Regular Article
Amino Acid Substitutions in theaSubunit Affect the ϵ Subunit of F1F0ATP Synthase fromEscherichia coli

https://doi.org/10.1006/abbi.1998.0995Get rights and content

Abstract

Amino acid substitutions at many positions in theasubunit of F1F0ATP synthase result in impaired proton translocation and altered catalytic activity. In this work, we demonstrate that amino acid substitutions in theasubunit affect the ϵ subunit. In mutant F1F0ATP synthases, the ϵ subunit was studied by determining its sensitivity to proteolysis and by chemical crosslinking under conditions of active turnover and in quiescent enzyme. Like native F1F0ATP synthase, the ϵ subunit in enzymes carrying either theaarg-210→ileoragly-218→aspsubstitutions proved resistant to trypsin digestion during ATP hydrolysis. In each case, the ϵ subunit was rapidly digested in the presence of a nonhydrolyzable ligand, but this did not result in the activation of hydrolytic activity typically seen in wild-type enzyme. In enzyme carrying theaala-217→argsubstitution, the trypsin digestion of the ϵ subunit occurred regardless of ligand and was accompanied by a limited hydrolytic activation. Relative to the native F1F0ATP synthase, theaala-217→argsubstitution resulted in reduced efficiency of crosslinking between the ϵ and β subunits using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. These observations indicate that the structural changes resulting from amino acid substitutions in theasubunit are propagated to the ϵ subunit and are specific to the individual substitutions.

References (35)

  • J. Weber et al.

    FEBS Lett.

    (1997)
  • S. Engelbrecht et al.

    FEBS Lett.

    (1997)
  • G.B. Cox et al.

    J. Mol. Biol.

    (1993)
  • Y. Zhang et al.

    J. Biol. Chem.

    (1994)
  • Y. Zhang et al.

    J. Biol. Chem.

    (1995)
  • S.D. Watts et al.

    J. Biol. Chem.

    (1996)
  • S.D. Watts et al.

    J. Biol. Chem.

    (1997)
  • R. Aggeler et al.

    Biochim. Biophys. Acta

    (1995)
  • R. Aggeler et al.

    J. Biol. Chem.

    (1996)
  • M. Kuki et al.

    J. Biol. Chem.

    (1988)
  • A.J. Rodgers et al.

    J. Biol. Chem.

    (1997)
  • S.D. Dunn et al.

    J. Biol. Chem.

    (1998)
  • B.D. Cain et al.

    J. Biol. Chem.

    (1989)
  • P.E. Hartzog et al.

    J. Biol. Chem.

    (1994)
  • F.I. Valiyaveetil et al.

    J. Biol. Chem.

    (1997)
  • R. Aggeler et al.

    J. Biol. Chem.

    (1997)
  • D.L. Foster et al.

    J. Biol. Chem.

    (1980)
  • Cited by (20)

    • Intrinsic uncoupling in the ATP synthase of Escherichia coli

      2008, Biochimica et Biophysica Acta - Bioenergetics
      Citation Excerpt :

      The possibility of modulating the coupling efficiency might require the existence of a structural device acting somehow as a clutch. The ɛ subunit appears to us as a possible candidate for this role, since it has frequently been indicated as a key regulatory and structural feature in the coupling mechanism (see e.g. [13–18,54] and for reviews [55–58]). In particular, the drastic changes in the ɛ trypsinization pattern induced by Pi [48] are consistent with Pi triggering the interconversion between two conformations, and the occurrence of drastically different conformations in this subunit has been confirmed by structural [59,60] and mutational studies [38,39].

    • The role of transmembrane span 2 in the structure and function of subunit a of the ATP synthase from Escherichia coli

      2003, Archives of Biochemistry and Biophysics
      Citation Excerpt :

      Assays were conducted in the presence of LDAO, as an indicator of the level of F1 ATPase present in the membrane preparations. Some mutations in ATP synthase subunits are known to cause variable inhibition of ATP hydrolysis due to the ε subunit [48,49]. Since LDAO relieves inhibition by ε [50], its inclusion in the assay medium will result in a rate of ATP hydrolysis that correlates with the amount of F1 present.

    • Genetic fusions of globular proteins to the ε subunit of the Eescherichia coli ATP synthase. Implications for in vivo rotational catalysis and ε subunit function

      2002, Journal of Biological Chemistry
      Citation Excerpt :

      In addition, Mendel-Hartvig and Capaldi (54) found that the nucleotide-dependent conformational change of ε in ATP synthase was blocked by treatment with DCCD, again implying that partial rotation of the N-terminal domain of ε, and the attached ring of c subunits, is an essential part of the change. Recently, Gardner and Cain (59) reported that nucleotide-dependent conformational changes in ε are impaired by mutations such as G218D in the a subunit. Furthermore, the residual ATPase activity of the aR210I mutant was uncoupled but DCCD-sensitive, like the larger ε fusions.

    View all citing articles on Scopus

    Funding for this investigation was provided by the United States Public Health Service Grant R01-GM43495.

    2

    To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, Box 100245, University of Florida, Gainesville, FL 32610. Fax: (352) 392-2953. E-mail:[email protected].

    View full text