results demonstrate that membrane protein topology once attained can be changed in a reversible manner in response to alterations in phospholipid composition, and may be subject to post-assembly proofreading to correct misfolded structures.     

Topology of polytopic membrane protein subdomains is dictated by membrane phospholipid composition

In Escherichia coli, the major cytoplasmic domain (C6) of the polytopic membrane protein lactose permease (LacY) is exposed to the opposite side of the membrane from a neighboring periplasmic domain (P7). However, these domains are both exposed on the periplasmic side of the membrane in a mutant of E.coli lacking phosphatidylethanolamine (PE) wherein LacY only mediates facilitated transport. When purified LacY was reconstituted into liposomes lacking PE or phosphatidylcholine (PC), C6 and P7 were on the same side of the bilayer. In liposomes containing PE or PC, C6 and P7 were on opposite sides of the bilayer. Only the presence of PE in the liposomes restored active transport function of LacY as opposed to restoration of only facilitated transport function in the absence of PE. These results were the same for LacY purified from PE-containing or PE-lacking cells, and are consistent with the topology and function of LacY assembled in vivo. Therefore, irrespective of the mechanism of membrane insertion, the subdomain topological orientation and function of LacY are determined primarily by membrane phospholipid composition.     

Lipids and topological rules of membrane protein assembly: balance between long and short range lipid-protein interactions

The N-terminal six-transmembrane domain (TM) bundle of lactose permease of Escherichia coli is uniformly inverted when assembled in membranes lacking phosphatidylethanolamine (PE). Inversion is dependent on the net charge of cytoplasmically exposed protein domains containing positive and negative residues, net charge of the membrane surface, and low hydrophobicity of TM VII acting as a molecular hinge between the two halves of lactose permease (Bogdanov, M., Xie, J., Heacock, P., and Dowhan, W. (2008) J. Cell Biol. 182, 925-935). Net neutral lipids suppress the membrane translocation potential of negatively charged amino acids, thus increasing the cytoplasmic retention potential of positively charged amino acids. Herein, TM organization of sucrose permease (CscB) and phenylalanine permease (PheP) as a function of membrane lipid composition was investigated to extend these principles to other proteins. For CscB, topological dependence on PE only becomes evident after a significant increase in the net negative charge of the cytoplasmic surface of the N-terminal TM bundle. High negative charge is required to overcome the thermodynamic block to inversion due to the high hydrophobicity of TM VII. Increasing the positive charge of the cytoplasmic surface of the N-terminal TM hairpin of PheP, which is misoriented in PE-lacking cells, favors native orientation in the absence of PE. PheP and CscB also display co-existing dual topologies dependent on changes in the charge balance between protein domains and the membrane lipids. Therefore, the topology of both permeases is dependent on PE. However, CscB topology is governed by thermodynamic balance between opposing lipid-dependent electrostatic and hydrophobic interactions.     

Lipid-protein interactions as determinants of membrane protein structure and function

To determine how the lipid environment affects membrane protein structure and function, strains of Escherichia coli were developed in which normal phospholipid composition can be altered or foreign lipids can be introduced. The properties of LacY (lactose permease) were investigated as a function of lipid environment. Assembly of LacY in membranes lacking PE (phosphatidylethanolamine) results in misorientation of the N-terminal six-TM (transmembrane domain) helical bundle with loss of energy-dependent uphill transport and retention of energy-independent downhill transport. Post-assembly introduction of PE results in nearly native orientation of TMs and restoration of uphill transport. Foreign lipids with no net charge can substitute for PE in supporting native LacY topology, but restoration of uphill transport is dependent on native topology and the proper folding of a solvent-exposed domain. Increasing the positive charge density of the cytoplasmically exposed surface of LacY counters TM misorientation in the absence of neutral lipids, demonstrating that charge interactions between these domains and the surface of the membrane bilayer are determinants of TM orientation. Therefore membrane protein organization or reorganization is determined either during initial assembly or post-insertionally through direct interactions between the protein and the lipid environment, which affects the topogenic potency of opposing charged residues as topological signals independent of the translocon.     

