Matthijs A. Kol*, Annemieke van Dalen, Anton I.P.M. de Kroon and

38 Pages·2003·0.37 MB·English

JBC Papers in Press. Published on April 24, 2003 as Manuscript M301875200
TRANSLOCATION OF PHOSPHOLIPIDS IS FACILITATED BY A SUBSET OF MEMBRANE-
SPANNING PROTEINS OF THE BACTERIAL CYTOPLASMIC MEMBRANE

Matthijs A. Kol*, Annemieke van Dalen, Anton I.P.M. de Kroon and Ben de Kruijff

Department Biochemistry of Membranes, Centre for Biomembranes and Lipid
Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH
Utrecht, The Netherlands

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*Corresponding author: Fax: [phone], Tel.: [phone], E-mail: ww
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Running title: phospholipid translocation induced by membrane proteins
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Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT:
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 (C -NBD)-phospholipids (Kol et al. Biochemistry (2001), 40(35),
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10500-6). Here, the dithionite reduction assay was used to measure transbilayer
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equilibration of C -NBD-phospholipids in proteoliposomes, composed of E.coli o
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phospholipids and a subset of bacterial membrane proteins. It is shown that two well- ed
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characterized integral proteins of the bacterial cytoplasmic membrane, leader http
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peptidase and the potassium channel KcsA, induce phospholipid translocation, most w
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likely by their transmembrane domains. In contrast, the ABC transporter from the rg
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E.coli inner membrane MsbA, a putative lipid flippase, did not mediate phospholipid es
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translocation, irrespective of the presence of ATP. OmpT, an outer membrane protein pril 1
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from E.coli did not facilitate flop either, demonstrating specificity of protein-mediated 0
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phospholipid translocation. The results are discussed in the light of phospholipid
transport across the E.coli inner membrane.
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INTRODUCTION
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. Since 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 (1-3) and the ER
membrane (4-7), exhibit rapid phospholipid flip-flop with similar characteristics,
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including limited sensitivity towards proteolysis, bidirectionality, energy- o
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independence, and phospholipid head group independence (recently reviewed in (8)). ed
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In contrast to flip-flop in biogenic membranes, phospholipid translocation in model http
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membranes composed of only lipids is very slow (9). It is therefore generally accepted w
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that phospholipid translocation is a protein-mediated process. In some membranes, rg
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this activity has been attributed to dedicated proteins (see e.g. (10)), however the es
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identities of the putative phospholipid translocators, or flippases, in the bacterial pril 1
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cytoplasmic membrane and in the ER remain obscure despite many efforts. 0
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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 NBD-phospholipid 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 α-helical model peptides. α-Helical
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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 E.coli has two membrane-spanning α-helices and
adopts an overall N /C topology in the inner membrane (IM) of E.coli (13). The
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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
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in membrane biogenesis, as it clips off the signal peptide of proteins that are ow
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translocated via the E.coli Sec-machinery. Moreover, its purification and functional ed
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reconstitution in proteoliposomes have been characterized (17,18). Taken together, ttp
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this renders Lep a excellent model protein to test our hypothesis. w
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The potassium channel KcsA is another well-characterized protein of the brg/
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bacterial cytoplasmic membrane. Its crystal structure has been determined (19). es
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Unlike Lep, KcsA is an oligomeric protein forming a stable homotetramer (20). Each ril 1
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monomer contains two membrane-spanning domains, and has an overall Nin/Cin 019
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 ABC-transporter proteins, or traffic ATPases. In bacteria, the
ABC transporters have a complex membrane organization with two hydrophobic
domains, each of them typically spanning the membrane 6 times (23). MsbA is a
homodimer, the 64 kDa monomer spanning the membrane with 6 TMHs (24,25). It
was shown to be an ATPase (26). The msbA gene was first discovered as a multicopy
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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 α-helical transmembrane segments, another principle structural
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motif by which integral membrane proteins span the bilayer is a β-barrel. In E.coli, β- o
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barrel proteins are exclusively found in the outer membrane. We included the E.coli ed
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protease OmpT, with known crystal structure (28), as a paradigm for the β-barrel http
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membrane proteins, to investigate whether phospholipid flop is facilitated by w
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membrane proteins with other membrane-spanning structures. rg
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All proteins tested were reconstituted in proteoliposomes composed of E.coli es
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phospholipids, and translocation of the phospholipid analogue C6NBD-PG was pril 1
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measured fluorimetrically by determining its susceptibility towards dithionite 0
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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.
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EXPERIMENTAL PROCEDURES

Materials
The E.coli total phospholipid extract was isolated from the wild-type strain W3899 as
described previously (11), or obtained from Avanti Polar Lipids (Alabaster, AL), and
purified on a silica column (11). All other lipids were obtained from Avanti and used
without further purification. Stock solutions were prepared in chloroform or ethanol,
stored under N at –20 ºC, and periodically checked for purity by thin layer
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chromatography.
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WALP23 (AcGWWL(AL)8WWANH2) was synthesized as described (29,30). ow
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Peptide H1, corresponding to the N-terminal residues 1-25 of Lep, ed
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AcNleANNleFALILVIATLVTGILWCVDKFNH2 and a positively charged derivative http
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with an N-terminal three amino-acid substitution named H1' w
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(AcGKKNleFALILVIATLVTGILWCVDKFNH2), were synthesized, essentially as brg/
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described for WALP23, on an Applied Biosystems 433A Peptide Synthesizer using es
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the FastMoc protocol on a 0.25 mmol scale. Norleucines (Nle) are isosteric pril 1
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substitutions for the methionine residues at positions 1 and 4, and were used because 0
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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 N at –20 °C.
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Sodium dithionite (technical grade) was from Aldrich (Steinheim, Germany). All
other chemicals used were analytical grade.

