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Nature Reviews Molecular Cell Biology (2019)

 

Abstract

Mitochondria are essential for the viability of eukaryotic cells as they perform crucial functions in bioenergetics, metabolism and signalling and have been associated with numerous diseases. Recent functional and proteomic studies have revealed the remarkable complexity of mitochondrial protein organization. Protein machineries with diverse functions such as protein translocation, respiration, metabolite transport, protein quality control and the control of membrane architecture interact with each other in dynamic networks. In this Review, we discuss the emerging role of the mitochondrial protein import machinery as a key organizer of these mitochondrial protein networks. The preprotein translocases that reside on the mitochondrial membranes not only function during organelle biogenesis to deliver newly synthesized proteins to their final mitochondrial destination but also cooperate with numerous other mitochondrial protein complexes that perform a wide range of functions. Moreover, these protein networks form membrane contact sites, for example, with the endoplasmic reticulum, that are key for integration of mitochondria with cellular function, and defects in protein import can lead to diseases.

Introduction

Mitochondria are a hallmark and the powerhouses of eukaryotic cells; they synthesize ATP via oxidative phosphorylation but are also deeply integrated into cellular metabolism and signalling pathways.

Mitochondria consist of two membranes and two aqueous compartments (Fig. 1). The surface area of the mitochondrial inner membrane is several-fold larger than that of the outer membrane; it therefore forms invaginations known as cristae, which contain the oxidative phosphorylation system, comprising the respiratory complexes I to IV and the F1F0-ATP synthase for ATP production. Only a small set of proteins are encoded by the mitochondrial genome, and these are typically hydrophobic membrane proteins that form core parts of the oxidative phosphorylation complexes of the mitochondrial inner membrane. Approximately 99% of mitochondrial proteins are encoded by nuclear genes and depend on specific targeting signals that direct them from the cytosol, where they are synthesized, to mitochondrial surface receptors and then into the proper mitochondrial subcompartments1,2.

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Mitochondria consist of four compartments: outer membrane (OM), intermembrane space (IMS), inner membrane (IM) and matrix. A large variety of functions have been assigned to mitochondrial proteins and protein complexes and are indicated in the figure: energy metabolism with respiration and synthesis of ATP; metabolism of amino acids, lipids and nucleotides; biosynthesis of iron–sulfur (Fe–S) clusters and cofactors; expression of the mitochondrial genome; quality control and degradation processes including mitophagy and apoptosis; signalling and redox processes; membrane architecture and dynamics; and the import and processing of precursor proteins that are synthesized on cytosolic ribosomes. AAA, ATP-dependent proteases of the inner membrane; E3, ubiquitin-protein ligase; ER, endoplasmic reticulum; mtDNA, mitochondrial DNA; TCA, tricarboxylic acid; Ub, ubiquitin.

Traditionally, research on mitochondria focused on bioenergetics, but studies in the past 15–20 years have revealed a greater than expected complexity and versatility of mitochondrial activities, integrating mitochondrial energetics with protein biogenesis, metabolic pathways, cellular signalling, stress responses and apoptosis. It is becoming increasingly evident that mitochondrial protein machineries, which have diverse functions, are physically and functionally connected rather than functioning as independent units. It is therefore important to understand the principles that govern the formation and maintenance of these complex networks.

In this Review, we first discuss recent proteomic studies that unveiled a large spectrum of mitochondrial protein types and functions. Quantitative proteomics revealed for the first time absolute numbers for the cellular abundance of all important mitochondrial machineries under respiratory and non-respiratory conditions. We then discuss how preprotein translocases are at the core of dynamic protein networks that link organelle biogenesis to energy metabolism, membrane morphology and dynamics. With this integrative view, we discuss how the mitochondrial protein import machinery is connected to mitochondrial stress responses, quality control mechanisms and diseases and discuss its role in the formation of membrane contact sites between mitochondria and other organelles, which are important for cell function.

Multifunctional mitochondria

Systematic analyses of the mitochondrial proteome have provided a comprehensive overview of the mitochondrial protein complement and of the large variety of functions performed by mitochondria (Fig. 1). Importantly, our understanding of mitochondrial activities has been shaped by the recent absolute quantification of the mitochondrial proteome.

Numerous and diverse functions of mitochondria

Textbooks typically describe mitochondria as comprising the respiratory complexes and the F1F0-ATP synthase in the inner membrane cristae and transporters and channels for metabolites and ions in both mitochondrial membranes and as the site of metabolic pathways, which are mainly localized to the matrix and inner membrane3 (Fig. 1). These include energy metabolism pathways, such as the tricarboxylic acid cycle, as well as amino acid, lipid and nucleotide metabolism pathways. Electrons derived from the oxidation of metabolites are fed into the respiratory chain, which generates an electrochemical gradient by pumping protons from the mitochondrial matrix into the intermembrane space. The resulting proton gradient is used to drive ATP synthesis by the F1F0-ATP synthase and to enable the import of precursor proteins and the transfer of some metabolites across the inner membrane.

Functions of mitochondria, which are essential for cell viability under all growth conditions, include the synthesis of iron–sulfur (Fe–S) clusters4 and mitochondrial protein import and maturation1,2. The mitochondrial matrix also contains a complete genetic system, which includes the mitochondrial genome, numerous factors that are crucial for the maintenance and regulation of the genome and mitochondrial ribosomes, which differ in size and composition from cytosolic ribosomes5. Proteins encoded by the mitochondrial genome are inserted into the inner membrane in a co-translational mechanism by coupling translating ribosomes to the oxidase assembly (OXA) insertase1,2,6.

Mitochondria form a dynamic network in most cell types that is continuously remodelled by fusion and fission of the organelles7,8. Several machineries have been identified that control mitochondrial membrane architecture and dynamics, which include factors that mediate fusion or fission, such as dynamin-related GTPases located at the outer and inner membranes, and membrane-shaping components. The mitochondrial contact site and cristae organizing system (MICOS) is crucial for maintaining the characteristic shape of inner membrane cristae9,10,11. MICOS and several other protein complexes form contact sites between the mitochondrial outer and inner membranes to promote the transfer of proteins, lipids and metabolites9,10,12,13.

Topics that are being intensively studied include quality control systems and the regulation of mitochondrial signalling (all of which have been recently reviewed14,15,16,17,18,19). Numerous cytosolic signalling cascades are connected to mitochondria under physiological and pathophysiological conditions. The metabolic activity of the organelle serves as a measure for mitochondrial fitness and quality14, and elaborate pathways for mitochondrial stress responses, selective degradation of damaged mitochondria by autophagy (mitophagy)15,16and programmed cell death (apoptosis) via mitochondria have been identified17. Mitochondria contain a set of internal proteases that are also involved in quality control and turnover of mitochondrial proteins18. In addition, mitochondria are a major site of cellular production of reactive oxygen species (ROS) and contain numerous redox pathways19.

