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All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane-adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel-fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large-scale lipid phase separation and protein segregation in intact, protein-crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.

The cyanide-insensitive alternative oxidase (AOX) is a non-proton-pumping ubiquinol oxidase that catalyzes the reduction of oxygen to water and is posttranslationally regulated by redox mechanisms and 2-oxo acids. Arabidopsis (Arabidopsis thaliana) possesses five AOX isoforms (AOX1A-AOX1D and AOX2). AOX1D expression is increased in aox1a knockout mutants from Arabidopsis (especially after restriction of the cytochrome c pathway) but cannot compensate for the lack of AOX1A, suggesting a difference in the regulation of these isoforms. Therefore, we analyzed the different AOX isoenzymes with the aim to identify differences in their posttranslational regulation. Seven tricarboxylic acid cycle intermediates (citrate, isocitrate, 2-oxoglutarate, succinate, fumarate, malate, and oxaloacetate) were tested for their influence on AOX1A, AOX1C, and AOX1D wild-type protein activity using a refined in vitro system. AOX1C is insensitive to all seven organic acids, AOX1A and AOX1D are both activated by 2-oxoglutarate, but only AOX1A is additionally activated by oxaloacetate. Furthermore, AOX isoforms cannot be transformed to mimic one another by substituting the variable cysteine residues at position III in the protein. In summary, we show that AOX isoforms from Arabidopsis are differentially fine-regulated by tricarboxylic acid cycle metabolites (most likely depending on the amino-terminal region around the highly conserved cysteine residues known to be involved in regulation by the 2-oxo acids pyruvate and glyoxylate) and propose that this is the main reason why they cannot functionally compensate for each other.

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane-adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel-fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large-scale lipid phase separation and protein segregation in intact, protein-crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase-separation process induced by low fluidity is biologically reversible.

The ATP synthase (FOF1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c10γϵ and the stator ab2α3β3δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled FOF1 complexes by affinity chromatography with the use of mutants synthesizing different sets of FOF1 subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that FOF1 is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H+-translocating unit as intermediate product. Surprisingly, during the biogenesis of FOF1, F1 subunit δ is the key player in generating stable FO. Subunit δ serves as clamp between ab2 and c10α3β3γϵ and guarantees that the open H+ channel is concomitantly assembled within coupled FOF1 to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H+-translocating enzymes. Background: FOF1 ATP synthases are rotary nanomachines transporting protons across the membrane during catalysis. Results: Escherichia coli FOF1 is assembled from subcomplexes with subunit δ functioning as clamp to generate stable FO. Conclusion: δ guarantees that the open proton channel is concomitantly assembled within coupled FOF1. Significance: We investigate how a proton-translocating unit is assembled while simultaneously maintaining the low membrane proton permeability essential for viability.

Escherichia coli F O F 1 ATP synthase, a rotary nanomachine, is composed of eight different subunits in a α 3 β 3 γδe ab 2 c 10 stoichiometry. Whereas F O F 1 has been studied in detail with regard to its structure and function, much less is known about how this multisubunit enzyme complex is assembled. Single-subunit atp deletion mutants are known to be arrested in assembly, thus leading to formation of partially assembled subcomplexes. To determine whether those subcomplexes are preserved in a stable standby mode, a time-delayed in vivo assembly system was developed. To establish this approach, we targeted the time-delayed assembly of membrane-integrated subunit a into preformed F O F 1 lacking subunit a (F O F 1 - a ) which is known to form stable subcomplexes in vitro . Two expression systems ( araBADp and T7 p-laco ) were adjusted to provide compatible, mutually independent, and sufficiently stringent induction and repression regimens. In detail, all structural atp genes except atpB (encoding subunit a ) were expressed under the control of araBADp and induced by arabinose. Following synthesis of F O F 1 - a during growth, expression was repressed by glucose/d-fucose, and degradation of atp mRNA controlled by real-time reverse transcription-PCR. A time-delayed expression of atpB under T7 p-laco control was subsequently induced in trans by addition of isopropyl-β-d-thiogalactopyranoside. Formation of fully assembled, and functional, F O F 1 complexes was verified. This demonstrates that all subunits of F O F 1 - a remain in a stable preformed state capable to integrate subunit a as the last subunit. The results reveal that the approach presented here can be applied as a general method to study the assembly of heteromultimeric protein complexes in vivo .

Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein ‘insertase’ YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) — a supercomplex of SecYEG–SecDF–YajC–YidC — is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC.

Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap - a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using F\"orster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively., Comment: 9 pages, 3 figures

Adenosine triphosphate (ATP) is the universal chemical energy currency for cellular activities provided mainly by the membrane enzyme FoF1-ATP synthase in bacteria, chloroplasts and mitochondria. Synthesis of ATP is accompanied by subunit rotation within the enzyme. Over the past 15 years we have developed a variety of single-molecule FRET (smFRET) experiments to monitor catalytic action of individual bacterial enzymes in vitro. By specifically labeling rotating and static subunits within a single enzyme we were able to observe three-stepped rotation in the F1 motor, ten-stepped rotation in the Fo motor and transient elastic deformation of the connected rotor subunits. However, the spatial and temporal resolution of motor activities measured by smFRET were limited by the photophysics of the FRET fluorophores. Here we evaluate the novel FRET donor mNeonGreen as a fusion to FoF1-ATP synthase and compare it to the previously used fluorophore EGFP. Topics of this manuscript are the biochemical purification procedures and the activity measurements of the fully functional mutant enzyme., 14 pages, 7 figures

The subunit c stoichiometry of Escherichia coli ATP synthase was studied by intermolecular cross-linking via oxidation of bi-cysteine-substituted subunit c ( c A21C/ c M65C). Independent of the carbon source used for growth and independent of the presence of other F o F 1 subunits, an equal pattern of cross-link formation stopping at the formation of decamers was obtained.

FoF1-ATP synthases are membrane-embedded protein machines that catalyze the synthesis of adenosine triphosphate. Using photoactivation-based localization microscopy (PALM) in TIR-illumination as well as structured illumination microscopy (SIM), we explore the spatial distribution and track single FoF1-ATP synthases in living E. coli cells under physiological conditions at different temperatures. For quantitative diffusion analysis by mean-squared-displacement measurements, the limited size of the observation area in the membrane with its significant membrane curvature has to be considered. Therefore, we applied a 'sliding observation window' approach (M. Renz et al., Proc. SPIE 8225, 2012) and obtained the one-dimensional diffusion coefficient of FoF1-ATP synthase diffusing on the long axis in living E. coli cells., 13 pages, 5 figures

Subunit b of the Escherichia coli ATP synthase was isolated by preparative gel electrophoresis, acetone precipitated and after ion-pair extraction redissolved in a buffer either containing n-dodecyl-β-d-maltoside or sodium cholate. The secondary structure of isolated subunit b was shown to be the same as within the FO complex, but was strongly dependent on the detergent used for replacement of the phospholipid environment. This was shown by an identical tryptic digestion pattern, which was strongly influenced by the detergent used for solubilization. An influence of the detergent n-dodecyl-β-d-maltoside on the secondary structure of the hydrophilic part of subunit b was also shown for the soluble part of the polypeptide comprising residues Val25 to Leu156 (bsol) using CD spectroscopy. In order to determine the secondary structure of subunit b in its native conformation, isolated subunit b was reconstituted into E. coli lipid vesicles and analyzed with CD spectroscopy. The resulting spectrum revealed a secondary structure composition of 80% α helix together with 14% β turn conformation. These results suggest that subunit b is not a rigid rod-like α helix simply linking F1 to FO, but rather provides an inherent flexibility for the storage of elastic energy within the second stalk generated by rotational movements within the F1FO complex.

The membrane-bound ATP synthase (F1Fo) from mitochondria, chloroplasts and bacteria plays a crucial role in energy-transducing reactions. In the case of Escherichia coli, the reversible, proton-translocating ATPase complex consists of two different entities, F1 and Fo. The water-soluble F1 part carries the catalytic sites for ATP synthesis and hydrolysis. It is associated with the membrane-embedded Fo complex, which functions as a proton channel and consists of subunits a, b and c present in a stoichiometry of 1:2:12. Subunit b was isolated by preparative gel electrophoresis, acetone-precipitated and renatured in a cholate-containing buffer. Reconstituted subunit b together with purified ac subcomplex is active in proton translocation and F1 binding, thereby demonstrating that subunit b had recovered its native conformation. Circular dichroism spectroscopy of subunit b reconstituted into liposomes revealed a rather high degree of α -helical conformation of 80 %. After addition of a His6-tag to the N terminus of subunit a, a stable ab2 subcomplex was purified instead of a single subunit a, arguing in favour of a direct interaction between these subunits. After addition of subunit c and reconstitution into phospholipid vesicles, an Fo complex was obtained exhibiting rates of proton translocation and F1 binding comparable with those of wild-type Fo. The epitopes of monoclonal antibodies against subunit c are located in the hydrophilic loop region (cL31–Q42) as mapped by enzyme-linked immunosorbent assay using overlapping synthetic heptapeptides. Binding studies revealed that all monoclonal antibodies (mAbs) bind to everted membrane vesicles irrespective of the presence or absence of F1. Although the hydrophilic region of subunit c, and especially the highly conserved residues cA40, cR41, cQ42 and cP43, are known to interact with subunits γ and ε of the F1 part, the mAb molecules have no effect on the function of Fo, either in proton translocation or in F1 binding. However, the F1 part and the mAb molecule(s) are bound simultaneously to the Fo complex, suggesting that not all c subunits are involved in the interaction with F1.

