Updated on 2021/08/26

写真b

 
YAMADA Yasuyuki
 
*Items subject to periodic update by Rikkyo University (The rest are reprinted from information registered on researchmap.)
Affiliation*
College of Science Department of Life Science
Graduate School of Science Field of Study: Life Science
Graduate School of Science Field of Study: Life Science
Title*
Professor
Degree
博士(理学) ( 3 1999   東京工業大学 ) / 修士(理学) ( 3 1996   東京工業大学 )
Contact information
Mail Address
Research Theme*
  • タンパク質の構造変化がどのように機能に影響するかを明らかにする。主にFoF1-ATP合成酵素を材料として、その活性調節の分子機構を生物物理学的手法、生化学的手法により研究している。ATP合成酵素の調節サブユニットがどのような変化をすることで、ATP合成酵素複合体の活性をどのように制御しているかを、分子レベルで理解することをめざしている。

  • Research Interests
  • ATP synthase

  • regulation

  • allosteric

  • Campus Career*
    • 4 2015 - Present 
      College of Science   Department of Life Science   Professor
    • 4 2015 - Present 
      Graduate School of Science   Field of Study: Life Science   Professor
    • 4 2015 - Present 
      Graduate School of Science   Field of Study: Life Science   Professor
    • 4 2014 - 3 2015 
      Graduate School of Science   Field of Study: Life Science   Associate Professor
    • 4 2013 - 3 2014 
      Graduate School of Science   Field of Study: Life Science   Associate Professor
    • 4 2008 - 3 2015 
      College of Science   Department of Life Science   Associate Professor
    • 4 2004 - 3 2008 
      College of Science   Department of Life Science   Lecturer
     

    Research Areas

    • Life Science / Functional biochemistry

    • Life Science / Biophysics

    Research History

    • 4 2015 - Present 
      RIKKYO UNIVERSITY   College of Science Department of Life Science   Professor

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    • 4 2008 - 3 2015 
      RIKKYO UNIVERSITY   College of Science Department of Life Science   Associate Professor

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    • 4 2004 - 3 2008 
      RIKKYO UNIVERSITY   College of Science Department of Life Science   Lecturer

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    • 4 2003 - 3 2004 
      The University of Tokyo   Institute of Industrial Science

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    • 4 1999 - 3 2003 
      日本学術振興会   特別研究員(PD)

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    • 4 1998 - 3 1999 
      日本学術振興会   特別研究員(DC2)

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    Education

    • 4 1996 - 3 1999 
      Tokyo Institute of Technology   Graduate School, Division of Life Science and Engineering

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      Country: Japan

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    • 4 1994 - 3 1996 
      Tokyo Institute of Technology   Graduate School, Division of Life Science and Engineering

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      Country: Japan

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    • 4 1990 - 3 1994 
      Tokyo Institute of Technology   Faculty of Life Science and Engineering

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      Country: Japan

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    Awards

    • 10 2008  
      日本生化学会  平成20年度 日本生化学会奨励賞 
       
      山田 康之

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      Award type:Award from Japanese society, conference, symposium, etc.  Country:Japan

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    Papers

    • Evolution of Ribosomal Protein S14 Demonstrated by the Reconstruction of Chimeric Ribosomes in Bacillus subtilis. Peer-reviewed International journal

      Genki Akanuma, Fujio Kawamura, Satoru Watanabe, Masaki Watanabe, Fumiya Okawa, Yousuke Natori, Hideaki Nanamiya, Kei Asai, Taku Chibazakura, Hirofumi Yoshikawa, Akiko Soma, Takashi Hishida, Yasuyuki Kato-Yamada

      Journal of bacteriology203 ( 10 )   21 4 2021

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      Authorship:Last author   Language:English   Publishing type:Research paper (scientific journal)  

      Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn2+ binding motif and is ancestral. The second and third are the C- short and C- long types, neither of which contain a Zn2+ binding motif and which are ca. 90 residues and 100 residues in length, respectively. In the present study, the C+ type S14 from Bacillus subtilis ribosomes (S14BsC+) were completely replaced by the heterologous C- long type of S14 from Escherichia coli (S14Ec) or Synechococcus elongatus (S14Se). Surprisingly, S14Ec and S14Se were incorporated fully into 70S ribosomes in B. subtilis However, the growth rates as well as the sporulation efficiency of the mutants harboring heterologous S14 were significantly decreased. In these mutants, the polysome fraction was decreased and the 30S and 50S subunits accumulated unusually, indicating that cellular translational activity of these mutants was decreased. In vitro analysis showed a reduction in the translational activity of the 70S ribosome fraction purified from these mutants. The abundance of ribosomal proteins S2 and S3 in the 30S fraction in these mutants was reduced while that of S14 was not significantly decreased. It seems likely that binding of heterologous S14 changes the structure of the 30S subunit, which causes a decrease in the assembly efficiency of S2 and S3, which are located near the binding site of S14. Moreover, we found that S3 from S. elongatus cannot function in B. subtilis unless S14Se is present.IMPORTANCE S14, an essential ribosomal protein, may have evolved to adapt bacteria to zinc-limited environments by replacement of a zinc-binding motif with a zinc-independent sequence. It was expected that the bacterial ribosome would be tolerant to replacement of S14 because of the previous prediction that the spread of C- type S14 involved horizontal gene transfer. In this study, we completely replaced the C+ type of S14 in B. subtilis ribosome with the heterologous C- long type of S14 and characterized the resulting chimeric ribosomes. Our results suggest that the B. subtilis ribosome is permissive for the replacement of S14, but coevolution of S3 might be required to utilize the C- long type of S14 more effectively.