Quantitative analysis of the mitochondrial proteome

The wide range of mitochondrial activities is challenging to study and has led to different views on how mitochondria are organized. We can envisage three stages as having led to our current understanding of mitochondrial functions. First, the original research on metabolism and ATP production established energetics and metabolism as hallmarks of mitochondria. Second, systematic proteomic studies on mitochondria, which began ~15 years ago, led to the identification of many new mitochondrial proteins. We currently estimate that mitochondria contain at least 1,000 (in yeast) to 1,500 (in humans) different proteins20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38. The functional classification of this high number of different proteins26,32 indicated that only up to 15% of the mitochondrial proteins are directly involved in energy metabolism, including the energy metabolizing pathways and all structural subunits of the oxidative phosphorylation system. It is estimated that 20–25% of the mitochondrial proteome maintains and regulates the mitochondrial genome, which encodes only ~1% of mitochondrial proteins. In addition, numerous mitochondrial components were found to be connected to a variety of cellular signalling pathways and membrane dynamics. Thus, mitochondria emerged as signalling platforms that are crucial in the regulation of cell function, extending their role well beyond that of cellular powerhouses. Lastly, a more refined understanding of the nature and functions of mitochondrial proteins in different conditions became possible with the recent systematic quantification of the majority of the mitochondrial proteome, which yielded the absolute copy numbers of mitochondrial proteins present in distinct cells26,39,40,41,42,43. Proteins involved in energy metabolism are by far the most abundant protein classes in respiring yeast mitochondria (Box 1). The ~15% of mitochondrial proteins with a direct role in energy metabolism and respiration mentioned above constitute more than half of the mitochondrial protein mass under respiratory conditions, reinforcing the original view of mitochondria as cellular powerhouses. Taking the various metabolic processes, oxidative phosphorylation and metabolite carriers and channels together, ~75% of the protein mass of respiring mitochondria is dedicated to metabolism and bioenergetics26.

These seemingly controversial views, of mitochondria as powerhouses versus mitochondria as organelles with a large number of different functions, must be combined to understand the cellular importance of mitochondria. On the basis of absolute protein mass —that is, from a quantitative perspective — metabolism and bioenergetics remain the major tasks of mitochondria (the powerhouses). However, other functions are of central importance for mitochondrial fitness, cellular growth and development. A striking example is the system for Fe–S cluster biosynthesis, which makes up <1% of the protein mass of respiring mitochondria but is essential for the viability of all eukaryotic cells4,26. Equally important are the machineries for mitochondrial membrane morphology and dynamics, which represent <1% of the mitochondrial protein mass26. These proteins have key roles in mitochondrial architecture, fusion and fission and are thus crucial for maintaining and remodelling the mitochondrial network under different growth conditions7,8. Mitochondria can thus be seen as super-powerhouses that, in addition to their predominant metabolic and energetic functions, are deeply integrated into cellular dynamics, signalling and biosynthetic pathways by performing a multitude of functions. We discuss below that these functions are not independent of one another, as the machineries and proteins involved are physically connected in large, dynamic networks.

Box 1 The mitochondrial proteome: from fermentation to respiration

Recent studies have revealed the absolute copy numbers of mitochondrial proteins per cell26,39,40,41,42,43 (see the figure). Proteins involved in energy metabolism are by far the most abundant classes of mitochondrial proteins26. The abundance of these proteins is strongly regulated by the growth conditions. When shifting yeast from fermentation to respiratory conditions, their protein levels are increased approximately threefold or more. The levels of proteins involved in signalling, redox processes, membrane dynamics and morphology are increased approximately twofold from fermentation to respiration, which is comparable to the increase in the total mitochondrial protein mass. Interestingly, proteins involved in two essential processes — protein import, maturation and turnover, and biosynthesis of iron–sulfur (Fe–S) clusters — are only mildly affected when cells are shifted from fermentation to respiration26. The overall abundance of proteins involved in mitochondrial gene expression and translation and in the metabolism of lipids, amino acids and nucleotides is only mildly affected by different growth conditions; however, differences can be observed for individual classes of protein. For example, proteins involved in lipid metabolism are considerably more expressed under respiratory conditions, whereas proteins involved in amino acid metabolism are more strongly expressed under fermentable conditions26.

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Plasticity of the mitochondrial proteome

The mitochondrial content of a cell can vary considerably under different growth conditions and between different organisms and tissues21,24,26,27,28,44. Systematic analyses of yeast mitochondria revealed that the total mitochondrial protein mass constitutes ~9% of the cellular protein mass in fermentable conditions (when a lower activity of mitochondria is required) but is more than double in respiratory growth conditions, reaching ~20% of the cellular protein mass26. The changes from fermentation to respiration are quite different for mitochondrial proteins belonging to different functional classes (Box 1). There is more than a threefold increase in the absolute copy number per cell for proteins directly involved in energy metabolism and respiration. Proteins functioning in signalling, redox processes and membrane dynamics are increased by approximately twofold, like the overall increase in mitochondrial mass26.

Remarkably, the protein classes that are involved in protein biogenesis and folding and biosynthesis of Fe–S clusters — the two mitochondrial processes that are essential for cell viability in all cell types and growth conditions — are only moderately altered in their overall copy numbers when yeast cells transition from fermentation to respiration26. This indicates that cells are well equipped for these systems in non-respiratory conditions and require only a slight increase in their abundance during respiration. For example, the protein import machinery imports more than double the amount of proteins during respiration, and so it is evident that the machinery works at a considerably lower capacity during fermentation. We conclude that the protein import machinery and the system for biosynthesis of Fe–S clusters are essential housekeeping systems of mitochondria and are not or are only moderately regulated by their copy number. Indeed, studies on the translocase of the outer membrane (TOM) complex — which is the main protein entry gate, consisting of receptor proteins on the cytosolic face (Tom70, Tom20 and Tom22) and a pore-forming protein (Tom40) — revealed that at least four different cytosolic signalling systems regulate TOM complex activity by phosphorylation, leading to a sophisticated pattern of stimulatory and inhibitory effects depending on the kinase and TOM subunits involved45,46,47,48. Having a stable set of housekeeping systems that are regulated by reversible modification, such as phosphorylation, has the advantage of enabling rapid responses to changing conditions. The system would be rather slow in adapting to different requirements if it was dependent on increased gene expression, translation and import. Similarly, inhibition is faster when achieved by covalent modification rather than by reducing protein copy numbers. For example, during fermentable growth, when less translocation of metabolites in and out of mitochondria is needed, the activity of Tom70, which is required for the import of metabolite carriers, is inhibited by phosphorylation, leading to an immediate decrease in metabolite carrier import into mitochondria48. Thus, the largely stable protein copy numbers in housekeeping systems and their regulation by post-translational modification enable greater flexibility during mitochondrial biogenesis and remodelling of the mitochondrial network.

Assembly of functional protein networks

Studies of protein import from the cytosol into mitochondria were originally based on the assumption that all proteins were transported to their final destination along one central pathway. However, the characterization of precursor proteins carrying different targeting signals revealed that mitochondria use at least five major protein import pathways, each one directed by a different type of targeting signal. The complexity of the system is even higher, as preprotein translocases do not operate as isolated units but are connected to numerous mitochondrial protein complexes involved in seemingly unrelated functions.

