Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli

Abstract

Over 35% of all proteins are integrated into cell membranes, and many others interact at the membrane surface. Therefore, determining how lipid-protein interactions govern protein structure and function is fundamental to understanding cellular processes at the molecular level. Although reconstitution of purified membrane proteins with lipids is necessary to study lipid-protein interactions, the complexity of lipid molecular forms that make up the lipidome has largely been ignored in most studies, which have employed lipid mixtures that rarely reflect the composition of host membranes.

Phosphatidylcholine (PC) has been extensively used in reconstitution studies because it spontaneously organizes into bilayers. However, use of PC for reconstitution of membrane proteins from bacterial sources that do not contain PC may not provide reliable information, because there are no data on the in vivo function of these proteins in membranes where PC replaces the major phospholipid phosphatidylethanolamine (PE). Therefore, a distinction between a biological preference for PE over PC and an artifact in a reconstituted system cannot be made.

The topological orientation and function of the 12 transmembrane (TM) domain-spanning membrane integrated permeases for lactose (LacY), phenylalanine (PheP), and γ-aminobutyrate (GabP) of Escherichia coli are dependent on membrane lipid composition (1). In mutants lacking PE (normally ca. 70% of total phospholipid) and containing only anionic phospholipids [primarily phosphatidylglycerol (PG) and cardiolipin (CL)], the N-terminal six TM helical bundle of LacY (see Fig. 1) and the N-terminal two-TM helical hairpin of PheP and GabP are inverted with respect to their orientation in PE-containing cells and the remaining TMs. These permeases do not carry out proton-coupled energy-dependent uphill transport of substrate in cells lacking PE, but still display energy-independent downhill transport. LacY reconstituted into total E. coli phospholipids carries out uphill transport with domains C6 and P7 on opposite sides of the membrane bilayer as observed in wild-type cells (9). Leaving out PE during reconstitution results in only downhill transport with domains C6 and P7 residing on the same side of the bilayer as observed in PE-lacking cells (9). Reconstitution into proteoliposomes where dioleoyl-PC (diC_18∶1PC) replaces PE results in wild-type topology and downhill transport (9) but not uphill transport (9–11). An ionizable amine or hydrogen bond donor capacity is also required for uphill transport because methylation of the amine of PE progressively reduces activity (10, 11). PE but not diC_18∶1PC or eukaryotic-derived PC also supports uphill transport by the multidrug transporter (LmrP) of Lactococcus lactis (12, 13), the leucine permease of Pseudomonas aeruginosa (14), the branched chain amino acid transporter of Streptococcus cremoris (15), the ABC transporter HorA from Lactococcus lactis (16), and the Ca²⁺ ATPase of the sarcoplasmic reticulum (17).

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Fig. 1.

Topological organization of LacY as a function of membrane lipid composition. TMs (Roman numerals), extramembrane domains (P for periplasmic and C for cytoplasmic as in +PE cells), N-terminus (NT) and C-terminus (CT) domains are indicated. The approximate positions and names of amino acids substituted by cysteine and used for topological analysis are indicated near the closed and open symbols in a cysteineless derivative of LacY used in this work. The conformation-sensitive (2, 3), PE-dependent (4–8) epitope in domain P7 recognized by mAb 4B1 is noted with the amino acids that are part of this epitope marked with an asterisk. (Top) Denotes orientation in +PE cells; (Bottom) denotes orientation in -PE cells with the upper side of each figure being the side that faces the cytoplasm. TM VII resides in the periplasm in -PE cells. Topological orientation of LacY is taken from ref. 1.

Another feature of LacY that is strongly correlated with uphill transport is the structure of the extramembrane domain P7 (3) that is exposed to the periplasm and connects TMs VII and VIII. The conformation-specific monoclonal antibody (mAb) 4B1 recognizes this domain in membranes and on Western blots of LacY that has been assembled in PE-containing but not in PE-lacking E. coli membranes (7). Binding of mAb 4B1 also inhibits uphill transport by LacY (2, 3). Epitope recognition is restored to LacY from PE-lacking membranes by exposure to PE but not diC_18∶1PC blotted to the same solid support prior to Western blot analysis (8). Recognition by mAb 4B1 appears to be a reliable indicator of the proper topological and structural organization of LacY in the vicinity of domain P7 (Fig. 1) and of LacY uphill transport function.

