Characterization of reconstituted Fo from wild-type Escherichia coli and identification of two other fluxes co-purifying with Fo.
We purified the ATPase Fo 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 Fo activity depends on both lipids and reconstitution methods. In asolectin vesicles, Fo has a single-channel conductance of about 0.2 fS. Additionally, the relatively impure Fo 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 (Hs). Unlike the Fo flux, these fluxes could not be blocked by the Fo inhibitor DCCD. The C, 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 Fo preparations displayed a 120 pS cation channel with characteristics consistent with C(L) flux. The Hs conducts only H+ but at a slower rate than the Fo. We were therefore able to: 1) quantitate the single-channel conductance of the Fo, 2) demonstrate that our Fo purification method co-purified other membrane proteins that have ion-conduction properties, and 3) show that certain lipids are necessary for functional reconstitution of Fo.
Cao
N J
NJ
Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Brusilow
W S
WS
Tomashek
J J
JJ
Woodbury
D J
DJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006859:Hydrogen; D008051:Lipid Bilayers; D008055:Lipids / Q000737:chemistry; D016012:Poisson Distribution; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D011522:Protons; D013997:Time Factors
D008051:Lipid Bilayers; D008055:Lipids; D011522:Protons; D006859:Hydrogen; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases