Journal
CELL BIOCHEMISTRY AND BIOPHYSICS
Volume 34, Issue 3, Pages 305-320Publisher
HUMANA PRESS INC
DOI: 10.1385/CBB:34:3:305
Keywords
F1Fo ATPase; reconstitution; H+ channel; proton channel; lipids; bilayer
Funding
- NIGMS NIH HHS [GM54108] Funding Source: Medline
- NIMH NIH HHS [MH50003] Funding Source: Medline
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM054108] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF MENTAL HEALTH [R01MH050003, R29MH050003] Funding Source: NIH RePORTER
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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.
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