      DOI: 10.1128/JB.00599-20

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    • ATP-binding affinity of the ε subunit of thermophilic F1-ATPase under label-free conditions. Peer-reviewed International journal

      Miria Fujiwara, Yasuyuki Kato-Yamada

      Biochemistry and biophysics reports21   100725 - 100725   3 2020

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

      The ε subunits of several bacterial F1-ATPases bind ATP. ATP binding to the ε subunit has been shown to be involved in the regulation of F1-ATPase from thermophilic Bacillus sp. PS3 (TF1). We previously reported that the dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C is 4.3 μM by measuring changes in the fluorescence of the dye attached to the ε subunit (Kato, S. et al. (2007) J. Biol. Chem. 282, 37618). However, we have recently noticed that this varies with the dye used. In this report, to determine the affinity for ATP under label-free conditions, we have measured the competitive displacement of 2'(3')-O-N'-methylaniloyl-aminoadenosine-5'-triphosphate (Mant-ATP), a fluorescent analog of ATP, by ATP. The dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C was determined to be 0.29 μM, which is one order of magnitude higher affinity than previously reported values.

      DOI: 10.1016/j.bbrep.2020.100725

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    • C-terminal regulatory domain of the ε subunit of Fo F1 ATP synthase enhances the ATP-dependent H+ pumping that is involved in the maintenance of cellular membrane potential in Bacillus subtilis. Peer-reviewed International journal

      Genki Akanuma, Tomoaki Tagana, Maho Sawada, Shota Suzuki, Tomohiro Shimada, Kan Tanaka, Fujio Kawamura, Yasuyuki Kato-Yamada

      MicrobiologyOpen8 ( 8 ) e00815   8 2019

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

      The ε subunit of Fo F1 -ATPase/synthase (Fo F1 ) plays a crucial role in regulating Fo F1 activity. To understand the physiological significance of the ε subunit-mediated regulation of Fo F1 in Bacillus subtilis, we constructed and characterized a mutant harboring a deletion in the C-terminal regulatory domain of the ε subunit (ε∆C ). Analyses using inverted membrane vesicles revealed that the ε∆C mutation decreased ATPase activity and the ATP-dependent H+ -pumping activity of Fo F1 . To enhance the effects of ε∆C mutation, this mutation was introduced into a ∆rrn8 strain harboring only two of the 10 rrn (rRNA) operons (∆rrn8 ε∆C mutant strain). Interestingly, growth of the ∆rrn8 ε∆C mutant stalled at late-exponential phase. During the stalled growth phase, the membrane potential of the ∆rrn8 ε∆C mutant cells was significantly reduced, which led to a decrease in the cellular level of 70S ribosomes. The growth stalling was suppressed by adding glucose into the culture medium. Our findings suggest that the C-terminal region of the ε subunit is important for alleviating the temporal reduction in the membrane potential, by enhancing the ATP-dependent H+ -pumping activity of Fo F1 .

      DOI: 10.1002/mbo3.815

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    • Magnesium Suppresses Defects in the Formation of 70S Ribosomes as Well as in Sporulation Caused by Lack of Several Individual Ribosomal Proteins. Peer-reviewed International journal

      Genki Akanuma, Kotaro Yamazaki, Yuma Yagishi, Yuka Iizuka, Morio Ishizuka, Fujio Kawamura, Yasuyuki Kato-Yamada

      Journal of bacteriology200 ( 18 )   15 9 2018

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      Individually, the ribosomal proteins L1, L23, L36, and S6 are not essential for cell proliferation of Bacillus subtilis, but the absence of any one of these ribosomal proteins causes a defect in the formation of the 70S ribosomes and a reduced growth rate. In mutant strains individually lacking these ribosomal proteins, the cellular Mg2+ content was significantly reduced. The deletion of YhdP, an exporter of Mg2+, and overexpression of MgtE, the main importer of Mg2+, increased the cellular Mg2+ content and restored the formation of 70S ribosomes in these mutants. The increase in the cellular Mg2+ content improved the growth rate and the cellular translational activity of the ΔrplA (L1) and the ΔrplW (L23) mutants but did not restore those of the ΔrpmJ (L36) and the ΔrpsF (S6) mutants. The lack of L1 caused a decrease in the production of Spo0A, the master regulator of sporulation, resulting in a decreased sporulation frequency. However, deletion of yhdP and overexpression of mgtE increased the production of Spo0A and partially restored the sporulation frequency in the ΔrplA (L1) mutant. These results indicate that Mg2+ can partly complement the function of several ribosomal proteins, probably by stabilizing the conformation of the ribosome.IMPORTANCE We previously reported that an increase in cellular Mg2+ content can suppress defects in 70S ribosome formation and growth rate caused by the absence of ribosomal protein L34. In the present study, we demonstrated that, even in mutants lacking individual ribosomal proteins other than L34 (L1, L23, L36, and S6), an increase in the cellular Mg2+ content could restore 70S ribosome formation. Moreover, the defect in sporulation caused by the absence of L1 was also suppressed by an increase in the cellular Mg2+ content. These findings indicate that at least part of the function of these ribosomal proteins can be complemented by Mg2+, which is essential for all living cells.

      DOI: 10.1128/JB.00212-18

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    • Mechanistic Insights into the Activation of Soluble Guanylate Cyclase by Carbon Monoxide: A Multistep Mechanism Proposed for the BAY 41-2272 Induced Formation of 5-Coordinate CO-Heme. Peer-reviewed International journal

      Ryu Makino, Yuji Obata, Motonari Tsubaki, Tetsutaro Iizuka, Yuki Hamajima, Yasuyuki Kato-Yamada, Keisuke Mashima, Yoshitsugu Shiro

      Biochemistry57 ( 10 ) 1620 - 1631   13 3 2018

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      Soluble guanylate cyclase (sGC) is a heme-containing enzyme that catalyzes cGMP production upon sensing NO. While the CO adduct, sGC-CO, is much less active, the allosteric regulator BAY 41-2272 stimulates the cGMP productivity to the same extent as that of sGC-NO. The stimulatory effect has been thought to be likely associated with Fe-His bond cleavage leading to 5-coordinate CO-heme, but the detailed mechanism remains unresolved. In this study, we examined the mechanism under the condition including BAY 41-2272, 2'-deoxy-3'-GMP and foscarnet. The addition of these effectors caused the original 6-coordinate CO-heme to convert to an end product that was an equimolar mixture of a 5- and a new 6-coordinate CO-heme, as assessed by IR spectral measurements. The two types of CO-hemes in the end product were further confirmed by CO dissociation kinetics. Stopped-flow measurements under the condition indicated that the ferrous sGC bound CO as two reversible steps, where the primary step was assigned to the full conversion of the ferrous enzyme to the 6-coordinate CO-heme, and subsequently followed by the slower second step leading a partial conversion of the 6-coordinate CO-heme to the 5-coordinate CO-heme. The observed rates for both steps linearly depended on CO concentrations. The unexpected CO dependence of the rates in the second step supports a multistep mechanism, in which the 5-coordinate CO-heme is led by CO release from a putative bis-carbonyl intermediate that is likely provided by the binding of a second CO to the 6-coordinate CO-heme. This mechanism provides a new aspect on the activation of sGC by CO.

      DOI: 10.1021/acs.biochem.7b01240

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    • Essential Role of the ε Subunit for Reversible Chemo-Mechanical Coupling in F1-ATPase. Peer-reviewed International journal

      Rikiya Watanabe, Makoto Genda, Yasuyuki Kato-Yamada, Hiroyuki Noji

      Biophysical journal114 ( 1 ) 178 - 187   9 1 2018

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      F1-ATPase is a rotary motor protein driven by ATP hydrolysis. Among molecular motors, F1 exhibits unique high reversibility in chemo-mechanical coupling, synthesizing ATP from ADP and inorganic phosphate upon forcible rotor reversal. The ε subunit enhances ATP synthesis coupling efficiency to > 70% upon rotation reversal. However, the detailed mechanism has remained elusive. In this study, we performed stall-and-release experiments to elucidate how the ε subunit modulates ATP association/dissociation and hydrolysis/synthesis process kinetics and thermodynamics, key reaction steps for efficient ATP synthesis. The ε subunit significantly accelerated the rates of ATP dissociation and synthesis by two- to fivefold, whereas those of ATP binding and hydrolysis were not enhanced. Numerical analysis based on the determined kinetic parameters quantitatively reproduced previous findings of two- to fivefold coupling efficiency improvement by the ε subunit at the condition exhibiting the maximum ATP synthesis activity, a physiological role of F1-ATPase. Furthermore, fundamentally similar results were obtained upon ε subunit C-terminal domain truncation, suggesting that the N-terminal domain is responsible for the rate enhancement.

      DOI: 10.1016/j.bpj.2017.11.004

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    • The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase. Peer-reviewed International journal

      Alexander Krah, Yasuyuki Kato-Yamada, Shoji Takada

      PloS one12 ( 5 ) e0177907   2017

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:PUBLIC LIBRARY SCIENCE  

      The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

      DOI: 10.1371/journal.pone.0177907

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    • Pressure adaptation of 3-isopropylmalate dehydrogenase from an extremely piezophilic bacterium is attributed to a single amino acid substitution. Peer-reviewed International journal

      Yuki Hamajima, Takayuki Nagae, Nobuhisa Watanabe, Eiji Ohmae, Yasuyuki Kato-Yamada, Chiaki Kato

      Extremophiles : life under extreme conditions20 ( 2 ) 177 - 86   3 2016

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:SPRINGER JAPAN KK  

      3-Isopropylmalate dehydrogenase (IPMDH) from the extreme piezophile Shewanella benthica (SbIPMDH) is more pressure-tolerant than that from the atmospheric pressure-adapted Shewanella oneidensis (SoIPMDH). To understand the molecular mechanisms of this pressure tolerance, we analyzed mutated enzymes. The results indicate that only a single mutation at position 266, corresponding to Ala (SbIPMDH) and Ser (SoIPMDH), essentially affects activity under higher-pressure conditions. Structural analyses of SoIPMDH suggests that penetration of three water molecules into the cleft around Ser266 under high-pressure conditions could reduce the activity of the wild-type enzyme; however, no water molecule is observed in the Ala266 mutant.