Phosphatidylethanolamine and monoglucosyldiacylglycerol are interchangeable in supporting topogenesis and function of the polytopic membrane protein lactose permease

To determine the specific role lipids play in membrane protein topogenesis in vivo, the orientation with respect to the membrane bilayer of Escherichia coli lactose permease (LacY) transmembrane (TM) domains and their flanking extramembrane domains was compared after assembly in native membranes and membranes with genetically modified lipid content using the substituted cysteine accessibility method for determining TM domain mapping. LacY assembled in the absence of the major membrane lipid phosphatidylethanolamine (PE) does not carry out uphill transport of substrate and displays an inverted orientation for the N-terminal six-TM domain helical bundle (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). Strikingly, the replacement of PE in vivo by the foreign lipid monoglucosyldiacylglycerol (MGlcDAG), synthesized by the Acholeplasma laidlawii MGlcDAG synthase, restored uphill transport and supported the wild type TM topology of the N-terminal helical bundle of LacY. An interchangeable role in defining membrane protein TM domain orientation and supporting function is played by the two most abundant lipids, PE and MGlcDAG, in gram-negative and gram-positive bacteria, respectively. Therefore, these structurally diverse lipids endow the membrane with similar properties necessary for the proper organization of protein domains in LacY that are highly sensitive to lipids as topological determinants.     

Molecular genetic and biochemical approaches for defining lipid-dependent membrane protein folding

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.     

Phospholipids as determinants of membrane protein topology. Phosphatidylethanolamine is required for the proper topological organization of the gamma-aminobutyric acid permease (GabP) of Escherichia coli

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 gamma-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 PE-containing 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. Pi, J. Pittard, A. J., and Dowhan, W. (2003) J. Biol. Chem. 278, 50128-50135), also an amino acid/polyamine/organocation family member but quite distinct from those observed with lactose permease (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116), a major facilitator superfamily member. Therefore, by extending the studies of similarities and differences in lipid responses among and between family groups, we may identify elements within the proteins that facilitate lipid responsiveness.     

A topological model for the general aromatic amino acid permease, AroP, of Escherichia coli.

The general aromatic amino acid permease, AroP, of Escherichia coli is responsible for the active transport of phenylalanine, tyrosine, and tryptophan. A proposed topological model for the AroP permease, consisting of 12 hydrophobic transmembrane spans connected by hydrophilic loops, is very similar to that of the closely related phenylalanine-specific permease. The validity of this model and its similarity to that of the PheP permease were investigated by studying fusion proteins of AroP permease and alkaline phosphatase. Based on the results obtained from the AroP-alkaline phosphatase sandwich fusions, we have significantly revised the proposed topological model for AroP in two regions. In this modified AroP topological model, the three charged residues E151, E153, and K160 are repositioned within the membrane in span 5. These three residues are conserved in a large family of amino acid transport proteins, and site-directed mutagenesis identifies them as being essential for transport activity. It is postulated that these residues together with E110 in transmembrane span 3 may be involved in a proton relay system.     

In vitro synthesis of lactose permease to probe the mechanism of membrane insertion and folding

Insertion and folding of polytopic membrane proteins is an important unsolved biological problem. To study this issue, lactose permease, a membrane transport protein from Escherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro. The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays. By exploiting this system it is possible to express the N-terminal six helices of the permease (N(6)) and probe changes in conformation during insertion into the membrane. Specifically, when N(6) remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable. Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N(6) is released from the ribosome or more helices are translated. Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N(6) is released from the ribosome and inserted into the membrane. Moreover, in contrast to full-length permease, N(6) is degraded by FtsH protease in vivo, and N(6) with a single Cys residue at position 148 does not react with N-ethylmaleimide. Taken together, the findings indicate that N(6) remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer. Furthermore, there is synergism between N(6) and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains.     

A single negatively charged residue affects the orientation of a membrane protein in the inner membrane of Escherichia coli only when it is located adjacent to a transmembrane domain

The orientation of membrane proteins is determined by the asymmetric distribution of charged residues in the sequences flanking the transmembrane domains. For the inner membrane of Escherichia coli, numerous studies have shown that an excess of positively charged residues defines a cytoplasmic domain of a membrane protein ("positive inside" rule). The role of negatively charged residues in establishing membrane protein topology, however, is not completely understood. To investigate the influence of negatively charged residues on this process in detail, we have constructed a single spanning chimeric receptor fragment comprising the N terminus and first transmembrane domain of the heptahelical G protein-coupled vasopressin V(2) receptor and the first cytoplasmic loop of the beta(2)-adrenergic receptor. When fused to alkaline phosphatase (PhoA), the receptor fragment inserted into the inner membrane of E. coli with its N terminus facing the cytoplasm (N(in)-C(out) orientation), although both membrane-flanking domains had rather similar topogenic determinants. The orientation of the receptor fragment was changed after the introduction of single glutamate residues into the N terminus. Orientation inversion, however, was found to be dependent on the location of the glutamate substitutions, which had to lie within a narrow window up to 6 residues distant from the transmembrane domain. These results demonstrate that a single negatively charged residue can play an active role as a topogenic determinant of membrane proteins in the inner membrane of E. coli, but only if it is located adjacent to a transmembrane domain.     

