Characterization of reconstituted F-o from wild-type Escherichia coli and identification of two other fluxes co-purifying with F-o

We purified the ATPase F-o sector from a nonoverexpressing strain of Escherichia coli, reconstituted it into lipid vesicles made of either asolectin or two different mixtures of purified lipids, and measured proton flux through the reconstituted proton channel. We measured single-channel conductances and found that F-o activity depends on both lipids and reconstitution methods. In asolectin vesicles, F-o has a single-channel conductance of about 0.2 fS. Additionally, the relatively impure F-o prepared from cells carrying single-copy ATPase genes allowed us to observe two other fluxes, a nonselective cation leak (C-L) and a slow H+ flux (H-s). Unlike the F-o flux, these fluxes could not be blocked by the F-o inhibitor DCCD. The C-L reduces the total apparent trapped volume inside vesicles and therefore must equilibrate both H+ and K+ in the vesicles that contain it. When reconstituted into bilayers, these F-o preparations displayed a 120 pS cation channel with characteristics consistent with C-L flux. The H-s conducts only H+ but at a slower rate than the F-o. We were therefore able to: 1) quantitate the single-channel conductance of the F-o, 2) demonstrate that our F-o purification method co-purified other membrane proteins that have ion-conduction properties, and 3) show that certain lipids are necessary for functional reconstitution of F-o.