      DOI: 10.1007/s00792-016-0811-4

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    • Ribosome dimerization is essential for the efficient regrowth of Bacillus subtilis. Peer-reviewed International journal

      Genki Akanuma, Yuka Kazo, Kazumi Tagami, Hirona Hiraoka, Koichi Yano, Shota Suzuki, Ryo Hanai, Hideaki Nanamiya, Yasuyuki Kato-Yamada, Fujio Kawamura

      Microbiology (Reading, England)162 ( 3 ) 448 - 458   3 2016

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:MICROBIOLOGY SOC  

      Ribosome dimers are a translationally inactive form of ribosomes found in Escherichia coli and many other bacterial cells. In this study, we found that the 70S ribosomes of Bacillus subtilis dimerized during the early stationary phase and these dimers remained in the cytoplasm until regrowth was initiated. Ribosome dimerization during the stationary phase required the hpf gene, which encodes a homologue of the E. coli hibernation-promoting factor (Hpf). The expression of hpf was induced at an early stationary phase and its expression was observed throughout the rest of the experimental period, including the entire 6 h of the stationary phase. Ribosome dimerization followed the induction of hpf in WT cells, but the dimerization was impaired in cells harbouring a deletion in the hpf gene. Although the absence of ribosome dimerization in these Hpf-deficient cells did not affect their viability in the stationary phase, their ability to regrow from the stationary phase decreased. Thus, following the transfer of stationary-phase cells to fresh LB medium, Δhpf mutant cells grew slower than WT cells. This observed lag in growth of Δhpf cells was probably due to a delay in restoring their translational activity. During regrowth, the abundance of ribosome dimers in WT cells decreased with a concomitant increase in the abundance of 70S ribosomes and growth rate. These results suggest that the ribosome dimers, by providing 70S ribosomes to the cells, play an important role in facilitating rapid and efficient regrowth of cells under nutrient-rich conditions.

      DOI: 10.1099/mic.0.000234

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    • High affinity nucleotide-binding mutant of the ε subunit of thermophilic F1-ATPase. Peer-reviewed International journal

      Yasuyuki Kato-Yamada

      Biochemical and biophysical research communications469 ( 4 ) 1129 - 32   22 1 2016

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      Authorship:Lead author, Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      Specific ATP binding to the ε subunit of thermophilic F1-ATPase has been utilized for the biosensors of ATP in vivo. I report here that the ε subunit containing R103A/R115A mutations can bind ATP with a dissociation constant at 52 nM, which is two orders of magnitude higher affinity than the wild type. The mutant retained specificity for ATP; ADP and GTP bound to the mutant with dissociation constants 16 and 53 μM, respectively. Thus, the mutant would be a good platform for various types of nucleotide biosensor with appropriate modifications.

      DOI: 10.1016/j.bbrc.2015.12.121

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    • Pressure effects on the chimeric 3-isopropylmalate dehydrogenases of the deep-sea piezophilic Shewanella benthica and the atmospheric pressure-adapted Shewanella oneidensis. Peer-reviewed International journal

      Yuki Hamajima, Takayuki Nagae, Nobuhisa Watanabe, Yasuyuki Kato-Yamada, Takeo Imai, Chiaki Kato

      Bioscience, biotechnology, and biochemistry78 ( 3 ) 469 - 71   2014

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:TAYLOR & FRANCIS LTD  

      The chimeric 3-isopropylmalate dehydrogenase enzymes were constructed from the deep-sea piezophilic Shewanella benthica and the shallow water Shewanella oneidensis genes. The properties of the enzymatic activities under pressure conditions indicated that the central region, which contained the active center and the dimer forming domains, was shown to be the most important region for pressure tolerance in the deep-sea enzyme.

      DOI: 10.1080/09168451.2014.890033

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    • Severe MgADP inhibition of Bacillus subtilis F1-ATPase is not due to the absence of nucleotide binding to the noncatalytic nucleotide binding sites. Peer-reviewed International journal

      Toru Ishikawa, Yasuyuki Kato-Yamada

      PloS one9 ( 9 ) e107197   2014

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:PUBLIC LIBRARY SCIENCE  

      F1-ATPase from Bacillus subtilis (BF1) is severely suppressed by the MgADP inhibition. Here, we have tested if this is due to the loss of nucleotide binding to the noncatalytic site that is required for the activation. Measurements with a tryptophan mutant of BF1 indicated that the noncatalytic sites could bind ATP normally. Furthermore, the mutant BF1 that cannot bind ATP to the noncatalytic sites showed much lower ATPase activity. It was concluded that the cause of strong MgADP inhibition of BF1 is not the weak nucleotide binding to the noncatalytic sites but the other steps required for the activation.

      DOI: 10.1371/journal.pone.0107197

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    • ε subunit of Bacillus subtilis F1-ATPase relieves MgADP inhibition. Peer-reviewed International journal

      Junya Mizumoto, Yuka Kikuchi, Yo-Hei Nakanishi, Naoto Mouri, Anrong Cai, Tokushiro Ohta, Takamitsu Haruyama, Yasuyuki Kato-Yamada

      PloS one8 ( 8 ) e73888   2013

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Public Library of Science  

      MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF1. We conclude that the ε subunit relieves BF1 from MgADP inhibition.

      DOI: 10.1371/journal.pone.0073888

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    • Hydrophobic shield on the molecular surface enhances thermal stability of ferredoxin of Cyanidioschyzon merolae Peer-reviewed

      Yuko Ueno, Yasuyuki Kato-Yamada, Takeo Imai

      Journal of Japanese Society for Extremophiles11   59 - 63   2012

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    • ATP binding to the ϵ subunit of thermophilic ATP synthase is crucial for efficient coupling of ATPase and H+ pump activities. Peer-reviewed International journal

      Fumitaka Kadoya, Shigeyuki Kato, Kei Watanabe, Yasuyuki Kato-Yamada

      The Biochemical journal437 ( 1 ) 135 - 40   1 7 2011

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:PORTLAND PRESS LTD  

      ATP binding to the ϵ subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ϵ subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282, 37618-37623]. In the present study, we report how ATP binding to the ϵ subunit affects ATPase and H+ pumping activities in the holoenzyme TFoF1. Wild-type TFoF1 showed significant H+ pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ϵ subunit, did not support efficient H+ pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H+ pumping activities. Similar uncoupling was observed when TFoF1 contained an ATP-binding-deficient ϵ subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ϵ subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ϵ subunit was in its extended-state conformation. The present study reveals a novel role of the ϵ subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1.

      DOI: 10.1042/BJ20110443

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    • Conformational transitions of subunit epsilon in ATP synthase from thermophilic Bacillus PS3. Peer-reviewed International journal

      Boris A Feniouk, Yasuyuki Kato-Yamada, Masasuke Yoshida, Toshiharu Suzuki

      Biophysical journal98 ( 3 ) 434 - 42   3 2 2010

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

      Subunit epsilon of bacterial and chloroplast F(O)F(1)-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit epsilon can adopt two conformations. In the "extended", inhibitory conformation, its two C-terminal alpha-helices are stretched along subunit gamma. In the "contracted", noninhibitory conformation, these helices form a hairpin. The transition of subunit epsilon from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59 degrees C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit epsilon and in the N-terminus of subunit gamma was used to follow the conformational transition in real time. It was found that ATP induced the conformational transition from the extended to the contracted state (half-maximum transition extent at 140 microM ATP). ADP could neither prevent nor reverse the ATP-induced conformational change, but it did slow it down. Acid residues in the DELSEED region of subunit beta were found to stabilize the extended conformation of epsilon. Binding of ATP directly to epsilon was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 microM) suggests that subunit epsilon probably adopts the extended state and strongly inhibits ATP hydrolysis only when the intracellular ATP level drops significantly below the normal value.

      DOI: 10.1016/j.bpj.2009.10.023

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    • Inhibition of thermophilic F1-ATPase by the ε subunit takes different path from the ADP-Mg inhibition. Peer-reviewed

      Takamitsu Haruyama, Yoko Hirono-Hara, Yasuyuki Kato-Yamada

      Biophysics (Nagoya-shi, Japan)6   59 - 65   2010

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

      The F1-ATPase, the soluble part of FoF1-ATP synthase, is a rotary molecular motor consisting of α3β3γδε. The γ and ε subunits rotate relative to the α3β3δ sub-complex on ATP hydrolysis by the β subunit. The ε subunit is known as an endogenous inhibitor of the ATPase activity of the F1-ATPase and is believed to function as a regulator of the ATP synthase. This inhibition by the ε subunit (ε inhibition) of F1-ATPase from thermophilic Bacillus PS3 was analyzed by single molecule measurements. By using a mutant ε subunit deficient in ATP binding, reversible transitions between active and inactive states were observed. Analysis of pause and rotation durations showed that the ε inhibition takes a different path from the ADP-Mg inhibition. Furthermore, the addition of the mutant ε subunit to the α3β3γ sub-complex was found to facilitate recovery of the ATPase activity from the ADP-Mg inhibition. Thus, it was concluded that these two inhibitions are essentially exclusive of each other.

      DOI: 10.2142/biophysics.6.59

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    • Modulation of nucleotide binding to the catalytic sites of thermophilic F(1)-ATPase by the epsilon subunit: implication for the role of the epsilon subunit in ATP synthesis. Peer-reviewed International journal

      Taichi Yasuno, Eiro Muneyuki, Masasuke Yoshida, Yasuyuki Kato-Yamada

      Biochemical and biophysical research communications390 ( 2 ) 230 - 4   11 12 2009

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      Effect of epsilon subunit on the nucleotide binding to the catalytic sites of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been tested by using alpha(3)beta(3)gamma and alpha(3)beta(3)gammaepsilon complexes of TF(1) containing betaTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the epsilon subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the epsilon subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.

      DOI: 10.1016/j.bbrc.2009.09.092

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    • Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Peer-reviewed International journal

      Hiromi Imamura, Kim P Huynh Nhat, Hiroko Togawa, Kenta Saito, Ryota Iino, Yasuyuki Kato-Yamada, Takeharu Nagai, Hiroyuki Noji

      Proceedings of the National Academy of Sciences of the United States of America106 ( 37 ) 15651 - 6   15 9 2009

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATL ACAD SCIENCES  

      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 epsilon subunit of the bacterial F(o)F(1)-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 muM 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(o)F(1)-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

      DOI: 10.1073/pnas.0904764106

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    • Role of the epsilon subunit of thermophilic F1-ATPase as a sensor for ATP. Peer-reviewed International journal

      Shigeyuki Kato, Masasuke Yoshida, Yasuyuki Kato-Yamada

      The Journal of biological chemistry282 ( 52 ) 37618 - 23   28 12 2007

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

      The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon 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 alpha(3)beta(3)gamma complex of TF(1). The ATPase activity of the resulting alpha(3)beta(3)gammaepsilon complexes was measured, and the extent of inhibition by the mutant epsilon 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 epsilon subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF(1) by fixing the epsilon subunit into the folded conformation.

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    • Structures of the thermophilic F1-ATPase epsilon subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1. Peer-reviewed International journal

      Hiromasa Yagi, Nobumoto Kajiwara, Hideaki Tanaka, Tomitake Tsukihara, Yasuyuki Kato-Yamada, Masasuke Yoshida, Hideo Akutsu

      Proceedings of the National Academy of Sciences of the United States of America104 ( 27 ) 11233 - 8   3 7 2007

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      Language:English   Publishing type:Research paper (scientific journal)  

      The epsilon subunit of bacterial and chloroplast F(o)F(1)-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound epsilon subunit from a thermophilic Bacillus PS3 at 1.9-A resolution. The C-terminal two alpha-helices were folded into a hairpin, sitting on the beta 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 epsilon subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 epsilon 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 epsilon C-terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of F(o)F(1)-ATP synthase.