Five major import pathways of precursor proteins into mitochondria

The presequence pathway is the best-characterized pathway, responsible for the import of ~60% of all mitochondrial proteins49. The precursor proteins carry amino-terminal targeting signals, termed presequences, which form positively charged amphipathic α-helices. These presequences are typically recognized by the TOM receptors Tom20 and Tom22 (refs50,51) on the mitochondrial surface and are transported through the main protein translocation channel of the outer membrane Tom40 (refs1,2,52,53) (Fig. 2). Upon passage across the outer membrane, the preproteins are engaged by the presequence translocase of the inner membrane (TIM23), which directs their transfer across the inner membrane54,55,56,57. The membrane potential (Δψ) across the inner membrane (negative on the inside) activates the TIM23 channel and drives the positively charged presequences towards the matrix58,59,60,61. The presequence translocase-associated motor (PAM) contains the mitochondrial heat shock protein 70 (mtHsp70) as the central ATP-driven chaperone, which binds the precursor proteins to promote their unidirectional movement62. Together with five co-chaperones, mtHsp70 translocates the polypeptide chain into the matrix63,64,65,66,67,68, where the presequences are removed by the mitochondrial processing peptidase (MPP)1,2,69. The matrix contains additional processing enzymes involved in quality control functions, as mitochondrial proteins can be degraded by the N-end rule pathwaydepending on whether they contain stabilizing or destabilizing amino acid residues at their amino termini49,70. The intermediate cleaving peptidase of 55 kDa (Icp55) and the octapeptidyl peptidase (Oct1) remove destabilizing amino acid residues from the amino termini of imported proteins after cleavage by MPP, thus generating amino termini that are less prone to degradation by matrix proteases49,71,72. With the help of mtHsp70 and other chaperones such as the Hsp60–Hsp10 chaperonin complex73,74, the proteins are then folded into their active form.

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Five major pathways of mitochondrial protein import have been identified. The protein import machineries have been well conserved from fungi (shown in this figure) to mammals (shown in Box 4). First, the presequence pathway transports presequence-carrying cleavable preproteins through the translocase of the outer membrane (TOM) and the presequence translocase of the inner membrane (TIM23) with the presequence translocase-associated motor (PAM). The membrane potential (Δψ) across the inner membrane (IM) activates the TIM23 channel and drives translocation of the positively charged presequences into the matrix. The presequences are removed by the mitochondrial processing peptidase (MPP), and additional proteolytic processing can occur by intermediate cleaving peptidase of 55 kDa (Icp55) or octapeptidyl peptidase (Oct1). IM proteins are either laterally released from the TIM23 complex or are transported via the matrix and inserted into the IM by the oxidase assembly protein 1 (Oxa1) insertase. IM proteins synthesized on mitochondrial ribosomes are also inserted by Oxa1. Second, cysteine-rich proteins destined for the intermembrane space (IMS) are imported through the TOM complex and are recognized by the mitochondrial IMS import and assembly protein (Mia40), which functions as an oxidoreductase to insert disulfide bonds into the imported proteins. The sulfhydryl oxidase Erv1 forms a disulfide relay with Mia40, transferring disulfides from Erv1 to Mia40 to imported proteins. Third, the precursors of non-cleavable IM proteins such as the carrier proteins are imported by the TOM complex, followed by transfer to the small TIM chaperones in the IMS and insertion into the IM by the TIM22 carrier translocase. Fourth, the precursors of outer membrane (OM) β-barrel proteins use the TOM complex and small TIM chaperones and are inserted into the OM by the sorting and assembly machinery (SAM). Fifth, many OM proteins with α-helical transmembrane segments are inserted into the membrane by the mitochondrial import (MIM) complex. α-Helical OM proteins typically do not use the Tom40 channel, but Tom70 can be involved in their recognition. The inset shows the absolute copy numbers of characteristic translocase components in a respiring yeast cell and, for comparison, the abundance of respiratory complexes, metabolite channels and carriers of the mitochondrial membranes26. The porin isoform porin 1 (Por1) of the OM is one of the most abundant mitochondrial proteins, whereas the isoform Por2 is one of the least abundant proteins. mtHsp70, mitochondrial heat shock protein 70.

 

Presequence-carrying precursors that become integrated into the inner mitochondrial membrane follow two distinct routes (Fig. 2). Those that have a hydrophobic sorting signal behind the matrix targeting signal become arrested in the TIM23 complex and are then released by the lateral gatekeeper Mgr2 into the inner membrane (stop transfer pathway)75,76. Other inner membrane proteins are first transported into the matrix and are incorporated into the inner membrane by the OXA insertase, which is also used by mitochondrially encoded proteins (conservative sorting)6,77,78,79,80.

Most other protein import pathways also use the TOM channel for preprotein translocation across the outer membrane81,82 (Fig. 2), but the dependence on the three TOM receptors Tom20, Tom22 and Tom70 and the mode of delivery from the cytosol to the TOM complex can differ83,84,85. The carrier pathway is dedicated to the import of hydrophobic multi-spanning inner membrane proteins. These proteins do not have amino-terminal presequences; they have internal targeting signals that contain hydrophobic elements but have not been fully characterized. Cytosolic chaperones of the Hsp90 and Hsp70 classes deliver these inner membrane precursor proteins to the receptor Tom70 (ref.86). After their release from the cytosolic chaperones, the precursors pass through the Tom40 channel in a loop formation and enter the intermembrane space87,88. Here, they are bound by small TIM chaperones, which prevent their aggregation89,90,91,92 and guide them to the carrier translocase of the inner membrane (TIM22) complex. The TIM22 complex operates in a Δψ-dependent manner to insert these multi-spanning proteins in the inner membrane93,94,95,96,97 (Fig. 2).

Many proteins of the mitochondrial intermembrane space contain characteristic cysteine motifs that become oxidized to form stabilizing disulfide bonds in the mature proteins. The mitochondrial intermembrane space import and assembly (MIA) system, which mediates the import and oxidative folding of intermembrane space proteins, consists of two main components: the oxidoreductase Mia40 (refs98,99) and the sulfhydryl oxidase Erv1 (ref.100) (Fig. 2). Upon passage through the Tom40 channel, Mia40 recognizes the precursor proteins that contain an intermembrane space sorting signal, typically consisting of a hydrophobic element flanked by a cysteine residue101,102,103. Mia40 forms transient disulfide bonds with these precursors and then transfers the disulfide bonds to them, which leads to intramolecular disulfide bond formation via oxidation and stabilization of the proteins104,105. At each transfer of a disulfide bond to a protein, cysteines of Mia40 become reduced and are re-oxidized by Erv1. In this disulfide relay, disulfide bonds are thus transferred from Erv1 to Mia40 to imported proteins.

