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Molecular & Cellular Proteomics 4:1653-1663, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Protein Complexes in the Archaeon Methanothermobacter thermautotrophicus Analyzed by Blue Native/SDS-PAGE and Mass Spectrometry^*,S

Murtada H. Farhoud^,, Hans J. C. T. Wessels^,, Peter J. M. Steenbakkers^,¶,||, Sandy Mattijssen^¶, Ron A. Wevers^**, Baziel G. van Engelen, Mike S. M. Jetten^¶, Jan A. Smeitink, Lambert P. van den Heuvel and Jan T. Keltjens^¶

From the Nijmegen Center for Mitochondrial and Metabolic Disorders, Radboud University Nijmegen Medical Center, Geert Grooteplein 10, 6500 HB Nijmegen, ^¶ Department of Microbiology, Faculty of Science, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, ^** Department of Pediatrics and Neurology, Radboud University Nijmegen Medical Center, Reinier Postlaan 4, 6525 GC Nijmegen, and Neuromuscular Center Nijmegen, Department of Neurology, Radboud University Nijmegen Medical Center, Reinier Postlaan 4, 6525 GC Nijmegen, The Netherlands

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N⁵-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.

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