Hybrid Rhodospirillum rubrum F0F1 ATP synthases containing spinach chloroplast F1 β or α and β subunits reveal the essential role of the α subunit in ATP synthesis and tentoxin sensitivity

Ward C. Tucker, Ziyun Du, Ray Hein, Mark L. Richter, Zippora Gromet-Elhanan

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Abstract

Trace amounts (~5%) of the chloroplast α subunit were found to be absolutely required for effective restoration of catalytic function to LiCl- treated chromatophores of Rhodospirillum rubrum with the chloroplast subunit (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072). To clarify the role of the α subunit in the rebinding of β, restoration of catalytic function, and conferral of sensitivity to the chloroplast-specific inhibitor tentoxin, LiCl-treated chromatophores were analyzed by immunoblotting before and after reconstitution with mixtures of R. rubrum and chloroplast α and β subunits. The treated chromatophores were found to have lost, in addition to most of their β subunits, approximately a third of the α subunits, and restoration of catalytic activity required rebinding of both subunits. The hybrid reconstituted with the R. rubrum α and chloroplast β subunits was active in ATP synthesis as well as hydrolysis, and both activities were completely resistant to tentoxin. In contrast, a hybrid reconstituted with both chloroplast α and β subunits restored only a MgATPase activity, which was fully inhibited by tentoxin. These results indicate that all three copies of the R. rubrum α subunit are required for proton-coupled ATP synthesis, whereas for conferral of tentoxin sensitivity at least one copy of the chloroplast α subunit is required together with the chloroplast subunit. The hybrid system was further used to examine the effects of amino acid substitution at position 83 of the β subunit on sensitivity to tentoxin.

Original languageEnglish (US)
Pages (from-to)906-912
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number2
DOIs
StatePublished - Jan 14 2000

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Rhodospirillum rubrum
Spinacia oleracea
Chloroplasts
Adenosine Triphosphate
Restoration
Chromatophores
Hybrid systems
Protons
Hydrolysis
Catalyst activity
Substitution reactions
tentoxin
Amino Acid Substitution
Amino Acids
Immunoblotting
Catalytic Domain

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Hybrid Rhodospirillum rubrum F0F1 ATP synthases containing spinach chloroplast F1 β or α and β subunits reveal the essential role of the α subunit in ATP synthesis and tentoxin sensitivity. / Tucker, Ward C.; Du, Ziyun; Hein, Ray; Richter, Mark L.; Gromet-Elhanan, Zippora.

In: Journal of Biological Chemistry, Vol. 275, No. 2, 14.01.2000, p. 906-912.

Research output: Contribution to journalArticle

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abstract = "Trace amounts (~5{\%}) of the chloroplast α subunit were found to be absolutely required for effective restoration of catalytic function to LiCl- treated chromatophores of Rhodospirillum rubrum with the chloroplast subunit (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072). To clarify the role of the α subunit in the rebinding of β, restoration of catalytic function, and conferral of sensitivity to the chloroplast-specific inhibitor tentoxin, LiCl-treated chromatophores were analyzed by immunoblotting before and after reconstitution with mixtures of R. rubrum and chloroplast α and β subunits. The treated chromatophores were found to have lost, in addition to most of their β subunits, approximately a third of the α subunits, and restoration of catalytic activity required rebinding of both subunits. The hybrid reconstituted with the R. rubrum α and chloroplast β subunits was active in ATP synthesis as well as hydrolysis, and both activities were completely resistant to tentoxin. In contrast, a hybrid reconstituted with both chloroplast α and β subunits restored only a MgATPase activity, which was fully inhibited by tentoxin. These results indicate that all three copies of the R. rubrum α subunit are required for proton-coupled ATP synthesis, whereas for conferral of tentoxin sensitivity at least one copy of the chloroplast α subunit is required together with the chloroplast subunit. The hybrid system was further used to examine the effects of amino acid substitution at position 83 of the β subunit on sensitivity to tentoxin.",
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