The mitochondrial outer membrane contains different classes of membrane protein: single-spanning and multi-spanning proteins with α-helical transmembrane segments and β-barrel proteins. The precursors of β-barrel proteins are initially translocated by the TOM complex106 to the intermembrane space and interact with small TIM chaperones like the carrier precursors92 (Fig. 2). Insertion of β-barrel precursors into the outer membrane is mediated by the sorting and assembly machinery (SAM)107,108,109 in a step-wise process that involves translocation into the SAM channel (formed by the Sam50 subunit) and lateral release into the lipid phase of the membrane110. The carboxy-terminal β-strand of these proteins functions as a β-signal that directs insertion via SAM111. α-Helical outer membrane proteins usually follow distinct import routes that do not involve the Tom40 channel. The sorting signal is typically contained within the α-helical transmembrane segments and flanking positively charged amino acid residues. Single-spanning proteins with an amino-terminal membrane anchor (signal-anchored proteins) and multi-spanning outer membrane proteins can use the mitochondrial import (MIM) channel for membrane insertion, assisted by Tom70 at least in the case of multi-spanning proteins112,113,114,115,116,117. In the case of single-spanning proteins with a carboxy-terminal membrane anchor (tail-anchored proteins) and some multi-spanning proteins, the lipid composition of the outer membrane seems to be important for membrane insertion, yet the exact molecular mechanism is unknown118,119,120,121. Several possibilities have been proposed, including protein-independent direct insertion into the phospholipid membrane, MIM complex-assisted insertion or the involvement of an unknown proteinaceous insertase of the outer membrane.

Abundance and versatility of import machineries

The absolute copy numbers of a variety of mitochondrial import components have been identified26 (Fig. 2). The TOM complex is the most abundant translocase, consistent with its role in feeding precursors into at least four distinct downstream translocase systems. The TIM23 complex is also abundant, as expected given the major role of the presequence import pathway. Interestingly, mtHsp70 is approximately ten times more abundant than Tim23 and other motor subunits such as Tim44. mtHsp70 plays a dual role: a small fraction of mtHsp70 molecules act in the TIM23-associated PAM to drive preprotein import, whereas the majority of mtHsp70 is dedicated to protein folding in the mitochondrial matrix2. Tim22 and Sam50 are present in quite low amounts. However, their major substrates are of high abundance, which underscores the importance and activity of these translocases. Inner membrane metabolite carriers, the substrates of the TIM22 complex, are highly abundant, and of note, one of the most abundant mitochondrial proteins, the outer membrane β-barrel metabolite channel porin 1 (Por1), is a substrate of the SAM complex. Thus, the TIM22 and SAM complexes are crucial for the biogenesis of mitochondrial metabolite carriers and channels that mediate the export of ATP and link mitochondrial and cellular metabolism.

The TOM complexes can form different types of dynamic supercomplex: a TOM–SAM supercomplex for efficient transfer of β-barrel precursors and a two-membrane-spanning TOM–TIM23–preprotein supercomplex122,123,124,125,126,127. TOM also interacts with the small TIM chaperones, and in mammals, TIM29 of the TIM22 complex was found to associate with TOM82,128,129. The differential abundance of the translocases (Fig. 2) indicates that TOM complexes are sufficiently abundant to form the different supercomplexes. Whether separate pools of TOM complexes exist for different import pathways or whether the TOM complexes are freely interchangeable in one large dynamic pool is currently a subject of debate. The translocation of several intermembrane space precursor proteins across the outer membrane depends on Tom40 but does not require the TOM receptor domains130,131. Competition experiments suggest that intermembrane space precursors and presequence-carrying precursors do not use the same TOM complexes, which is in support of the view that there may be distinct pools of the TOM complex130. We speculate that in addition to the full-size TOM complex, which comprises the Tom40 channels, all three receptors and three small Tom proteins82, mitochondria might also contain simpler forms of TOM complex. These simpler TOM complexes may just contain the Tom40 channel and possibly some of the small Tom subunits and may be dedicated, for example, to the import of intermembrane space precursors that are recognized by Mia40 and do not depend on classical TOM receptors. The differential phosphorylation of TOM complexes by cytosolic signalling cascades also contributes to the heterogeneity of TOM complexes and suggests that cellular signalling pathways control the activity of distinct import routes45,46,47,48.

In metazoans, there are two forms of the presequence translocase, which are differentially distributed across tissues: one form contains the stably expressed housekeeping subunit TIM17B, found in skeletal muscle, and the other form contains the stress-regulated subunit TIM17A, found in the brain132. Under stress conditions, the levels of TIM17A decrease as a result of reduced synthesis and increased degradation by the ATP-dependent AAA protease of the inner membrane that is exposed to the intermembrane space (iAAA protease) (Fig. 3). This decrease in TIM17A levels promotes a mitochondrial unfolded protein response (UPRmt)133. Thus, whereas the overall abundance of the mitochondrial protein import machinery in rapidly growing unicellular organisms like yeast is quite stable under different metabolic conditions, the abundance of tissue-specific isoforms varies in metazoans, suggesting that additional regulatory mechanisms operate in multicellular organisms132,133,134.

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Supercomplexes of the mitochondrial respiratory chain are integrated into functional networks with the presequence translocase of the inner membrane (TIM23) (see Box 2) and the ATP-dependent AAA proteases of the inner membrane (IM). The ATP-dependent AAA proteases degrade not only several IM proteins but also selected proteins of the matrix, intermembrane space (IMS) and outer membrane (OM), functioning as a quality control system of mitochondria. Several respiratory chain–AAA linker proteins, AAA–substrate adaptor proteins and assembly factors for respiratory supercomplexes were identified in fungi. The coenzyme Q (CoQ) biosynthetic complex on the matrix side of the IM provides CoQ for the respiratory chain and further enzymes. The precursors of the CoQ complex are imported by the translocase of the outer membrane (TOM) and TIM machineries. Proteolytic processing in the matrix can involve two steps, as it does for the precursor of Coq5. The mitochondrial respiratory chain is a main source for the generation of reactive oxygen species (ROS), which can exert harmful effects but also function in signalling. Targeting of the cytosolic translation machinery by ROS leads to decreased protein synthesis, providing a link between the status of the respiratory chain and protein biogenesis. ΔμH+, electrochemical proton gradient; CI, complex I; CII, complex II; CIII, complex III; CIV, complex IV; iAAA, IMS-exposed AAA; mAAA, matrix-exposed AAA; MPP, mitochondrial processing peptidase; Oct1, octapeptidyl peptidase; PAM, presequence translocase-associated motor.

 

Elements of different import pathways can be combined to create new pathways. For example, a large domain of the single-spanning outer membrane protein Om45 is exposed to the intermembrane space. The Om45 precursor is first transported by the presequence pathway through the TOM complex and interacts with the TIM23 complex but then escapes into the intermembrane space to be inserted into the outer membrane by a topologically opposite action of the MIM complex135,136. Cleavable carboxy-terminal targeting signals are another example of the versatility of the mitochondrial import machinery, as they are imported via the presequence pathway and removed by matrix or intermembrane space peptidases, followed by differential sorting to intramitochondrial destinations137,138,139. Thus, although import pathways are classified into five major pathways, import mechanisms are much more diverse and versatile, and we expect that the systematic analysis of the large number of substrates will uncover new import routes and possibly also new translocases.

