Quality control of the mitochondrial proteome.

Mitochondria contain about 1,000-1,500 proteins that fulfil multiple functions. Mitochondrial proteins originate from two genomes: mitochondrial and nuclear. Hence, proper mitochondrial function requires synchronization of gene expression in the nucleus and in mitochondria and necessitates efficient import of mitochondrial proteins into the organelle from the cytosol. Furthermore, the mitochondrial proteome displays high plasticity to allow the adaptation of mitochondrial function to cellular requirements. Maintenance of this complex and adaptable mitochondrial proteome is challenging, but is of crucial importance to cell function. Defects in mitochondrial proteostasis lead to proteotoxic insults and eventually cell death. Different quality control systems monitor the mitochondrial proteome. The cytosolic ubiquitin-proteasome system controls protein transport across the mitochondrial outer membrane and removes damaged or mislocalized proteins. Concomitantly, a number of mitochondrial chaperones and proteases govern protein folding and degrade damaged proteins inside mitochondria. The quality control factors also regulate processing and turnover of native proteins to control protein import, mitochondrial metabolism, signalling cascades, mitochondrial dynamics and lipid biogenesis, further ensuring proper function of mitochondria. Thus, mitochondrial protein quality control mechanisms are of pivotal importance to integrate mitochondria into the cellular environment.

Mitochondrial complexome reveals quality-control pathways of protein import.

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.

Mitochondrial protein translocation-associated degradation.

Mitochondrial biogenesis and functions depend on the import of precursor proteins via the 'translocase of the outer membrane' (TOM complex). Defects in protein import lead to an accumulation of mitochondrial precursor proteins that induces a range of cellular stress responses. However, constitutive quality-control mechanisms that clear trapped precursor proteins from the TOM channel under non-stress conditions have remained unknown. Here we report that in Saccharomyces cerevisiae Ubx2, which functions in endoplasmic reticulum-associated degradation, is crucial for this quality-control process. A pool of Ubx2 binds to the TOM complex to recruit the AAA ATPase Cdc48 for removal of arrested precursor proteins from the TOM channel. This mitochondrial protein translocation-associated degradation (mitoTAD) pathway continuously monitors the TOM complex under non-stress conditions to prevent clogging of the TOM channel with precursor proteins. The mitoTAD pathway ensures that mitochondria maintain their full protein-import capacity, and protects cells against proteotoxic stress induced by impaired transport of proteins into mitochondria.

Structure of the mitochondrial import gate reveals distinct preprotein paths.

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins1-4. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex5-9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 ß-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes1-3. Each Tom40 ß-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer.

A novel import route for an N-anchor mitochondrial outer membrane protein aided by the TIM23 complex.

The membrane topology of Om45 in the yeast mitochondrial outer membrane (OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N-terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence-containing proteins, and the membrane potential (ΔΨ). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex.

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

The majority of mitochondrial precursor proteins are imported through the Tom40 ß-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for ß-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here, we demonstrate that the α-helical outer membrane protein Mco6 co-assembles with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery. MCO6 and MDM10 display a negative genetic interaction, and a mco6-mdm10 yeast double mutant displays reduced levels of the TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and show assembly defects of the TOM complex. Thus, this work uncovers a role of the SAMMco6 complex for the biogenesis of the mitochondrial outer membrane.

The mitochondrial Hsp70 controls the assembly of the F1FO-ATP synthase.

The mitochondrial F1FO-ATP synthase produces the bulk of cellular ATP. The soluble F1 domain contains the catalytic head that is linked via the central stalk and the peripheral stalk to the membrane embedded rotor of the FO domain. The assembly of the F1 domain and its linkage to the peripheral stalk is poorly understood. Here we show a dual function of the mitochondrial Hsp70 (mtHsp70) in the formation of the ATP synthase. First, it cooperates with the assembly factors Atp11 and Atp12 to form the F1 domain of the ATP synthase. Second, the chaperone transfers Atp5 into the assembly line to link the catalytic head with the peripheral stalk. Inactivation of mtHsp70 leads to integration of assembly-defective Atp5 variants into the mature complex, reflecting a quality control function of the chaperone. Thus, mtHsp70 acts as an assembly and quality control factor in the biogenesis of the F1FO-ATP synthase.

Mitochondrial protein import clogging as a mechanism of disease.

Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in ADP/ATP translocase 1 (ANT1), and its yeast homolog ADP/ATP carrier 2 (Aac2), cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis, and cell viability independent of ANT1's nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/ANT1 causes severe clogging primarily at the translocase of the outer membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger ANT1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy ANT1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.

Inside our cells, compartments known as mitochondria generate the chemical energy required for life processes to unfold. Most of the proteins found within mitochondria are manufactured in another part of the cell (known as the cytosol) and then imported with the help of specialist machinery. For example, the TOM and TIM22 channels provide a route for the proteins to cross the two membrane barriers that separate the cytosol from the inside of a mitochondrion. ANT1 is a protein that is found inside mitochondria in humans, where it acts as a transport system for the cell's energy currency. Specific mutations in the gene encoding ANT1 have been linked to degenerative conditions that affect the muscles and the brain. However, it remains unclear how these mutations cause disease. To address this question, Coyne et al. recreated some of the mutations in the gene encoding the yeast equivalent of ANT1 (known as Aac2). Experiments in yeast cells carrying these mutations showed that the Aac2 protein accumulated in the TOM and TIM22 channels, creating a 'clog' that prevented other essential proteins from reaching the mitochondria. As a result, the yeast cells died. Mutant forms of the human ANT1 protein also clogged up the TOM and TIM22 channels of human cells in a similar way. Further experiments focused on mice genetically engineered to produce a "super-clogger" version of the mouse equivalent of ANT1. The animals soon developed muscle and neurological conditions similar to those observed in human diseases associated with ANT1. The findings of Coyne et al. suggest that certain genetic mutations in the gene encoding the ANT1 protein cause disease by blocking the transport of other proteins to the mitochondria, rather than by directly affecting ANT1's nucleotide trnsport role in the cell. This redefines our understanding of diseases associated with mitochondrial proteins, potentially altering how treatments for these conditions are designed.

