The reconstituted Escherichia coli MsbA protein displays lipid flippase activity - PubMed (original) (raw)

Comparative Study

. 2010 Jul 1;429(1):195-203.

doi: 10.1042/BJ20100144.

Affiliations

Comparative Study

The reconstituted Escherichia coli MsbA protein displays lipid flippase activity

Paul D W Eckford et al. Biochem J. 2010.

Abstract

The MsbA protein is an essential ABC (ATP-binding-cassette) superfamily member in Gram-negative bacteria. This 65 kDa membrane protein is thought to function as a homodimeric ATP-dependent lipid translocase or flippase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. We have previously shown that purified MsbA from Escherichia coli displays high ATPase activity, and binds to lipids and lipid-like molecules, including lipid A, with affinity in the low micromolar range. Bacterial membrane vesicles isolated from E. coli overexpressing His6-tagged MsbA displayed ATP-dependent translocation of several fluorescently NBD (7-nitrobenz-2-oxa-1,3-diazole)-labelled phospholipid species. Purified MsbA was reconstituted into proteoliposomes of E. coli lipid and its ability to translocate NBD-labelled lipid derivatives was characterized. In this system, the protein displayed maximal lipid flippase activity of 7.7 nmol of lipid translocated per mg of protein over a 20 min period for an acyl chain-labelled PE (phosphatidylethanolamine) derivative. The protein showed the highest rates of flippase activity when reconstituted into an E. coli lipid mixture. Substantial flippase activity was also observed for a variety of other NBD-labelled phospholipids and glycolipids, including molecules labelled on either the headgroup or the acyl chain. Lipid flippase activity required ATP hydrolysis, and was dependent on the concentration of ATP and NBD-lipid. Translocation of NBD-PE was inhibited by the presence of the putative physiological substrate lipid A. The present paper represents the first report of a direct measurement of the lipid flippase activity of purified MsbA in a reconstituted system.

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Figures

Figure 1

Figure 1. ATP-dependent translocation of NBD–PE by reconstituted MsbA

Proteoliposomes of E. coli lipid containing MsbA and 0.3% NBD–PE (16:0, 6:0) were incubated at 37 °C in the presence or absence of ATP and a regenerating system for 20 min. After termination of the reaction, NBD–PE fluorescence emission (excitation at 464 nm, emission at 536 nm) was monitored at 20 °C until a stable baseline was established. After 3 min, 4 mM dithionite was added (indicated by an interruption in the trace), and after a relatively stable baseline was again reached, 1% (w/v) Triton X-100 was added to establish the background fluorescence. Traces consist of raw fluorescence data (1 point per s) presented as a scatter plot, and were normalized to the fluorescence intensity recorded prior to dithionite addition, which was taken as 100%. The vertical arrows represent the total fluorescence of NBD–PE in both leaflets (Fi+o), and the inner leaflet (Fi). The Figure shows a representative fluorescence trace. Experiments with reconstituted proteoliposomes of the same lipid composition gave highly reproducible traces.

Figure 2

Figure 2. MsbA-mediated NBD–PE flippase activity in E. coli membrane vesicles and reconstituted proteoliposomes

(A) NBD–PE (16:0, 6:0) was incorporated into membrane vesicles isolated from E. coli overexpressing MsbA, which were incubated at 37 °C for 0–20 min in the presence (●) and absence (○) of 5 mM ATP and a regenerating system (added at time zero). The distribution of NBD–lipid between the two bilayer leaflets was determined using dithionite quenching. Experiments were also carried out using membrane vesicles isolated from cells transformed with empty vector (inset). Similar experiments were carried out (B) using E. coli lipid proteoliposomes containing NBD–PE (16:0, 6:0) and MsbA. Data are also shown for liposomes of E. coli lipid and NBD–PE (16:0, 6:0) alone with no incorporated MsbA (Δ) in the absence of ATP. (C) Egg PC proteoliposomes containing NBD–PE and MsbA. (D) E. coli lipid/egg PC 1:1 (w/w) proteoliposomes containing NBD–PE and MsbA. Inset graphs show ATP-dependent movement of NBD–lipids, calculated by normalizing the curves in the presence of ATP (●) to those in the absence of ATP (○). Data points are the means of duplicate determinations; error bars represent the range and, where not visible, they fall within the symbols.

