Translocation of Phospholipids Is Facilitated by a Subset of Membrane-spanning Proteins of the Bacterial Cytoplasmic Membrane
2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês
10.1074/jbc.m301875200
ISSN1083-351X
AutoresMatthijs Kol, Annemieke van Dalen, Anton I.P.M. de Kroon, Ben de Kruijff,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe mechanism by which phospholipids are transported across biogenic membranes, such as the bacterial cytoplasmic membrane, is unknown. We hypothesized that this process is mediated by the presence of the membrane-spanning segments of inner membrane proteins, rather than by dedicated flippases. In support of the hypothesis, it was demonstrated that transmembrane α-helical peptides, mimicking the membrane-spanning segments, mediate flop of 2–6-(7-nitro-2,1,3-benzoxadiazol-4-yl) aminocaproyl (C6-NBD)-phospholipids (Kol, M. A., de Kroon, A. I., Rijkers, D. T., Killian, J. A., and de Kruijff, B. (2001) Biochemistry 40, 10500–10506). Here the dithionite reduction assay was used to measure transbilayer equilibration of C6-NBD-phospholipids in proteoliposomes, composed of Escherichia coli phospholipids and a subset of bacterial membrane proteins. It is shown that two well characterized integral proteins of the bacterial cytoplasmic membrane, leader peptidase and the potassium channel KcsA, induce phospholipid translocation, most likely by their transmembrane domains. In contrast, the ATP-binding cassette transporter from the E. coli inner membrane MsbA, a putative lipid flippase, did not mediate phospholipid translocation, irrespective of the presence of ATP. OmpT, an outer membrane protein from E. coli, did not facilitate flop either, demonstrating specificity of protein-mediated phospholipid translocation. The results are discussed in the light of phospholipid transport across the E. coli inner membrane. The mechanism by which phospholipids are transported across biogenic membranes, such as the bacterial cytoplasmic membrane, is unknown. We hypothesized that this process is mediated by the presence of the membrane-spanning segments of inner membrane proteins, rather than by dedicated flippases. In support of the hypothesis, it was demonstrated that transmembrane α-helical peptides, mimicking the membrane-spanning segments, mediate flop of 2–6-(7-nitro-2,1,3-benzoxadiazol-4-yl) aminocaproyl (C6-NBD)-phospholipids (Kol, M. A., de Kroon, A. I., Rijkers, D. T., Killian, J. A., and de Kruijff, B. (2001) Biochemistry 40, 10500–10506). Here the dithionite reduction assay was used to measure transbilayer equilibration of C6-NBD-phospholipids in proteoliposomes, composed of Escherichia coli phospholipids and a subset of bacterial membrane proteins. It is shown that two well characterized integral proteins of the bacterial cytoplasmic membrane, leader peptidase and the potassium channel KcsA, induce phospholipid translocation, most likely by their transmembrane domains. In contrast, the ATP-binding cassette transporter from the E. coli inner membrane MsbA, a putative lipid flippase, did not mediate phospholipid translocation, irrespective of the presence of ATP. OmpT, an outer membrane protein from E. coli, did not facilitate flop either, demonstrating specificity of protein-mediated phospholipid translocation. The results are discussed in the light of phospholipid transport across the E. coli inner membrane. Biological membranes are composed of proteins and lipids, the latter organized as a bilayer. Cellular growth requires influx of new membrane components into the membranes. Because the synthesis of phospholipids, the major lipid constituents of most biological membranes, is generally confined to one leaflet of biogenic membranes (i.e. membranes containing phospholipid biosynthetic enzymes), transport of phospholipids to the other membrane leaflet is required. It has been shown that biogenic membranes, such as bacterial plasma membranes (1Huijbregts R.P. de Kroon A.I. de Kruijff B. Biochim. Biophys. Acta. 1996; 1280: 41-50Google Scholar, 2Huijbregts R.P. de Kroon A.I. de Kruijff B. J. Biol. Chem. 1998; 273: 18936-18942Google Scholar, 3Hrafnsdottir S. Nichols J.W. Menon A.K. Biochemistry. 1997; 36: 4969-4978Google Scholar) and the endoplasmic reticulum membrane (4Herrmann A. Zachowski A. Devaux P.F. Biochemistry. 1990; 29: 2023-2027Google Scholar, 5Marx U. Lassmann G. Holzhutter H.G. Wustner D. Muller P. Hohlig A. Kubelt J. Herrmann A. Biophys. J. 2000; 78: 2628-2640Google Scholar, 6Buton X. Morrot G. Fellmann P. Seigneuret M. J. Biol. Chem. 1996; 271: 6651-6657Google Scholar, 7Buton X. Herve P. Kubelt J. Tannert A. Burger K.N. Fellmann P. Muller P. Herrmann A. Seigneuret M. Devaux P.F. Biochemistry. 