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.     

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.     

Effect of the delta subunit on assembly and proton permeability of the F0 proton channel of Escherichia coli F1F0 ATPase.

During the assembly of the Escherichia coli proton-translocating ATPase, the subunits of F1 interact with F0 to increase the proton permeability of the transmembrane proton channel. We tested the involvement of the delta subunit in this process by partially and completely deleting uncH (delta subunit) from a plasmid carrying the genes for the F0 subunits and delta and testing the effects of those F0 plasmids on the growth of unc+ and unc mutant E. coli strains. We found that the delta subunit was required for inhibition of growth of unc+ cells. We also tested membranes isolated from unc-deleted cells containing F0 plasmids for F1-binding ability. In unc-deleted cells, these plasmids produced F0 in amounts comparable to those found in normal unc+ E. coli cells, while having only small effects on cell growth. These studies demonstrate that the delta subunit plays an important role in opening the F0 proton channel but that it does not serve as a temporary plug of F0 during assembly, as had been previously speculated (S. Pati and W. S. A. Brusilow, J. Biol. Chem. 264:2640-2644, 1989).     

Effect of an uncE ribosome-binding site mutation on the synthesis and assembly of the Escherichia coli proton-translocating ATPase

Plasmid pRPG54, which carries the genes for the eight subunits of the proton-translocating ATPase of Escherichia coli, has been found to carry a single base change of a G to an A in the ribosome-binding site for uncE, the gene which codes for the N,N'-dicyclohexylcarbodiimide-binding subunit c of the Fo. This noncoding region mutation both lowers expression of uncE by a factor of 2-3 and affects the function of the ATPase, specifically of the Fo sector. The presence of the mutation results in a decrease in the proton permeability of the Fo or of the entire F1Fo-ATPase complex when either is synthesized from genes on a multicopy plasmid. Expression of uncE from an F1Fo plasmid carrying the wild type ribosome binding site results in increased membrane proton permeability and decreased ability of the resultant ATPase to couple a transmembrane proton gradient to ATP synthesis both in vitro and in vivo. Also, although an Fo plasmid carrying the correct ribosome-binding site causes harmful, F1-dependent proton permeability in unc+ cells (Brusilow, W. S. S. (1987) J. Bacteriol. 169, 4984-4990), an identical plasmid carrying the mutation does not, even though it still codes for a functional reconstitutable Fo. The results show a relationship between the relative level of expression of uncE from a multicopy plasmid and the assembly pathway, proton permeability, and energy-coupling characteristics of the ATPase.     

Purification and reconstitution of active chloroplast F0

Chloroplast F0 (CF0) was purified from the ATP synthase by Zwittergent 3-12 treatment and DEAE-Trisacryl anion exchange chromatography. Purified CF0 contains four subunits corresponding to subunits I, II, III, and IV. CF0 mediated proton translocation across the membrane after incorporation into asolectin liposomes. The CF0-mediated proton transport was inhibited by N,N'-dicyclohexylcarbodiimide and the binding of chloroplast coupling factor 1 (CF1). Rebinding of CF1 to CF0 liposomes resulted in reconstitution of N,N'-dicyclohexylcarbodiimide and uncoupler sensitive energy-transducing activities. Like CF0 in native thylakoid membranes, purified CF0 bound CF1 as well as CF1 deficient in either the delta or epsilon subunits.     

Assembly of the F0 proton channel of the Escherichia coli F1F0 ATPase: low proton conductance of reconstituted Fo sectors synthesized and assembled in the absence of F1.

We have previously proposed that during assembly of the Escherichia coli F1F0 ATPase, the proton permeability of the Fo sector of the E. coli F1F0 ATPase is increased significantly by interactions with F1 subunits [Pati, S., & Brusilow, W.S.A. (1989) J. Biol. Chem 264, 2640-2644]. To test this model for Fo assembly, we purified F0 sectors synthesized in the presence and absence of F1 subunits and measured the abilities of these different preparations to bind purified F1 ATPase and to conduct protons when reconstituted into liposomes. The results of these studies demonstrated significant differences in proton-conducting abilities of the different Fo preparations. Fo sectors synthesized in the presence of F1 subunits were more permeable to protons than those synthesized in the absence of F1 subunits.     

