Tim17 is a central, membrane-embedded subunit of the mitochondrial protein import machinery. In this study, we show that Tim17 contains a pair of highly conserved cysteine residues that form a structural disulfide bond exposed to the intermembrane space (IMS). This disulfide bond is critical for efficient protein translocation through the TIM23 complex and for dynamic gating of its preprotein-conducting channel. The disulfide bond in Tim17 is formed during insertion of the protein into the inner membrane. Whereas the import of Tim17 depends on the binding to the IMS protein Mia40, the oxidoreductase activity of Mia40 is surprisingly dispensable for Tim17 oxidation. Our observations suggest that Tim17 can be directly oxidized by the sulfhydryl oxidase Erv1. Thus, import and oxidation of Tim17 are mediated by the mitochondrial disulfide relay, though the mechanism by which the disulfide bond in Tim17 is formed differs considerably from that of soluble IMS proteins.

The mitochondrial inner membrane harbors a large number of metabolite carriers. The precursors of carrier proteins are synthesized in the cytosol and imported into mitochondria by the translocase of the outer membrane (TOM) and the carrier translocase of the inner membrane (TIM22). Molecular chaperones in the cytosol and intermembrane space bind to the hydrophobic precursors to prevent their aggregation. We report that the major metabolite channel of the outer membrane, termed porin or voltage-dependent anion channel (VDAC), promotes efficient import of carrier precursors. Porin interacts with carrier precursors arriving in the intermembrane space and recruits TIM22 complexes, thus ensuring an efficient transfer of the precursors to the inner membrane translocase. Porin channel mutants impaired in metabolite transport are not disturbed in carrier import into mitochondria. We conclude that porin serves distinct functions as outer membrane channel for metabolites and as coupling factor for protein translocation into the inner membrane.

Phosphatidylserine decarboxylase 1 (Psd1p) catalyzes the formation of the majority of phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae. Psd1p is localized to mitochondria, anchored to the inner mitochondrial membrane (IMM) through membrane spanning domains and oriented towards the mitochondrial intermembrane space. We found that Psd1p harbors at least two inner membrane-associated domains, which we named IM1 and IM2. IM1 is important for proper orientation of Psd1p within the IMM (Horvath et al., J. Biol. Chem. 287 (2012) 36744-55), whereas it remained unclear whether IM2 is important for membrane-association of Psd1p. To discover the role of IM2 in Psd1p import, processing and assembly into the mitochondria, we constructed Psd1p variants with deletions in IM2. Removal of the complete IM2 led to an altered topology of the protein with the soluble domain exposed to the matrix and to decreased enzyme activity. Psd1p variants lacking portions of the N-terminal moiety of IM2 were inserted into IMM with an altered topology. Psd1p variants with deletions of C-terminal portions of IM2 accumulated at the outer mitochondrial membrane and lost their enzyme activity. In conclusion we showed that IM2 is essential for full enzymatic activity, maturation and correct integration of yeast Psd1p into the inner mitochondrial membrane.

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.

The mitochondrial outer membrane contains integral proteins with α-helical membrane anchors or a transmembrane β-barrel. The translocase of the outer membrane (TOM) cooperates with the sorting and assembly machinery (SAM) in the import of β-barrel proteins, whereas the mitochondrial import (MIM) complex inserts precursors of multi-spanning α-helical proteins. Single-spanning proteins constitute more than half of the integral outer membrane proteins; however, their biogenesis is poorly understood. We report that the yeast MIM complex promotes the insertion of proteins with N-terminal (signal-anchored) or C-terminal (tail-anchored) membrane anchors. The MIM complex exists in three dynamic populations. MIM interacts with TOM to accept precursor proteins from the receptor Tom70. Free MIM complexes insert single-spanning proteins that are imported in a Tom70-independent manner. Finally, coupling of MIM and SAM promotes early assembly steps of TOM subunits. We conclude that the MIM complex is a major and versatile protein translocase of the mitochondrial outer membrane.