Figure 3

Figure 3. Time course of translocation of NBD–PE by MsbA

Proteoliposomes of E. coli lipid containing 0.3% NBD–PE (16:0, 6:0) and reconstituted MsbA were incubated for 0–90 min in the presence or absence of 5 mM ATP and a regenerating system, or with 5 mM ATP and a regenerating system (arrow at time zero) for the first 45 min, after which an additional aliquot of ATP/regenerating system was added (arrow at 45 min) and the sample was incubated for a further 45 min (▼, +2ATP). Data points (●) represent the difference in the means of duplicate determinations in the presence and absence of ATP. Error bars represent the range and, where not visible, they fall within the symbols.

Figure 4

Figure 4. Characterization of MsbA-mediated translocation of NBD–PE

(A) ATP dependence of NBD–PE (16:0, 6:0) translocation. E. coli lipid proteoliposomes were incubated at 37 °C for 20 min in the presence of 0–20 mM ATP and a regenerating system. The transbilayer distribution of NBD–PE was determined, and the data were normalized to the value with 20 mM ATP, which was taken as 100%. Data points represent means±range for duplicate determinations. (B) Effect of nucleotides, Vi, AlFx and BeFx on translocation of NBD–PE. E. coli lipid proteoliposomes were incubated at 37 °C for 20 min in the absence of ATP, or in the presence of the regenerating system+5 mM ATP, AMP, GTP or AMP-PNP, 5 mM ATP+100 or 200 μM Vi, or 5 mM ATP+800 μM AlFx or 800 μM BeFx. The transbilayer distribution of NBD–PE was determined, and the data were normalized to the value with 5 mM ATP, which was taken as 100%. Data points represent means±range for duplicate determinations. (C) Dependence of translocation on the concentration of NBD–PE in the bilayer. Reconstituted proteoliposomes of E. coli lipid containing increasing amounts of NBD–PE were incubated for 20 min at 37 °C, and the extent of translocation was determined. Data were normalized to the extent of translocation with 0.6% NBD–PE, which was taken as 100%, and are represented as the means±range for duplicate determinations.

Figure 5

Figure 5. Time course of the MsbA-mediated translocation of four NBD-labelled lipids

Proteoliposomes of E. coli lipid containing reconstituted MsbA and 0.3% (A) NBD–PS (16:0, 6:0), (B) NBD–PE (18:1) (C) NBD–PG (16:0, 6:0) and (D) NBD–C12-SM were incubated for 0–20 min in the presence or absence of 5 mM ATP and a regenerating system. Data points represent the ATP-dependent movement of NBD–lipid, calculated from the difference in the means of duplicate determinations in the presence and absence of ATP. Error bars represent the range and, where not visible, they fall within the symbols.

Figure 6

Figure 6. Inhibition of ATP-dependent MsbA-mediated NBD–PE translocation by bacterial lipids

Net translocation of NBD–PE (16:0, 6:0) in proteoliposomes of E. coli lipid and MsbA was assessed in the presence of various concentrations of (A) lipid A, (B) RaLPS and (C) ReLPS, relative to a control without added lipid (taken as 100%), and a control with added lipid but without ATP (taken as 0%), or (D) incorporation of various concentrations of lipid A during preparation of proteoliposomes. The transbilayer distribution of NBD–PE was determined after incubation with 5 mM ATP and a regenerating system for 20 min at 37 °C. Data points represent means±range for duplicate determinations; where not visible, error bars fall within the symbols.

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