2002; 41: 13106-13115Google Scholar), exhibit rapid phospholipid flip-flop with similar characteristics, including limited sensitivity toward proteolysis, bidirectionality, energy independence, and phospholipid head group independence (recently reviewed in Ref. 8Kol M.A. de Kruijff B. de Kroon A.I. Semin. Cell Dev. Biol. 2002; 13: 163-170Google Scholar). In contrast to flip-flop in biogenic membranes, phospholipid translocation in model membranes composed of only lipids is very slow (9Kornberg R.D. McConnell H.M. Biochemistry. 1971; 10: 1111-1120Google Scholar). It is therefore generally accepted that phospholipid translocation is a protein-mediated process. In some membranes, this activity has been attributed to dedicated proteins (see e.g. Ref. 10Bevers E.M. Comfurius P. Dekkers D.W. Zwaal R.F. Biochim. Biophys. Acta. 1999; 1439: 317-330Google Scholar); however, the identities of the putative phospholipid translocators, or flippases, in the bacterial cytoplasmic membrane and in the endoplasmic reticulum remain obscure despite many efforts. Based on the general characteristics of flip-flop, we hypothesized that the mere presence of α-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 α-helical stretches of transmembrane proteins, induces flop of C6-NBD-PL 1The 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-β-d-maltoside; IM, inner membrane; Lep, Leader peptidase from E. coli; LPS, lipopolysaccharide; LUVETs, large unilamellar vesicles prepared by extrusion technique; OG, n-octyl-β-d-glucopyranoside; PG, phosphatidylglycerol; -Pi, inoganic phosphate; PL, phospholipid(s); TLE, total phospholipid extract of E. coli; TMH, transmembrane helix. analogues in model membranes (11Kol M.A. de Kroon A.I. Rijkers D.T. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 10500-10506Google Scholar, 12Kol M.A. Van Laak A.N. Rijkers D.T. Killian J.A. De Kroon A.I. De Kruijff B. Biochemistry. 2003; 42: 231-237Google Scholar), supporting this hypothesis. Bacterial membrane proteins display a large diversity in structure and organization, more than can be accounted for by α-helical model peptides. α-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 α-helices and adopts an overall Nout/Cout topology in the inner membrane (IM) of E. coli (13Laws J.K. Dalbey R.E. EMBO J. 1989; 8: 2095-2099Google Scholar). The large C-terminal catalytic domain is in close contact with the periplasmic leaflet of the IM, interacting with phospholipids (14van Klompenburg W. Paetzel M. de Jong J.M. Dalbey R.E. Demel R.A. von Heijne G. de Kruijff B. FEBS Lett. 1998; 431: 75-79Google Scholar, 15van den Brink-van der Laan E. Dalbey R.E. Demel R.A. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 9677-9684Google Scholar). Lep is an essential protein (16Date T. J. Bacteriol. 1983; 154: 76-83Google Scholar) 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 (17Ohno-Iwashita Y. Wickner W. J. Biol. Chem. 1983; 258: 1895-1900Google Scholar, 18van Dalen A. Killian A. de Kruijff B. J. Biol. Chem. 1999; 274: 19913-19918Google Scholar). 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 (19Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Google Scholar). Unlike Lep, KcsA is an oligomeric protein forming a stable homotetramer (20Cortes D.M. Perozo E. Biochemistry. 1997; 36: 10343-10352Google Scholar). 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 (21Valiyaveetil F.I. Zhou Y. MacKinnon R. Biochemistry. 2002; 41: 10771-10777Google Scholar, 22van Dalen A. Hegger S. Killian J.A. de Kruijff B. FEBS Lett. 2002; 525: 33-38Google Scholar). 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 (23Putman M. van Veen H.W. Konings W.N. Microbiol. Mol. Biol. Rev. 2000; 64: 672-693Google Scholar). MsbA is a homodimer, the 64-kDa monomer spanning the membrane with six TMHs (24Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Google Scholar, 25Chang G. Roth C.B. Science. 2001; 293: 1793-1800Google Scholar). It was shown to be an ATPase (26Doerrler W.T. Raetz C.R. J. Biol. Chem. 2002; 277: 36697-36705Google Scholar). The msbA gene was first discovered as a multicopy suppressor of mutations in htrB (24Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Google Scholar), 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 (27Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R. J. Biol. Chem. 1998; 273: 12466-12475Google Scholar). Based on these observations and on the recently resolved crystal structure (25Chang G. Roth C.B. Science. 