Yasuyuki Kato-Yamada

Direct Observation of the Rotation of ε Subunit in F1-ATPase

Rotation of the ε subunit in F1-ATPase from thermophilic Bacillusstrain PS3 (TF1) was observed under a fluorescence microscope by the method used for observation of the γ subunit rotation (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299–302). The α3β3γε complex of TF1 was fixed to a solid surface, and fluorescently labeled actin filament was attached to the ε subunit through biotin-streptavidin. In the presence of ATP, the filament attached to ε subunit rotated in a unidirection. The direction of the rotation was the same as that observed for the γ subunit. The rotational velocity was slightly slower than the filament attached to the γ subunit, probably due to the experimental setup used. Thus, as suggested from biochemical studies (Aggeler, R., Ogilvie, I., and Capaldi, R. A. (1997)J. Biol. Chem. 272, 19621–19624), the ε subunit rotates with the γ subunit in F1-ATPase during catalysis.

Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators

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 ε 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 μM 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 FoF1-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

Highly coupled ATP synthesis by F1-ATPase single molecules

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. At 10 Hz, the mechanochemical coupling efficiency was low for the α3β3γ subcomplex (F1-ɛ), but reached up to 77% after reconstitution with the ɛ-subunit (F1+ɛ). We provide here direct evidence that F1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the ɛ-subunit has an essential function during ATP synthesis.

Structures of the thermophilic F1-ATPase ε subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1

The ε subunit of bacterial and chloroplast FoF1-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound ε subunit from a thermophilic Bacillus PS3 at 1.9-Å resolution. The C-terminal two α-helices were folded into a hairpin, sitting on the β 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 ε subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 ε 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 ε C-terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of FoF1-ATP synthase.

ε Subunit, an Endogenous Inhibitor of Bacterial F1-ATPase, Also Inhibits F0F1-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980)Biochemistry 19, 526–531), the ε subunit, an endogenous inhibitor of bacterial F1-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F0F1-ATPase. However, we report here that the ε subunit is exerting inhibition in F0F1-ATPase. We prepared a C-terminal half-truncated ε subunit (εΔC) of the thermophilicBacillus PS3 F0F1-ATPase and reconstituted F1- and F0F1-ATPase containing εΔC. Compared with F1- and F0F1-ATPase containing intact ε, those containing εΔC showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F0F1-ATPase containing εΔC was capable of ATP-driven H+ pumping. The time-course of pumping at low ATP concentration was faster than that by the F0F1-ATPase containing intact ε. Thus, the comparison with noninhibitory εΔC mutant shed light on the inhibitory role of the intact ε subunit in F0F1-ATPase.

The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the epsilon subunit.

In F1-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the β subunit moves and contacts the rotor γ subunit when the nucleotide fills the catalytic site, and the acidic nature of the motif was previously assumed to play a critical role in rotation. Our previous work, however, disproved the assumption (Hara, K. Y., Noji, H., Bald, D., Yasuda, R., Kinosita, K., Jr., and Yoshida, M. (2000) J. Biol. Chem. 275, 14260–14263), and the role of this motif remained unknown. Here, we found that the ε subunit, an intrinsic inhibitor, was unable to inhibit the ATPase activity of a mutant thermophilic F1-ATPase in which all of the five acidic residues in the DELSEED motif were replaced with alanines, although the ε subunit in the mutant F1-ATPase assumed the inhibitory form. In addition, the replacement of basic residues in the C-terminal region of the ε subunit by alanines caused a decrease of the inhibitory effect. Partial replacement of the acidic residues in the DELSEED motif of the β subunit or of the basic residues in the C-terminal α-helix of the ε subunit induced a partial effect. We here conclude that the ε subunit exerts its inhibitory effect through the electrostatic interaction with the DELSEED motif of the β subunit.

Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase

It has been proposed that C-terminal two α-helices of the ϵ subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F1 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 γ subunit and C terminus of the ϵ subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5′-Adenyl-β,γ-imidodiphosphate, ADP + \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{AlF}_{4}^{-}\) \end{document}, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic β 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 ϵ 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 ϵ 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 ϵ subunit acts as a sensor of ATP concentration in the cell.

Movement of the Helical Domain of the ε Subunit Is Required for the Activation of Thermophilic F1-ATPase

The inhibitory effect of ε subunit in F1-ATPase from thermophilic Bacillus PS3 was examined focusing on the structure-function relationship. For this purpose, we designed a mutant for ε subunit similar to the one constructed by Schulenberg and Capaldi (Schulenberg, B., and Capaldi, R. A. (1999) J. Biol. Chem. 274, 28351–28355). We introduced two cysteine residues at the interface of N-terminal β-sandwich domain (S48C) and C-terminal α-helical domain (N125C) of ε subunit. The α3β3γε complex containing the reduced form of this mutant ε subunit showed suppressed ATPase activity and gradual activation during the measurement. This activation pattern was similar to the complex with the wild type ε subunit. The conformation of the mutant ε subunit must be fixed and similar to the reported three-dimensional structure of the isolated ε subunit, when the intramolecular disulfide bridge was formed on this subunit by oxidation. This oxidized mutant ε subunit could form the α3β3γε complex but did not show any inhibitory effect. The complex was converted to the activated state, and the cross-link in the mutant ε subunit in the complex was efficiently formed in the presence of ATP-Mg, whereas no cross-link was observed without ATP-Mg, suggesting the conformation of the oxidized mutant ε subunit must be similar to that in the activated state complex. A non-hydrolyzable analog of ATP, 5′-adenylyl-β,γ-imidodiphosphate, could stimulate the formation of the cross-link on the ε subunit. Furthermore, the cross-link formation was stimulated by nucleotides even when this mutant ε subunit was assembled with a mutant α3β3γ complex lacking non-catalytic sites. These results indicate that binding of ATP to the catalytic sites induces a conformational change in the ε subunit and triggers transition of the complex from the suppressed state to the activated state.

Role of the ϵ Subunit of Thermophilic F1-ATPase as a Sensor for ATP

The ϵ subunit of F1-ATPase from the thermophilic Bacillus PS3 (TF1) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the ϵ subunit remains to be determined. To address this question, 11 mutants of the ϵ subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the α3β3γ complex of TF1. The ATPase activity of the resulting α3β3γϵ complexes was measured, and the extent of inhibition by the mutant ϵ subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the ϵ subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF1 by fixing the ϵ subunit into the folded conformation.

Isolated ε subunit of Bacillus subtilis F1-ATPase binds ATP

Previously, we demonstrated ATP binding to the isolated ε subunit of F1‐ATPase from thermophilic Bacillus PS3 [Kato‐Yamada Y., Yoshida M. (2003) J. Biol. Chem. 278, 36013]. However, whether it is a general feature of the ε subunit from other sources is yet unclear. Here, using a sensitive method to detect weak interactions between fluorescently labeled ε subunit and nucleotide, it was shown that the ε subunit of F1‐ATPase from Bacillus subtilis also bound ATP. The dissociation constant for ATP binding at room temperature was calculated to be 2 mM, which may be suitable for sensing cellular ATP concentration in vivo.