The inability of PC to support the full function of several transport systems as well as the native structure of domain P7 of LacY raises several questions of broad significance to studies of membrane protein structure, function, and lipid interaction in reconstituted systems. Is lack of functional and structural support an artifact of in vitro reconstitution or the use of an inappropriate PC species? Can PC replace PE in vivo with respect to structure and function of LacY? Are conclusions thus far made concerning lipid-protein interactions based on the ineffectiveness of PC valid? To address these questions we report on the orientation of TMs, transport function, and the recognition by mAb 4B1 after assembly of LacY in a mutant of E. coli where PC replaces PE.

Results

Transport Function of LacY in -PE + PC Cells.

We analyzed transport function of wild-type LacY expressed from a plasmid in strain AL95 without additional plasmids (-PE) or with either plasmid pDD72 (pssA⁺, +PE) or plasmid pAC-PCS_lp-Sp-Gm (-PE + PC). Choline and Mg²⁺ were present in all growth media. Consistent with previous results (18), LacY assembled in -PE cells was unable to catalyze uphill transport of the nonmetabolizable substrate methyl-β-D-galactopyranoside (TMG) (Fig. 2A). Surprisingly -PE + PC cells showed the same rate and level of uphill transport as +PE cells (Fig. 2A), which were confirmed by inhibition of accumulation of TMG by a protonophore (Fig. 2A). Substrate entry measured in -PE + PC cells was via LacY as evidenced by lack of TMG and lactose uptake by AL95/pAC-PCS_lp-Sp-Gm without a plasmid copy of LacY. Comparative rates of uphill transport are difficult to assess especially with multiple copies of lacY because 10% induction of single copy lacY results in 50% of the uphill transport rate after full induction of single copy lacY (24). However, the rate of downhill substrate transport was significantly lower than that observed in +PE cells (Fig. 2B), which appears to be due to a 5-fold decrease in LacY relative to that in +PE cells as detected by Western blot analysis (Fig. 2B Inset); the rate of downhill transport by LacY is proportional to the number of carriers in the membrane (24). Similar transport results were observed using the H205C derivative of LacY (Fig. S3).

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Fig. 2.

Transport function of wild-type LacY in whole cells. Uphill transport of TMG (A) or downhill transport of lactose (B) normalized to total cell protein was determined as a function of time by AL95/pDD72 (closed squares, +PE), AL95/pAC-PCS_lp-Sp-Gm (open squares -PE + PC), or AL95 (open circles -PE), all induced for expression of a plasmid-borne copy of the wild-type lacY gene. Uphill transport in -PE + PC cells was verified by addition of a protonophore (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) to the assay of TMG uptake (open diamonds), which reduced the uptake to the level of the -PE results (open circles). No uptake of TMG or lactose by AL95/pAC-PCS_lp-Sp-Gm (closed diamonds, -PE + PC ΔLacY) demonstrated that uphill and downhill transport in -PE + PC cells (open squares) were mediated by LacY. Standard deviations for the average of two determinations are shown except for lines where uptake was essentially zero (open diamonds, open circles, and closed diamonds). The reduced level (one-fifth) of LacY in -PE + PC cells relative to the level of LacY in +PE cells (25 μg of protein per lane) is shown by Western blot analysis (B Inset) using anti-LacY polyclonal antibody.

Topological Orientation of TMs of LacY in -PE + PC Cells.