Respiratory chain interactions link bioenergetics, biogenesis and quality control

The respiratory chain complexes of the mitochondrial inner membrane are the core of a large protein network that connects bioenergetics to mitochondrial biogenesis, regulation and turnover processes (Fig. 3). Respiratory complexes I (NADH:ubiquinone oxidoreductase), III (cytochrome c reductase) and IV (cytochrome c oxidase) assemble into large I–III–IV supercomplexes, also termed respirasomes140,141,142,143,144. The assembly into such supercomplexes is now generally accepted, but there are different views about their functions141,143. The supercomplexes may influence the assembly and stability of respiratory complexes, regulate the activity of the complexes and/or reduce the formation of ROS. Various factors involved in the formation of respiratory supercomplexes have been reported (Fig. 3), but whether they mainly function in the assembly of individual respiratory complexes or in the formation of supercomplexes remains to be elucidated141,143,145,146,147,148,149.

Not only are preprotein translocases in crosstalk with each other, they also form physical contacts with other mitochondrial machineries, including machineries involved in mitochondrial energy metabolism. The TIM23 complex forms a hub in the sorting of preproteins at the inner membrane cooperating with import complexes TOM and PAM and forms supercomplexes with the respiratory complexes III and IV as well as with the ADP/ATP carrier (Box 2; Fig. 3). These interactions of the TIM23 complex support protein import under energy-limiting conditions150,151,152 and can also promote the assembly of respiratory complexes153,154,155 (Box 2). The respiratory complexes as well as the ADP/ATP carrier are several-fold more abundant than the TIM23 complex26 (Fig. 2), and thus, only a fraction of them are engaged in the interaction with TIM23. Respiratory complexes are preferentially located in cristae membranes, yet a smaller fraction is found in the inner boundary membrane, which is adjacent to the outer membrane156, and can thus interact with the TIM23 complexes. TIM23 and respiratory complexes seem to form dynamic, non-permanent supercomplexes150,151.

A link between the mitochondrial respiratory chain and the machinery for protein synthesis was found by analysing the effects of mitochondrially generated ROS. The respiratory chain is a major source of ROS, and stress conditions and dysfunction of the respiratory chain can lead to increased ROS production that causes oxidative damage in proteins, DNA and membranes. As ROS have signalling functions, mitochondrially produced ROS can signal the functional state of mitochondria19,143,157. ROS were found to target redox-sensitive cysteine residues (redox switches) of the cytosolic translation apparatus, including the ribosome and translation factors157. When ROS production is increased, translation efficiency is decreased in a reversible manner (Fig. 3). The respiratory chain thus participates in controlling cytosolic protein synthesis to decrease the protein load under mitochondrial stress conditions157.

Coenzyme Q (CoQ), also termed ubiquinone, is a central molecule for the function of the respiratory chain158,159. CoQ is a redox-active lipid that mediates electron transfer from respiratory complexes I and II to complex III (Fig. 3) and functions as a cofactor of many enzymes. Several components of CoQ biosynthesis were recently identified using systematic mass spectrometry profiling at the proteomic, lipidomic and metabolomic levels26,71,158,159. The CoQ biosynthetic complex, which is located at the matrix side of the mitochondrial inner membrane, contains numerous enzymes involved in CoQ biosynthesis. All protein subunits of this dynamic CoQ biosynthetic complex are encoded by nuclear genes and are imported by the TOM and TIM machineries158,159. For example, the precursor of the methyltransferase Coq5 is processed twice. A first cleavage by MPP generates an unstable intermediate and a second cleavage by Oct1 generates the stable mature Coq5 enzyme (Fig. 3). Disturbance of processing by Oct1 leads to CoQ deficiency and respiratory defects71. Thus, import and specific processing of Coq precursors by the mitochondrial protein import machinery are functionally linked to the CoQ biosynthetic complex. A recent study showed that the machinery for assembly of Fe–S clusters is associated with respiratory chain supercomplexes160, underscoring a close connection between respiratory functions and cofactor biogenesis.

The mitochondrial inner membrane carries two large ATP-dependent protease complexes, the iAAA protease and the matrix-exposed mAAA protease18,161,162,163. These proteases are main elements of a quality control system for protein processing and turnover in mitochondria. AAA proteases cleave or degrade different inner membrane proteins such as some subunits of respiratory complexes and preprotein translocases and are also involved in the quality control and turnover of selected matrix, intermembrane space and outer membrane proteins (Fig. 3). In fungi, the adaptor proteins Mgr1 and Mgr3 associate with the iAAA protease and promote substrate recognition by the protease161,163. A recent proteomic study of yeast mitochondria identified the respiratory chain-interacting proteins Rci37 and Rci50 and demonstrated that they also interacted with the mAAA protease and the iAAA protease26, respectively, revealing specific connections between respiratory complexes III and IV and the inner membrane quality control system.

The functional network of the mitochondrial respiratory chain thus includes respiratory supercomplexes and machineries for protein biogenesis, cofactor biosynthesis and mitochondrial quality control.

Box 2 Mitochondrial presequence translocase and respiratory chain assembly

Interaction network of the presequence translocase

The translocase of the inner membrane (TIM23) complex is a central junction in the presequence import pathway and interacts with several partner complexes in a dynamic manner: with the translocase of the outer membrane (TOM) complex during preprotein transfer from the outer membrane (OM) to the inner membrane (IM), forming a TOM–TIM–preprotein supercomplex; with the ATP-driven presequence translocase-associated motor (PAM); with respiratory chain complexes III (CIII) and IV (CIV), which generate an electrochemical proton gradient (ΔμH+) driving preprotein insertion150,151; and with the ADP/ATP carrier, which also supports preprotein translocation152,234 (see the figure, part a; interactions depicted by double-headed arrows). The translocase subunit Tim21 functions as a dynamic coupling factor that interacts with TOM and the respiratory supercomplex CIII–CIV in an alternating manner. In fully active, respiring yeast mitochondria, the activity of the presequence translocase drives preprotein import efficiently. However, when the respiratory activity is decreased, coupling of the translocase to machineries involved in bioenergetics is beneficial to maintain the energy-dependent action of the translocase150,151,152. Preprotein translocases in the immediate vicinity of proton pumping respiratory complexes likely experience an increased proton motive force (localized proton gradients), and thus, under energy-limiting conditions, preprotein insertion into the IM is still possible150,151, ensuring that the biogenesis of respiratory complexes continues even in conditions of limited food supply.

Coupling of presequence translocase to respiratory chain assembly

The characterization of respiratory chain biogenesis in human mitochondria revealed a further level of cooperation between the protein import machinery and the respiratory chain, which is mediated by the mitochondrial translation regulation assembly intermediate of cytochrome c oxidase (MITRAC)153,154,155. MITRAC comprises several assembly intermediate complexes of the respiratory chain and plays a dual role (see the figure, part b). It links the presequence translocase to respiratory chain assembly intermediates via the TIM21-mediated transfer of imported proteins from TIM23 to MITRAC154. Moreover, MITRAC cooperates with the machineries for mitochondrial protein synthesis and insertion (oxidase assembly (OXA) insertion machinery) by adapting the efficiency of mitochondrial translation to the import of nuclear-encoded partner proteins (translational plasticity). MITRAC assembly factors bind to partially synthesized membrane-inserted proteins, such as the highly hydrophobic COX1 protein, and delay their synthesis until the appropriate partner proteins have been imported to ensure a proper balance of nuclear and mitochondrially encoded subunits155. The functions of human TIM14 (also known as DNAJC19 or PAM18) and ROMO1 (also known as MGR2) in the TIM23–PAM machinery have not been defined so far (indicated by dashed borders).

