Stiffness of γ subunit of F1-ATPase

F₁-ATPase is a molecular motor in which the γ subunit rotates inside the α₃β₃ ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F₁-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F₁ and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F₁ from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the α₃β₃ ring, and the complex of the external part of the γ subunit and the α₃β₃ ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F_o and F₁-ATPase.     

Does F1-ATPase subunit gamma turn in the wrong direction?

Analyzing the direction of F1-ATPase subunit gamma rotation, its shape and non-random distribution of surface residues, a mechanism is proposed for how gamma induces the closing/opening of the catalytic sites at beta/alpha interfaces: by keeping contact with the mobile domain of subunits beta at the 'jaw' (D386, the seven consecutive hydrophobic residues and D394/E395), rotating gamma works as a screw conveyer within the barrel of (alpha,beta)3. Mutations of the conveyer contacts are predicted to inhibit. Rotating wheel cartoons illustrate enzyme turnover and conformational changes. Steric clashes, polar interactions and also substrate limitations lead to specific stops. Because it is constructed as a stepper, gamma prevents uncoupling at high energy charge.     

Does F1-ATPase subunit γ turn in the wrong direction?

Analyzing the direction of F₁-ATPase subunit γ rotation, its shape and non-random distribution of surface residues, a mechanism is proposed for how γ induces the closing/opening of the catalytic sites at β/α interfaces: by keeping contact with the mobile domain of subunits β at the ‘jaw’ (D386, the seven consecutive hydrophobic residues and D394/E395), rotating γ works as a screw conveyer within the barrel of (α,β)₃. Mutations of the conveyer contacts are predicted to inhibit. Rotating wheel cartoons illustrate enzyme turnover and conformational changes. Steric clashes, polar interactions and also substrate limitations lead to specific stops. Because it is constructed as a stepper, γ prevents uncoupling at high energy charge.     

Cyclic AMP-dependent phosphorylation of the precursor to beta subunit of mitochondrial F1-ATPase: a physiological mistake?

By using the purified rat liver protein for reference in electrophoresis and peptide mapping experiments, I have identified the beta subunit of mitochondrial F1-ATPase and its cytoplasmic precursor in two-dimensional gel patterns of proteins from S49 mouse lymphoma cells. The beta subunit precursor is a substrate for cAMP-dependent phosphorylation during its synthesis. Normally, both nonphosphorylated and phosphorylated forms of beta subunit precursor are processed rapidly to the smaller, more acidic forms of mature beta subunit. When processing is inhibited with valinomycin, both nonphosphorylated and phosphorylated forms of beta subunit precursor are stabilized. Nonphosphorylated beta subunit is one of the most stable of cellular proteins, but the phosphorylated form is eliminated within minutes of processing. This suggests that phosphorylated beta subunit is recognized as aberrant and excluded from assembly into the ATPase complex. These results argue that cAMP-dependent phosphorylation of the beta subunit precursor is a physiological mistake that is remedied after mitochondrial import and processing.     

On the role of excitonic interactions in carotenoid–phthalocyanine dyads and implications for photosynthetic regulation

In two recent studies, energy transfer was reported in certain phthalocyanine–carotenoid dyads between the optically forbidden first excited state of carotenoids (Car S₁) and phthalocyanines (Pcs) in the direction Pc → Car S₁ (Kloz et al., J Am Chem Soc 133:7007–7015, 2011) as well as in the direction Car S₁ → Pc (Liao et al., J Phys Chem A 115:4082–4091, 2011). In this article, we show that the extent of this energy transfer in both directions is closely correlated in these dyads. This correlation and the additional observation that Car S₁ is instantaneously populated after Pc excitation provides evidence that in these compounds excitonic interactions can occur. Besides pure energy transfer and electron transfer, this is the third type of tetrapyrrole–carotenoid interaction that has been shown to occur in these model compounds and that has previously been proposed as a photosynthetic regulation mechanism. We discuss the implications of these models for photosynthetic regulation. The findings are also discussed in the context of a model in which both electronic states are disordered and in which the strength of the electronic coupling determines whether energy transfer, excitonic coupling, or electron transfer occurs.

     

Binding of phytopolyphenol piceatannol disrupts β/γ subunit interactions and rate-limiting step of steady-state rotational catalysis in Escherichia coli F1-ATPase

In observations of single molecule behavior under V(max) conditions with minimal load, the F(1) sector of the ATP synthase (F-ATPase) rotates through continuous cycles of catalytic dwells (～0.2 ms) and 120° rotation steps (～0.6 ms). We previously established that the rate-limiting transition step occurs during the catalytic dwell at the initiation of the 120° rotation. Here, we use the phytopolyphenol, piceatannol, which binds to a pocket formed by contributions from α and β stator subunits and the carboxyl-terminal region of the rotor γ subunit. Piceatannol did not interfere with the movement through the 120° rotation step, but caused increased duration of the catalytic dwell. The duration time of the intrinsic inhibited state of F(1) also became significantly longer with piceatannol. All of the beads rotated at a lower rate in the presence of saturating piceatannol, indicating that the inhibitor stays bound throughout the rotational catalytic cycle. The Arrhenius plot of the temperature dependence of the reciprocal of the duration of the catalytic dwell (catalytic rate) indicated significantly increased activation energy of the rate-limiting step to trigger the 120° rotation. The activation energy was further increased by combination of piceatannol and substitution of γ subunit Met(23) with Lys, indicating that the inhibitor and the β/γ interface mutation affect the same transition step, even though they perturb physically separated rotor-stator interactions.     

The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase

The crystal structure of mitochondrial F₁-ATPase indicatesthat the and subunits fold into a structure defined by threedomains: the top -barrel domain, the middle nucleotide-binding domain,and the C-terminal -helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The -barrel domains of the and subunits form a crown structure at the top ofF₁, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the -barrel domain in the and subunits of the yeast Saccharomyces cerevisiae F₁,with regard to its folding and assembly, was investigated. The -barreldomains of yeast F₁ and subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the -barrel domain of the subunit formed a largeaggregate structure, while the domain of the subunit waspredominately a monomer or dimer. However, coexpression of the -barreldomain of subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the -barrel domain of the and subunits interact specifically with each other and thatthese interactions prevent the aggregation of the -barrel domain of the subunit. These results mimic in vivo results and suggest thatthe interactions of the -barrel domains may be critical during thefolding and assembly of F₁.     

Amino acid substitutions in the ε-subunit of the F1F0-ATPase of Escherichia coli

A mutant strain of Escherichia coli was isolated in which Gly-48 of the mature ε-subunit of the energy-transducing adenosine triphosphatase was replaced by Asp. This amino acid substitution caused inhibition of ATPase activity (about 70%), loss of ATP-dependent proton translocation and lowered oxidative phosphorylation, but did not affect proton translocation through the F₀. Purified F₁-ATPase from the mutant strain bound to stripped membranes with the same affinity as the normal F₁-ATPase. Partial revertant strains were isolated in which Pro-47 of the ε-subunit was replaced by Ser or Thr. Pro-47 and Gly-48 are predicted to be residues 2 and 3 in a Type II β-turn and the Gly-48 to Asp substitution is predicted to cause a change from a Type II to a Type I or III β-turn. Space-filling models of the β-turn (residues 46–49) in the normal, mutant and partial revertant ε-subunits indicate that the peptide oxygen between Pro-47 and Gly-48 is in a different position to the peptide oxygen between Pro-47 and Asp-48 and that the substitution of Pro-47 by either Ser or Thr restores an oxygen close to the original position. It is suggested that the peptide oxygen between Pro-47 and Gly-48 of the ε-subunit is involved either structurally in inter-subunit H-bonding or directly in proton movements through the F₁-ATPase.     