The antigenic determinants of mAbs against subunit c of the Escherichia coli ATP synthase were mapped by ELISA using overlapping synthetic heptapeptides. All epitopes recognized are located in the hydrophilic loop region and are as follows: 31-LGGKFLE-37, 35-FLEGAAR-41, 36-LEGAAR-41 and 36-LEGAARQ-42. Binding studies with membrane vesicles of different orientation revealed that all mAbs bind to everted membrane vesicles independent of the presence or absence of the F1 part. Although the hydrophilic region of subunit c and particularly the highly conserved residues A40, R41, Q42 and P43 are known to interact with subunits gamma and epsilon of the F1 part, the mAb molecules have no effect on the function of F0. Furthermore, it could be demonstrated that the F1 part and the mAb molecule(s) are bound simultaneously to the F0 complex suggesting that not all c subunits are involved in F1 interaction. From the results obtained, it can be concluded that this interaction is fixed, which means that subunits gamma and epsilon do not switch between the c subunits during catalysis and furthermore, a complete rotation of the subunit c oligomer modified with mAb(s) along the stator of the F1F0 complex, proposed to be composed of at least subunits b and delta, seems to be unlikely.

FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/β cleft, with bI close to subunit α, whereas bII is proximal to subunit β. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-β, α-bI-bII-β, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase. Background: The peripheral stator stalk of Escherichia coli ATP synthase contains two b subunits. Results: Using disulfide bond formation, one b subunit was cross-linked to a, α, and δ and the other to β. Conclusion: The b subunits adopt distinct positions within the stator to generate stability. Significance: The different positions imply different roles in counteracting the torque generated by the rotor.

Two conserved charged amino acids of the N-terminal 'crown' region of the alpha subunit of E. coli-F(1), alpha-D36 and alpha-R40 were exchanged for chemically related (alpha-D36--E, alpha-R40--K) or unrelated amino acids (alpha D-36--K, alpha R40--G), respectively, by employing oligonucleotide-directed mutagenesis. ATP formation and ATP hydrolyzing activity of isolated plasma membrane vesicles was strongly inhibited in mutant HS2 (alpha-D36--K), but only slightly affected in the other mutants. The inhibition is not due to a lower content of F0F1 in HS2. In this mutant the extent of the proton gradient generated by ATP hydrolysis was more than 80% inhibited; in all other transformants much smaller effects were observed. The proton gradient established by NADH oxidation was 33% decreased in HS2, but was decreased to a lesser extent in all other mutants. After blockage of F0 by DCCD treatment, the same NADH-induced proton gradient was obtained in all transformants including HS2. This and the fact that the activity of NADH oxidation was unchanged indicate increased proton leakiness of F0F1 carrying the alpha-D36--K mutation. In F1 alpha-D36 is located in a domain contacting the beta subunit in the vicinity of the arginine beta-R52. The effect of alpha-D36--K replacement on catalysis and coupling thus may be due to an electrostatic repulsive effect in the crown region which alters the alpha and beta interaction.

The antigenic determinants of mAbs against subunit c of the Escherichia coli ATP synthase were mapped by ELISA using overlapping synthetic heptapeptides. All epitopes recognized are located in the hydrophilic loop region and are as follows: 31-LGGKFLE-37, 35-FLEGAAR-41, 36-LEGAAR-41 and 36-LEGAARQ-42. Binding studies with membrane vesicles of different orientation revealed that all mAbs bind to everted membrane vesicles independent of the presence or absence of the F1 part. Although the hydrophilic region of subunit c and particularly the highly conserved residues A40, R41, Q42 and P43 are known to interact with subunits γ and e of the F1 part, the mAb molecules have no effect on the function of F0. Furthermore, it could be demonstrated that the F1 part and the mAb molecule(s) are bound simultaneously to the F0 complex suggesting that not all c subunits are involved in F1 interaction. From the results obtained, it can be concluded that this interaction is fixed, which means that subunits γ and e do not switch between the c subunits during catalysis and furthermore, a complete rotation of the subunit c oligomer modified with mAb(s) along the stator of the F1F0 complex, proposed to be composed of at least subunits b and δ, seems to be unlikely.

The DNA encoding the subunits of the ATP synthase (F1F0) of Streptomyces lividans 66 strain 1326 was identified using oligodeoxyribonucleotide probes derived from the N-terminal sequence of subunit gamma of the F1 complex. The complete nucleotide sequence of the operon was determined. The atp operon contains nine genes, atpIBEFHAGDC, encoding the eight structural components of the ATP synthase complex and the i protein, a polypeptide of unknown function. The gene order found is identical to that in other non-photosynthetic eubacteria. The determination of the N-terminal amino acid (aa) sequences of the F1 subunits alpha, beta, gamma, delta and epsilon allowed us to identify the translational start points and to define the primary structures of the proteins. The aa sequence deduced for subunit delta revealed an N-terminal extension of about 90 aa, which is not present in any delta subunit or OSCP (oligomycin sensitivity-conferral protein) of other species studied so far. The phylogenetic relationship of eu- and archaebacteria was investigated using sequencing data of the highly conserved beta subunit of different ATP synthases including that of S. lividans. The calculations revealed that S. lividans beta does not form a phylogenetic group together with the Gram+ taxa of low G+C contents, but is more closely related to the beta subunit of Rhodobacteria.

