Synthesis of a functional F0 sector of the Escherichia coli H+-ATPase does not require synthesis of the alpha or beta subunits of F1.
The uncB, E, F, and H genes of the Escherichia coli unc operon were cloned behind the lac promoter of plasmid pUC9, generating plasmid pBP101. These unc loci code, respectively, for the chi, omega, and psi subunits of the F0 sector and the delta subunit of the F1 sector of the H+-ATP synthase complex. Induction of expression of the four unc genes by the addition of isopropyl-beta-D-thiogalactoside resulted in inhibition of growth. During isopropyl-beta-D-thiogalactoside induction, the three subunits of F0 were integrated into the cytoplasmic membrane with a resultant increase in H+ permeability. A functional F0 was formed from plasmid pBP101 in a genetic background lacking all eight of the unc structural genes coding the F1F0 complex. In the unc deletion background, a reasonable correlation was observed between the amount of F0 incorporated into the membrane and the function measured, i.e., high-affinity binding of F1 and rate of F0-mediated H+ translocation. This correlation indicates that most or all of the F0 assembled in the membrane is active. Although the F0 assembled under these conditions binds F1, only partial restoration of NADH-dependent or ATP-dependent quenching of quinacrine fluorescence was observed with these membranes. Proteolysis of a fraction of the psi subunit may account for this partial deficiency. The experiments described demonstrate that a functional F0 can be assembled in vivo in E. coli strains lacking genes for the alpha, beta, gamma, and epsilon subunits of F1.
Fillingame
R H
RH
Porter
B
B
Hermolin
J
J
White
L K
LK
D001426:Bacterial Proteins / Q000378:metabolism; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D009243:NAD / Q000494:pharmacology; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D009243:NAD; D006180:Proton-Translocating ATPases