From the Department Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Received for publication, February 21, 2003 , and in revised form, April 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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Based on the general characteristics of flip-flop, we hypothesized that the mere presence of -helical stretches of transmembrane proteins is sufficient for flop to occur, rendering the elusive flippases redundant. We showed previously that the presence of synthetic transmembrane peptides, mimicking the
-helical stretches of transmembrane proteins, induces flop of C6-NBD-PL1 analogues in model membranes (11, 12), supporting this hypothesis.
Bacterial membrane proteins display a large diversity in structure and organization, more than can be accounted for by -helical model peptides.
-Helical membrane proteins often span the bilayer with several TMHs, whereas the model peptides are single-spanning. Additionally, membrane proteins usually have domains outside the membrane, and in some cases form oligomers. Here we report on phospholipid translocation, induced by a subset of well characterized membrane proteins with different membrane organizations, briefly described below.
Leader peptidase (Lep) from Escherichia coli has two membrane-spanning -helices and adopts an overall Nout/Cout topology in the inner membrane (IM) of E. coli (13). The large C-terminal catalytic domain is in close contact with the periplasmic leaflet of the IM, interacting with phospholipids (14, 15). Lep is an essential protein (16) involved in membrane biogenesis, as it clips off the signal peptide of proteins that are translocated via the E. coli Sec machinery. Moreover, its purification and functional reconstitution in proteoliposomes have been characterized (17, 18). Taken together, this renders Lep an excellent model protein to test our hypothesis.
The potassium channel KcsA is another well characterized protein of the bacterial cytoplasmic membrane. Its crystal structure has been determined (19). Unlike Lep, KcsA is an oligomeric protein forming a stable homotetramer (20). Each monomer contains two membrane-spanning domains and has an overall Nin/Cin topology. The interaction of KcsA with lipids has been characterized, as well as the role lipids play in its membrane assembly (21, 22).
The E. coli inner membrane protein MsbA was chosen as a representative of the large superfamily of ATP-binding cassette transporter proteins, or traffic ATPases. In bacteria, the ATP-binding cassette transporters have a complex membrane organization with two hydrophobic domains, each of them typically spanning the membrane six times (23). MsbA is a homodimer, the 64-kDa monomer spanning the membrane with six TMHs (24, 25). It was shown to be an ATPase (26). The msbA gene was first discovered as a multicopy suppressor of mutations in htrB (24), which encodes a protein involved in the synthesis of lipopolysaccharide (LPS). Overexpression of msbA was shown to complement the htrB phenotype by restoring transport of non-mature LPS precursors. In a temperature-sensitive msbA strain, LPS precursors and phospholipids accumulate in the inner membrane at the non-permissive temperature (27). Based on these observations and on the recently resolved crystal structure (25), MsbA was suggested to be a (phospho)lipid flippase, which provided an extra rationale for testing the capacity of this protein to induce phospholipid translocation.
Apart from -helical transmembrane segments, another principal structural motif by which integral membrane proteins span the bilayer is a
-barrel. In E. coli,
-barrel proteins are exclusively found in the outer membrane. We included the E. coli protease OmpT, with known crystal structure (28), as a paradigm for the
-barrel membrane proteins to investigate whether phospholipid flop is facilitated by membrane proteins with other membrane-spanning structures.
All proteins tested were reconstituted in proteoliposomes composed of E. coli phospholipids, and translocation of the phospholipid analogue C6NBD-PG was measured fluorimetrically by determining its susceptibility toward dithionite reduction. Evidence is presented that a subset of integral membrane proteins of the bacterial inner membrane facilitates phospholipid translocation via their transmembrane -helices. The efficiency of flop induced by different proteins varied. Moreover, data are presented that argue against MsbA being an ATP-dependent phospholipid flippase.
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EXPERIMENTAL PROCEDURES |
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WALP23 (AcGWWL(AL)8WWA-NH2) was synthesized as described (29, 30). Peptide H1, corresponding to the N-terminal residues 125 of Lep, AcNleANNleFALILVIATLVTGILWCVDKF-NH2, and a positively charged derivative with an N-terminal 3-amino acid substitution named H1' (AcGKKNleFALILVIATLVTGILWCVDKF-NH2) were synthesized, essentially as described for WALP23, on an Applied Biosystems 433A Peptide Synthesizer using the FastMoc protocol on a 0.25 mmol scale. Norleucines (Nle) are isosteric substitutions for the methionine residues at positions 1 and 4 and were used because the latter are sensitive to oxidation. Stock solutions of the peptides in trifluoroethanol (0.48 mM) were prepared on the basis of weight and stored under N2 at 20 °C. Sodium dithionite (technical grade) was from Aldrich. All other chemicals used were analytical grade.