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    • gammaepsilon Sub-complex of thermophilic ATP synthase has the ability to bind ATP. Peer-reviewed International journal

      Satoshi Iizuka, Shigeyuki Kato, Masasuke Yoshida, Yasuyuki Kato-Yamada

      Biochemical and biophysical research communications349 ( 4 ) 1368 - 71   3 11 2006

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      Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

      The isolated epsilon subunit of F(1)-ATPase from thermophilic Bacillus PS3 (TF(1)) binds ATP [Y. Kato-Yamada, M. Yoshida, J. Biol. Chem. 278 (2003) 36013]. The obvious question is whether the ATP binding concern with the regulation of ATP synthase activity or not. If so, the epsilon subunit even in the ATP synthase complex should have the ability to bind ATP. To check if the ATP binding to the epsilon subunit within the ATP synthase complex may occur, the gammaepsilon sub-complex of TF(1) was prepared and ATP binding was examined. The results clearly showed that the gammaepsilon sub-complex can bind ATP.

      DOI: 10.1016/j.bbrc.2006.09.001

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    • Isolated epsilon subunit of Bacillus subtilis F1-ATPase binds ATP. Peer-reviewed International journal

      Yasuyuki Kato-Yamada

      FEBS letters579 ( 30 ) 6875 - 8   19 12 2005

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      Authorship:Lead author, Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE BV  

      Previously, we demonstrated ATP binding to the isolated epsilon 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 epsilon subunit from other sources is yet unclear. Here, using a sensitive method to detect weak interactions between fluorescently labeled epsilon subunit and nucleotide, it was shown that the epsilon 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.

      DOI: 10.1016/j.febslet.2005.11.036

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    • Real-time monitoring of conformational dynamics of the epsilon subunit in F1-ATPase. Peer-reviewed International journal

      Ryota Iino, Tomoe Murakami, Satoshi Iizuka, Yasuyuki Kato-Yamada, Toshiharu Suzuki, Masasuke Yoshida

      The Journal of biological chemistry280 ( 48 ) 40130 - 4   2 12 2005

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

      It has been proposed that C-terminal two alpha-helices of the epsilon subunit of F1-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 gamma subunit and C terminus of the epsilon 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-beta,gamma-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic beta 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 epsilon subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 degrees 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 epsilon subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 degrees C reached to 0.67 mm, a consistent value to assume that the epsilon subunit acts as a sensor of ATP concentration in the cell.

      DOI: 10.1074/jbc.M506160200

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    • Highly coupled ATP synthesis by F1-ATPase single molecules. Peer-reviewed International journal

      Yannick Rondelez, Guillaume Tresset, Takako Nakashima, Yasuyuki Kato-Yamada, Hiroyuki Fujita, Shoji Takeuchi, Hiroyuki Noji

      Nature433 ( 7027 ) 773 - 7   17 2 2005

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

      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 alpha3beta3gamma subcomplex (F1-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F1+epsilon)). We provide here direct evidence that F1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the epsilon-subunit has an essential function during ATP synthesis.

      DOI: 10.1038/nature03277

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    • Planar lipid bilayer reconstitution with a micro-fluidic system. International journal

      Hiroaki Suzuki, Kazuhito Tabata, Yasuyuki Kato-Yamada, Hiroyuki Noji, Shoji Takeuchi

      Lab on a chip4 ( 5 ) 502 - 5   10 2004

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      Language:English   Publishing type:Research paper (scientific journal)  

      A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 microm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.

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    • Planar lipid bilayer reconstitution with a micro-fluidic system Peer-reviewed

      H Suzuki, K Tabata, Y Kato-Yamada, H Noji, S Takeuchi

      LAB ON A CHIP4 ( 5 ) 502 - 505   2004

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:ROYAL SOC CHEMISTRY  

      A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 mm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.

      DOI: 10.1039/b405967k

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    • Isolated epsilon subunit of thermophilic F1-ATPase binds ATP. Peer-reviewed International journal

      Yasuyuki Kato-Yamada, Masasuke Yoshida

      The Journal of biological chemistry278 ( 38 ) 36013 - 6   19 9 2003

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)  

      F1-ATPase, a soluble part of the F0F1-ATP synthase, has subunit structure alpha3beta3gammadeltaepsilon in which nucleotide-binding sites are located in the alpha and beta subunits and, as believed, in none of the other subunits. However, we report here that the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus strain PS3 can bind ATP. The binding was directly demonstrated by isolating the epsilon subunit-ATP complex with gel filtration chromatography. The binding was not dependent on Mg2+ but was highly specific for ATP; however, ADP, GTP, UTP, and CTP failed to bind. The epsilon subunit lacking the C-terminal helical hairpin was unable to bind ATP. Although ATP binding to the isolated epsilon subunits from other organisms has not been detected under the same conditions, a possibility emerges that the epsilon subunit acts as a built in cellular ATP level sensor of F0F1-ATP synthase.

