A functional His-tagged c subunit of the Escherichia coli F-type ATPase/Synthase.

The c subunit of the Escherichia coli F0 has been tagged with a hexahistidine motif at its C-terminus. The tagged subunit is capable of forming functional F0 complexes that translocate protons in the absence of the F1 complex. In the presence of F1, the two sectors associate and display all biochemical activities of the wildtype enzyme: DCCD-inhibitable ATPase activity, ATP synthase activity, and ATP-dependent proton pumping. The enzyme can be solubilized and purified as an intact complex under native conditions on immobilized-metal affinity chromatography (IMAC) resin. The purified complex can be reincorporated into liposomes and demonstrates ATP-dependent proton pumping activity. Hexahistine tags placed at the N-terminus, in contrast, were all inactive. These experiments demonstrate the feasibility of tagging the c subunit for further studies of the F0 and suggest an important role for the N-terminus of the c subunit in either assembly or function of the protein.

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.

Stoichiometry of energy coupling by proton-translocating ATPases: a history of variability.

One of the central energy-coupling reactions in living systems is the intraconversion of ATP with a transmembrane proton gradient, carried out by proton-translocating F- and V-type ATPases/synthases. These reversible enzymes can hydrolyze ATP and pump protons, or can use the energy of a transmembrane proton gradient to synthesize ATP from ADP and inorganic phosphate. The stoichiometry of these processes (H(+)/ATP, or coupling ratio) has been studied in many systems for many years, with no universally agreed upon solution. Recent discoveries concerning the structure of the ATPases, their assembly and the stoichiometry of their numerous subunits, particularly the proton-carrying proteolipid (subunit c) of the F(O) and V(0) sectors, have shed new light on this question and raise the possibility of variable coupling ratios modulated by variable proteolipid stoichiometries.

Identification of an intragenic ribosome binding site that affects expression of the uncB gene of the Escherichia coli proton-translocating ATPase (unc) operon.

The uncB gene codes for the a subunit of the Fo proton channel sector of the Escherichia coli F1 Fo ATPase. Control of expression of uncB appears to be exerted at some step after translational initiation. Sequence analysis by the perceptron matrices (G. D. Stormo, T. D. Schneider, L. Gold, and A. Ehrenfeucht, Nucleic Acids Res. 10:2997-3011, 1982) identified a potential ribosome binding site within the uncB reading frame preceding a five-codon reading frame which is shifted one base relative to the uncB reading frame. Elimination of this binding site by mutagenesis resulted in a four- to fivefold increase in expression of an uncB'-'lacZ fusion gene containing most of uncB. Primer extension inhibition (toeprint) analysis to measure ribosome binding demonstrated that ribosomes could form an initiation complex at this alternative start site. Two fusions of lacZ to the alternative reading frame demonstrated that this site is recognized by ribosomes in vivo. The results suggest that expression of uncB is reduced by translational frameshifting and/or a translational false start at this site within the uncB reading frame.

Effects of carbon source on expression of F0 genes and on the stoichiometry of the c subunit in the F1F0 ATPase of Escherichia coli.

Expression of the genes for the membrane-bound F0 sector of the Escherichia coli F1F0 proton-translocating ATPase can respond to changes in metabolic conditions, and these changes are reflected in alterations in the subunit stoichiometry of the oligomeric F0 proton channel. Transcriptional and translational lacZ fusions to the promoter and to two F0 genes show that, during growth on the nonfermentable carbon source succinate, transcription of the operon and translation of uncB, encoding the a subunit of F0, are higher than during growth on glucose. In contrast, translation of the uncE gene, encoding the c subunit of F0, is higher during growth on glucose than during growth on succinate. Translation rates of both uncB and uncE change as culture density increases, but transcription rates do not. Quantitation of the c stoichiometry shows that more c subunits are assembled into the F1F0 ATPase in cells grown on glucose than in cells grown on succinate. E. coli therefore appears to have a mechanism for regulating the composition and, presumably, the function of the ATPase in response to metabolic circumstances.

Anabolic steroids induce injury and apoptosis of differentiated skeletal muscle.

Apoptosis is an active form of cellular death, or suicide, which plays an important physiologic role during organ development and in cellular turnover in differentiated tissues. Apoptosis has also been demonstrated to occur in several organs in response to hypoxic/ischemic, oxidative, or drug-induced injury and is thus involved in disease pathogenesis. However, it is generally assumed that apoptosis does not occur in differentiated skeletal muscle. Apoptosis has been demonstrated in differentiated myocardial muscle, neonatal skeletal muscle, and skeletal myoblasts in response to injury. We therefore studied differentiated murine C2 skeletal muscle cells that have been injured by supraphysiologic doses (>10 microM) of an anabolic steroid, stanozolol. Stanozolol-injured muscle cells exhibited pathologic features suggestive of apoptosis: cytoplasmic shrinkage and chromatin condensation. Muscle cells also showed positive in situ nick-end labeling of nuclear chromatin, indicating DNA strand breakage. Staining with the DNA-binding dye 33342 (bisbenzimide) also showed chromatin changes characteristic of apoptotic nuclei. Total protein levels measured at 4 and 24 hr post-stanozolol injury was not significantly decreased, indicating absence of cell lysis. Cellular ATP levels (nmol ATP/mg protein) of stanozolol-injured muscle cells, measured 4 and 24 hr postinjury, also did not change significantly. In contrast, necrotic muscle cells, injured by the calcium ionophore A23187 (2 microM), showed a progressive decline in total protein and ATP levels. This study supports two other histologic studies that showed evidence of apoptosis in differentiated skeletal muscle fibers. Our data further suggest that during the early stages of apoptosis, but not necrosis, cellular energy metabolism is preserved.

