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The reconstituted Escherichia coli MsbA protein displays lipid flippase activity
- Source :
- Biochemical Journal
- Publication Year :
- 2010
- Publisher :
- Portland Press Ltd., 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.
- Subjects :
- DTE, dithioerythritol
PG, phosphatidylglycerol
medicine.disease_cause
Biochemistry
ABC, ATP-binding-cassette
NBD–GlcCer, NBD–C6-glucosylceramide
Lipid A
PC, phosphatidylcholine
chemistry.chemical_compound
ATP hydrolysis
Pgp, P-glycoprotein
Translocase
Phospholipid Transfer Proteins
lipid A
Phospholipids
ATP-binding-cassette (ABC) superfamily
DLS, dynamic light scattering
0303 health sciences
Escherichia coli Proteins
030302 biochemistry & molecular biology
PS, phosphatidylserine
ReLPS, deep rough chemotype LPS
AlFx, aluminium fluoride
NBD, 7-nitrobenz-2-oxa-1,3-diazole
Vi, sodium orthovanadate
LPS, lipopolysaccharide
lipids (amino acids, peptides, and proteins)
NBD–LacCer, NBD–C6-lactosylceramide
Research Article
DM, n-dodecyl-β-D-maltoside
Phospholipid
OG, octyl-β-D-glucopyranoside
Biology
PE, phosphatidylethanolamine
03 medical and health sciences
Bacterial Proteins
Escherichia coli
medicine
MsbA
Molecular Biology
030304 developmental biology
Phosphatidylethanolamine
SM, sphingomyelin
lipid flippase
NB, nucleotide-binding
Cell Biology
Flippase
BeFx, beryllium fluoride
RaLPS, LPS from the Ra mutant of Escherichia coli
Enzyme Activation
chemistry
Membrane protein
Ni-NTA, Ni2+-nitrilotriacetate
phosphatidylethanolamine
reconstitution
biology.protein
ATP-Binding Cassette Transporters
AMP-PNP, adenosine 5′-[β,γ-imido]triphosphate
TM, transmembrane
Subjects
Details
- ISSN :
- 14708728 and 02646021
- Volume :
- 429
- Database :
- OpenAIRE
- Journal :
- Biochemical Journal
- Accession number :
- edsair.doi.dedup.....7c318af1ba8a2bc1367e2eb6f558a3b3
- Full Text :
- https://doi.org/10.1042/bj20100144