2001; 293: 1793-1800Google Scholar), 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 α-helical transmembrane segments, another principal structural motif by which integral membrane proteins span the bilayer is a β-barrel. In E. coli, β-barrel proteins are exclusively found in the outer membrane. We included the E. coli protease OmpT, with known crystal structure (28Vandeputte-Rutten L. Kramer R.A. Kroon J. Dekker N. Egmond M.R. Gros P. EMBO J. 2001; 20: 5033-5039Google Scholar), as a paradigm for the β-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 α-helices. The efficiency of flop induced by different proteins varied. Moreover, data are presented that argue against MsbA being an ATP-dependent phospholipid flippase. Materials—The E. coli total phospholipid extract was isolated from the wild-type strain W3899 as described previously (11Kol M.A. de Kroon A.I. Rijkers D.T. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 10500-10506Google Scholar), or obtained from Avanti Polar Lipids (Alabaster, AL), and purified on a silica column (11Kol M.A. de Kroon A.I. Rijkers D.T. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 10500-10506Google Scholar). All other lipids were obtained from Avanti and used without further purification. Stock solutions were prepared in chloroform or ethanol, stored under N2 at –20 °C, and periodically checked for purity by thin layer chromatography. WALP23 (AcGWWL(AL)8WWA-nh2) was synthesized as described (29Killian J.A. Salemink I. de Planque M.R. Lindblom G. Koeppe R.E. Greathouse D.V. Biochemistry. 1996; 35: 1037-1045Google Scholar, 30de Planque M.R. Greathouse D.V. Koeppe II, R.E. Schafer H. Marsh D. Killian J.A. Biochemistry. 1998; 37: 9333-9345Google Scholar). Peptide H1, corresponding to the N-terminal residues 1–25 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 Proteins—Leader peptidase with a C-terminal His5 tag was produced in E. coli strain MC1061 carrying the p827 plasmid as described (31van Klompenburg W. Whitley P. Diemel R. von Heijne G. de Kruijff B. Mol. Membr. Biol. 1995; 12: 349-353Google Scholar) 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 × g, and the resulting supernatant was centrifuged at 90,500 × 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 (Δ2-75) was produced and purified as described (15van den Brink-van der Laan E. Dalbey R.E. Demel R.A. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 9677-9684Google Scholar) and stored as a stock of 0.1–0.2 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(λDE3) carrying a pT7-KcsA plasmid, essentially as described previously (20Cortes D.M. Perozo E. Biochemistry. 1997; 36: 10343-10352Google Scholar, 32van Dalen A. Schrempf H. Killian J.A. de Kruijff B. EMBO Rep. 2000; 1: 340-346Google Scholar). Cells were grown for 2 h after addition of isopropyl-β-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 (26Doerrler W.T. Raetz C.R. J. Biol. Chem. 2002; 277: 36697-36705Google Scholar) 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 β-mercaptoethanol at –20 °C. OmpTE211K/R218E, an OmpT mutant with reduced autoproteolytic activity 2M. Egmond, personal communication. produced and purified as described (33Kramer R.A. Brandenburg K. Vandeputte-Rutten L. Werkhoven M. Gros P. Dekker N. Egmond M.R. Eur. J. Biochem. 2002; 269: 1746-1752Google Scholar), 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 (11Kol M.A. de Kroon A.I. Rijkers D.T. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 10500-10506Google Scholar), 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 Proteoliposomes—Reconstitution of Lep into proteoliposomes was performed by octyl glucoside dilution as described (18van Dalen A. Killian A. de Kruijff B. J. Biol. Chem. 1999; 274: 19913-19918Google Scholar, 34Ohno-Iwashita Y. Wolfe P. Ito K. Wickner W. Biochemistry. 1984; 23: 6178-6184Google Scholar). 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 × 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 × g) and resuspended in 400 μl of buffer. KcsA was reconstituted as described (22van Dalen A. Hegger S. Killian J.A. de Kruijff B. FEBS Lett. 2002; 525: 33-38Google Scholar), based on a published protocol (15van den Brink-van der Laan E. Dalbey R.E. Demel R.A. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 9677-9684Google Scholar). 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. (26Doerrler W.T. Raetz C.R. J. Biol. Chem. 2002; 277: 36697-36705Google Scholar) with minor modifications. LUVETs (5 mm TLE-Pi) were prepared in 50 mm HEPES, 50 mm NaCl, 2 mm β-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.Kingma, R. L. (2002) Outer Membrane Phospholipase A: Studies on Structure-Function Relationships. Ph.D. thesis, Utrecht University, The NetherlandsGoogle Scholar). 