Localization of a conformational energy-coupling determinant near the C terminus of the beta subunit of the F1F0-ATPase.

Escherichia coli mutants in the beta subunit of the F1F0-ATPase can be complemented with the beta subunit from the obligate aerobe Bacillus megaterium. It has been shown that cells carrying such hybrid ATPases have an unusual energy-coupling phenotype. Although they are able to grow on minimal succinate medium, and therefore carry a functional ATP synthase, they are defective in the ability to grow anaerobically, indicating some defect in ATP-driven proton pumping (Scarpetta, M., Hawthorne, C. A., and Brusilow, W. S. A. (1991) J. Biol. Chem. 266, 18567-18572). In this study, chimeric beta subunits were constructed consisting of the E. coli or the B. megaterium beta subunit carrying the C-terminal 18% of the other's beta subunit. The phenotypes of an E. coli beta mutant complemented with these chimeric subunits showed that the energy-coupling defect was located in this C-terminal region. The E. coli beta subunit carrying the B. megaterium C-terminal region displayed the energy-coupling defect, while the B. megaterium beta subunit carrying the E. coli C-terminal region did not. In ATP-dependent fluorescence quenching assays, membranes isolated from cells displaying the energy-coupling defect also pumped protons less well than membranes isolated from cells that were able to grow anaerobically. These results demonstrate that the C terminus of the beta subunit is involved in the conformational coupling pathway, which, through the polypeptide backbone of the beta subunit, physically links ATP synthesis or hydrolysis to the energy of proton translocation.

The effects of an atpE ribosome-binding site mutation on the stoichiometry of the c subunit in the F1F0 ATPase of Escherichia coli.

We tested the hypothesis that the stoichiometry of the c subunit in the F0 sector of the Escherichia coli F1F0 ATPase is dependent upon the level of atpE gene expression. F0 was purified from cells carrying plasmids encoding the F0 subunits with and without a ribosome-binding site mutation preceding atpE, the gene which codes for the c subunit. Subunit-specific antibodies were used to quantitate the relative amounts of the b and c subunits. The decreased expression of atpE resulted in a significantly decreased amount of the c subunit in the purified F0. Immunoblot quantitation of the amounts of b and c subunits in F1F0 precipitated by anti-F1 antiserum also showed that the mutation produced significant differences in the stoichiometry of subunit c. The amount of c subunit assembled into the F1F0 synthesized from a plasmid carrying the atpE ribosome binding site mutation was 2-5 times less than the amount found in the F1F0 synthesized from a wild-type plasmid. Therefore, the stoichiometry of the c subunit assembled into the F1F0 complex appears to be variable, depending on the expression of atpE.

Effects of the uncI gene on expression of uncB, the gene coding for the a subunit of the F1F0 ATPase of Escherichia coli.

The eight genes coding for the subunits of the E. coli F1F0 ATPase are preceded by a gene, designated uncI. A homologous gene, or a gene coding for an analagous protein, is found preceding the ATPase genes of several microorganisms. No function for the 1 gene has been described. Using lac fusions to measure gene expression in vivo, we tested the effects of deleting uncI on the expression of the adjacent gene uncB, which codes for the a subunit of the F0 sector of the ATPase. Deleting uncI reduced the expression of three uncB'-'lacZ fusion genes in vivo, but had no effect on the expression of two uncB'-'lacZ fusion genes containing a relatively smaller amount of the uncB coding region. The uncI deletion also reduced the relative synthesis of the a subunit in vitro. The I gene therefore appears to specifically affect the expression of uncB or the synthesis of the a subunit at some step after translational initiation of uncB.

Construction and function of chimeric beta subunits containing regions from the beta subunits of the F1F0 ATPases of Escherichia coli and Bacillus megaterium.

The highly conserved beta subunit of the Escherichia coli F1F0 ATPase was divided into three sections, each of which was exchanged with the homologous section of the beta subunit of the obligate aerobe Bacillus megaterium. Plasmids coding for the resultant six chimeric beta subunits varied in their abilities to complement two E. coli beta mutants as measured by testing transformed cells for aerobic growth on a nonfermentable carbon source or anaerobic growth on rich medium containing glucose. Two chimeras were able to restore both growth on succinate and anaerobic growth on rich medium. The genetic results corresponded to increased levels of membrane-bound ATPase and ATP synthase activities. These chimeric subunits were therefore capable of being assembled into functional E. coli ATPase complexes. The results indicate that chimeric beta subunits can be used to analyze assembly of the beta subunit and that the final 181 amino acids of the beta subunit might contain a region involved in functional energy coupling.

