Abstract
Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain.
Highlights
We argue that the effect is attributed to proton exchange between the proton pump (bo[3] oxidase) and the ATP synthase along the membrane surface
In addition to being regulated by the lipid composition, such interactions may be regulated through up- and down-regulation of small proteins that mediate more specific interactions between the components of the respiratory chain[49]
Summary
F1FO ATP synthase was expressed from plasmid pBWU13-β His in E. coli strain DK8 and purified as described[39]. Quinol-type bo[3] oxidase was expressed from plasmid pETcyo in E. coli strain C43 and purified as described (see[50] [51]). The lipid stock solutions were mixed at different ratios (see Figure legends) and the chloroform was evaporated under nitrogen followed by vacuum evaporation. The lipid mixture was re-suspended at a 10 mg/ml lipid concentration in a buffer containing 20 mM HEPES pH 7.5, 2.5 mM MgSO4, 50 g/l sucrose and subjected to at least 6 freeze-thaw cycles (1 min in liquid nitrogen, at 30 °C until thawed followed by 30 s vortexing). Liposomes were formed by extrusion (> 20 times) of the mixture through 100 nm or 200 nm Nuclepore membranes (Whatman Ltd)
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