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 Assay—All procedures were performed as described previously (11Kol M.A. de Kroon A.I. Rijkers D.T. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 10500-10506Google Scholar). 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 Assay—Proteoliposomes (∼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 Calculations—Fluorescence measurements were performed as described (11Kol M.A. de Kroon A.I. Rijkers D.T. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 10500-10506Google Scholar), 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, flop(%)=[1−(F180/F0)]·100 (Eq. 1) and flip(%)=(F180/F0)·100(Eq. 2) with F0 and F180 the fluorescence intensities after 0 and 180 s of reaction with dithionite. Equation 3 theoretically describes the process of flip or flop in a vesicle assuming that the inward and outward rate constants are equal (36Fattal E. Nir S. Parente R.A. Szoka F.C. Biochemistry. 1994; 33: 6721-6731Google Scholar), flip-flop(t)=0.5·(1−e−Kflip-flop•t)(Eq. 3) with flip-flop(t) the relative amount of flip or flop (depending on the experiment) at time t, the factor 0.5 representing the theoretical maximum value of flip-flop (50% redistribution of the probe), Kflip-flop the apparent first order rate constant of flip-flop, and t the time of incubation. 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, flip-flop(t)=Coffset+(0.5−Coffset)(1−e−Kflip-flop·t)(Eq. 4) Miscellaneous—Phospholipids were quantified according to Rouser et al. (37Rouser G. Fleischer S. Yamamoto A. Lipids. 1970; 5: 494-496Google Scholar). 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 (26Doerrler W.T. Raetz C.R. J. Biol. Chem. 2002; 277: 36697-36705Google Scholar). In this study we investigated the capacity of membrane proteins to induce helix-mediated phospholipid translocation in proteoliposomes, using short chain NBD-labeled phospholipids as probe molecules. Proteoliposomes are generally prepared by removal of detergent from a mixed micelle solution of lipids, protein, and detergent, in contrast to the previously used peptide-containing model membranes that were prepared by extrusion (11Kol M.A. de Kroon A.I. Rijkers D.T. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 10500-10506Google Scholar, 12Kol M.A. Van Laak A.N. Rijkers D.T. Killian J.A. De Kroon A.I. De Kruijff B. Biochemistry. 2003; 42: 231-237Google Scholar). Residual detergent in the proteoliposomes might compromise membrane integrity, in which case the dithionite reduction assay would not work, or might cause artifacts in phospholipid translocation. Therefore, the model peptide WALP23 was incorporated in vesicles by reconstitution from detergent and by extrusion of multilamellar vesicles prepared from a mixed peptide-lipid film. The resulting proteoliposomes and LUVETs, with and without WALP23 incorporated, were compared with respect to their permeability to dithionite, and the occurrence of flop of C6NBD-PG. The peptide/phospholipid ratios before (1:1000 mol/mol) and after vesicle preparation were similar for the two protocols (not shown). In both procedures C6NBD-PG was present during vesicle preparation and thus symmetrically incorporated in both membrane leaflets. Fig. 1A shows that vesicles prepared by the extrusion protocol and prepared by dilution from octyl glucoside have similar C6NBD-PG pools protected from dithionite reduction (∼35 and ∼40%, respectively), irrespective of the incorporation of WALP23. The quenching of the C6NBD-PG in the outer leaflet is completed in ∼3 min, as was shown previously (11Kol M.A. de Kroon A.I. Rijkers D.T. Killian J.A. de Kruijff B. Biochemistry. 2001; 40: 10500-10506Google Scholar). These data demonstrate that both reconstitution protocols yield vesicles that are sealed to dithionite and unilamellar. The slightly smaller protected C6NBD-PG pool in vesicles prepared by detergent dilution suggests that these vesicles are relatively small. 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 h–1, 4 times higher than the flop rate constant at 25 °C (12Kol M.A. Van Laak A.N. Rijkers D.T. Killian J.A. De Kroon A.I. De Kruijff B. Biochemistry. 2003; 42: 231-237Google Scholar), 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 (12Kol M.A. Van Laak A.N. Rijkers D.T. Killian J.A. De Kroon A.I. De Kruijff B. Biochemistry. 2003; 42: 231-237Google Scholar). 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 detergen
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