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    • The role of the beta DELSEED motif of F-1-ATPase - Propagation of the inhibitory effect of the epsilon subunit Peer-reviewed

      KY Hara, Y Kato-Yamada, Y Kikuchi, T Hisabori, M Yoshida

      JOURNAL OF BIOLOGICAL CHEMISTRY276 ( 26 ) 23969 - 23973   6 2001

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

      In F-1-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the beta subunit moves and contacts the rotor gamma 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 E subunit, an intrinsic inhibitor, was unable to inhibit the ATPase activity of a mutant thermophilic F-1-ATPase in which all of the five acidic residues in the DELSEED motif were replaced with alanines, although the epsilon subunit in the mutant F-1-ATPase assumed the inhibitory form. In addition, the replacement of basic residues in the C-terminal region of the epsilon subunit by alanines caused a decrease of the inhibitory effect. Partial replacement of the acidic residues in the DELSEED motif of the beta subunit or of the basic residues in the C-terminal alpha -helix of the epsilon subunit induced a partial effect. We here conclude that the epsilon subunit exerts its inhibitory effect through the electrostatic interaction with the DELSEED motif of the beta subunit.

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    • Movement of the helical domain of the epsilon subunit is required for the activation of thermophilic F-1-ATPase Peer-reviewed

      Y Kato-Yamada, M Yoshida, T Hisabori

      JOURNAL OF BIOLOGICAL CHEMISTRY275 ( 46 ) 35746 - 35750   11 2000

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

      The inhibitory effect of epsilon subunit in F-1-ATPase from thermophilic Bacillus PS3 was examined focusing on the structure-function relationship. For this purpose, we designed: a mutant for epsilon 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 beta -sandwich domain (S48C) and C-terminal cu-helical domain (N125C) of epsilon subunit, The alpha (3)beta (3)gamma epsilon complex containing the reduced form of this mutant epsilon subunit showed suppressed ATPase activity and gradual activation during the measurement. This activation pattern was similar to the complex with the wild type epsilon subunit, The conformation of the mutant epsilon subunit must be fixed and similar to the reported three-dimensional structure of the isolated epsilon subunit, when the intramolecular disulfide bridge was formed on this subunit by oxidation. This oxidized mutant epsilon subunit could form the alpha (3)beta (3)gamma epsilon complex but did not show any inhibitory effect. The complex was converted to the activated state, and the cross-link in the mutant epsilon 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 epsilon subunit must be similar to that in the activated state complex. A non-hydrolyzable analog of ATP, 5'-adenylyl-beta,gamma -imidodiphosphate, could stimulate the formation of the cross-link on the epsilon subunit, Furthermore, the cross-link formation was stimulated by nucleotides even when this mutant epsilon subunit was assembled with a mutant alpha (3)beta (3)gamma complex lacking non-catalytic sites. These results indicate that binding of ATP to the catalytic sites induces a conformational change in the epsilon subunit and triggers transition of the complex from the suppressed state to the activated state.

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    • epsilon Subunit, an endogenous inhibitor of bacterial F-1-ATPase, also inhibits F0F1-ATPase Peer-reviewed

      Y Kato-Yamada, D Bald, M Koike, K Motohashi, T Hisabori, M Yoshida

      JOURNAL OF BIOLOGICAL CHEMISTRY274 ( 48 ) 33991 - 33994   11 1999

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

      Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F-1-ATPase, has long been thought not to inhibit activity of the holo enzyme, F0F1-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F0F1-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(Delta C)) of the thermophilic Bacillus PS3 F0F1-ATPase and reconstituted F-1- and F0F1-ATPase containing epsilon(Delta C). Compared with F-1- and F0F1-ATPase containing intact epsilon, those containing epsilon(Delta C) 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 epsilon(Delta C) 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 epsilon. Thus, the comparison with noninhibitory epsilon(Delta C) mutant shed light on the inhibitory role of the intact epsilon subunit in F0F1-ATPase.

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    • Direct observation of the rotation of epsilon subunit in F-1-ATPase Peer-reviewed

      Y Kato-Yamada, H Noji, R Yasuda, K Kinosita, M Yoshida

      JOURNAL OF BIOLOGICAL CHEMISTRY273 ( 31 ) 19375 - 19377   7 1998

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

      Rotation of the epsilon subunit in F-1-ATPase from thermophilic Bacillus strain PS3 (TF1) was observed under a fluorescence microscope by the method used for observation of the gamma subunit rotation (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302). The alpha(3)beta(3)gamma epsilon complex of TF, was fixed to a solid surface, and fluorescently labeled actin filament was attached to the epsilon subunit through biotin-streptavidin. In the presence of ATP, the filament attached to epsilon subunit rotated in a unidirection. The direction of the rotation was the same as that observed for the gamma subunit. The rotational velocity was slightly slower than the filament attached to the gamma 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 epsilon subunit rotates with the gamma subunit in F-1-ATPase during catalysis.

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    • Thermophilic F-1-ATPase is activated without dissociation of an endogenous inhibitor, epsilon subunit Peer-reviewed

      Y Kato, T Matsui, N Tanaka, E Muneyuki, T Hisabori, M Yoshida

      JOURNAL OF BIOLOGICAL CHEMISTRY272 ( 40 ) 24906 - 24912   10 1997

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

      Subunit complexes (alpha(3) beta(3) gamma, alpha(3) beta(3) gamma delta, alpha(3) beta(3) gamma epsilon, and alpha(3) beta(3) gamma delta epsilon) of thermophilic F-1-ATPase were prepared, and their catalytic properties were compared to know the role of delta and epsilon subunits in catalysis. The presence of delta subunit in the complexes had slight inhibitory effect on the ATPase activity, The effect of epsilon subunit was more profound, The (-epsilon) complexes, alpha(3) beta(3) gamma and alpha(3) beta(3) gamma delta, initiated ATP hydrolysis without a lag. In contrast, the (+epsilon) complexes, alpha(3) beta(3) gamma epsilon and alpha(3) beta(3) gamma delta epsilon, started hydrolysis of ATP (<700 mu M) with a lag phase that was gradually activated during catalytic turnover. As ATP concentration increased, the lag phase of the (+epsilon) complexes became shorter, and it was not observed above 1 mM ATP, Analysis of binding and hydrolysis of the ATP analog, 2',3'-O-(2,4,6-trinitrophenyl) ATP, suggested that the (+epsilon) complexes bound substrate only slowly. Differing from Escherichia coli F-1-ATPase, the activation of the (+epsilon) complexes from the lag phase was not due to dissociation of epsilon subunit since the re-isolated activated complex retained epsilon subunit. This indicates that there are two alternative forms of the (+epsilon) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.

