Role of the delta subunit in enhancing proton conduction through the F0 of the Escherichia coli F1F0 ATPase.

We studied the effect of the delta subunit of the Escherichia coli F1 ATPase on the proton permeability of the F0 proton channel synthesized and assembled in vivo. Membranes isolated from an unc deletion strain carrying a plasmid containing the genes for the F0 subunits and the delta subunit were significantly more permeable to protons than membranes isolated from the same strain carrying a plasmid containing the genes for the F0 subunits alone. This increased proton permeability could be blocked by treatment with either dicyclohexyl-carbodiimide or purified F1, both of which block proton conduction through the F0. After reconstitution with purified F1 in vitro, both membrane preparations could couple proton pumping to ATP hydrolysis. These results demonstrate that an interaction between the delta subunit and the F0 during synthesis and assembly produces a significant change in the proton permeability of the F0 proton channel.

Synthesis and assembly of the F0 proton channel from F0 genes cloned into bacteriophage lambda and integrated into the Escherichia coli chromosome.

The promoter region and the first four genes of the Escherichia coli proton-translocating ATPase (unc) operon, uncIBEF, were cloned into bacteriophage lambda, enabling this region to be recombined into an unc-deleted E. coli chromosome at the lambda att site. The resultant E. coli strain, carrying single-copy F0 genes, was tested for synthesis and assembly of functional F0 proton channels. Membranes isolated from this strain contained all three F0 subunits and were capable of binding purified F1 and reconstituting F1F0-dependent energy coupling activities. The presence of these F0 sectors did not affect cell growth or membrane proton permeability assayed by fluorescence quenching. When compared with wild type membranes, membranes from the single-copy F0 strain contained less a and b subunits. When the single-copy lambda F0 strain was transformed with an F1 plasmid, the cells became phenotypically and biochemically Unc+, with membrane-bound ATPase and ATP synthase activities that were 50-60% of wild type. The results demonstrate that F0 produced from single-copy genes in the absence of F1 is membrane-bound and functional (i.e. reconstitutable) but not freely permeable to protons. The presence of F1 genes and/or subunits during F0 synthesis and assembly both increases the relative amounts of membrane-bound a and b subunits and produces an F0 sector more like that found in wild type cells than is produced from the single-copy F0 genes alone.

Effects of inducing expression of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase.

To evaluate whether expression of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase is sufficient to cause membrane proton permeability, plasmids carrying different combinations of the uncB, E, and F genes, encoding the a, c, and b subunits of the F0 sector, cloned behind the inducible lac promoter in pUC9 or pUC18, were constructed. The effects of inducing F0 synthesis in an unc deletion strain were monitored by measuring cell growth rate, quantitating F0 subunits by immunoblotting, and measuring the ability of membranes to maintain a respiration-induced proton gradient and to bind F1 and carry out energy-coupling reactions. The levels of functional reconstitutable F0 in membranes could be increased four- to sixfold with no change in cellular growth rate or membrane proton permeability (assayed by fluorescence quenching). These results were obtained in uninduced cultures, so the F0 genes were presumably being transcribed from some promoter besides lac. Induction of transcription of all three F0 genes produced increased amounts of F0 subunits in membranes as determined by immunoblot and F1-binding assays, but, when reconstituted with F1, the F0 in membranes isolated from induced cultures was significantly less functional than the F0 in membranes isolated from uninduced cultures. Such induction did result in growth inhibition, but there was no correlation between growth inhibition and either increased membrane proton permeability or the presence of functional, reconstitutable F0.

Molecular analysis of an anion pump: purification of the ArsC protein.

The ars operon of resistance plasmid R773 encodes an anion-translocating ATPase which catalyzes extrusion of the oxyanions arsenite, antimonite, and arsenate, thus providing resistance to the toxic compounds. Although both arsenite and arsenate contain arsenic, they have different chemical properties. In the absence of the arsC gene the pump transports arsenite and antimonite, oxyanions with the +III oxidation state of arsenic or antimony. The complex neither transports nor provides resistance to arsenate, the oxyanion of the +V oxidation state of arsenic. The arsC gene encodes a 16-kDa polypeptide, the ArsC protein, which alters the substrate specificity of the pump to allow for recognition and transport of the alternate substrate arsenate. The arsC gene was cloned behind a strong promoter and expressed at high levels. The ArsC protein was purified and crystallized.