Isolation and Purification of ProteinsLeader peptidase with a C-terminal His5 tag was produced in E. coli strain MC1061 carrying the p827 plasmid as described (31) with minor modifications as detailed below. After induction of Lep, cells were harvested and washed with 0.9% (w/v) NaCl. Subsequently the cells were resuspended at 1 g wet weight per 5 ml in 10 mM Tris/HCl, pH 8, 5 mM EDTA, supplemented with 1 mM phenylmethylsulfonyl fluoride, 300 ng/ml leupeptin, 10 µg/ml lysozyme, and incubated on ice for 1 h while stirring. The resulting spheroplasts were disrupted by sonication 10 times for 10 s on ice, using the Branson microtip at maximum allowed power. Residual intact cells and spheroplasts were removed by low spin centrifugation for 10 min at 3,000 x g, and the resulting supernatant was centrifuged at 90,500 x g for 1 h at 4 °C. The pellet containing the inner membranes was solubilized in 10 mM Tris/HCl, pH 8, 1% (w/v) octyl glucoside, 10 mM imidazole, and 100 mM NaCl. Undissolved material was removed by repeating the previous centrifugation step. The supernatant was loaded on a Ni2+-nitrilotriacetic acid column with 7-ml column volume (Qiagen, Valencia, CA). After washing the column with 5 volumes of the aforementioned buffer (10 mM imidazole), Lep was eluted with 60 mM imidazole and stored at a concentration of
0.25 mg/ml at 20 °C. The P2 domain of leader peptidase (
2-75) was produced and purified as described (15) and stored as a stock of 0.10.2 mg/ml in 20 mM Tris/HCl, pH 7.4, at 20 °C.
KcsA with an N-terminal His6 tag was overproduced and purified in E. coli strain BL21(DE3) carrying a pT7-KcsA plasmid, essentially as described previously (20, 32). Cells were grown for 2 h after addition of isopropyl-
-D-thiogalactopyranoside and harvested. The membrane fraction was isolated as described above. The membrane pellet was solubilized in 10 mM HEPES, 100 mM NaCl, 5 mM KCl, 10 mM imidazole, and 1 mM dodecylmaltoside (DDM) and applied to a Ni2+-nitrilotriacetic acid column. After washing the column with
5 volumes of 10 mM and
5 volumes of 50 mM imidazole in the above buffer, respectively, the protein was eluted with buffer containing 300 mM imidazole and stored at a concentration of 0.64 mg/ml at 4 °C.
His-tagged MsbA was a kind gift from Drs. William Doerrler and Christian Raetz (26) and was stored at a concentration of 0.4 mg/ml in 0.1% (w/v) DDM, 200 mM imidazole, 50 mM HEPES, 500 mM NaCl, 5 mM MgCl2, 10% (w/v) glycerol, and 5 mM
-mercaptoethanol at 20 °C.
OmpTE211K/R218E, an OmpT mutant with reduced autoproteolytic activity2 produced and purified as described (33), was generously supplied by Gerard-Jan de Roon and Dr. Maarten Egmond and stored as a stock of 2.6 mg/ml in 10 mM Tris/HCl, 1% n-octyl-oligo-oxoethylene, pH 8.3, at 20 °C.
Preparation of Large Unilamellar Vesicles by Extrusion (LUVETs) Vesicles with and without the model peptides H1 and WALP23 were prepared as described previously (11), except for omitting K3Fe(CN)6. Briefly, a mixed film was prepared consisting of E. coli lipid extract (TLE), the indicated amount of peptide, and C6NBD-PG at 0.2 mol % of PL-Pi. The lipid film was hydrated with buffer Z (50 mM triethanolamine, 10 mM KCl, 1 mM EDTA, pH 7.5) to a final concentration of 5 mM phospholipid. After repetitive freezing and thawing, and subsequent extrusion through 200-nm membrane filters (Anotop 10, Whatman, Maidstone UK), unilamellar, sealed vesicles symmetrically labeled with C6NBD-PG were obtained.
Preparation of ProteoliposomesReconstitution of Lep into proteoliposomes was performed by octyl glucoside dilution as described (18, 34). A lipid film containing TLE (typically 2 µmol of PL-Pi) and C6NBD-PG was mixed with octyl glucoside in buffer and Lep from the stock solution to yield a mixed micelle solution of TLE, Lep (1:1000), C6NBD-PG (0.5%), and octyl glucoside (10:1, 1.2% w/v) (molar ratios with respect to the PL-Pi content of the TLE), typically in a volume of 500 µl. The micelles were diluted with buffer Z at a rate of 10 ml/h to a volume of 24 ml and incubated overnight at 4 °C under continuous stirring. The resulting symmetrically NBD-labeled proteoliposomes were collected by ultracentrifugation at 293,000 x g for 90 min at 4 °C and resuspended in a small volume of buffer Z.
The model peptide WALP23 was reconstituted by octyl glucoside dilution following the same procedure, starting from a mixed lipid film containing the peptide at a 1:1000 molar ratio with respect to PL-Pi.
To check whether the method of reconstitution influences the properties of the proteoliposomes with respect to phospholipid translocation, a second protocol was also used. LUVETs composed of TLE (5 mM PL-Pi) prepared in buffer Z were solubilized with octyl glucoside (OG) (1% (w/v) final concentration) resulting in an optically clear solution. Lep was added (1:1000 molar ratio with respect to PL-Pi), and the detergent was removed using Bio-Beads SM (Bio-Rad). Briefly, the mixed micelle solution was incubated for 30 min under gentle rotation at room temperature;
80 mg/ml Bio-Beads was added, and incubation was continued for 2 h. Next, the solution was added to 80 mg/ml fresh Bio-Beads and again incubated for 2 h under rotation. Subsequently, the solution was incubated overnight at 4 °C, again with fresh Bio-Beads. The vesicles were collected by centrifugation (1 h at 435,000 x g) and resuspended in 400 µl of buffer.
KcsA was reconstituted as described (22), based on a published protocol (15). Briefly, LUVETs (5 mM TLE-Pi) containing 0.5% C6NBD-PG with respect to total PL-Pi, prepared in 10 mM HEPES, 100 mM NaCl, 5 mM KCl were solubilized by adding Triton X-100 to a final concentration of 8 mM. The tetrameric protein was added at a molar ratio of 1:1000 or 1:2000 with respect to PL-Pi, as indicated, typically in a final volume of 700 µl. Detergent was removed using Bio-Beads as above.
MsbA was reconstituted according to Doerrler et al. (26) with minor modifications. LUVETs (5 mM TLE-Pi) were prepared in 50 mM HEPES, 50 mM NaCl, 2 mM -mercaptoethanol, pH 7.5, and solubilized with 0.2% (w/v) DDM from a 20% (w/v) stock in H2O, yielding an optically clear solution. Protein was added to a 1:1000 molar ratio with respect to PL-Pi, in a final volume of 350 µl. Following dilution to 1 ml, detergent was removed with Bio-Beads as above.
OmpT was reconstituted as described for OMPLA, another E. coli outer membrane protein (35). LUVETs prepared from TLE (5 mM PL-Pi) and containing C6NBD-PG (0.5% of total PL-Pi) in buffer Z were solubilized with OG added from a 20% (w/v) stock solution in buffer Z to a final concentration of 1% (w/v) and supplemented with OmpT at a 1:1000 molar ratio with respect to PL-Pi to form mixed micelles, which were subsequently incubated with Bio-Beads to remove detergent as above.
Flop AssayAll procedures were performed as described previously (11). The LUVETs or proteoliposomes, symmetrically labeled with C6NBD-PG, were incubated with 25 mM sodium dithionite (Na2S2O4) for 5 min to reduce and thereby quench the fluorescent NBD label in the outer membrane leaflet, followed by gel filtration to remove excess dithionite. The resulting asymmetrically labeled vesicles were incubated at a concentration of 3 mM PL-Pi at the temperature indicated. At different time points aliquots of vesicles were taken, and the in-out translocation (flop) of NBD-phospholipids was measured by determining the amount of NBD-phospholipid susceptible to reduction by 8 mM sodium dithionite in 3 min at 20 °C.