Hybrid Fo complexes of the ATP synthases of spinach chloroplast (CFo) and Escherichia coli (EFo) were investigated. Immunoprecipitations with polyclonal antibodies against the different Fo subunits clearly revealed that hybrid Fo complexes derived from CFo subunit III and EFo subunits a and b were formed in vivo. In addition, the ATPase activities of the hybrid ATP synthase, measured in everted cytoplasmic membranes of an atpE mutant strain transformed with the atpH gene coding for CFo III, were comparable to activities obtained for the same mutant strain complemented with the atpE gene (EFo c). Nevertheless, CFo III was not able to replace EFo c functionally, since the strain containing the hybrid ATP synthase was not able to grow on succinate. In order to investigate the reason for this lack of function, hybrid proteolipids of CFo III and EFo c were constructed. Only a chimaeric protein comprising the seven N-terminal amino acid residues from CFo III and the remaining part of EFo c was able to replace wild-type EFo c, whereas hybrid proteins with 13 and 33 N-terminal amino acids of CFo III were not functional. The results suggested that a network of interactions between the subunits essential for proton translocation and/or coupling of the F1 part exists, which was optimized for each species during evolution, although the overall structure of FoF1 complexes has been conserved.

The H(+)-translocating F0F1-ATPase from Escherichia coli (EF0F1) was purified and reconstituted into preformed reverse-phase liposomes prepared from egg yolk phosphatidylcholine/phosphatidic acid. The EF0F1 liposomes were energized by an acid/base transition (pHout = 8.3; pHin = 5.0) and a superimposed K+/valinomycin diffusion potential ([K+]out = 100 mM; [K+]in = 0.6 mM) yielding a maximum rate (turnover number) of ATP synthesis of 27 +/- 8 mol ATP . mol EF0F1(-1) . s-1), i.e. 27 +/- 8 s-1. This reaction was inhibited by NH4Cl or by addition of the F0F1 inhibitor N,N'-dicyclohexylcarbodiimide. The rate of ATP synthesis measured as a function of the phosphate and ADP concentrations, can be described by Michaelis-Menten kinetics with a Km of 0.7 +/- 0.2 mM for phosphate ([ADP] = 200 microM) and a Km of 27 +/- 7 microM for ADP ([phosphate] = 5 mM), respectively.

To characterize the role of supernumerary subunits of the mammalian FoF1 ATP synthase, crossreconstitution of mitochondrial and bacterial FoF1 complexes has been carried out. Escherichia coli F1 (EcF1) can be reconstituted with F1-stripped everted membranes of E. coli (UPEc) and of bovine heart mitochondria (USMP). Bovine heart mitochondrial F1 (BHF1) can also be reconstituted with both membranes. Both EcF1 and BHF1, when reconstituted with UPEc, exhibited oligomycin-insensitive ATP-hydrolase activity. Subunits of the mammalian Fo, in particular FoI-PVP protein, F6 and oligomycin-sensitivity-conferring protein (OSCP) conferred oligomycin sensitivity to the catalytic activity of EcF1 or BHF1 reconstituted with UPEc. Reaction of N, N′-dicyclohexylcarbodiimide and development of inhibition of passive H+ conduction was, in UPEc, considerably slower and exhibited a lower apparent affinity than in USMP. The ATP hydrolase activity of UPEc+EcF1 or UPEc+BHF1 was, also, less sensitive to inhibition by N, N′-dicyclohexylcarbodiimide than USMP+EcF1 or USMP+BHF1. Addition of mitochondrial FoI-PVP to UPEc enhanced the sensitivity of H+ conduction to oligomycin. FoI-PVP and OSCP added to UPEc, promoted inhibition by N, N′-dicyclohexylcarbodiimide of passive H+ conduction and increased its binding affinity to subunit c of E. coli Fo. The presence of FoI-PVP and OSCP also promoted inhibition by N, N′-dicyclohexylcarbodiimide of the ATP-hydrolase activity of EcF1 or BHF1 reconstituted with UPEc.

After incorporation of the purified Na(+)-translocating F0F1-ATPase from Propionigenium modestum into preformed phospholipid vesicles the synthesis of ATP from ADP and inorganic phosphate could be observed under conditions where a valinomycin-mediated K+ diffusion potential (delta psi) and/or a Na+ concentration gradient (delta pNa) were imposed. This reaction was not inhibited by the protonophore carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP). Furthermore, the delta pNa-driven ATP synthesis was stimulated by FCCP. In contrast, the addition of the Na+/H+ antiporter monensin or of the F0F1 inhibitors N,N'-dicyclohexylcarbodiimide and venturicidin abolished the synthesis of ATP completely. Finally, delta pNa alone was able to elicit ATP synthesis, when a Na+ concentration gradient of sufficient magnitude was applied. In this case ATP synthesis occurred above a threshold level of approximately 120 mV and, furthermore, delta psi and delta pNa appear to be equivalent as driving forces for this process. Therefore, the data provide firm evidence for the concept that delta"mu Na+ is the primary driving force for the synthesis of ATP in P. modestum.

In the presence of urea, Helicobacter pylori survived for at least 3 h at pH 1. Under these conditions, the cells maintained their cytoplasmic pH at 5.8. De novo protein synthesis during acid shock was not essential for survival of H. pylori at pH 1.

The ATP synthase (F1F0 ) of Escherichia coli consists of two structurally and functionally distinct entities. The F1 part is composed of five subunits α, β, γ, δ and ϵ (3:3:1:1:1) and carries the catalytic centres of the enzyme. The membrane-bound F0complex functions as a proton channel and consists of the three subunits a, b and c (l:2:10±l). Subunit c (8288 Mr) exhibits a hairpin-like structure within the membrane. A conserved acidic residue (Asp-61) in the C-terminal hydrophobic segment is absolutely required for proton translocation through F0, whereas the hydrophilic loop region is necessary for F, binding. Expression of the chloroplast proteolipid together with subunits a and b of E. coli did not produce an active F0 hybrid complex. Therefore, the construction of hybrid c subunits consisting of parts of the proteolipid from both organisms is in progress to determine those parts of subunit c that are essential for a functional interplay with subunits a and b. Subunit a (30276 Mr), which is also involved in proton translocation, is an extremely hydrophobic protein with 5–8 membrane-spanning helices. Studies with alkaline phosphatase fusion proteins resulted in controversial conclusions about the localization of the N and C termini of the protein. A foreign epitope (13 amino acids) has been inserted into the N-or C-terminal region of subunit a without affecting the function of Fo. Binding studies with a monoclonal antibody against this epitope are now under investigation to determine the orientation of subunit a. Subunit b (17265 Mr) is anchored in the membrane by its apolar N-terminal region, whereas the hydrophilic part protrudes into the cytoplasm. Studies with proteases and truncated b′ subunits revealed that the C-terminal part of subunit b is involved in binding of F1 to F0 and is necessary for correct assembly of F0.

Incubation of F1-stripped everted membrane vesicles with antibodies against subunit b of the ATP synthase from Escherichia coli resulted in an inhibition of the binding of F1 to F0, whereas the proton translocation remained unaffected. Incubation of unstripped everted membrane vesicles with anti-b antibodies resulted in a partial loss of F1, and the remaining membrane-bound ATP-hydrolyzing activity is uncoupled from proton translocation. Similar results were obtained when F(ab')2 or Fab fragments were used. The immunoblot analysis of truncated b' subunits different in length showed that the antigenic determinants are located in the carboxyl-terminal half of the polypeptide chain.

The Fo complex of the ATP synthase (F1Fo) of Escherichia coli contains only two cysteinyl residues, Cys21, of the two copies of subunit b. Modification of Cys21 with the hydrophobic maleimide N-(7-dimethylamino-4-methyl-coumarinyl)maleimide resulted in impairment of Fo functions [Schneider, E. & Altendorf, K. (1985) Eur. J. Biochim. 153, 105-109]. We replaced this residue (via cassette mutagenesis) by Ser, Gly, Ala, Thr, Asp and Pro. None of the replacements resulted in detectable alterations of the function of the ATP synthase, making a functional role for these sulfhydryl residues unlikely. Due to its high tolerance towards amino acid substitutions, the region around Cys21 seems not to be a protein-protein contact area.

The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified. The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ion-exchange chromatography and gel filtration. The purified F1 complex from S. lividans (SLF1) consists of five subunits alpha, beta, gamma, delta and epsilon with molecular masses of 58,000, 50,000, 36,000, 28,000 and 13,000, respectively and exhibits immunological cross-reactivity with the F1 portion purified from Escherichia coli (ECF1). The enzymatic properties of SLF1 were determined by the use of microtiter-plate-based assay and compared with data obtained for ECF1. ATPase activity of SLF1 (specific activity: 20-30 U/mg) was only observed in the presence of high concentrations of Ca2+ (10mM). Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca(2+)-stimulated activity of SLF1. SLF1 was re-bound to F1-stripped membranes of S. lividans, but not to F1-stripped membrane vesicles of E. coli. In contrast, ECF1 could be cross-reconstituted with F1-stripped membranes of S. lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed.

The uncI gene is the most poorly expressed gene of the unc operon of Escherichia coli. We examined how the structural features of the translational initiation region (TIR) of this gene relate to the efficiency of its expression. A set of various TIR sequences was created for the uncI gene by coupling synthetic oligodeoxynucleotides to the 5' part of the plasmid-borne gene. Analyses of transcription and translation of the resulting constructs revealed that weak translational initiation efficiency of the uncI TIR sequence is a rate-controlling factor for expression. Moreover, the identification of an endonucleotytic cleavage site within the 5' part of the uncI mRNA by Northern blotting lends further support to the notion that instability of the uncI cistron within the polycistronic unc mRNA represents a further mode of control. The analysis of the roles of the different Shine-Dalgarno regions and putative start codons within the translational initiation region of the uncI gene revealed that the Shine-Dalgarno region III and the GUG codon (codon 4 of the uncI coding sequence described by Walker, J. E., Saraste, M., and Gay, N. J. (1984) Biochim. Biophys. Acta 768, 164-200) define the main site of initiation. However, manipulations of the translational initiation region led to selection of an initiation codon further upstream. The relationship between mRNA sequence and higher order structure on the one hand, and initiation site selection and initiation efficiency on the other, was analyzed.