Impaired proton conductivity resulting from mutations in the a subunit of F1F0 ATPase in Escherichia coli

Mutations in the uncB gene which encodes the a subunit of F1F0-ATPase in Escherichia coli were isolated and characterized. Eight mutations caused premature polypeptide chain termination. Two mutations were single amino acid substitutions resulting in the replacements of serine 206 with leucine (ser-206----leu) and histidine 245 with tyrosine (his-245----tyr). The ser-206----leu mutation does not alter F1 binding and allows ATP driven membrane energization at a low level. Stripping of F1 from membranes containing the ser-206----leu mutation does not render the membranes permeable to protons indicating impaired proton conductivity. The his-245----tyr mutation also blocks Fo-mediated proton conduction but has normal F1 binding properties. F1 bound to membranes with both ser-206----leu and his-245----tyr mutant a subunits is sensitive to dicyclohexylcarbodiimide. Apparently, both missense mutations impair proton conduction without altering assembly of the F1F0-ATPase complex. The direct involvement of the a subunit in proton translocation is discussed.     

Proton leakiness caused by cloned genes for the F0 sector of the proton-translocating ATPase of Escherichia coli: requirement for F1 genes.

To study expression of uncG, the gene coding for the gamma subunit of the Escherichia coli proton-translocating ATPase, deletions were made in the intergenic region between uncA, the gene coding for the alpha subunit, and uncG. Two deletions which fused uncA and uncG coded for alpha-gamma fusion polypeptides which were synthesized well both in vitro and in vivo, demonstrating that uncG expression is normally controlled by nucleotides in the intergenic region. Multicopy plasmids carrying these fusion genes and the genes for the other subunits of the ATPase had a harmful effect on the growth of E. coli. The effect was overcome by N,N'-dicyclohexylcarbodiimide, indicating that the cells probably leaked protons. The deleterious effect was eliminated by making a nonpolar deletion in the upstream F0 gene uncB, or by cloning each of the uncA-uncG fusion genes onto a separate plasmid, removed from the F0 genes, thus demonstrating that the fusion genes were not primarily responsible for the proton permeability. A plasmid which carried F0 genes and the gene for the delta subunit caused deleterious proton leakiness in unc+ cells but not in cells from which the unc operon was deleted. The proton leakiness caused by these different plasmids was therefore due to the production of a leaky F0 proton channel and required the presence of F1 genes. The results support a model for ATPase assembly in which F1 genes or polypeptides are involved in the formation or opening of the F0 proton channel.     

Assembly of a functional F0 of the proton-translocating ATPase of Escherichia coli

We have investigated both structural and functional assembly of the F0 portion of the Escherichia coli proton-translocating ATPase in vivo. Fractionation of E. coli minicells containing plasmids which code for parts of the unc operon shows that each of the F0 peptides a, b, and c insert into the cytoplasmic membrane independent of each other and without the polypeptides which form the F1 portion of the complex alpha, beta, gamma, delta, and epsilon. Assays of membrane energization indicate that, while formation of a functional proton channel requires the presence of all three F0 polypeptides a, b and c, they are not sufficient. Synthesis of both the alpha and beta subunits of the F1 are required for formation of a functional proton channel.     

Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits.

A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed.     

F0F1-ATPase from Vibrio alginolyticus. Subunit composition and proton pumping activity.

An F0F1-ATPase was isolated from the membranes of the marine bacterium Vibrio alginolyticus. Homology between the subunits of the F0-complexes from E. coli and V. alginolyticus was found using antibodies against subunits a, b and c of the E. coli F0F1-ATPase. The F0F1-complex from V. alginolyticus was reconstituted into proteoliposomes, which were competent in ATP-dependent proton uptake. This process was inhibited by triphenyltin, DCCD, and venturicidin. Na+ did not affect proton translocation.     

Novel streptococcal mutants defective in the regulation of H+-ATPase biosynthesis and in F0 complex

In Streptococcus faecalis (faecium), the cytoplasmic pH is regulated by proton extrusion via a proton translocating F1F0-ATPase; the level of this enzyme increases in response to cytoplasmic acidification (Kobayashi, H., Suzuki, T., and Unemoto, T. (1986) J. Biol. Chem. 261, 627-630). We describe here two novel acid-sensitive mutants, designated AS8 and AS17, that contain ATPase activity but fail to grow on acid media. Our data suggested that in mutant AS17, acidification of the cytoplasm stimulates synthesis of the F0 sector of the ATPase but not the F1 sector. The accumulation in the plasma membrane of F0 sectors devoid of F1 results in enhanced proton permeability, and as a consequence mutant AS17 is unable to regulate the cytoplasmic pH in acid media. The genetic defect may reside in a gene that regulates expression of the F1F0-ATPase. Mutant AS8 does not generate a proton motive force. Our results suggest that the F1F0-ATPase can hydrolyze ATP but fails to translocate protons due to a defect in one of the subunits of the F0 sector.     