Flip AssayProteoliposomes (2 mM PL-Pi) were incubated 30 min on ice with 0.1 mol % NBD-PL, added from an
1 mM stock solution in ethanol, to allow the probe to incorporate in the outer membrane leaflet. The concentration of ethanol in the vesicle suspension never exceeded 0.2% (v/v). Phospholipid out-in translocation (flip) was initiated by shifting the vesicles to 37 °C. At different time points, aliquots were assayed for the transmembrane distribution of C6NBD-PG as described for the flop assay. The pool of C6NBD-PG protected against reduction by dithionite was taken as the amount of flip.
Fluorescence Measurements and CalculationsFluorescence measurements were performed as described (11), in buffer Z unless noted otherwise, using an SLM Aminco SPF 500C spectrofluorometer (excitation 460 nm and emission 534 nm). The percentage of NBD-phospholipid in the outer (flop assay) or inner (flip assay) leaflet at different time points was calculated according to Equations 1 and 2, respectively,
![]() | (Eq. 1) |
![]() | (Eq. 2) |
![]() | (Eq. 3) |
In our system, flip-flop at t = 0 variably deviated from the theoretical value of 0%. To be able to compare flip-flop rate constants in vesicles of different compositions, Coffset was introduced as a constant equal to the amount of accessible NBD label at t = 0 in control vesicles without peptide or protein (0.03 < Coffset < 0.1, depending on the experiment). The apparent first order flip-flop rate constants (Kflip-flop) were calculated by a least squares fit to Equation 4,
![]() | (Eq. 4) |
MiscellaneousPhospholipids were quantified according to Rouser et al. (37). Phospholipid compositions and concentrations are presented based on lipid phosphorus (PL-Pi). C6NBD-PL contents of the membranes are presented as the mol % with respect to PL-Pi, before reduction with dithionite. Aliquots of proteoliposomes were mixed with sample buffer and heated to 95 °C before subjecting the samples to SDS-PAGE. The Coomassie-stained SDS gels were quantitated using bovine serum albumin as a standard, on a Bio-Rad GS-700 Densitometer using the Quantity One software (Bio-Rad) to check the efficiency of reconstitution of the proteins. In the case of KcsA, proteoliposomes mixed with sample buffer were directly subjected to electrophoresis, to allow detection of the tetrameric protein. The extent of incorporation of WALP23 was checked by measuring Trp fluorescence in buffer Z containing 1% (w/v) octyl glucoside, (excitation 280 nm and emission 350 nm). ATPase activity was determined as described (26).
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RESULTS |
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Subsequently, the vesicles were treated with dithionite to quench the NBD fluorescence in the outer membrane leaflet. After removal of excess dithionite, the vesicles were incubated at 37 °C and assayed at different time points for the reappearance of C6NBD-PG in the outer leaflet. Fig. 1B shows that at 37 °C efficient flop occurs in vesicles containing WALP23 prepared by reconstitution from detergent, albeit at a slightly slower rate than in WALP23-containing LUVETs. The apparent first order flop rate constant at 37 °C in LUVETs containing WALP23 at a 1:1000 molar ratio was determined according to Equation 4 and found to be 0.8 h1, 4 times higher than the flop rate constant at 25 °C (12), indicating that flop rates increase with temperature. The spontaneous translocation of C6NBD-PG in vesicles without peptide was slightly increased at 37 °C (Fig. 1B) as compared with 25 °C (12). Together, these data clearly demonstrate that reconstitution from detergent yields vesicles in which the dithionite reduction assay can be used to measure peptide-induced flop and that any residual detergent does not enhance flop.
Next, we investigated whether leader peptidase (Lep), an inner membrane protein of E. coli, reconstituted by dilution from OG could induce flop. Reconstitution of Lep (starting from a 1:1000 protein/lipid molar ratio) results in protein-containing vesicles (Fig. 2A, inset). Recovery of phospholipid and protein after reconstitution are 70 and
80%, respectively. Addition of dithionite (Fig. 2A) reveals that the reconstituted vesicles are sealed and unilamellar, with a protected pool of C6NBD-PG of 32 ± 3%, similar to that of vesicles without protein, which have a protected pool of 35 ± 3%. Fig. 2B shows that the presence of Lep (triangles) enhances flop of C6NBD-PG in vesicles composed of an E. coli lipid extract. The flop rate in vesicles containing Lep was 3-fold higher than that in the absence of protein (Fig. 2B, circles). Because flip-flop in E. coli has been show to be bi-directional (1), we also investigated Lep-induced flip in proteoliposomes. To this end, the proteoliposomes, prepared by detergent removal with Bio-Beads, were incubated on ice with C6NBD-PG, added from a stock solution in ethanol. After incorporation of the probe into the outer leaflet, the proteoliposomes were shifted to 37 °C to allow phospholipid translocation. Fig. 2B (diamonds) shows that flip of C6NBD-PG induced by Lep is comparable with flop (triangles). Flip proceeds at a rate
2.5 times higher than flip in vesicles without protein (not shown). Thus, phospholipid translocation mediated by Lep proceeds in both directions.