Expression of subunit III of the ATP synthase from spinach chloroplasts in Escherichia coli has been achieved. Although the protein is inserted into the bacterial cytoplasmic membrane, formation of a functional Fo complex was not observed.

The uncI gene, the first gene of the unc operon, has been cloned into an expression vector carrying the lambda PRPL promoters in tandem orientation and the gene cI857 coding for the thermolabile repressor. Linkage of the uncI gene to an efficient ribosome binding site (the translational initiation region of the uncE gene) resulted in 10-20-fold increased gene expression. The i protein has been extracted from overproducing cells using chloroform/methanol and purified to homogeneity by ion exchange chromatography. Analyzing the products of the uncI gene encoded by different plasmids, we provide evidence that, in contrast to the previously reported data (Walker, J. E., Saraste, M., and Gay, N. J. (1984) Biochim. Biophys. Acta 768, 164-200), the chromosome-encoded i protein contains the N-terminal sequence Ser-Val-Ser-Leu-Val-Ser-Arg and has a molecular weight of 13,504.

P-type ATPases are ubiquitously abundant enzymes involved in active transport of charged residues across biological membranes. The KdpB subunit of the prokaryotic Kdp-ATPase (KdpFABC complex) shares characteristic regions of homology with class II-IV P-type ATPases and has been shown previously to be misgrouped as a class IA P-type ATPase. Here, we present the NMR structure of the AMP-PNP-bound nucleotide binding domain KdpBN of the Escherichia coli Kdp-ATPase at high resolution. The aromatic moiety of the nucleotide is clipped into the binding pocket by Phe(377) and Lys(395) via a pi-pi stacking and a cation-pi interaction, respectively. Charged residues at the outer rim of the binding pocket (Arg(317), Arg(382), Asp(399), and Glu(348)) stabilize and direct the triphosphate group via electrostatic attraction and repulsion toward the phosphorylation domain. The nucleotide binding mode was corroborated by the replacement of critical residues. The conservative mutation F377Y produced a high residual nucleotide binding capacity, whereas replacement by alanine resulted in low nucleotide binding capacities and a considerable loss of ATPase activity. Similarly, mutation K395A resulted in loss of ATPase activity and nucleotide binding affinity, even though the protein was properly folded. We present a schematic model of the nucleotide binding mode that allows for both high selectivity and a low nucleotide binding constant, necessary for the fast and effective turnover rate realized in the reaction cycle of the Kdp-ATPase.

The addition of a His6 tag to the N terminus of subunit a of the F0 complex of the Escherichia coli ATP synthase allowed the purification of an ab2 subcomplex after solubilization of membranes with n-dodecyl-β-d-maltoside and subsequent nickel-nitrilotriacetic acid affinity chromatography. After co-reconstitution of the ab2 subcomplex with purified subunit c, passive proton translocation rates as well as coupled ATPase activities after binding of F1 were measured that were comparable with those of wild type F0. The interaction between subunits a and b, which has been shown to be stoichiometric and functional, is not triggered by any cross-linking reagent and therefore reflects subunit interactions occurring within the F0 complex in vivo.

In this review we discuss recent work from our laboratory concerning the structure and/or function of the F 0 subunits of the proton-translocating ATP synthase of Escherichia coli . For the topology of subunit a a brief discussion gives (i) a detailed picture of the C-terminal two-thirds of the protein with four transmembrane helices and the C terminus exposed to the cytoplasm and (ii) an evaluation of the controversial results obtained for the localization of the N-terminal region of subunit a including its consequences on the number of transmembrane helices. The structure of membrane-bound subunit b has been determined by circular dichroism spectroscopy to be at least 75% α-helical. For this purpose a method was developed, which allows the determination of the structure composition of membrane proteins in proteoliposomes. Subunit b was purified to homogeneity by preparative SDS gel electrophoresis, precipitated with acetone, and redissolved in cholate-containing buffer, thereby retaining its native conformation as shown by functional coreconstitution with an ac subcomplex. Monoclonal antibodies, which have their epitopes located within the hydrophilic loop region of subunit c , and the F 1 part are bound simultaneously to the F 0 complex without an effect on the function of F 0 , indicating that not all c subunits are involved in F 1 interaction. Consequences on the coupling mechanism between ATP synthesis/hydrolysis and proton translocation are discussed.

Using antibodies raised against the purifiedi protein, the expression of the chromosomaluncI gene was demonstrated. Thei protein was identified as a component of the cytoplasmic membrane and shown to be present in preparations of F0 or F1F0. The protein is not associated with the F1 moiety.