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Mitochondrial membrane architecture and membrane contact sites

Contact sites between the mitochondrial outer and inner membranes and between the outer membrane and the endoplasmic reticulum (ER) are crucial elements of a large network of membrane contact sites that functions in protein and lipid biogenesis, mitochondrial membrane architecture and dynamics, metabolite and ion transport and mitochondrial inheritance (Fig. 4). Preprotein translocases form central building blocks of this ER–mitochondria organizing network (ERMIONE)164. The TOM and SAM complexes of the outer membrane interact with the large MICOS complex of the inner membrane9,10,11,13,165,166,167,168. MICOS is enriched at crista junctions169,170, and its largest subunit, Mic60, plays an important role in the formation of outer–inner membrane contact sites. In addition, Mic60 transiently interacts with the receptor and oxidoreductase Mia40 of the intermembrane space assembly machinery9. MICOS thus helps to position the downstream machineries MIA and SAM close to the import channel TOM and promotes the efficient import of cysteine-rich precursors into the intermembrane space and of β-barrel precursors into the outer membrane9,165.

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The mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane (IM) and the protein translocases translocase of the outer membrane (TOM) and sorting and assembly machinery (SAM) of the outer membrane (OM) form the core of a large endoplasmic reticulum (ER)–mitochondria organizing network (ERMIONE) that includes multiple dynamic interactions: with the ER–mitochondria encounter structure (ERMES); with further ER–mitochondria contact sites that involve the receptor Tom70 and inositol trisphosphate (InsP3) receptors or the lipid transfer protein Lam6, as well as with vacuole–mitochondria contact sites (including Tom40 and the bridging protein Vps39); with the kinase PTEN-induced putative kinase 1 (PINK1) and the metabolite channel porin; with the mitochondrial intermembrane space (IMS) protein import and assembly system (Mia40); with respiratory chain complexes, the F1F0-ATP synthase and the fusion protein optic atrophy 1 (OPA1) of the IM; and with mitochondrial DNA (mtDNA) nucleoids (with the mtDNA packaging factor, termed mitochondrial transcription factor A (TFAM))262 of the matrix. Most components shown have been functionally conserved from yeast to humans; proteins that have been characterized in fungi only are indicated by a dashed border. In sum, ERMIONE forms a membrane-spanning system for the coordination of protein and lipid biogenesis, energetics, inheritance and quality control of mitochondria. CI, complex I; CII, complex II; CIII, complex III; CIV, complex IV.

 

The MICOS–SAM–TOM core of ERMIONE interacts with other mitochondrial machineries, which results in a large and complex network of functional interactions (Fig. 4). Most ERMIONE-interacting partners have been identified biochemically (via direct binding) or genetically (identifying synthetic growth defects).

The mechanisms that regulate the interactions in this complex network and their functional importance are the subject of intensive research and are being gradually revealed. In the inner membrane, Mic10, a core component of MICOS171,172, and its partner protein Mic27 are in dynamic contact with the dimeric F1F0-ATP synthase that shapes cristae rims, leading to a crosstalk between the two major membrane-shaping machineries of the inner membrane, MICOS and F1F0-ATP synthase144,173,174. Assembly of the Mic10-containing subcomplex of MICOS is linked to respiratory complexes and the mitochondria-specific dimeric phospholipid cardiolipin175,176. In addition, MICOS is connected to the machineries for mitochondrial fusion, including the inner membrane fusion protein optic atrophy 1 (termed OPA1 in mammals and Mgm1 in yeast)144,177,178. Studies of mutants have uncovered a functional link between MICOS and nucleoid aggregation and inheritance of mitochondrial DNA (mtDNA)179,180, but the underlying molecular mechanisms require further analysis. At the outer membrane, the SAM complex not only directly interacts with a fraction of TOM complexes in TOM–SAM supercomplexes125,127 but also exchanges Mdm10 with the ER–mitochondria encounter structure(ERMES) that links the mitochondrial outer membrane to the ER (Fig. 4). The outer membrane β-barrel protein Mdm10 is a subunit of both SAM, where it functions in TOM biogenesis, and ERMES, where it contributes to lipid transfer and maintenance of mitochondrial morphology181,182,183,184. The shuttling of Mdm10 between SAM and ERMES is regulated by the small protein Tom7. Tom7 has a dual localization: it is mainly located in the TOM complex but also functions outside the TOM complex as a regulatory factor that promotes Mdm10 transfer to ERMES185,186,187,188. Non-assembled Tom7 retards TOM assembly by shifting Mdm10 from the SAM form to the ERMES form, which constitutes a regulatory mechanism that is active when an excess of non-assembled TOM subunits accumulate in mitochondria. The major outer membrane metabolite channel porin, also termed voltage-dependent anion channel (VDAC), interacts with MICOS as well as the TOM complex10,189, linking metabolite transport to ERMIONE. Tom70, one of the receptors of the TOM complex, is also found outside the TOM complex and has crucial roles in forming ER–mitochondria contact sites (Fig. 4). Tom70 and its isoform Tom71 interact with Lam6 (also known as Ltc1), a lipid transfer protein that is anchored to membranes through lipid-binding sites and regulates contact sites between mitochondria, ER and other organelles190,191. Mammalian TOM70 also interacts with inositol trisphosphate (inositol-1,4,5-trisphosphate) receptors of the ER to promote Ca2+ transfer from the ER to mitochondria192. Moreover, Tom40 participates in the formation of vacuole–mitochondria contact sites, known as vacuole and mitochondria patch (vCLAMP), involving the vacuolar GTPase Ypt7 and the bridging protein Vps39 (also known as Vam6)190,193. Furthermore, vesicles can be released from the mitochondrial outer membrane to direct selected cargo to lysosomal degradation194,195,196 and Tom70 and Tom71 were found to be required for the formation of at least some mitochondria-derived vesicles197, linking the transport machinery to quality control and degradation systems. The AAA-type ATPase Msp1 promotes the extraction of mistargeted proteins from the outer membrane198,199. Upon accumulation of non-imported precursor proteins, Msp1 is recruited to Tom70 via the peripheral membrane protein Cis1 (also known as Atg31), leading to removal of non-imported proteins and their degradation by the proteasome200. Lastly, as discussed below, TOM and MICOS are involved in the accumulation of PTEN-induced putative kinase 1 (PINK1) at the outer membrane of dysfunctional or damaged mitochondria, which promotes their removal by mitophagy15,16,201,202.

