The subunit composition of mitochondrial NADH:ubiquinone oxidoreductase (complex I) from Pichia pastoris.

TitleThe subunit composition of mitochondrial NADH:ubiquinone oxidoreductase (complex I) from Pichia pastoris.
Publication TypeJournal Article
Year of Publication2010
AuthorsBridges, HR, Fearnley, IM, Hirst, J
JournalMol Cell Proteomics
Volume9
Issue10
Pagination2318-26
Date Published2010 Oct
ISSN1535-9484
KeywordsAmino Acid Sequence, Chromatography, High Pressure Liquid, Electron Transport Complex I, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Pichia, Recombinant Proteins, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Abstract

Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. It is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from Pichia pastoris, using a combination of proteomic and bioinformatic approaches. Forty-one subunits were identified in P. pastoris complex I, comprising the 14 core (conserved) subunits and 27 supernumerary subunits; seven of the core subunits are mitochondrial encoded. Three of the supernumerary subunits (named NUSM, NUTM, and NUUM) have not been observed previously in any species of complex I. However, homologues to all three of them are present in either Yarrowia lipolytica or Pichia angusta complex I. P. pastoris complex I has 39 subunits in common with Y. lipolytica complex I, 37 in common with N. crassa complex I, and 35 in common with the bovine enzyme. The mitochondrial encoded subunits (translated by the mold mitochondrial genetic code) retain their N-α-formyl methionine residues. At least eight subunits are N-α-acetylated, but the N-terminal modifications of the nuclear encoded subunits are not well-conserved. A combination of two methods of protein separation (SDS-PAGE and HPLC) and three different mass spectrometry techniques (peptide mass fingerprinting, tandem MS and molecular mass measurements) were required to define the protein complement of P. pastoris complex I. This requirement highlights the need for inclusive and comprehensive strategies for the characterization of challenging membrane-bound protein complexes containing both hydrophilic and hydrophobic components.

DOI10.1074/mcp.M110.001255
Alternate JournalMol. Cell Proteomics
Citation Key10.1074/mcp.M110.001255
PubMed ID20610779
PubMed Central IDPMC2953923
Grant ListMC_U105663141 / / Medical Research Council / United Kingdom