The F1F0-ATPase of Escherichia coli. The substitution of alanine by threonine at position 25 in the c-subunit affects function but not assembly

A mutant strain of Escherichia coli carrying a mutation in the uncE gene which codes for the c-subunit of the F1F0-ATPase has been isolated and examined. The mutant allele, designated uncE513, results in alanine at position 25 of the c-subunit being replaced by threonine. The mutant F1F0-ATPase appears to be fully assembled and is partially functional with respect to oxidative phosphorylation. The ATPase activity of membranes from the mutant strain is resistant to the inhibitor dicyclohexylcarbodiimide, but this is due to the F1-ATPase being lost from the membranes in the presence of the inhibitor. Mutant membranes from which the F1-ATPase has been removed have a greatly reduced proton permeability compared with similarly treated normal membranes. The results are discussed in relation to a previously proposed mechanism of oxidative phosphorylation.     

Mitochondrial F0F1 H+-ATP synthase. Characterization of F0 components involved in H+ translocation

The membrane F0 sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F1-depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin-sensitive proton conduction, or recombined with purified F1 to give the oligomycin-sensitive F0F1-ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDS-polyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F0 polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD-binding protein, the nuclear encoded protein [(1987) J. Mol. Biol. 197, 89-100], characterized as an intrinsic component of F0 (F0I, PVP protein [(1988) FEBS Lett. 237,9-14]) [corrected] is involved in H+ translocation and the sensitivity of this process to the F0 inhibitors, DCCD and oligomycin.     

Structural interpretations of F(0) rotary function in the Escherichia coli F(1)F(0) ATP synthase

F(1)F(0) ATP synthases are known to synthesize ATP by rotary catalysis in the F(1) sector of the enzyme. Proton translocation through the F(0) membrane sector is now proposed to drive rotation of an oligomer of c subunits, which in turn drives rotation of subunit gamma in F(1). The primary emphasis of this review will be on recent work from our laboratory on the structural organization of F(0), which proves to be consistent with the concept of a c(12) oligomeric rotor. From the NMR structure of subunit c and cross-linking studies, we can now suggest a detailed model for the organization of the c(12) oligomer in F(0) and some of the transmembrane interactions with subunits a and b. The structural model indicates that the H(+)-carrying carboxyl of subunit c is located between subunits of the c(12) oligomer and that two c subunits pack in a front-to-back manner to form the proton (cation) binding site. The proton carrying Asp61 side chain is occluded between subunits and access to it, for protonation and deprotonation via alternate entrance and exit half-channels, requires a swiveled opening of the packed c subunits and stepwise association with different transmembrane helices of subunit a. We suggest how some of the structural information can be incorporated into models of rotary movement of the c(12) oligomer during coupled synthesis of ATP in the F(1) portion of the molecule.     

The proton pore in the Escherichia coli F0F1-ATPase: substitution of glutamate by glutamine at position 219 of the alpha-subunit prevents F0-mediated proton permeability

Three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli were produced by site-directed mutagenesis. These mutations directed the substitution of Glu-219 by Gln, or of Lys-203 by Ile, or of Glu-196 by Ala. Strains carrying either the Lys-203 or Glu-196 substitutions showed growth characteristics indistinguishable from the coupled control strain. Properties of membrane preparations from these strains were also similar to those from the coupled control strain. The substitution of Glu-219 by Gln resulted in a strain which was unable to utilise succinate as sole carbon source and had a growth-yield characteristic of an uncoupled strain. Membrane preparations of the Glu-219 mutant were proton impermeable and the F1-ATPase activity was inhibited by about 50% when membrane-bound. The results are discussed with reference to a previously proposed intramembranous proton pore involving subunits a and c.     

Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit

The subunit arrangement of the F0 sector of the Escherichia coli ATP synthase is examined using hydrophilic and hydrophobic (cleavable) cross-linking reagents and the water-soluble labeling reagent [35S] diazoniumbenzenesulfonate ( [35S]DABS). Cross-linking is performed on purified ATP synthase and inverted minicell membranes. ATP synthase incorporated into liposomes is labeled with [35S]DABS. Three cross-linked products involving the F0 subunits (a, b, and c) are observed with the purified ATP synthase in solution: a-b, b2, and c2 dimers. A cross-link between the F0 and F1 is detected and occurs between the a and beta subunits. A cross-linker independent association between the b and beta subunits is also evident, suggesting that the two subunits are close enough to form a disulfide bridge. A cross-linking reagent stable to reducing agents produces a b-beta dimer, as detected by immunoblotting with anti-beta serum. The c subunit does not cross-link with any F1 polypeptide. Minicell membranes containing ATP synthase polypeptides radioactively labeled in vivo similarly show b2 and c2 dimers after cross-linking. [35S]DABS labels the a and b, but not c, subunits, showing that the a and b, but not c, subunits possess hydrophilic domains. Thus, certain domains of subunits a and b extend from the membrane and are in close proximity to one another and the F1 catalytic subunit beta.