Thus, the mitochondrial membranes contain at least two large protein networks, both containing TOM complexes: the TOM–TIM23–respiratory chain–AAA network, which couples protein import to bioenergetics and quality control mechanisms, and the MICOS–SAM–TOM–ER network (ERMIONE), which links protein biogenesis to membrane contact sites and membrane morphology. Whereas MICOS is enriched at crista junctions, TOM–TIM23–preprotein supercomplexes are preferentially found ~30–60 nm away from crista junctions124. It remains to be determined whether these two large networks function independently of each other or whether they exchange components as a means of coordinating protein biogenesis, energetics, membrane morphology and quality control. Although substantial future work will be required to fully understand how ERMIONE functions at the molecular level, the identification of these networks clearly demonstrates that mitochondrial machineries do not function as stand-alone units but are intimately linked to each other.

Protein import and pathophysiology

The efficiency of protein import into mitochondria is a sensitive indicator of the energetic state and the fitness of mitochondria. Various disorders of mitochondrial respiration and metabolism lead to reduction in the inner membrane potential203,204. As the membrane potential is crucial for protein translocation into and across the inner membrane, the import of preproteins is diminished1,2. Defects of protein homeostasis in the mitochondrial matrix by the accumulation of misfolded proteins also lead to a reduced protein import, likely by disturbing the mtHsp70 import motor205. The impaired activity of the mitochondrial protein import machinery under stress conditions or in mitochondrial diseases is a direct indicator of impaired mitochondrial functions and can induce stress responses or lead to the removal of damaged mitochondria by mitophagy (Box 3).

A mild disturbance of mitochondrial protein import can trigger the activation of the UPRmt as a result of the failure to import the transcription factor ATFS-1 (also known as ATF5) into the mitochondria, leading to its transport into the nucleus and a transcriptional stress response206,207 to rescue partially damaged mitochondria (Box 3). The stress-induced decrease in TIM17A levels also leads to decreased mitochondrial protein import and promotes the induction of a UPRmt133. In addition, accumulation of mitochondrial precursor proteins in the cytosol leads to an attenuation of cytosolic protein synthesis and activation of the proteasome to clear the mistargeted proteins from the cytosol208,209,210,211,212. This process is known as unfolded protein response activated by mistargeted mitochondrial proteins (UPRam) or mitochondrial precursor over-accumulation stress (mPOS).

Upon severe damage of mitochondrial protein import, the kinase PINK1 is not imported, processed and degraded but associates with the TOM complex as full-length protein, initiating a cascade that leads to removal of damaged mitochondria by mitophagy15,16,201,202 (Box 3). As mutations in PINK1 are linked to Parkinson disease, it has been proposed that insufficient mitophagy may be one of the causes underlying the development of the disease201. Recently, PINK1 and MIC60 of the MICOS complex were found to interact transiently, suggesting a crosstalk between PINK1 accumulation and inner membrane cristae remodelling213,214 (Fig. 4).

A recent study in yeast has suggested that the mitochondrial protein import machinery removes misfolded proteins from the cytosol and transports them to degradation inside mitochondria215. This process, which was named mitochondria as guardian in cytosol (MAGIC), requires further investigation to define its relevance for cellular protein homeostasis (proteostasis) and its relation to stress responses that are initiated by a decreased mitochondrial protein import efficiency such as UPRmt, UPRam and the PINK1–parkin pathway.

Proteolytic processing of precursor proteins also plays a role in mitochondrial quality control. In one mechanism, the removal of destabilizing amino-terminal amino acid residues by the processing enzyme Icp55 or Oct1 stabilizes imported proteins against proteolytic degradation49,71,72 (Fig. 2). In another mechanism, imported proteins are differentially processed, yielding two or more isoforms with distinct amino termini. For example, the inner membrane fusion protein OPA1 is first processed by MPP, generating a long isoform, and further processing by inner membrane proteases such as AAA proteases and OMA1 in mammals generates short isoforms216,217,218. The balance between long and short isoforms, which is important for membrane fusion and fission, is modulated by stress and the energetic state of the inner membrane (that is, mitochondrial activity)216,217,218.

The impaired processing of preproteins has been linked to mitochondrial dysfunctions in Alzheimer disease219. The matrix peptidasome degrades presequences and other peptides such as Alzheimer-linked amyloid-β peptides. Upon accumulation of amyloid-β peptides in mitochondria, the degradation of presequences is slowed down competitively, leading to an inhibition of processing peptidases. As a consequence, proteins imported into mitochondria are retained in precursor or intermediate forms that cannot fold properly and are prone to rapid degradation. The accumulation of amyloid-β peptides thus causes numerous changes in mitochondrial protein composition, providing possible explanations for a wide variety of mitochondrial alterations observed in Alzheimer disease.

Studies in recent years have provided increasing evidence for the involvement of mitochondrial protein import and processing in the pathogenesis of human diseases. At present, however, there are different views on whether mitochondrial dysfunctions are directly or indirectly involved in the development of major neurodegenerative diseases such as Parkinson disease and Alzheimer disease220. In Box 4, we provide an overview of more rare diseases and disorders that have been linked to specific components of the mitochondrial machineries for protein import and maturation, suggesting an involvement in disease pathogenesis. The diseases mostly affect the nervous system and other tissues with a high energy demand such as heart, muscles and kidney. On a mechanistic level, defects in preprotein targeting, the presequence pathway, processing and folding, the MIA pathway and the carrier pathway have been observed (Box 4).

The elaborate networks between preprotein translocases and other mitochondrial machineries have mostly been studied under physiological conditions. We expect that these networks will play an important role in understanding the mechanistic basis of mitochondrial stress responses and pathogenesis of diseases, exemplified by the role of TOM and MICOS in the accumulation of PINK1 at the outer membrane and the subsequent removal of damaged mitochondria by mitophagy213,221,222,223.

Box 3 Quality control pathways associated with the protein import machinery

Mitochondrial unfolded protein response (UPRmt)

The stress-activated transcription factor ATFS-1 contains mitochondrial and nuclear localization signals. The factor is imported into healthy mitochondria and degraded. When mitochondrial import is impaired, the transcription factor accumulates in the cytosol, is translocated into the nucleus and induces expression of chaperones, proteases and further factors to promote recovery of impaired mitochondria206,207.

Unfolded protein response activated by mistargeted mitochondrial proteins (UPRam)

Upon disturbance of mitochondrial protein import, precursor proteins accumulating in the cytosol trigger a stress response, the UPRam (also known as mitochondrial precursor over-accumulation stress (mPOS)), that reduces the efficiency of cytosolic protein synthesis and increases the activity of the proteasome, thus reducing the accumulation of mistargeted proteins in the cytosol208,209.

The PINK1–parkin pathway

The mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) has been identified in familial cases of Parkinson disease. In healthy mitochondria, PINK1 is imported by the presequence pathway and processed by mitochondrial processing peptidase (MPP) and the presenilin-associated rhomboid-like protease PARL, followed by release into the cytosol and degradation by the proteasome. When protein import or processing by the presequence pathway is disturbed, unprocessed PINK1 accumulates at the translocase of the outer membrane (TOM) complex221,222,223 (Fig. 4), where it phosphorylates ubiquitin and the E3 ubiquitin ligase parkin, triggering the removal of damaged mitochondria by mitophagy.

Mitochondria as guardian in cytosol (MAGIC)

Some aggregation-prone or misfolded cytosolic proteins may be imported into mitochondria and degraded215, suggesting a role of mitochondria in cytosolic proteostasis.

