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

Abstract

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 proteomewide 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 N 5 -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.

Sequence-specific RNA binding mediated by the RNase PH domain of components of the exosome

“…Binding to the wider PH pore side of the complex could not be ruled out. Furthermore, stoichiometry of exosomal subunits varies in archaeal species (Evguenieva-Hackenberg et al 2003;Farhoud et al 2005) and the existence of numerous independent complexes cannot be excluded. In fact, a recent immunolocalization study highlighted the possible existence of numerous subexosomal complexes in Drosophila cells (Graham et al 2006).…”

Immunogold Localization of Key Metabolic Enzymes in the Anammoxosome and on the Tubule-Like Structures of Kuenenia stuttgartiensis

“…The PageRuler Plus prestained protein ladder (Thermo Scientific, USA) was used as a molecular weight marker. Subunit identification of all enzymes was performed by MALDI-TOF MS as described previously (1,26). In brief, gel plugs were picked from visible protein bands and subjected to tryptic digestion and MALDI-TOF MS analysis on a Bruker III mass spectrometer (Bruker Daltonik, Germany).…”

A New Addition to the Cell Plan of Anammox Bacteria: "Candidatus Kuenenia stuttgartiensis" Has a Protein Surface Layer as the Outermost Layer of the Cell

“…The peptides were extracted from the gel pieces by using a mix of 0.05% trifluoroacetic acid and 50% acetonitrile. For MALDI-TOF MS, the samples were applied onto a MALDI plate using ␣-cyano-hydroxy cinnamic acid as matrix and analyzed by using a Bruker Biflex III MALDI-TOF MS instrument (44). For LC-MS/ MS, the gel pieces were analyzed as described previously (45).…”