Adenosine 5-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the subunit of the bacterial FoF1-ATP synthase sandwiched by the cyan-and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 M to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of F oF1-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment. fluorescent indicator ͉ FRET ͉ live imaging ͉ oxidative phosphorylation A denosine 5Ј-triphosphate (ATP) is the ubiquitous energy currency of all living organisms. The high phosphatetransfer potential of ATP is used for many biological processes, including muscle contraction, synthesis and degradation of biological molecules, and membrane transport. In addition, it has been suggested that ATP acts as an intracellular or extracellular signaling molecule in cellular processes, such as insulin secretion (1), neurotransmission (2), cell motility (3), and organ development (4). However, it has been difficult to precisely understand how ATP controls cellular processes and how the intracellular ATP level is regulated at the single cell level, because the conventional ATP quantification methods can only provide the averaged ATP level of an ensemble of cells based on cell extract analysis. Moreover, the distribution pattern of ATP between different intracellular compartments is unclear. Several attempts have been made to monitor ATP levels real-time in individual cells; however, these methods present several problems. For example, in chemiluminescence imaging from cells expressing firefly luciferase (5), chemiluminescence by luciferase depends not only on the intracellular ATP level but also on the luciferase concentration, as well as the other substrates, oxygen, and luciferin. Moreover, pH also affects luciferase activity. Another drawback of this method is that the intracellular ATP level could be perturbed because of ATP consumption. Furthermore, the dim luminescence of luciferase requires longer exposure time for image acquisition, making real-time observation cumbersome. Other approaches include measurement of the ion channel activity (6) or conformational change (7) of the ATP-s...

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The subunit of bacterial and chloroplast FoF1-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound subunit from a thermophilic Bacillus PS3 at 1.9-Å resolution. The C-terminal two ␣-helices were folded into a hairpin, sitting on the ␤ sandwich structure, as reported for Escherichia coli. A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the Cterminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of F oF1-ATP synthase.ATP hydrolysis ͉ ATP-binding motif ͉ ATPase regulation ͉ ATP synthase ͉ F1 rotation

It has been proposed that C-terminal two ␣-helices of the ⑀ subunit of F 1 -ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F 1 from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the ␥ subunit and C terminus of the ⑀ subunit, respectively. High FRET (extended form) of F 1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5-Adenyl-␤,␥-imidodiphosphate, ADP ؉ AlF 4 ؊ , ADP ؉ NaN 3 , and GTP also caused the retracted form, indicating that ATP binding to the catalytic ␤ subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the ⑀ subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65°C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated ⑀ subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65°C reached to 0.67 mM, a consistent value to assume that the ⑀ subunit acts as a sensor of ATP concentration in the cell.A rotary motor F 1 -ATPase (F 1 ) 2 is a water-soluble portion of F 0 F 1 -ATP synthase, which catalyzes ATP synthesis/hydrolysis coupled with a transmembrane proton translocation (1, 2). F 1 has a subunit structure of ␣ 3 ␤ 3 ␥␦⑀; ␣ and ␤ subunits have a non-catalytic and catalytic nucleotide binding sites, respectively; ␥ subunit rotates in the ␣ 3 ␤ 3 ring; ␦ subunit connects the ring to the stator part of F 0 ; and ⑀ subunit rotates together with ␥ subunit as a body. The ⑀ subunit (ϳ14 kDa) has a regulatory function and consists of N-terminal ␤-sandwitch and C-terminal two ␣-helices (3, 4).Previous structural studies of F 1 indicated two conformations of the ⑀ subunit with different arrangement of the two ␣-helices, that is, retracted folded-hairpin form and partly extended form (Fig. 1, A and B) (5-8). Cross-linking studies suggested the third conformation with fully extended ␣-helices 3 (Fig. 1C) (9). Biochemical data have indicated that the ⑀ subunit adopts the extended form in the absence of nucleotide or in the presence of ADP, in which ATPase activity is inhibited, and that ATP counteracts ADP by favoring the retracted form, which is a noninhibitory conformation (9). Thus, it appears that the regulatory function of the ⑀ subunit is dependent on the drastic conformational transition that is affected by nucleotide and other factors. However, previous studies have not provided kinetic information on how these dynamic conformational transitions occur in the enzyme at work. Fluorescence resonance energy transfer (FRET) is a powerful technique that enables us to probe conformational...

F 1 -ATPase, a soluble part of the F 0 F 1 -ATP synthase, has subunit structure ␣ 3 ␤ 3 ␥␦⑀ in which nucleotide-binding sites are located in the ␣ and ␤ subunits and, as believed, in none of the other subunits. However, we report here that the isolated ⑀ subunit of F 1 -ATPase from thermophilic Bacillus strain PS3 can bind ATP. The binding was directly demonstrated by isolating the ⑀ subunit-ATP complex with gel filtration chromatography. The binding was not dependent on Mg 2؉ but was highly specific for ATP; however, ADP, GTP, UTP, and CTP failed to bind. The ⑀ subunit lacking the C-terminal helical hairpin was unable to bind ATP. Although ATP binding to the isolated ⑀ subunits from other organisms has not been detected under the same conditions, a possibility emerges that the ⑀ subunit acts as a built in cellular ATP level sensor of F 0 F 1 -ATP synthase.F 0 F 1 -ATPase/synthase (F 0 F 1 ) 1 catalyzes ATP synthesis coupled with the proton flow across the membrane through mechanical rotation of the central shaft subunits relative to the surrounding stator subunits (1, 2). F 1 is the water-soluble portion of F 0 F 1 and has ATP hydrolysis activity by itself. It consists of five kinds of subunits with a stoichiometry of ␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 in which the catalytic nucleotide-binding sites are located on the ␤ subunits and the non-catalytic nucleotidebinding sites are on the ␣ subunits. The ␥ subunit inserts its long coiled-coil helices into the central cavity of the ␣ 3 ␤ 3 cylinder (3), and ATP hydrolysis occurring in the ␣ 3 ␤ 3 drives rotation of the ␥ subunit along with the ⑀ subunit that is associated with the ␥ subunit (reviewed in Ref. 2).Responding to the varying energy supply for ATP synthesis in living organisms, the activity of F 0 F 1 must be regulated. Eukaryotic organellar F 0 F 1 has developed unique regulatory systems; mitochondrial F 0 F 1 has a specific ATPase inhibitor protein and its cofactor proteins (4, 5), and chloroplast F 0 F 1 is regulated by the reversible formation of a disulfide bond in the ␥ subunit (reviewed in Ref. 6). More ubiquitous is the inhibition by the ⑀ subunit, which was noticed since the early stage of studies of ATP synthase (7-9). The ⑀ subunit is a small subunit of 130 -140 residues consisting of two distinct domains, an N-terminal ␤-sandwich domain and a C-terminal helical hairpin domain (10, 11). Accumulating biochemical and structural studies have revealed that the ⑀ subunit can adopt at least two different conformational states in F 1 and F 0 F 1 , "down"-state and "up"-state (12)(13)(14)(15)(16)(17)(18)(19). The structures of the isolated ⑀ from Escherichia coli represent the down-state conformation that does not exhibit the inhibitory effect. The exact conformation of the upstate ⑀ subunit in F 1 and F 0 F 1 is not known, but it is certain that the C-terminal helical hairpin in the down state is opened in the up state (19) and comes in contact with the ␣ and ␤ subunits (20). The up-state ⑀ exerts the inhibitory effect on ATP hydrolysis activity but, interestingly, ...