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Isolation of a complete A1AO ATP synthase comprising nine subunits from the hyperthermophile Methanococcus jannaschii

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Abstract

Archaeal A1AO ATP synthase/ATPase operons are highly conserved among species and comprise at least nine genes encoding structural proteins. However, all A1AO ATPase preparations reported to date contained only three to six subunits and, therefore, the study of this unique class of secondary energy converters is still in its infancy. To improve the quality of A1AO ATPase preparations, we chose the hyperthermophilic, methanogenic archaeon Methanococcus jannaschii as a model organism. Individual subunits of the A1AO ATPase from M. jannaschii were produced in E. coli, purified, and antibodies were raised. The antibodies enabled the development of a protocol ensuring purification of the entire nine-subunit A1AO ATPase. The ATPase was solubilized from membranes of M. jannaschii by Triton X-100 and purified to apparent homogeneity by sucrose density gradient centrifugation, ion exchange chromatography, and gel filtration. Electron micrographs revealed the A1 and AO domains and the central stalk, but also additional masses which could represent a second stalk. Inhibitor studies were used to demonstrate that the A1 and AO domains are functionally coupled. This is the first description of an A1AO ATPase preparation in which the two domains (A1 and AO) are fully conserved and functionally coupled.

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Acknowledgments

This work was supported by a grant from the Deutsche Forschungsgemeinschaft. We are indebted to Holger Kavermann and Volker Kuhle for their contributions to the expression studies. We thank Thorsten Lemker for his support in protein purification.

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Authors and Affiliations

  1. Microbiology Section, Department of Biology I, Ludwig-Maximilians-Universität München, Maria-Ward-Strasse 1a, 80638, Munich, Germany

    Astrid Lingl & Volker Müller

  2. Lehrstuhl für Mikrobiologie, Universität Regensburg, Regensburg, Germany

    Harald Huber & Karl O. Stetter

  3. Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Göttingen, Germany

    Frank Mayer

  4. Max-Planck-Institut für Biochemie, Martinsried, Germany

    Josef Kellermann

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  1. Astrid Lingl
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  2. Harald Huber
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  3. Karl O. Stetter
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  4. Frank Mayer
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  5. Josef Kellermann
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  6. Volker Müller
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Correspondence to Volker Müller.

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Communicated by G. Antranikian

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Lingl, A., Huber, H., Stetter, K.O. et al. Isolation of a complete A1AO ATP synthase comprising nine subunits from the hyperthermophile Methanococcus jannaschii . Extremophiles 7, 249–257 (2003). https://doi.org/10.1007/s00792-003-0318-7

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  • DOI: https://doi.org/10.1007/s00792-003-0318-7

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