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    • The regulatory functions of the gamma and epsilon subunits from chloroplast CF1 are transferred to the core complex, alpha(3)beta(3), from thermophilic bacterial F-1 Peer-reviewed

      T Hisabori, Y Kato, K Motohashi, P KrothPancic, H Strotmann, T Amano

      EUROPEAN JOURNAL OF BIOCHEMISTRY247 ( 3 ) 1158 - 1165   8 1997

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      Language:English   Publishing type:Research paper (scientific journal)   Publisher:SPRINGER VERLAG  

      The expression plasmids for the subunit gamma (gamma(c)) and the subunit epsilon (epsilon(c)) of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli, Both expressed subunits were obtained as inclusion bodies, When recombinant gamma(c) was mixed with recombinant alpha and beta subunits of F-1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (alpha(3) beta(3) gamma(c)) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCl2, which gamma(c). Hence, this complex has induces formation of a disulfide bond between two cysteine residues in similar modulation characteristics as CF1.
      The effects of recombinant epsilon(c) and epsilon subunit from TF1 (epsilon(t)) on alpha(3) beta(3) gamma(c) were also investigated. epsilon(c) strongly inhibited the ATP hydrolysis activity of chimeric alpha(3) beta(3) gamma(c) complex but epsilon(t) did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium, The addition of epsilon(c) or epsilon(t) to the alpha(3) beta(3) gamma(t) complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity.
      These results suggest that (a) the specific regulatory function of gamma(c) can be transferred to the bacterial subunit complex: (b) the interaction between the gamma(c) subunit and epsilon(c) strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the alpha(3) beta(3) core.

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    • ANALYSIS OF TIME-DEPENDENT CHANGE OF ESCHERICHIA-COLI F1-ATPASE ACTIVITY AND ITS RELATIONSHIP WITH APPARENT NEGATIVE COOPERATIVITY Peer-reviewed

      Y KATO, T SASAYAMA, E MUNEYUKI, M YOSHIDA

      BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS1231 ( 3 ) 275 - 281   10 1995

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      Authorship:Lead author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE BV  

      Except for the case of gradual activation of EF(1) (F-1-ATPase from Escherichia coli) caused by the dissociation of the epsilon subunit [Laget, P.P. and Smith, J.B. (1979) Arch. Biochem. Biophys. 197, 83-89], EF(1) has long been thought not to show a time-dependent change in activity [Senior, A.E. et al. (1992) Arch. Biochem. Biophys. 297, 340-344]. Here, we report the time-dependent inactivation and activation of EF(1), which are apparently similar to those of mitochondrial F-1-ATPases [Vasilyeva, E.A. et al, (1982) Biochem. J. 202, 15-23]. Analysis of these changes as a function of ATP concentrations in relation to negative cooperativity revealed that the initial inactivation phase was attributable to the decrease in the V-max associated with the low K-m (around 10 mu M), and the following activation, probably due to the dissociation of the epsilon subunit, corresponded to the increase in the V-max associated with the high K-m (in the order of 100 mu M). Thus, the time-dependent change in EF(1) activity is closely related to the apparent negative cooperativity (multiple K-m values) of ATP hydrolysis.

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    Misc.

    • On the regulatory roole of the epsilon subunit in ATP synthase Invited

      Yasuyuki KATO-YAMADA

      Seikagaku81 ( 11 )   11 2009

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      Authorship:Lead author, Last author, Corresponding author   Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (other)   Publisher:Japanese Biochemical Society  

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    Professional Memberships

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      American Society for Biochemistry and Molecular Biology

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      日本生化学会

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      日本生物物理学会

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    Research Projects

    • ATP合成酵素のATPモーターとプロトンモーターをつなぐ分子内クラッチ

      日本学術振興会  科学研究費助成事業 

      山田 康之

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      4 2015 - Present

      Authorship:Principal investigator  Grant type:Competitive

      ATP合成酵素に見られる、条件的脱共役状態の分子機構を明らかにする。

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    • 枯草菌ATP合成酵素の活性調節の包括的理解

      日本学術振興会  科学研究費助成事業 

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      4 2011 - 3 2016

      Grant type:Competitive

      枯草菌ATP合成酵素の活性調節機構の解明

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    • ATP合成酵素の回転モータ制御の分子機構

      文部科学省  科学研究費助成事業 

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      4 2006 - 3 2011

      Grant type:Competitive

      代表:久堀徹

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    • ATP合成酵素のεサブユニットへのATP結合と活性調節

      日本学術振興会  科学研究費助成事業 

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      4 2006 - 3 2008

      Grant type:Competitive

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    Industrial property rights

    • Fluorescently labeled fusion protein for assaying adenosine triphosphate.

      Hiroyuki Noji, Hiromi Imamura, Ryota Iino, Yasuyuki Yamada

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      Patent/Registration no:US Patent 08524447  Date issued:3 9 2013

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