 

Box 4 Disorders and diseases associated with distinct steps of human mitochondrial protein import and maturation

Mitochondrial targeting

Mutations in mitochondrial targeting signals can impair import of individual proteins, causing pyruvate dehydrogenase E1α deficiency235 or mitochondrial aspartyl-tRNA synthetase import defect linked to leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL)236. L-Alanine:glyoxylate aminotransferase resides in peroxisomes in humans; however, mutations can generate a mitochondrial targeting signal, leading to mistargeting to mitochondria and primary hyperoxaluria type 1 (refs237,238). Similarly, in a form of renal Fanconi syndrome, a mutation generates a mitochondrial targeting signal in a peroxisomal protein involved in fatty acid oxidation, causing its mistargeting to mitochondria and disturbance of mitochondrial energy production in the proximal tubule239.

Mitochondrial intermembrane space import

A mutation in the ERV1 gene, which encodes the disulfide relay component GFER (also known as ALR), causes impairment of FAD cofactor binding, resulting in myopathy with cataract and combined respiratory chain deficiency240,241.

Mitochondrial presequence import pathway

Mutations in TIMM50, the gene encoding the presequence translocase receptor translocase of the inner membrane 50 (TIM50), lead to an impaired import by the presequence pathway, reduced levels of oxidative phosphorylation components, increased reactive oxygen species (ROS) production, severe epileptic encephalopathy, 3-methylglutaconic aciduria and lactic acidosis242,243. Mutations in the DNAJC19 gene, encoding the human mitochondrial import inner membrane (IM) translocase subunit TIM14 (also known as DNAJC19 or PAM18), cause dilated cardiomyopathy with ataxia, anaemia and testicular dysgenesis244,245. As TIM14 is mainly associated with prohibitin complexes affecting cardiolipin metabolism, cardiolipin alteration is likely involved in disease pathogenesis246. DNAJC15 (also known as methylation-controlled J protein (MCJ)), another human TIM14 homologue247, has been linked to tumorigenesis. TIM14 and DNAJC15 have been connected to distinct presequence translocase forms132, yet their exact relevance for protein import needs further analysis (indicated by a dashed border). A mutation in the MAGMAS gene, encoding the human TIM16 J-like co-chaperone (also known as PAM16) of the presequence translocase-associated import motor is linked to a severe spondylodysplastic dysplasia248.

Precursor protein processing

Mutations in the genes encoding the mitochondrial processing peptidase (MPP) subunits α (PMPCA) and β (PMPCB) cause defects in preprotein processing, which are linked to cerebellar ataxia249or early childhood neurodegeneration with cerebellar atrophy250. Mutations in the MIPEP gene, encoding the human octapeptidyl peptidase, cause left ventricular non-compaction cardiomyopathy with hypotonia and developmental delay251. Mutations in XPNPEP3, the gene encoding the human intermediate cleaving peptidase, are linked to nephronophthisis-like cystic kidney disease252,253.

Chaperones for precursor protein folding

Mutations in HSPD1, encoding the chaperonin HSP60, lead to neurodegenerative disorders, spastic paraplegia and mitochondrial chaperonin-60 disease254,255. A mutation in the HSPE1 gene, encoding the co-chaperonin HSP10, is associated with infantile spasms and developmental delay256.

Membrane protein transfer chaperone

Mutations in the DDP1 gene (also known as TIMM8A), encoding a small TIM chaperone (subunit TIM8A), cause deafness dystonia syndrome, which is also termed Mohr–Tranebjaerg syndrome257,258.

Carrier translocase

Mutations in the acyl glycerol kinase AGK gene lead to cataracts, cardiomyopathy and skeletal myopathy (Sengers syndrome)259. AGK plays a dual role as a lipid kinase and as subunit of the human TIM22 carrier translocase, linking lipid metabolism and protein import to Sengers syndrome260,261.

ΔΨ, membrane potential; IMS, inner membrane space; MIA, mitochondrial intermembrane space import and assembly; mtHSP70, mitochondrial heat shock protein 70 kDa; OM, outer membrane; TOM, translocase of the outer membrane.

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Conclusions and perspectives

We have discussed that mitochondrial preprotein translocases, respiratory complexes, metabolite transporters, proteases, morphology complexes and membrane contact sites do not function as independent machineries but are physically and functionally connected in large dynamic networks. The protein translocases represent an essential housekeeping system of mitochondria. Not only are the translocases responsible for importing ~1,000–1,500 different proteins; they also form stable building blocks of the mitochondrial protein networks.

The rapid progress in identifying connections between machineries of different functions11,26,144,158,159,224,225 indicates that we have not reached saturation in the analysis of mitochondrial protein networks. In addition to the experimentally established connections described in this Review, interesting further network candidates include scaffold protein complexes that locally organize the protein–lipid composition of the inner membrane, such as the prohibitin ring complexes and stomatin-like protein 2, which associates with protease complexes and regulates the processing of PINK1 and OPA1 (refs11,226); lipid biosynthesis and remodelling enzymes; and cytosolic machineries that are involved in transferring preproteins, lipids or metabolites to mitochondria. Whereas several contact sites between mitochondria and other cell organelles have been identified recently, we have only a limited understanding of the interplay between cytosolic proteins or protein complexes and the mitochondrial outer membrane. This includes the potential involvement of specialized pools of cytosolic ribosomes in protein delivery to mitochondria227, the role of cytosolic chaperones, co-chaperones and potential targeting factors in cytosol–mitochondria crosstalk86,228,229,230, the rerouting of mitochondrial preproteins from the surface of the ER to mitochondria231 and the emerging evidence that numerous mitochondrial proteins possess a dual function and localization26,232.

Important questions concern the dynamics, regulation and turnover of the protein networks. It is likely that partner complexes in networks are turned over at different rates. Examples are the stress-regulated degradation of the TIM17A isoform of metazoan presequence translocases133 and the selective degradation of the outer membrane proteins Tom22 and porin-associated Om45 by the iAAA protease163(Fig. 3), whereas the other subunits of the complexes are turned over by different proteolytic machineries. The differential control of the networks by mitochondrial proteolytic systems, the cytosolic ubiquitin–proteasome system and mitophagy, as well as the role of lipids in establishing and maintaining the networks, will become central topics of research.

The large number of distinct functions observed in mitochondrial protein networks may give the initial impression that collaborations of protein machineries have developed in a random manner. The mechanistic studies performed so far, however, indicate that the interactions are highly specialized and specifically regulated, such as those between presequence translocase and respiratory supercomplexes and those between MICOS, TOM, SAM and the ER-mitochondria contact sites. To date, the studies have been mainly performed in yeast and partially in human mitochondria, which both belong to the same supergroup of eukaryotes, opisthokonts, including fungal and metazoan kingdoms. As the characterization of the mitochondrial protein import machinery in different supergroups yielded remarkable insight into core machineries and the high variability of transport complexes233, a systematic analysis of mitochondrial protein networks in the five eukaryotic supergroups will represent a rich source for defining core principles and variable parts of mitochondrial organization.

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