Membrane-bound ATP synthases (F0F1-ATPases) of bacteria serve two important physiological functions. The enzyme catalyzes the synthesis of ATP from ADP and inorganic phosphate utilizing the energy of an electrochemical ion gradient. On the other hand, under conditions of low driving force, ATP synthases function as ATPases, thereby generating a transmembrane ion gradient at the expense of ATP hydrolysis. The enzyme complex consists of two structurally and functionally distinct parts: the membrane-integrated ion-translocating F0 complex and the peripheral F1 complex, which carries the catalytic sites for ATP synthesis and hydrolysis. The ATP synthase of Escherichia coli, which has been the most intensively studied one, is composed of eight different subunits, five of which belong to F1, subunits alpha, beta, gamma, delta, and epsilon (3:3:1:1:1), and three to F0, subunits a, b, and c (1:2:10 +/- 1). The similar overall structure and the high amino acid sequence homology indicate that the mechanism of ion translocation and catalysis and their mode of coupling is the same in all organisms.

The rate of ATP synthesized by the ATP synthase (F,,F,-ATPase) is limited by the rate of energy production via the respiratory chain, when measured in everted membrane vesicles of an Eschericliia coli atp wild-type strain. After energization of the membranes with NADH, fractional inactivation of F,,F, by the covalent inhibitor N,N'-dicyclohexylcarbodiimide allowed the rate of ATP synthedmol 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"C, the optimal growth temperature of E. coli. In parallel, the amount of F,,F, complexes present in membrane vesicles was determined by immnnoquantitation to be 3.3 t 0.3 % of the membrane protein for cells grown in rich medium and 6.6 t 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 5 40 s~' could be determined in the presence of 5 8 active F,F, 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.Keywords: F,,F, ATP synthase ; oxidative phosphorylation ; turnover number; dicyclohexylcarbodiimide : Escherichia coli.ATP synthases (F,F,-ATPases) are found in energy-transducing membranes of mitochondria, chloroplasts and bacteria, where they catalyze the synthesis of ATP from ADP and inorganic phosphate using an electrochemical gradient of protons built up by respiration or photosynthesis. In bacteria. the enzyme can also function in the reverse direction by coupling the hydrolysis of ATP to the translocation of protons. The multisubunit enzyme complex consists of the globular, water-soluble F, complex and the membrane-integrated F, complex linked by a stalk. The F, complex carries the catalytic sites for ATP synthesis and hydrolysis, whereas the F,, complex constitutes a path for protons across the membrane. Energy released by proton translocation through F,, is relayed to the catalytic sites in F, by conformational changes. In Escherichia coli, the F, complex is composed of the three different polypeptides a, b, and c in a stoichiometry of 1 : 2: 10 t 1, whereas the F, complex consists of five subunits with a stoichiometry of a&& (Fillingame, 1990, 1992: Capaldi et al., 1994Deckers-Hebestreit and Altendorf, 1996). Recently, the F, part of bovine heart mitochondria was crystallized and the structures of the subunits a, / l , and part of 7 , have been solved by X-ray diffraction at 0.28-nm resolution (Abrahams et al., 1994).F,F,-ATPases from different sources reveal great similarities in subunit composition, structure and function. Hence, one could Enzyme. ATP synthase (F,,F,) (EC 3.6.1.34).Bertani; SMP, submitochondrial particles.imply that also the catalytic mechanisms and the turnover numbers of the enzyme c...

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.

Cholate-solubilized F, complexes of the ATP synthase (F,F,) from Escherichia coli were studied by application of conventional transmission electron microscopy and electron spectroscopic imaging (ESI) of negatively stained samples. Using the ESI mode, the structural organization of the F, complex (diameter of 7.5 5 0.5 nm) could be observed in more detail and defined projections could be distinguished. Projection A appears as a deltoid-like structure with bilateral symmetry. Projection B has an overall trapezoidal shape with some similarity in shape to the letter W. Applying the ESI mode to the uc complex dissolved in cholate-containing buffer, an elongated structure consisting of two intensity maxima could be observed. Simulations with models of the F, and the ac complex revealed that the projections observed can be obtained by tilting and rotating a model in which subunit a and the two copies of subunit b are located outside the subunit c oligomer. This view of structural organization was supported by results obtained with F, complexes decorated with monoclonal antibodies against subunits a, b or c.

The H+-translocating F,F,-ATPase from Escherichia coli (EF,F,) was purified and reconstituted into preformed reverse-phase liposomes prepared from egg yolk phosphatidylcholine/phosphatidic acid. The EF,F, liposomes were energized by an acidhase transition (pH,,, = 8. Membrane-bound ATP synthases (Fa,-ATPases ; H+-ATPase) synthesize ATP from ADP and inorganic phosphate utilizing the energy of an electrochemical gradient of protons built up by respiration or photosynthesis. In bacteria, the enzyme also functions in the reverse direction generating a transmembrane proton gradient driven by hydrolysis of ATP.