In vivo membrane assembly of the E.coli polytopic protein, melibiose permease, occurs via a Sec-independent process which requires the protonmotive force.

To investigate the mechanism of polytopic membrane protein insertion in Escherichia coli, we have examined the protein and energy requirements for in vivo membrane assembly of the prototypic 12 transmembrane domain sugar co-transporter, melibiose permease (MelB). MelB membrane assembly was analyzed both kinetically, by pulse labeling experiments, and functionally by measuring the activity of the inserted permease. Strikingly, the rate of MelB membrane assembly is decreased approximately 4-fold upon dissipation of the transmembrane electrochemical proton gradient, delta(mu)H+, indicative of a strong requirement for delta(mu)H+. Interestingly, selective dissipation of either the electrical (delta(psi)) or the chemical (delta(pH)) component of delta(mu)H+ demonstrates that either form of energy is required for MelB membrane assembly. In contrast, MelB membrane assembly does not require SecA, SecY or SecE, all three proteins which are strictly required for protein translocation. Neither the rate of MelB membrane assembly nor the amount of functional permease is affected by inactivation or depletion of these Sec proteins. These results strongly suggest that polytopic membrane proteins such as MelB insert into the cytoplasmic membrane by a mechanism fundamentally different from protein translocation.     

Topology of the phenylalanine-specific permease of Escherichia coli.

The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on sequence analysis of the putative protein in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm had been proposed (J. Pi, P. J. Wookey, and A. J. Pittard, J. Bacteriol. 173:3622-3629, 1991). This topological model of PheP has been further examined by generating protein fusions with alkaline phosphatase. Twenty-five sandwich fusion proteins have been constructed by inserting the 'phoA gene at specific sites within the pheP gene. In general, the PhoA activities of the fusions support a PheP topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, alterations to the model, affecting spans III and VI, were indicated by this analysis and were supported by additional site-directed mutagenesis of some of the residues involved.     

Co-operation between different targeting pathways during integration of a membrane protein

Membrane protein assembly is a fundamental process in all cells. The membrane-bound Rieske iron-sulfur protein is an essential component of the cytochrome bc₁ and cytochrome b₆f complexes, and it is exported across the energy-coupling membranes of bacteria and plants in a folded conformation by the twin arginine protein transport pathway (Tat) transport pathway. Although the Rieske protein in most organisms is a monotopic membrane protein, in actinobacteria, it is a polytopic protein with three transmembrane domains. In this work, we show that the Rieske protein of Streptomyces coelicolor requires both the Sec and the Tat pathways for its assembly. Genetic and biochemical approaches revealed that the initial two transmembrane domains were integrated into the membrane in a Sec-dependent manner, whereas integration of the third transmembrane domain, and thus the correct orientation of the iron-sulfur domain, required the activity of the Tat translocase. This work reveals an unprecedented co-operation between the mechanistically distinct Sec and Tat systems in the assembly of a single integral membrane protein.     

Phospholipid-assisted protein folding: phosphatidylethanolamine is required at a late step of the conformational maturation of the polytopic membrane protein lactose permease.

Previously we presented evidence that phosphatidylethanolamine (PE) acts as a molecular chaperone in the folding of the polytopic membrane protein lactose permease (LacY) of Escherichia coli. Here we provide more definitive evidence supporting the chaperone properties of PE. Membrane insertion of LacY prevents its irreversible aggregation, and PE participates in a late step of conformational maturation. The temporal requirement for PE was demonstrated in vitro using a coupled translation-membrane insertion assay that allowed the separation of membrane insertion from phospholipid-assisted folding. LacY was folded properly, as assessed by recognition with conformation-specific monoclonal antibodies, when synthesized in the presence of PE-containing inside-out membrane vesicles (IOVs) or in the presence of IOVs initially lacking PE but supplemented with PE synthesized in vitro either co- or post-translationally. The presence of IOVs lacking PE and containing anionic phospholipids or no addition of IOVs resulted in misfolded or aggregated LacY, respectively. Therefore, critical folding steps occur after membrane insertion dependent on the interaction of LacY with PE to prevent illicit interactions which lead to misfolding of LacY.     