The antigenic determinants of mAbs against subunit a of the Escherichia coli ATP synthase were mapped by ELISA using overlapping synthetic decapeptides. For two of the mAbs the epitopes are E4NMTPQD10 (GDH 14-5C6) and V29DPQ32 (GDH 8-8B3). Binding of these mAbs to membrane vesicles of different orientation revealed that both epitopes are accessible in vesicles with inside-out orientation. These results demonstrate that at least the N-terminal amino acids 1-32 of subunit a are located at the cytoplasmic side of the membrane. A further determination of the topology of subunit a was performed by inserting the reporter epitope DYKDDDDK (FLAG epitope) at different positions of the polypeptide chain. 10 of 13 insertions led to a functional F0F1 ATP synthase and allowed specific detection of the modified subunit a by immunoblotting using an mAb against the FLAG epitope. In addition, polyclonal anti-FLAG IgG was applied for the recognition of the mutant FLAG epitope DYKDDVDK. Cells carrying this mutant FLAG epitope at the C terminus of subunit a were able to grow on succinate as sole carbon and energy source, revealing a functional ATP synthase, in contrast to those carrying the original FLAG epitope at the same position. Binding studies with membrane vesicles of different orientation and anti-FLAG Ig demonstrated that both termini of the protein are located at the cytoplasmic side of the membrane, indicating that an even number of membrane-spanning segments is present in subunit a. In addition, insertion of two FLAG epitopes in tandem after K66, or one epitope after H95, and Q181 revealed that the polypeptide regions including these residues are accessible from the cytoplasmic surface of the membrane. These results support the view that the polypeptide chain of subunit a traverses the membrane six times.

The rate of ATP synthesized by the ATP synthase (F0F1-ATPase) is limited by the rate of energy production via the respiratory chain, when measured in everted membrane vesicles of an Escherichia coli atp wild-type strain. After energization of the membranes with NADH, fractional inactivation of F0F1 by the covalent inhibitor N,N'-dicyclohexylcarbodiimide allowed the rate of ATP synthesis/mol remaining active ATP synthase complexes to increase; the active ATP synthase complexes were calculated using ATP hydrolysis rates as the defining parameter. In addition, variation of the assay temperature revealed an increase of the ATP synthesis rate up to a temperature of 37 degrees C, the optimal growth temperature of E. coli. In parallel, the amount of F0F1 complexes present in membrane vesicles was determined by immunoquantitation to be 3.3 +/- 0.3% of the membrane protein for cells grown in rich medium and 6.6 +/- 0.3% for cells grown in minimal medium with glycerol as sole carbon and energy source. Based on these data, a turnover number for ATP synthesis of 270 +/- 40 s(-1) could be determined in the presence of 5% active F0F1 complexes. Therefore, these studies demonstrate that the ATP synthase complex of E. coli has, with respect to maximum rates, the same capacity as the corresponding enzymes of eukaryotic organells.

Subunit c of the membrane-integrated, proton-translocating F0 portion of the ATP synthase (F1F0) from Escherichia coli has been isolated under nondenaturing conditions (Schneider, E., and Altendorf, K. (1985) EMBO J. 4, 515-518) and antibodies have been raised in rabbits. The primary antisera did not recognize the antigen when present in the same buffer as used for the immunization. Surprisingly, in one of the three antisera a strong antibody binding was observed when intact F0, a.c complex or reconstituted subunit c was provided as the antigen. Incorporation of subunit c into liposomes together with subunits a and b forming an active, H+-translocating complex was not required for the recognition by the antiserum. Subunit c prepared by chloroform/methanol extraction or by chromatography in the presence of sodium dodecyl sulfate was not recognized by the anti-c antiserum when incorporated into liposomes.

FOF1-ATP synthases catalyse ATP formation from ADP and Pi by using the free energy supplied by the transmembrane electrochemical potential of the proton. The δ subunit of F1 plays an important role at the interface between the channel portion Fo and the catalytic portion F1. In chloroplasts it can plug the protonic conductance of CFo and in Escherichia coli it is required for binding of EF1 to EFo. We wanted to know whether or not δ of one species was effective between Fo and F1 of the other species and vice versa. To this end the respective coupling membrane (thylakoids, everted vesicles from E. coli) was (partially) depleted of F1 and purified F1, F1(-δ), and δ were added in various combinations to the F1-depleted membranes. The efficiency or reconstitution was measured in thylakoids via the rate of phenazinemethosulfate-mediated cyclic photophosphorylation and in E. coli everted vesicles via the degree of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching. Addition of CF1 to partially CF1-depleted thylakoid vesicles restored photophosphorylation to the highest extent. CF1(-δ)+chloroplast δ, EF1, EF1(-δ)+E. coliδ were also effective but to lesser extent. CF1(-δ)+E. coliδ and EF1(-δ)+chloroplast δ restored photophosphorylation to a small but still significant extent. With F1-depleted everted vesicles prepared by repeated EDTA treatment of E. coli membranes, addition of CF1, CF1(-δ)+chloroplast δ and CF1(-δ)+E. coliδ gave approximately half the extent of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching as compared to EF1 or EF1(-δ)+E. coliδ by energization of the vesicles with NADH, while EF1(-δ)+chloroplast δ was ineffective. All ‘mixed’ combinations were probably reconstitutively active only by plugging the protonic leak through the exposed Fo (structural reconstitution) rather than by catalytic activity. Nevertheless, the cross-reconstitution is stunning in view of the weak sequence similarity between chloroplast δ and E. coliδ. It favors a role of δ as a conformational transducer rather than as a proton conductor between F0 and F1.