LacY with cysteine replacements in otherwise cysteineless LacY (Fig. 1) was expressed in -PE + PC cells and reactivity with 3-(N-maleimidylpropionyl) biocytin (MPB) in intact cells (periplasmic exposed) or during cell disruption by sonication (cytoplasmic and periplasmic exposed) was used to establish TM orientation based on derivatization of cysteines in flanking extramembrane domains (25, 26). As shown in Fig. 3, cysteines residing within normal cytoplasmic domains C2, C4, C6, C8, or C10 were labeled only after cell disruption indicating cytoplasmic exposure, whereas cysteines in normal periplasmic domains P1, P7, or P9 were labeled whether or not cells were disrupted indicating periplasmic exposure in these cells. However, the cysteines residing within normal periplasmic domains P3 or P5 were not accessible to MPB at pH 7.5. Lack of biotinylation can be due to location within a TM or proximal environmental effects, which affect thiol pK_a or accessibility (26). Increasing solution pH should favor alkylation of an extramembrane cysteine or disruption of local secondary structure without exposure of cysteines in TMs (27). P3 and P5 cysteines were fully labeled at pH 9.1 without sonication, indicating periplasmic exposure. The H205C substitution in domain C6 remained inaccessible to MBP at pH 9.1 without sonication, which verified retention of cell impermeability to MBP (Fig. S4B). None of the cysteines in the normal cytoplasmic N-terminal (NT) domain were labeled by exposure to pH 7.5, 9.1, or 10.5 either before or during sonication, which indicates a significant departure from LacY expressed in +PE cells where labeling occurs at pH 7.5 (6). Presence of sufficient amounts of LacY with the L5C substitution to be detected by MBP labeling was shown using Western blot analysis with polyclonal antibody (Fig. S4A). Although the disposition of domains NT and TM I remains uncertain, aberrant organization of domains NT through TM II was previously shown not to affect uphill transport (27). Thus the gross topology of LacY assembled in -PE + PC cells starting at least from domain P1 is the same as in wild-type cells and is consistent with topology determined after reconstitution of LacY into -PE + diC_18∶1PC proteoliposomes containing PG and CL that did not support uphill transport (9).

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Fig. 3.

Mapping the topology of LacY in -PE + PC cells. All samples were treated with MPB without sonication (-) or during sonication (+). AL95/pAC-PCS_lp-Sp-Gm (-PE + PC) cells expressing single cysteine replacements in the indicated extramembrane loop/amino acid were grown and processed as described in Methods subsequent to Western blotting using avidin-HRP to detect the biotin moiety linked to cysteines (see Fig. 1 for approximate positions of substitutions) that were accessible to MPB during labeling. MBP labeling was performed at pH 7 except for P3/I103C and P5/T163C, which were labeled at pH 9.1.

Conformation of Functionally Important Domain P7.

The native conformation of an epitope within periplasmic domain P7 (Fig. 1), as determined by the conformation-specific mAb 4B1, is strongly correlated with LacY uphill transport (3, 4, 6, 7). Binding of mAb 4B1 to LacY inhibits uphill transport (2, 3). Consistent with uphill transport function, LacY assembled in -PE + PC and +PE cells (Fig. 4A, right and center lanes, respectively) was strongly recognized by mAb 4B1 after SDS PAGE and Western blotting. LacY assembled in -PE cells (left lane) was very weakly recognized if at all.

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Fig. 4.

Western and Eastern-Western blotting of LacY used to probe the conformation of domain P7. (A) Inner membrane preparations (duplicate samples of 10 μg of protein) from cells with the indicated lipid composition and expressing a plasmid-borne copy of wild-type LacY were analyzed by Western blot analysis using conformation-specific mAb 4B1. (B–E) Inner membrane preparations from cells with the indicated phospholipid composition and expressing a plasmid-borne copy of wild-type LacY were subjected to SDS PAGE. The region of the gel containing LacY (molecular weight 31,000–33,000) was transferred to the area of a nitrocellulose sheet onto which the indicated phospholipids were transferred from a TLC plate (Eastern blot) followed by Western blotting analysis using mAb 4B1. Single samples contained 10 μg of membrane protein and duplicate samples contained 5 (Left) or 10 (Right) μg of membrane protein. Preblotted phospholipids were as follows: (B) no lipid (none), total lipid extract from -PE + PC cells, diC_18∶1PC (Avanti); (C) PE (Avanti) purified from E. coli (+PE), 70% diC_18∶1PC (Avanti) supplemented with 30% of total lipid extract from -PE cells containing approximately equal amounts of PG and CL, total lipid extract from -PE + PC cells, PC isolated from -PC + PE cells by TLC (EcPC); (D) total lipid extract from -PE + PC cells, PC isolated from -PC + PE cells by DE52 chromatography either alone (EcPC) or 70% PC supplemented with E. coli PG (3%) and CL (27%) from Avanti (EcPC + PG + CL); (E) preblotted PCs (Avanti) with the indicated fatty acid composition are shown. One fatty acid denotes both acyl chains are identical. Two fatty acids indicate fatty acids at positions sn-1 and sn-2, respectively. PCs were supplemented with E. coli PG plus CL (Avanti) in the ratio as indicated for D. None indicates no lipid preblotted. Fatty acids for lanes 1 and 2 were Δ9 trans and Δ9 cis, respectively.

