Macromolecular complexes are essential players in numerous biological processes. They are often large, dynamic, and rather labile; approaches to study them are scarce. Covering masses up to ∼30 MDa, we separated the native complexome of rat heart mitochondria by blue-native and large-pore blue-native gel electrophoresis to analyze its constituents by mass spectrometry. Similarities in migration patterns allowed hierarchical clustering into interaction profiles representing a comprehensive analysis of soluble and membrane-bound complexes of an entire organelle. The power of this bottom-up approach was validated with well-characterized mitochondrial multiprotein complexes. TMEM126B was found to comigrate with known assembly factors of mitochondrial complex I, namely CIA30, Ecsit, and Acad9. We propose terming this complex mitochondrial complex I assembly (MCIA) complex. Furthermore, we demonstrate that TMEM126B is required for assembly of complex I. In summary, complexome profiling is a powerful and unbiased technique allowing the identification of previously overlooked components of large multiprotein complexes.

No abstract

Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.

A catalytically active subcomplex of respiratory chain complex I lacks 14 of its 42 subunits yet retains half of its proton-pumping capacity, indicating that its membrane arm has two pump modules.

Mitochondrial complex I is the largest and most complicated enzyme of the oxidative phosphorylation system. It comprises a number of so-called accessory subunits of largely unknown structure and function. Here we studied subunit NB4M [NDUFA6, LYR motif containing protein 6 (LYRM6)], a member of the LYRM family of proteins. Chromosomal deletion of the corresponding gene in the yeast Yarrowia lipolytica caused concomitant loss of the mitochondrial acyl carrier protein subunit ACPM1 from the enzyme complex and paralyzed ubiquinone reductase activity. Exchanging the LYR motif and an associated conserved phenylalanine by alanines in subunit NB4M also abolished the activity and binding of subunit ACPM1. We show, by single-particle electron microscopy and structural modeling, that subunits NB4M and ACPM1 form a subdomain that protrudes from the peripheral arm in the vicinity of central subunit domains known to be involved in controlling the catalytic activity of complex I. M itochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase, EC 1.6.5.3) is a 1-MDa membrane protein complex with a central function in cellular energy conversion (1). Redox-linked proton translocation by complex I contributes to the electrochemical proton gradient across the inner mitochondrial membrane that drives ATP synthesis by ATP synthase. Complex I dysfunction caused by mutations or toxins is associated with a number of neuromuscular and neurodegenerative human disorders, such as Parkinson's disease. Fourteen central subunits are conserved from bacteria to humans that harbor the core function of energy conversion. In eukaryotes, a substantial fraction of the mass of the holoenzyme is contributed by so-called accessory subunits (2). A broad range of functions from structural reinforcement to regulation of the enzyme complex has been suggested for the accessory subunits, but in most cases, their specific role remains unresolved. Electron microscopy (EM) (3) and X-ray crystallographic analysis of mitochondrial complex I at 6.3 Å resolution (4) revealed the arrangement of functional modules within the L-shaped complex of a highly hydrophobic membrane arm consisting of the proximal and distal pump-modules (P P and P D modules) and a hydrophilic peripheral arm extruding into the mitochondrial matrix that comprises the NADH oxidation and ubiquinone reduction modules (N and Q modules). However, information on the structure and position of individual accessory subunits is still very limited.In this study, we focused on accessory complex I subunit NB4M (alternative designations NDUFA6 and B14). NB4M/ NDUFA6 belongs to the Complex1_LYR family of LYRM proteins (5-7) that is characterized by a motif comprising an N-terminal leucine-tyrosine-arginine sequence located upstream of several conserved arginines and an invariant phenylalanine. The human genome contains at least 11 proteins of the LYRM superfamily, and the mammalian complex I subunit NDUFA6 is identical to LYR motif containing protein 6 (LYRM6). Several other LYRM proteins were shown ...