Complexome Profiling Identifies TMEM126B as a Component of the Mitochondrial Complex I Assembly Complex

Abstract

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.

Assembly factors for the membrane arm of human complex I

“…Therefore, NDUFA3, NDUFA8, and NDUFA13 also are parts of the same assembly intermediate. They were accompanied by assembly factors NDUFAF3 and NDUFAF4, both involved in formation of the 315-kDa assembly intermediate (30) reported to comigrate with TMEM126B in native gel profiles (27). Here, isoforms 1 and 5 of TMEM126B were found with relative abundances similar to these extrinsic assembly factor proteins.…”

“…The SILAC ratios of some subunits of complex I were unchanged, indicating that they were not enriched in the complex purified with C3orf1-FL (Dataset S2), and that therefore they are not associated with the 315-kDa subcomplex. They include NDUFA9, which has been reported previously to be a component of the 315-kDa subcomplex (33), although this observation was not corroborated (27). Three other proteins, TMEM126A, HSPA9, and GHITM, also accumulated with C3orf1-FL, but to a much lesser extent than the complex-Irelated proteins listed above (Dataset S2).…”

“…Assembly factors (NDUFAF2-4 and FOXRED1) have been identified via human mutations that impair function (29,30,38), by homology with an assembly factor for complex I in Neurospora crassa (NDUFAF1) or by copurification with other known assembly factors (ECSIT and ACAD9) (31,32). The BN-PAGE comigration pattern of TMEM126B with NDUFAF1, ECSIT, and ACAD9 led to its characterization as a complex I assembly factor (27). How these assembly factors function is not known, but they may stabilize the subcomplexes and help to join them to other subcomplexes to build the complete enzyme.…”

“…As the analyses were performed in the presence of n-dodecyl-β-D-maltoside, which dissociates respiratory complexes into individual enzymes, the increase in complexes III and IV is not due to their release from supercomplexes. In their experiments on TMEM126B, Heide et al reported an increase in complex III, but not of complex IV (27).…”

“…NDUFA11 has the characteristics of an intrinsic assembly factor for complex I and together with C3orf1 and another extrinsic assembly factor TMEM126B (27), they probably help to assemble the membrane arm of the complex.…”