Previously, we showed that E. coli-derived PE or total E. coli phospholipids when blotted to the surface of nitrocellulose sheets (Eastern blot) prior to transfer by electroblotting (Western blot) of LacY from SDS polyacrylamide gels restored recognition by mAb 4B1 of LacY derived from -PE cells (8). DiC_18∶1PC with or without PG and CL supplementation showed very weak or no restoration of mAb 4B1 recognition and even resulted in loss of recognition for LacY initially made in +PE cells (8). However, recognition by mAb 4B1 of LacY synthesized in -PE cells was restored in the Eastern-Western blotting procedure using total phospholipids from -PE + PC cells (Fig. 4B), which is consistent with observed support of uphill transport. However, diC_18∶1PC alone (Fig. 4B) or supplemented with PG and CL (Fig. 4C) showed little or no restoration of recognition by mAb 4B1. To rule out factors other than lipids in these extracts, PC made in E. coli was isolated by DE52 column chromatography or after separation by TLC. As shown in Fig. 4 C and D, purified E. coli PC alone was very weak in supporting proper refolding. However, this lipid supported proper refolding when mixed with purified E. coli PG plus CL (Avanti Polar Lipids) at a ratio mimicking the phospholipid composition of -PE + PC cells (Fig. 4D). Synthetic diC_18∶1PC weakly supported proper refolding either used alone or in ternary mixtures with E. coli PG plus CL (Fig. 4 B or C, respectively).

Next LacY expressed in -PE cells was subjected to Eastern-Western blotting using synthetic PCs with different fatty acid compositions. As shown in Fig. 4E, synthetic C_16∶0-C_18∶1-PC alone (lane 4) was weak in assisting proper refolding of domain P7 unless mixed with E. coli PG plus CL (Avanti Polar Lipids) at a ratio mimicking the phospholipid composition of -PE + PC cells (lane 5). DiC_16∶0PC (Fig. 4E, lane 9) and diC_{16∶1Δ9-trans}PC (Fig. 4E, lane 1) supported refolding of domain P7 if mixed with E. coli PG plus CL, whereas diC_{16∶1Δ9-cis}PC did not (Fig. 4E, lane 2). In order to test the acyl chain positional specificity of phospholipid-assisted refolding of domain P7, C_16∶0-C_18∶1PC and C_18∶1-C_16∶0PC were compared (Fig. 4E, lanes 7 and 8, respectively) and were shown to be equally effective. CL or PG alone was as effective as PG plus CL in supporting refolding in the presence of C_16∶0-C_18∶1PC (Fig. S5A), and the latter with anionic lipids was as effective as lipids from -PE + PC cells or E. coli PE alone (Fig. S5B).

Discussion

A major approach to understanding how lipid-protein interactions govern cell function is to study purified membrane proteins reconstituted with a variety of natural or foreign lipids differing in physical and chemical properties. A limitation of this approach has been the inability to verify in vitro observations in an in vivo system. Development of E. coli strains in which membrane lipid composition can be systematically manipulated (1) now makes this possible. A primary aim of this study was to determine the molecular basis for the ability of the foreign lipid PC to support native topological organization and downhill transport (9) but not uphill transport (9–11) or the structure of domain P7 of E. coli LacY (8). The differences between PE and PC in supporting function of several secondary transport proteins has focused on the mode of direct interaction between the lipid head groups and specific amino acids of the mature protein during catalysis (12, 13, 16, 28), which, based on our results, appears not to be the case. Our results suggest that the major effect of the lipid environment occurs during folding of the protein into its final structure (i.e., P7 conformation) rather than specific lipid-protein interactions after native structure is attained.