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To investigate which part of Lep is responsible for inducing flop, the first transmembrane stretch of Lep (designated H1) was chemically synthesized and incorporated in LUVETs. As was shown for WALP23 in Fig. 1, also the presence of this membrane-spanning -helical stretch induced very efficient translocation of C6NBD-PG (Fig. 2B, squares). Previously, we reported that a positively charged model peptide (AcGKK(LA)8-KKA-NH2, KALP23) was particularly efficient in inducing flop of C6NBD-PG in vesicles composed of E. coli phospholipids (11). To investigate whether introducing positively charged lysine residues in the membrane-water interface would influence the efficiency of flop induced by H1, a derivative (H1'), in which the first three amino acids are replaced by the first three amino acids of KALP23 (GKK), was also tested. The N-terminal modification did not have a significant effect on the translocation efficiency, indicating that primarily the transmembrane part causes phospholipid translocation. It has been reported (14, 15) that the water-soluble P2 domain of Lep interacts with membranes. To investigate whether this contributes to Lep-induced phospholipid translocation, the P2 domain was added to LUVETs at a high protein/lipid ratio (1:50 mol/mol), at only one, or at both sides of the model membrane. P2 was added either after formation of the vesicles thus being present at the outer leaflet only, or during and after vesicle formation, in which case P2 molecules have access to both membrane leaflets. Irrespective of the presence of P2 at one or at both membrane leaflets, no significant flop of C6NBD-PG was observed during 90 min of incubation (data not shown), again indicating that the transmembrane segment(s) of Lep are responsible for flop. In conclusion, phospholipid translocation induced by Lep is bi-directional and likely to be mediated by the transmembrane stretch(es) of this protein.
To investigate whether Lep is unique in its ability to induce phospholipid translocation, we tested another bacterial inner membrane protein, the tetrameric potassium channel KcsA. The quality of the proteoliposomes with and without KcsA was checked as above. After overnight removal of Triton X-100 by Bio-Beads, 60% of the phospholipids and
70% of the protein were recovered, and sealed vesicles were obtained, with a protected C6NBD-PG pool of 34% in the presence (1:1000 tetramer/lipid molar ratio) and 42% in the absence of protein. Upon reconstitution, KcsA retains the stable tetramer configuration (Fig. 3, inset). At a low protein (tetramer)/lipid ratio (Fig. 3, triangles, 1:2000), flop of C6NBD-PG is somewhat enhanced as compared with vesicles without protein (circles). When the protein is incorporated at a tetramer/lipid ratio of 1:1000, the flop rate is 4-fold increased compared with vesicles without protein, comparable with the rate of flop induced by Lep at a 1:1000 monomer/PL-Pi ratio (Fig. 2). These results show that induction of passive translocation of NBD-PL is not a unique property of leader peptidase but that flop is also facilitated by an oligomeric membrane protein. The rates of translocation for the various proteins and peptides are summarized in Table I.