A two-step mitochondrial import pathway couples the disulfide relay with matrix complex I biogenesis.

Mitochondria critically rely on protein import and its tight regulation. Here, we found that the complex I assembly factor NDUFAF8 follows a two-step import pathway linking IMS and matrix import systems. A weak targeting sequence drives TIM23-dependent NDUFAF8 matrix import, and en route, allows exposure to the IMS disulfide relay, which oxidizes NDUFAF8. Import is closely surveyed by proteases: YME1L prevents accumulation of excess NDUFAF8 in the IMS, while CLPP degrades reduced NDUFAF8 in the matrix. Therefore, NDUFAF8 can only fulfil its function in complex I biogenesis if both oxidation in the IMS and subsequent matrix import work efficiently. We propose that the two-step import pathway for NDUFAF8 allows integration of the activity of matrix complex I biogenesis pathways with the activity of the mitochondrial disulfide relay system in the IMS. Such coordination might not be limited to NDUFAF8 as we identified further proteins that can follow such a two-step import pathway.

Fidelity of organellar protein targeting.

The majority of cellular proteins are targeted to organelles. Cytosolic ribosomes produce these proteins as precursors with cleavable or non-cleavable targeting sequences that direct them to receptor proteins on the organelle surface. Multiple targeting factors ensure cellular sorting of the precursor proteins. In co-translational protein import, the ribosome-nascent chain complex is transported to the organellar protein translocase to couple protein synthesis and protein import. In post-translational mode, targeting factors like molecular chaperones guide the precursor proteins from ribosomes to the cell organelle. Defects in protein targeting and import cause mistargeting of proteins to different cellular compartments and challenge the balance of cellular proteostasis. Specific dislocases and degradation machineries remove such mislocalized proteins from the membrane to allow retargeting or their proteasomal turnover. In this review, we discuss targeting and quality control factors that ensure fidelity of protein targeting to mitochondria.

Versatility of Preprotein Transfer from the Cytosol to Mitochondria.

Mitochondrial biogenesis requires the import of a large number of precursor proteins from the cytosol. Although specific membrane-bound preprotein translocases have been characterized in detail, it was assumed that protein transfer from the cytosol to mitochondria mainly involved unselective binding to molecular chaperones. Recent findings suggest an unexpected versatility of protein transfer to mitochondria. Cytosolic factors have been identified that bind to selected subsets of preproteins and guide them to mitochondrial receptors in a post-translational manner. Cotranslational import processes are emerging. Mechanisms for crosstalk between protein targeting to mitochondria and other cell organelles, in particular the endoplasmic reticulum (ER) and peroxisomes, have been uncovered. We discuss how a network of cytosolic machineries and targeting pathways promote and regulate preprotein transfer into mitochondria.

Respiratory chain supercomplexes associate with the cysteine desulfurase complex of the iron-sulfur cluster assembly machinery.

Mitochondria are the powerhouses of eukaryotic cells. The activity of the respiratory chain complexes generates a proton gradient across the inner membrane, which is used by the F1FO-ATP synthase to produce ATP for cellular metabolism. In baker's yeast, Saccharomyces cerevisiae, the cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) associate in respiratory chain supercomplexes. Iron-sulfur clusters (ISC) form reactive centers of respiratory chain complexes. The assembly of ISC occurs in the mitochondrial matrix and is essential for cell viability. The cysteine desulfurase Nfs1 provides sulfur for ISC assembly and forms with partner proteins the ISC-biogenesis desulfurase complex (ISD complex). Here, we report an unexpected interaction of the active ISD complex with the cytochrome bc1 complex and cytochrome c oxidase. The individual deletion of complex III or complex IV blocks the association of the ISD complex with respiratory chain components. We conclude that the ISD complex binds selectively to respiratory chain supercomplexes. We propose that this molecular link contributes to coordination of iron-sulfur cluster formation with respiratory activity.

Recruitment of Cytosolic J-Proteins by TOM Receptors Promotes Mitochondrial Protein Biogenesis.

Mitochondria possess elaborate machineries for the import of proteins from the cytosol. Cytosolic factors like Hsp70 chaperones and their co-chaperones, the J-proteins, guide proteins to the mitochondrial surface. The translocase of the mitochondrial outer membrane (TOM) forms the entry gate for preproteins. How the proteins are delivered to mitochondrial preprotein receptors is poorly understood. We identify the cytosolic J-protein Xdj1 as a specific interaction partner of the central receptor Tom22. Tom22 recruits Xdj1 to the mitochondrial surface to promote import of preproteins and assembly of the TOM complex. Additionally, we find that the receptor Tom70 binds a different cytosolic J-protein, Djp1. Our findings suggest that cytosolic J-proteins target distinct TOM receptors and promote the biogenesis of mitochondrial proteins.