Biosynthesis of PC in an E. coli mutant lacking PE resulted in restoration of the wild-type zwitterionic and anionic phospholipid levels to 70% and 30%, respectively. The PC species displayed a similar fatty acid distribution as found in PE, which is significantly different from that used in reconstitution studies. Cells remained dependent on divalent cation for viability consistent with the postulated role of divalent cations in inducing the nonbilayer phase for CL (which was highly elevated compared to wild-type cells), the only potentially non-bilayer-prone lipid in -PE cells (29). Tryptophan prototrophy of -PE + PC cells suggests that PC corrects the uphill transport defect in other secondary transporters (1). Topological organization of LacY was near wild type, which extended the in vitro results where only the orientation of the C6-TM VII-P7 domain of LacY was probed (9). However, cysteines in periplasmic domains were less accessible to MPB, and the NT domain was completely inaccessible, indicating incomplete native structure. LacY exhibited downhill transport, but contrary to in vitro results also exhibited uphill transport, which correlated with proper folding of domain P7 as determined by mAb 4B1. PC containing at least one saturated fatty acid, but not diC_18∶1PC as previously shown (8), in the presence of PG or CL restored the conformation of domain P7 of LacY assembled in PE-lacking cells.

PE, PC, GlcDAG (20), and GlcGlcDAG (21) are mostly interchangeable in their ability to support near native topological organization of LacY. These lipids vary in size, structure, charge, and hydrogen-bonding properties of their head groups as well as in their effects on the physical properties of the membrane. The common feature among these lipids is a net neutral head group, which has been proposed to dilute the high negative surface charge density contributed by PG and CL that results in aberrant topological organization of LacY, PheP, and GabP in the absence of PE (1, 27). PE and GlcDAG are nonbilayer, whereas PC and GlcGlcDAG are bilayer-prone lipids, and only the head group of PC cannot act as a hydrogen bond donor, which indicates these properties are not important in supporting native topology.

The most surprising result was that LacY carried out uphill transport when expressed in cells in which PE was replaced by an equivalent amount of PC contrary to in vitro results (9–11). GlcDAG (20) but not GlcGlcDAG (21) also supports uphill transport in vivo. PE is interchangeable with glycolipids in supporting uphill transport for the branched chain amino acid transporter of S. cremoris (15) and the eukaryotic Ca²⁺ ATPase (17). These results demonstrate that the ethanolamine head group of PE is not specifically required for uphill transport carried out by several transporters but does not explain why PC does not substitute for PE in reconstituted systems.

The conformation of the P7 domain as indicated by recognition with mAb 4B1 is directly correlated with the ability of LacY to carry out uphill transport that requires protonation of the gluamate carboxyl at position 325, which displays a pK_a above 9 (30). Binding of mAb 4B1 to LacY inhibits uphill transport in spheroplasts and in proteoliposomes (31) concomitant with conformational changes in several TMs (32). An E325D substitution prevents uphill transport and reduces the rate of substrate exchange above pH 8.5 instead of above 9.5 as observed with E325 (33). Binding of mAb 4B1 reduces these exchange rates at one pH unit lower for the E325D mutant (30). Therefore structural changes within P7 induced by binding of mAb 4B1, and by extrapolation folding defects in P7 due to lack of PE, result in a conformational change that lowers the pK_a of an essential acidic residue at position 325, which is involved in proton-coupled uphill transport. These results are consistent with studies on LmrP in which the average pK_a of acidic residues was 6.5 when reconstituted in the presence of PE but only 4.5 in the presence of egg yolk PC (12), which does not support uphill transport. There were also pH-dependent conformational changes associated with acidic group ionization in PE but not in PC proteoliposomes.