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Although helix-mediated flop of phospholipids may provide an important route for phospholipid translocation in the E. coli inner membrane, the existence of dedicated phospholipid transporters in the E. coli inner membrane cannot be excluded. Recently, MsbA was proposed as a candidate lipid flippase (25). MsbA is a membrane-spanning E. coli inner membrane protein with six TMH per monomer, which is functional as a homodimer and hydrolyzes ATP at the cytosolic side of the E. coli inner membrane. This is also the site where phospholipids destined for translocation are synthesized. Because ATP can be conveniently added to pre-existing proteoliposomes (i.e. outside the liposomes), we tested the ability of MsbA to induce flip (rather than flop) of C6NBD-PL. The inset of Fig. 4 represents a Coomassie-stained SDS-PAGE gel, showing that the protein is present in the vesicle fraction after reconstitution. The reconstitution efficiency of the proteoliposomes was 42 ± 5% based on phospholipid and 53 ± 9% based on MsbA. Fig. 4 shows flip of C6NBD-PG in vesicles without (circles) and with protein at an 1:900 protein/PL-Pi molar ratio (diamonds) in the absence of ATP (open symbols). No MsbA-mediated flip of C6NBD-PG was observed, in contrast to flip induced by Lep and KcsA. Addition of ATP did not initiate flip mediated by MsbA (black symbols) nor did it influence flip in vesicles without protein as expected. In addition, no significant protein-induced flip of C6NBD-phosphatidylethanolamine was observed in TLE vesicles (not shown), irrespective of the presence of ATP, which was hydrolyzed at a rate of 10.4 ± 3.0 nmol·min1·mg protein1 during the assay (i.e. at 37 °C), in agreement with data reported (26). These data indicate that MsbA by itself is not capable of ATP-dependent phospholipid translocation and that not all E. coli inner membrane proteins induce passive phospholipid translocation.
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So far, it has been demonstrated that phospholipid translocation is facilitated by a subset of proteins with -helical membrane-spanning segments. To investigate further whether the
-helical stretches are necessary for translocation, the
-barrel protein OmpT was also tested. The protected pool of C6NBD-PG in the reconstituted proteoliposomes containing OmpT was
37% and
50% in the control vesicles without OmpT. Recovery of phospholipid and protein was somewhat lower than for the other proteins, around 20%. In the presence of OmpT, we observed a slightly higher offset of flop, which is most likely due to dithionite permeation. The presence (Fig. 5 inset, lane 2) of OmpT in a model membrane did not result in increased flop of C6NBD-PG (Fig. 5, diamonds) as compared with vesicles without OmpT (Fig. 5, circles). This confirms the observation that facilitating flip-flop is not a general property of membrane proteins and is consistent with the hypothesis that phospholipid translocation is mediated by transmembrane
-helices.
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DISCUSSION |
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We showed that the E. coli inner membrane protein leader peptidase, an essential protein involved in membrane biogenesis, induces phospholipid translocation. It has been suggested that in membrane biogenesis, lipid and protein assembly might be functionally coupled (38). Thus, one might speculate that these two transport processes converge at Lep. However, the present study indicates that translocation of phospholipids is not directly linked to the protein transport machinery, as the potassium channel KcsA also induces flop.
In studies with model peptides, we generally observed rapid flop of C6NBD-PG at low peptide/phospholipid molar ratios (see e.g. Fig. 1 and Ref. 11). Here we also show that a naturally occurring membrane-spanning helix, H1 of Lep, induces efficient translocation. This indicates that the specific structure of the model peptides with Leu-Ala repeats as a hydrophobic core is not a prerequisite for efficient phospholipid translocation, validating their use as representatives of membrane-spanning helices of proteins. These observations, taken together with the fact that leader peptidase facilitates translocation whereas its water-soluble P2 domain does not, indicate that flop is induced by the transmembrane segments.
The proteins Lep and KcsA, incorporated at similar protein/phospholipid molar ratios as the membrane-spanning peptides, show a moderate effect on phospholipid translocation, compared with the model TMHs. The method of vesicle preparation could have some effect on the flop rates, as demonstrated by the different flop rates induced by WALP23 in vesicles prepared by detergent removal and extrusion, but this effect alone cannot account for the differences between peptides and proteins observed in this study. We speculate that the model peptides, being single helices and typically smaller than 3 kDa, interact more dynamically with the lipid bilayer than membrane proteins, which span the membrane more often and are significantly larger. Therefore, the peptides may cause more disturbances in the lipid-helix interface. Additionally, helix-helix interactions in membrane proteins could effectively reduce the "surface" available as flop sites, as will be discussed shortly. Thus, the monomeric Lep could be expected to have more flop sites per TMH available than the tetrameric KcsA, and our data show that indeed Lep exhibits more translocating activity per monomer than KcsA. MsbA, a dimer with a total of 12 helices packed in the membrane, did not induce phospholipid translocation, consistent with this idea.
