Mitochondrial complexome reveals quality-control pathways of protein import

Abstract

Mitochondria have crucial roles in cellular energetics, metabolism, signalling and quality control1–4. They contain around 1,000 different proteins that often assemble into complexes and supercomplexes such as respiratory complexes and preprotein translocases1,3–7. The composition of the mitochondrial proteome has been characterized1,3,5,6; however, the organization of mitochondrial proteins into stable and dynamic assemblies is poorly understood for major parts of the proteome1,4,7. Here we report quantitative mapping of mitochondrial protein assemblies using high-resolution complexome profiling of more than 90% of the yeast mitochondrial proteome, termed MitCOM. An analysis of the MitCOM dataset resolves >5,200 protein peaks with an average of six peaks per protein and demonstrates a notable complexity of mitochondrial protein assemblies with distinct appearance for respiration, metabolism, biogenesis, dynamics, regulation and redox processes. We detect interactors of the mitochondrial receptor for cytosolic ribosomes, of prohibitin scaffolds and of respiratory complexes. The identification of quality-control factors operating at the mitochondrial protein entry gate reveals pathways for preprotein ubiquitylation, deubiquitylation and degradation. Interactions between the peptidyl-tRNA hydrolase Pth2 and the entry gate led to the elucidation of a constitutive pathway for the removal of preproteins. The MitCOM dataset—which is accessible through an interactive profile viewer—is a comprehensive resource for the identification, organization and interaction of mitochondrial machineries and pathways.

Role of the small protein Mco6 in the mitochondrial sorting and assembly machinery

“…Our recent high-resolution mitochondrial blue native PAGE complexome profiling uncovers a co-migration of the S. cerevisiae yeast protein Mco6 (Yjl127c-b/YJ127_YEAST) at ~180 kDa with all four known subunits of the sorting and assembly machinery (SAM), Sam35, Sam37, Sam50 and Mdm10 (Figures 1A and S1A). 40 . In addition, Mco6 migrates in a lower molecular mass form of ~100 kDa (Figures 1A and S1A).…”

“…The assembly of imported Mco6 was followed by blue native PAGE and Mco6 predominantly assembled into complexes of approximately ~300 kDa and ~160 kDa as assessed by the soluble blue native PAGE marker proteins (Figure 1C), which reflects the migration profile of endogenous Mco6 in the complexome profiling data set. 40…”

“…(A) Mitochondrial blue native PAGE complexome profiles of Mdm10, Mco6, Sam35, Sam37 and Sam50, derived from the complexome data of Schulte et al 40 See also Figure S1A.…”

“…The β-barrel Tom40 assembles with the a-helical membrane proteins Tom5, Tom6, Tom7, Tom22 and interacts with the receptors Tom20, Tom70 and Om14. 40,46–48 After identification of the a-helical membrane proteins of the TOM complex, the specific complex formation between β-barrel membrane proteins and a-helical membrane proteins was regarded as a special case. Meanwhile the porin β-barrel membrane protein was found to interact with the a-helical membrane proteins Om14 and Om45 and the β-barrel protein Mdm10 interacts as subunit of the ER-mitochondria encounter structure (ERMES) with the a-helical outer membrane proteins Tom7 and Gem1(Miro-1) 23,25–27,49…”

“…(A) Individual blue native PAGE yeast complexome profiles of Sam50, Sam35, Sam37, Mdm10 and Mco6 related to Figure 1A, derived from the complexome data of Schulte et al (Extended Data Figures 5 and 8a and Supplementary Tables 1 and 2, see: http://www.complexomics.org). 40 …”