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 deletions in the uncA-uncG intergenic regions on expression of uncG, the gene for the gamma subunit of the Escherichia coli F1Fo-ATPase.

The gamma subunit of the E. coli F1Fo-ATPase is coded for by uncG. This gene is poorly expressed compared to uncA (alpha subunit), which precedes uncG in the unc operon. The genes are separated by a 50-nucleotide intergenic region. We examined the effects of a set of deletions in this region on the relative expression of uncA'-'lacZ and uncG'-'lacZ translational fusion genes located either in the chromosomal unc operon or at the chromosomal lambda att site. The gene for the alpha subunit was expressed 3-6-times better than the gene for the gamma subunit, depending upon chromosomal location. Deletion analysis revealed that the uncA-uncG intergenic region significantly affects expression of uncG, but the Shine-Dalgarno region is not absolutely required for expression of uncG. Different deletions resulted in either increased or decreased expression of uncG.

Assembly of the Escherichia coli F1F0 ATPase, a large multimeric membrane-bound enzyme.

The F1F0 proton translocating ATPase of Escherichia coli is a large membrane-bound enzyme complex consisting of more than 20 polypeptides that are encoded by the unc operon. Besides being a system for analysing the enzymology of ATP synthesis and energy coupling, the ATPase is a model system for determining how large oligomeric membrane-bound proteins are synthesized and assembled. The assembly of the ATPase involves differential gene expression and assembly of the subunits within the membrane and with each other. This review discusses the influence of F1 subunits on the assembly and proton permeability of the F0 proton channel, and the possible advantages to assembly of the particular arrangement of genes in the unc operon.

Protein-lipid interactions of the proteolipid c subunit of the Escherichia coli proton-translocating adenosinetriphosphatase.

Interactions between Escherichia coli membrane phospholipids and the hydrophobic c subunit of the F1F0 proton-translocating ATPase were characterized. Extraction of E. coli membranes with a neutral mixture of chloroform and methanol and subsequent separation steps produced several protein-containing fractions. The protein-containing fraction most soluble in organic solvents contained subunit c and a lipid fraction enriched in phosphatidylglycerol compared to total E. coli membrane phospholipids. Other ATPase subunits and some additional proteins extracted from the membranes by this procedure could be separated from the c subunit by subsequent extraction. The purified and delipidated c subunit contained fatty acids which were released upon treatment with boron trifluoride methanol. Furthermore, deleting and restoring the genes for the F0 subunits changed the composition of extractable membrane phospholipid and fatty acids, indicating that the F0 plays a significant structural role in the membrane.

Regulation of the Escherichia coli uncH gene by mRNA secondary structure and translational coupling.

The uncH gene is one of the most poorly-expressed genes of the proton-translocating ATPase (unc) operon of Escherichia coli. We constructed in-frame lacZ fusions to uncH and used site-directed mutagenesis to decrease the stability of the putative mRNA secondary structure in the Shine and Dalgarno region for this gene. These mutations significantly increased the expression of uncH. We also used the unc-lac fusions to show that the insertion of stop codons and a frameshift mutation in uncF, the gene preceding uncH, caused a 10-fold reduction in uncH expression. Hybridization of total cellular RNA with a lacZ-specific probe indicated that transcriptional polarity could not account for the observed decrease in gene expression. These results demonstrate that uncH expression is controlled by mRNA sequences around the translational initiation region, and is translationally coupled to uncF, even in cases where the putative mRNA secondary structure is weakened or eliminated.

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.

Characterization of semi-uncoupled hybrid Escherichia coli-Bacillus megaterium F1F0 proton-translocating ATPases.

Cloned atp genes for the proton-translocating ATPase of the obligate aerobe Bacillus megaterium have been demonstrated to be capable of complementing Escherichia coli ATPase (unc) mutants (Hawthorne, C. A., and Brusilow, W. S. A. (1986) J. Biol. Chem. 261, 5245-5248). To determine the minimum subunit requirements for cross-species complementation, we constructed all combinations of B. megaterium atpA, G, D, and C genes (coding for the alpha, gamma, beta, and epsilon subunits, respectively) and tested their abilities to complement two uncA (alpha subunit) and two uncD (beta subunit) mutants of E. coli. The results indicated that complementation of either uncD mutant required atpD (beta) only. Complementation of one of the uncA (alpha) mutants required atpA, G, and D (alpha, gamma, and beta) and possibly atpE (epsilon) as well. The other uncA mutant was not complemented by any combination of B. megaterium ATPase genes. Complementation of a beta mutant by atpD (beta) or atpD and C (beta epsilon) produced cells which could grow aerobically on a nonfermentable carbon source (succinate) but not anaerobically on rich medium containing glucose. These E. coli therefore had become obligate aerobes. The ability to grow anaerobically could be restored to the mutant complemented by atpD alone by growth at pH 7.5 or pH 8 in the presence of 0.1 M potassium.

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.

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).