Using the Eastern-Western procedure, we demonstrated that lipid mixtures that support uphill transport were able to restore native conformation of the P7 domain, as demonstrated by regain of recognition by mAb 4B1 of LacY assembled in -PE cells. Strong recognition was observed using total lipids from -PE + PC cells and purified E. coli-derived PC reconstituted with E. coli PG plus CL, whereas weak or no recognition was observed with diC_18∶1PC in all lipid mixtures or with E. coli-derived PC alone. Further analysis using PCs with different fatty acid compositions showed that reconstitution of the epitope occurred with PCs (always in the presence of E. coli PG and CL) containing at least one saturated fatty acid or Δ9 trans fatty acids; cis unsaturated fatty acids impart distinctly different physical properties to phospholipids as compared to saturated or trans unsaturated fatty acids. Unlike E. coli PC, bilayer-prone PE species (E. coli PE and PE species with saturated fatty acids) alone as opposed to non-bilayer-prone PE (those with two unsaturated fatty acids) alone supported epitope formation (8). Mixing of non-bilayer-prone PE species with bilayer-prone PG or CL supported epitope formation. Although functional PC species are all bilayer prone, PG or CL was still required to support epitope formation. The missing component contributed by the anionic lipids might be the ability of their head groups to be hydrogen bond donors in the case of PC or provide an overall bilayer phase in the case of unsaturated non-bilayer-prone PE species. The requirement for PG or CL appears to be their anionic or hydrogen-bonding properties rather than specific structural features (Fig. S5A), which indicates that the high CL content of -PE + PC cells is not a factor. Cells in which GlcDAG replaced PE display the native conformation of the P7 domain (Fig. S6) and carry out uphill transport by LacY (20). GlcGlcDAG (Fig. S6) only weakly supports the P7 native conformation and does not support uphill transport (21), which may be due to its larger head group.

The lack of phospholipid head group specificity and the variability with respect to fatty acid composition in support of uphill transport and the structure of the P7 domain strongly indicate that specific lipid head group protein interactions in the folded state are not the defining lipid requirement. Rather the attainment of native structure of LacY facilitated during folding by the proper physical and chemical properties of the lipid environment is of primary importance. Native folding of domain P7 (resulting in recognition by mAb 4B1), which has long-range effects on structure, appears to occur by transient interaction with a subset of lipid species (now including certain species of PC) during a late step of initial assembly or postassembly (4, 6). Conformational memory within P7 remains after SDS treatment and removal of SDS and PE during electroblotting, which indicates a transient molecular chaperone role for lipid during protein folding (5, 7, 8). The epitope recognized by mAb 4B1 consists of Phe-247, Phe-250, and Gly-254 (Fig. 1) on one face of a short α-helical segment (3). Although PE, PC, and GlcDAG have diverse structural and chemical properties, they share the potential to interact through π bonding with the face of aromatic residues. Primary (PE) and quaternary (PC) amines have been observed to engage in π-bonding with the indole ring of Phe in the structure of cytochrome c oxidase (34) and within a cage formed by three aromatic residues in the structure of human PC transfer protein (35). Protein-carbohydrate interactions also rely on aromatic stacking interactions with sugar rings (36). Interestingly the analogous P7 domain of LmrP is also rich in Phe (37) and may also be responsible for transient interaction with the head group of PE during folding. Taken together these results strongly suggest that the lipid environment affects the conformation of the P7 domain of LacY during initial folding, which has long-range indirect effects on critical acidic residues involved in proton-dependent uphill transport of substrate.

Our results emphasize the necessity of verifying conclusions about lipid-protein interactions derived from in vitro observations under in vivo conditions. The physical and chemical properties of the lipid bilayer determine the proper overall conformation of LacY that supports native transport. Although direct interaction between specific lipid head groups and protein functional groups may be important, possibly transiently during protein folding, current results do not support such an interaction directly in support of native transport function. Therefore, the molecular basis for lipid requirements by LacY is more complex than previously thought and is not based solely on the nature of the lipid head group but involves the physical and chemical properties of a multicomponent membrane bilayer. Although E. coli PC supports LacY function, full native structure is still not attained, emphasizing the need to use native lipid composition in reconstituting membrane proteins. The availability of mAb 4B1 has demonstrated that different lipid environments have a dramatic effect on the structure of protein domains and that use of nonnative lipid mixtures can seriously compromise structural studies on membrane proteins. Our results also point out the shortcomings in current molecular simulations of lipid bilayers that include only single lipid species and not more complex lipid mixtures.

Methods

Additional procedures and results can be found in SI Text.

Supplementary Material

Acknowledgments.

Footnotes

References