Estimating loop-helix interfaces in a polytopic membrane protein by deletion analysis.

Insertions of amino acids into transmembrane helices of polytopic membrane proteins disrupt helix-helix interactions with loss of function, while insertions into loops have little effect on transmembrane helices and therefore little effect on activity [Braun, P., Persson, B., Kaback, H. R., and von Heijne, G. (1997) J. Biol. Chem. 272, 29566-29571]. Here the inverse approach, amino acid deletion, is utilized systematically to approximate loop-helix boundaries in the lactose permease of Escherichia coli. Starting with deletion mutants in the periplasmic loop between helices VII and VIII (loop VII/VIII), which has been defined by immunological analysis and nitroxide-scanning electron paramagnetic resonance spectroscopy, it is shown that mutants with single or multiple deletions in the central portion of the loop retain significant transport activity, while deletion of amino acid residues near the loop-helix boundaries or within the flanking helices leads to complete inactivation. Results consistent with hydropathy analysis are obtained with loops VI/VII, VIII/IX, and IX/X and the flanking helices. In contrast, deletion analysis of loops III/IV, IV/V, and V/VI and the flanking helices indicates that this region of the permease differs from hydropathy predictions. More specifically, evidence is presented supporting the contention that Glu126 and Arg144 which are charge paired and critical for substrate binding are within helices IV and V, respectively.     

Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAM(TM)): application to lipid-specific membrane protein topogenesis

We provide an overview of lipid-dependent polytopic membrane protein topogenesis, with particular emphasis on Escherichia coli strains genetically altered in their lipid composition and strategies for experimentally determining the transmembrane organization of proteins. A variety of reagents and experimental strategies are described including the use of lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by substituted cysteine site-directed chemical labeling. Employing strains in which lipid composition can be controlled temporally during membrane protein synthesis and assembly provides a means to observe dynamic changes in protein topology as a function of membrane lipid composition.     

Slow translocon gating causes cytosolic exposure of transmembrane and lumenal domains during membrane protein integration

Integral membrane proteins are cotranslationally inserted into the endoplasmic reticulum via the protein translocation channel, or translocon, which mediates the transport of lumenal domains, retention of cytosolic domains and integration of transmembrane spans into the phospholipid bilayer. Upon translocon binding, transmembrane spans interact with a lateral gate, which regulates access to membrane phospholipids, and a lumenal gate, which controls the translocation of soluble domains. We analyzed the in vivo kinetics of integration of model membrane proteins in Saccharomyces cerevisiae using ubiquitin translocation assay reporters. Our findings indicate that the conformational changes in the translocon that permit opening of the lumenal and lateral channel gates occur less rapidly than elongation of the nascent polypeptide. Transmembrane spans and lumenal domains are therefore exposed to the cytosol during integration of a polytopic membrane protein, which may pose a challenge to the fidelity of membrane protein integration.     

Reconstitution of an active lactose carrier in vivo by simultaneous synthesis of two complementary protein fragments.

Escherichia coli lactose permease mediates the proton-driven translocation of galactosides across the cytoplasmic membrane. To define regions important for membrane insertion as well as for biological function, we constructed plasmids encoding different portions of the lactose carrier. Among several lacY deletions, two were obtained that encoded mutant proteins with complementary amino acid sequences. The truncated polypeptide Y71/1 (amino acid residues 1 to 71) comprises the first two alpha-helices predicted for the intact protein, and polypeptide delta Y4-69 carries an internal deletion of this region. Regulated coexpression of these lacY-DNA segments governed by separate but identical lacOP control regions resulted in functional complementation with the following characteristics. (i) Simultaneous synthesis of both incomplete proteins restored transport activity in transport-negative cells, measured as accumulation of [14C]lactose. (ii) Under complementing conditions, but not in the absence of the smaller N-terminal protein, specific radiolabeling of the larger polypeptide by N-ethylmaleimide was prevented by substrate. (iii) The presence of the complementing N-terminal polypeptide was also required for the detection of the larger C-terminal protein by antibodies directed against the C terminus of lactose permease, indicating a stabilizing effect contributed by the smaller N-terminal fragment. Thus, coexpression of lacY mutant genes encoding two nonoverlapping portions of the lactose carrier resulted in reconstitution of a two-subunit protein in the cytoplasmic membrane exhibiting biological properties of intact lactose permease.