Functional compatibility between the F1 and F0 parts of ATP synthases from Escherichia coli (EF1F0) and the thermophilic bacterium PS3 (TF1F0) was analyzed. F1-stripped everted membrane vesicles from both organisms bound the homologous or heterologous F1 part to the same extent. Titration of the reconstituted membrane vesicles with dicyclohexylcarbodiimide revealed a similar sensitivity of the homologous and hybrid F1F0 complexes towards the inhibitor. Furthermore, the heterologous enzymes exhibited ATP-dependent H+ translocation comparable to that of homologous F1F0. Antisera raised against EF1 or subunits a, b, and c of EF0 were analyzed for cross-reactivity with TF1 and TF0. Common antigenic sites have been detected with immunoblot analysis for subunit beta and subunit c of EF1F0 and the corresponding subunits from TF1F0. A weak binding of the anti-a and anti-b antisera with the TF0 part has been observed in an enzyme-linked immunosorbent assay. Based on these findings the structural and functional relationship between the mesophilic and thermophilic ATP synthase complexes is discussed.

Everted membrane vesicles of the Gram-positive eubacterium Streptomyces lividans were prepared and the ATP synthase (F1Fo) was characterized in its membrane-bound form. In addition, the F1Fo complex was solubilized, purified, and functionally reconstituted into phospholipid vesicles. The enzyme complex is similar with respect to subunit composition to those of other eubacterial ATP synthases. Whereas the F1 part only exhibits ATPase activity in the presence of CaCl2 (Hensel, M., Deckers-Hebestreit, G. and Altendorf, K. (1991) Eur. J. Biochem. 202, 1313–1319), the membrane-bound ATPase is also moderately stimulated by high concentrations of Mg2+ ions (20 mM). In contrast, the physiological functions of the ATP synthase, i.e., ATP-driven H+ translocation and ATP synthesis are strictly dependent on Mg2+ ions. The biochemical properties of the ATP synthase of S. lividans show distinct similarity to the enzyme complex of rhodobacteria and bacilli. The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide, venturicidin, and tributyltin, typical inhibitors of F1Fo-ATPases, which react with the membrane-bound Fo complex. In addition, the ATPase activity is highly sensitive towards oligomycin, a feature which is only shared by the ATP synthase of rhodobacteria and mitochondria.

F1-stripped everted membrane vesicles of the ATP synthase-overproducing Escherichia coli strain KY 7485 were treated with trypsin for different lengths of time. Subsequently, the Fo complex was isolated and analyzed by sodium dodecyl sulfate-gel electrophoresis, as well as immunoblotting using antibodies raised against subunit b. By these techniques 3 degradation products with apparent molecular masses of about 16 kDa could be detected in accordance with previous findings (Perlin, D.S., and Senior, A.E. (1985) Arch. Biochem. Biophys. 236, 603-611). Labeling of isolated trypsin-treated Fo fractions with the thiol-specific reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide, which has been demonstrated recently to specifically modify subunit b (Schneider, E., and Altendorf, K. (1985) Eur. J. Biochem. 153, 105-109) revealed that the 16-kDa digestion products were degraded into two stable fragments of 12 and 8.3 kDa. These polypeptides do not react with the anti-b antibodies. Treatment of purified liposome-integrated Fo with trypsin resulted in a similar cleavage pattern. In both cases protease digestion inhibited F1 binding while proton-translocating activity remained unaffected. However, liposomes reconstituted with Fo isolated from trypsin-treated membranes were impaired in both binding of F1 and proton translocation. These activities could be restored when reconstitution was carried out in the presence of native subunit b. From this we conclude that the C-terminal region of subunit b is necessary for proper reconstitution of Fo into liposomes.

Antisera have been raised against denatured and non-denatured subunits a, b and c of the F0 complex of the ATP synthase from Escherichia coli. The subunit specificity of the antibodies has been established with immunoblot analysis or enzyme-linked immunosorbent assay (ELISA). In inside-out oriented membrane vesicles the binding avidities of both sets of antisera, against denatured and non-denatured subunits of F0, were similar in the presence as well as in the absence of the F1 part. F1-depleted everted membrane vesicles always produced an efficient binding of the different antisera. In the presence of F1 no antibody recognition could be observed with the anti-a antisera, while anti-b and anti-c antisera showed strong binding. However, a higher membrane protein concentration was necessary for the same antibody binding as in F1-stripped vesicles. In membrane vesicles with right-side-out orientation the recognition of the three F0 subunits was dependent on the antisera set used. Antisera raised against denatured subunits showed no binding to the membrane vesicles, only in case of anti-(dodecylsulfate-denatured b) antiserum could a slight affinity be detected. An antigen-antibody recognition with all three F0 subunits occurred when the antisera against non-denatured subunits were incubated with membrane vesicles of right-side-out orientation. The membrane protein concentration which was necessary to produce a significant binding was 10-100-fold higher compared to that of F1-depleted everted membrane vesicles.