Isolation and purification of proteins
Leader peptidase with a C-terminal His -tag, was produced in E.coli strain
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MC1061 carrying the p827 plasmid as described (31) with minor modifications as
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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 PMSF, 300 ng/mL
leupeptin, 10 µg/mL lysozyme, and incubated on ice for 1 h while stirring. The
resulting spheroplasts were disrupted by sonication for 10 x 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) octylglucoside,
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10 mM imidazole, and 100 mM NaCl. Undissolved material was removed by ow
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repeating the previous centrifugation step. The supernatant was loaded on a Ni2+ NTA ed
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column with ~7 mL column volume (Qiagen, Valencia CA, USA). After washing the ttp
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column with 5 volumes of the aforementioned buffer (10 mM imidazole), Lep was w
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eluted with 60 mM imidazole and stored at a concentration of ~0.25 mg/mL at –20 brg/
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°C. The P2 domain of leader peptidase (∆2-75) was produced and purified as st o
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described (15), and stored as a stock of 0.1–0.2 mg/mL in 20 mM Tris/HCl pH 7.4 at ril 1
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–20 °C. 19
KcsA with an N-terminal His -tag was overproduced and purified in E.coli
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strain BL21(λDE3) carrying a pT7-KcsA plasmid, essentially as described previously
(20,32). Cells were grown for 2 h after addition of isopropyl-β-D-
thiogalactopyranoside (IPTG) 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+ NTA 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
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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 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 MgCl , 10 % (w/v) glycerol
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and 5 mM β-mercaptoethanol at -20 °C.
OmpT , an OmpT mutant with reduced autoproteolytic activity (Dr.
E211K/R218E
Maarten Egmond, personal communication) produced and purified as described (33),
was generously supplied by Gerard-Jan de Roon and Dr. Maarten Egmond and stored
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as a stock of 2.6 mg/mL in 10 mM Tris/HCl, 1 % n-octyl-oligo-oxoethylene, pH 8.3 w
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at –20 °C. d fro
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Preparation of large unilamellar vesicles by extrusion (LUVETs) .jb
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Vesicles with and without the model peptides H1 and WALP23 were prepared b/
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as described previously (11), except for omitting K3Fe(CN)6. Briefly, a mixed film st o
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was prepared consisting of E.coli lipid extract (TLE), the indicated amount of peptide ril 1
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and C6NBD-PG at 0.2 mol% of PL-Pi. The lipid film was hydrated with buffer Z (50 19
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 C NBD-PG, were obtained.
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Preparation of proteoliposomes
Reconstitution of Lep into proteoliposomes was performed by octylglucoside
dilution as described (18,34). A lipid film containing TLE (typically 2 µmol PL-Pi)
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and C NBD-PG, was mixed with octylglucoside in buffer and Lep from the stock
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solution, to yield a mixed micelle solution of TLE, Lep (1:1000), C NBD-PG (0.5 %)
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and octylglucoside (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 octylglucoside dilution
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following the same procedure, starting from a mixed lipid film containing the peptide w
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at a 1:1000 molar ratio with respect to PL-Pi. d fro
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To check whether the method of reconstitution influences the properties of the ttp
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proteoliposomes with respect to phospholipid translocation, a second protocol was w
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also used. LUVETs composed of TLE (~5 mM PL-Pi) prepared in buffer Z were b/
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solubilized with octylglucoside (OG) (1% (w/v) final concentration (f.c.)) resulting in st o
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an optically clear solution. Lep was added (1:1000 molar ratio with respect to PL-Pi), ril 1
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and the detergent was removed using Bio-Beads SM (Bio-Rad Laboratories, Hercules, 1
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CA, USA). Briefly, the mixed micelle solution was incubated for 30 min under gentle
rotation at room temperature, ~80 mg/mL biobeads was added, and incubation was
continued for 2 h. Next, the solution was added to 80 mg/mL of fresh biobeads and
again incubated for 2 h under rotation. Subsequently, the solution was incubated
overnight at 4 ºC, again with fresh biobeads. The vesicles were collected by
centrifugation (1 h at 435,000 g) and resuspended in 400 µL buffer.
KcsA was reconstituted as described (22), based on a published protocol (15).
Briefly, LUVETs (~5 mM TLE-Pi) containing 0.5% C NBD-PG with respect to total
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PL-Pi, prepared in 10 mM HEPES, 100 mM NaCl, 5 mM KCl were solubilized by
adding Triton X100 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 biobeads 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 β-mercaptoethanol pH 7.5, and solubilized with 0.2 % (w/v) DDM from
a 20 % (w/v) stock in H O, yielding an optically clear solution. Protein was added to a
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1:1000 molar ratio with respect to PL-Pi, in a final volume of 350 µL. Following o
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dilution to 1 mL, detergent was removed with biobeads as above. ed
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OmpT was reconstituted as described for OMPLA, another E.coli outer http
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membrane protein (35). LUVETs prepared from TLE (~5 mM PL-Pi) and containing w
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C NBD-PG (0.5 % of total PL-Pi) in buffer Z were solubilized with OG added from a rg
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20% (w/v) stock solution in buffer Z to a final concentration of 1% (w/v), and es
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supplemented with OmpT at a 1:1000 molar ratio with respect to PL-Pi to form mixed pril 1
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micelles, which were subsequently incubated with biobeads to remove detergent as 0
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above.

Flop assay
All procedures were performed as described previously (11): The LUVETs or
proteoliposomes, symmetrically labeled with C NBD-PG, were incubated with 25
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mM sodium dithionite (Na S O ) for 5 min to reduce and thereby quench the
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fluorescent NBD-label in the outer membrane leaflet, followed by gel filtration to
remove excess dithionite. The resulting asymmetrically labeled vesicles were
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