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Lipid bilayer composition influences small multidrug transporters.

Charalambous K, Miller D, Curnow P, Booth PJ - BMC Biochem. (2008)

Bottom Line: Both EmrE and TBsmr are found to exhibit a similar dependence on lipid composition, with phosphatidylethanolamine increasing methyl viologen transport.These findings show that the physical state of the membrane modifies drug transport and that substrate translocation is dependent on in vitro lipid composition.Multidrug transport activity seems to respond to alterations in the lateral forces exerted upon the transport proteins by the bilayer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK. k.charalambous@mail.cryst.bbk.ac.uk

ABSTRACT

Background: Membrane proteins are influenced by their surrounding lipids. We investigate the effect of bilayer composition on the membrane transport activity of two members of the small multidrug resistance family; the Escherichia coli transporter, EmrE and the Mycobacterium tuberculosis, TBsmr. In particular we address the influence of phosphatidylethanolamine and anionic lipids on the activity of these multidrug transporters. Phosphatidylethanolamine lipids are native to the membranes of both transporters and also alter the lateral pressure profile of a lipid bilayer. Lipid bilayer lateral pressures affect membrane protein insertion, folding and activity and have been shown to influence reconstitution, topology and activity of membrane transport proteins.

Results: Both EmrE and TBsmr are found to exhibit a similar dependence on lipid composition, with phosphatidylethanolamine increasing methyl viologen transport. Anionic lipids also increase transport for both EmrE and TBsmr, with the proteins showing a preference for their most prevalent native anionic lipid headgroup; phosphatidylglycerol for EmrE and phosphatidylinositol for TBsmr.

Conclusion: These findings show that the physical state of the membrane modifies drug transport and that substrate translocation is dependent on in vitro lipid composition. Multidrug transport activity seems to respond to alterations in the lateral forces exerted upon the transport proteins by the bilayer.

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Related in: MedlinePlus

MV transport by EmrE. Radiolabelled, 14C MV2+ transport into E. coli lipid vesicles by EmrE over time. EmrE reconstituted into E. coli lipid vesicles extruded to (■), 50 nm; (○), 200 nm and (▲), sonicated lipid vesicles. Control data is shown for (□), absence of a pH gradient and (◆), absence of protein in 50 nm lipid vesicles. Errors are shown as first standard deviation of 3 measurements on different protein preparations (with each value used for each protein preparation, for each data point shown, also being the average of 3 measurements on that particular preparation).
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Figure 1: MV transport by EmrE. Radiolabelled, 14C MV2+ transport into E. coli lipid vesicles by EmrE over time. EmrE reconstituted into E. coli lipid vesicles extruded to (■), 50 nm; (○), 200 nm and (▲), sonicated lipid vesicles. Control data is shown for (□), absence of a pH gradient and (◆), absence of protein in 50 nm lipid vesicles. Errors are shown as first standard deviation of 3 measurements on different protein preparations (with each value used for each protein preparation, for each data point shown, also being the average of 3 measurements on that particular preparation).

Mentions: EmrE was reconstituted, via OG, into E. coli lipid vesicles that were initially prepared by sonication or extrustion to give differing sizes (50 and 200 nm in diameter). As shown in figure 1 increased assay reproducibility is observed when vesicles were formed by extrusion rather than sonication. In addition 50 nm diameter lipid vesicles were chosen over 200 nm lipid vesicles, since it was possible to determine both a linear regime of transport to determine an "initial rate of transport" as well as saturation of transport (by loss of pH gradient or substrate accumulation). The initial rate of MV transport by EmrE in 50 nm E. coli lipid vesicles was ~250 nmol. min-1. mg-1.


Lipid bilayer composition influences small multidrug transporters.

Charalambous K, Miller D, Curnow P, Booth PJ - BMC Biochem. (2008)

MV transport by EmrE. Radiolabelled, 14C MV2+ transport into E. coli lipid vesicles by EmrE over time. EmrE reconstituted into E. coli lipid vesicles extruded to (■), 50 nm; (○), 200 nm and (▲), sonicated lipid vesicles. Control data is shown for (□), absence of a pH gradient and (◆), absence of protein in 50 nm lipid vesicles. Errors are shown as first standard deviation of 3 measurements on different protein preparations (with each value used for each protein preparation, for each data point shown, also being the average of 3 measurements on that particular preparation).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2605743&req=5

Figure 1: MV transport by EmrE. Radiolabelled, 14C MV2+ transport into E. coli lipid vesicles by EmrE over time. EmrE reconstituted into E. coli lipid vesicles extruded to (■), 50 nm; (○), 200 nm and (▲), sonicated lipid vesicles. Control data is shown for (□), absence of a pH gradient and (◆), absence of protein in 50 nm lipid vesicles. Errors are shown as first standard deviation of 3 measurements on different protein preparations (with each value used for each protein preparation, for each data point shown, also being the average of 3 measurements on that particular preparation).
Mentions: EmrE was reconstituted, via OG, into E. coli lipid vesicles that were initially prepared by sonication or extrustion to give differing sizes (50 and 200 nm in diameter). As shown in figure 1 increased assay reproducibility is observed when vesicles were formed by extrusion rather than sonication. In addition 50 nm diameter lipid vesicles were chosen over 200 nm lipid vesicles, since it was possible to determine both a linear regime of transport to determine an "initial rate of transport" as well as saturation of transport (by loss of pH gradient or substrate accumulation). The initial rate of MV transport by EmrE in 50 nm E. coli lipid vesicles was ~250 nmol. min-1. mg-1.

Bottom Line: Both EmrE and TBsmr are found to exhibit a similar dependence on lipid composition, with phosphatidylethanolamine increasing methyl viologen transport.These findings show that the physical state of the membrane modifies drug transport and that substrate translocation is dependent on in vitro lipid composition.Multidrug transport activity seems to respond to alterations in the lateral forces exerted upon the transport proteins by the bilayer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK. k.charalambous@mail.cryst.bbk.ac.uk

ABSTRACT

Background: Membrane proteins are influenced by their surrounding lipids. We investigate the effect of bilayer composition on the membrane transport activity of two members of the small multidrug resistance family; the Escherichia coli transporter, EmrE and the Mycobacterium tuberculosis, TBsmr. In particular we address the influence of phosphatidylethanolamine and anionic lipids on the activity of these multidrug transporters. Phosphatidylethanolamine lipids are native to the membranes of both transporters and also alter the lateral pressure profile of a lipid bilayer. Lipid bilayer lateral pressures affect membrane protein insertion, folding and activity and have been shown to influence reconstitution, topology and activity of membrane transport proteins.

Results: Both EmrE and TBsmr are found to exhibit a similar dependence on lipid composition, with phosphatidylethanolamine increasing methyl viologen transport. Anionic lipids also increase transport for both EmrE and TBsmr, with the proteins showing a preference for their most prevalent native anionic lipid headgroup; phosphatidylglycerol for EmrE and phosphatidylinositol for TBsmr.

Conclusion: These findings show that the physical state of the membrane modifies drug transport and that substrate translocation is dependent on in vitro lipid composition. Multidrug transport activity seems to respond to alterations in the lateral forces exerted upon the transport proteins by the bilayer.

Show MeSH
Related in: MedlinePlus