Immunological detection of the mitochondrial F1-ATPase alpha subunit in the matrix of rat liver peroxisomes. A protein involved in organelle biogenesis?

Rat liver peroxisomes contain in their matrix the alpha-subunit of the mitochondrial F1-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No beta-subunit of the mitochondrial F1-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature alpha-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the alpha-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.     

Regulation of ATP hydrolysis by the ε subunit, ζ subunit and Mg-ADP in the ATP synthase of Paracoccus denitrificans

F₁F_O-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.     

The inhibitor peptide of the mitochondrial F1 · F0-ATPase interacts with calmodulin and stimulates the calmodulin-dependent Ca2+-ATPase of erythrocytes

The binding of calmodulin to the mitochondrial F₁ · F₀-ATPase has been studied. [^125I]Iodoazidocalmodulin binds to the ε-subunit and to the endogeneous ATPase inhibitor peptide in a Ca²⁺-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca²⁺-ATPase of erythrocytes has also been analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca²⁺-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the two mechanisms of activation are different. These in vitro effects of the two regulatory proteins may reflect a common origin of the two ATPases considered and/or of the regulatory proteins.     

A novel role of proteasomal β1 subunit in tumorigenesis

p27^Kip1 is a key cell-cycle regulator whose level is primarily regulated by the ubiquitin–proteasome degradation pathway. Its β1 subunit is one of seven β subunits that form the β-ring of the 20S proteasome, which is responsible for degradation of ubiquitinated proteins. We report here that the β1 subunit is up-regulated in oesophageal cancer tissues and some ovarian cancer cell lines. It promotes cell growth and migration, as well as colony formation. β1 binds and degrades p27^Kip1directly. Interestingly, the lack of phosphorylation at Ser¹⁵⁸ of the β1 subunit promotes degradation of p27^Kip1. We therefore propose that the β1 subunit plays a novel role in tumorigenesis by degrading p27^Kip1.     

Removal of the ϵ subunit from Escherichia coli F1-ATPase using monoclonal anti-ϵ antibody affinity chromatography

The usefulness of two monoclonal antibodies, ϵ-1 and ϵ-4, which recognize the ϵ subunit of Escherichia coli F₁-ATPase, for removing that subunit from ATPase was assessed. The ϵ subunit is a tightly bound, but dissociable, inhibitor of the ATPase. ϵ-1 binds ϵ with 10-fold higher affinity than ϵ-4. ϵ-1 recognizes a site on ϵ which is hidden by the quaternary structure of ATPase, while ϵ-4 can recognize ϵ when it is part of ATPase. Each antibody was purified and coupled to Sepharose to generate affinity columns. Solutions of ATPase in a buffer which was designed to reduce the affinity of ϵ for the enzyme were pumped through the columns and the degree of ϵ depletion was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blotting. Neither column retained ATPase significantly. At low ATPase concentrations and low flow rates, the ϵ-1 column was more efficient than the ϵ-4 column, removing in excess of 95% of the ϵ in a single passage compared with 93% removal by the ϵ-4 column. At higher protein concentrations or flow rates, however, the performance of the ϵ-1 column was substantially poorer, while that of the ϵ-4 column was much less affected. Very little ϵ emerged from the ϵ-4 column before most of the measured ϵ-binding capacity was filled. A second passage through the ϵ-4 column reduced residual ϵ to less than 2% of that which was originally present. Pure, active ϵ was eluted from either column by 1 m NH₄OH, pH 11. The relatively poor performance of ϵ-1 is discussed in terms of the low availability of the epitope and the tendency of the ϵ-depleted complex to compete with ϵ-1 for residual ϵ subunit. From consideration of these factors it appears likely that antibodies which recognize exposed epitopes will generally be more effective than antibodies which recognize cryptic epitopes in removing spontaneously dissociable subunits from protein complexes.