Based on several observations outlined in the Introduction, MsbA has been suggested to play a role in phospholipid transport. However, phospholipid translocation in the bacterial cytoplasmic membrane has been shown to proceed without an energy source (1, 39). Here we showed that MsbA was vesicleassociated after reconstitution and that it hydrolyzed ATP in proteoliposomes, as was reported previously (26). Therefore, we consider it likely that MsbA was functionally reconstituted. However, no MsbA-mediated flip in the presence or absence of ATP was observed. Possibly, MsbA needs a co-factor to be functional in phospholipid flip-flop. Alternatively, MsbA could be involved in transport of (phospho)lipids to the outer membrane or in translocation/transport of LPS, as has been suggested previously (27, 40). The data presented here argue against MsbA being a phospholipid flippase.
The E. coli outer membrane -barrel protein OmpT did not induce flop in TLE vesicles, consistent with our finding that
-helical membrane-spanning segments facilitate phospholipid translocation and with the fact that under normal conditions glycerophospholipids are absent from the outer leaflet of the outer membrane. The lipid asymmetry, with LPS exclusively in the outer leaflet, has been suggested to be important to preserve the barrier function of the outer membrane (41). However, it should be noted that reconstitution of OmpT in a glycerophospholipid double layer may not adequately reflect the properties of this protein.
Helix-mediated passive phospholipid flip-flop is consistent with the reported lack of energy requirement and lack of sensitivity toward treatment with alkylating agents reported for this process in vitro in isolated inner membrane vesicles of E. coli (1). However, the induction of phospholipid flip-flop is restricted to a subset of membrane proteins. Based on our previous studies (12), we proposed that translocation of phospholipids occurs near a transmembrane -helix, where a "flop site" is created by the helix through the disturbance of the lipid-lipid interactions. The present findings lead to a refined model depicted in Fig. 6. Membrane-spanning
-helical segments induce flop of phospholipids through flop sites, which occur near the helix through its dynamic interaction with the membrane (Fig. 6A). When more helices are assembled, both the surface available as a potential flop site (Fig. 6C) and the dynamics of the helices are reduced (Fig. 6B), resulting in less efficient flop. The presence of residual detergent in proteoliposomes may partially fill up the peptide-induced defects, resulting in lower flop rates.
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To address the question whether phospholipid translocation in E. coli in vivo could be accounted for by the presence of TMHs of integral membrane proteins, the number of TMHs per phospholipid in the E. coli inner membrane was previously estimated to be 1:9. Based on this ratio, the peptide-mediated translocation half-time at low peptide concentrations measured in model systems was extrapolated to that in the E. coli IM (11). However, the membrane protein Lep is substantially less efficient than a peptide in inducing flop, indicating that the above extrapolation may have overestimated the rate of helix-induced flop rate in the E. coli IM. When the observed translocation half-time of C6NBD-PG induced by reconstituted Lep is extrapolated to the expected TMH/lipid ratio of the inner membrane of E. coli, a translocation half-time of
6 min could be expected, which is sufficient to sustain growth at a doubling time of
30 min. This estimate does not take into account proteins like MsbA that do not induce phospholipid translocation. On the other hand signal peptides, for example, of exported membrane proteins might induce additional phospholipid translocation before they are degraded. Finally, care should be taken when translocation rates of phospholipid analogues are compared with those of their natural counterparts. For example, Huijbregts et al. (1) demonstrated that flop of NBD-phosphatidylethanolamine in the E. coli inner membrane has a translocation half-time of around 7 min, while its natural homologue redistributes within 30 s (2). Marx et al. (5) and Colleau et al. (42) observed different flop rates for NBD-PL versus the spin-labeled phospholipid analogues in rat liver microsomes and erythrocyte membranes, respectively. The studies cited above suggest that flop of C6-NBD-PL generally proceeds at a somewhat lower rate. This is according to expectation, because the rather bulky, relatively hydrophilic NBD group has to cross the hydrophobic interior of the bilayer.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 3130-2532465; Fax: 3130-2533969; E-mail: m.a.kol{at}chem.uu.nl.
1 The abbreviations used are: C6-NBD-PL, 1 palmitoyl-2,6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl-sn-glycero-3-phospholipid; DDM, n-dodecyl--D-maltoside; IM, inner membrane; Lep, Leader peptidase from E. coli; LPS, lipopolysaccharide; LUVETs, large unilamellar vesicles prepared by extrusion technique; OG, n-octyl-
-D-glucopyranoside; PG, phosphatidylglycerol; -Pi, inoganic phosphate; PL, phospholipid(s); TLE, total phospholipid extract of E. coli; TMH, transmembrane helix.
2 M. Egmond, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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