The presequence pathway is the best characterized pathway, responsible for the import of ~60% of all mitochondrial proteins (7). The precursor proteins in this pathway carry a cleavable N-terminal presequence that functions as a targeting signal (7–9). This unique feature of the pathway distinguishes it from all the others, where the precursor proteins do not have cleavable presequences, but possess different kinds of internal targeting signals. Presequence-carrying precursors to the mitochondrial matrix are typically recognized by the translocase of the outer membrane (TOM) (9–11), passaged through the IM by the translocase of the inner membrane (TIM23) (12–16), and driven into the matrix assisted by the presequence translocase-associated motor (PAM) (Figure 1) (17–24). The membrane potential (Δψ) across the IM is essential for the activation of the TIM23 channel and the translocation of presequences into the matrix (25–28). The presequences are cleaved by the mitochondrial processing peptidase (MPP) (3, 29–31), and additional proteolytic processing occurs by intermediate cleaving peptidases (7, 32–34). Presequence-carrying precursors that integrate into the IM follow two distinct routes (Figure 1). IM proteins are either directly released laterally from the TIM23 complex (35–37) or transported first into the matrix, followed by further insertion into the IM with the help of the oxidase assembly protein 1 (Oxa1) insertase (38–41). Oxa1 also is responsible for insertion of IM proteins synthesized on mitochondrial ribosomes (42).

The presequences are located at the N-termini of preproteins and typically consist of ~15–50 amino acids. An essential characteristic of mitochondrial presequences is the formation of an amphipathic α-helix that is specifically recognized by mitochondrial import receptors and other mitochondrial import components during preprotein translocation by TOM, TIM23, and PAM (3).

The TOM complex is the main gate for precursors entering mitochondria. The TOM complex is composed of Tom20, Tom22, and Tom70 as receptors, β-barrel protein Tom40 forming the channel, and three small associated proteins, Tom5, Tom6, and Tom7 (11, 43–45). Tom20 and Tom22 function cooperatively as the receptors for presequence-carrying precursors (9, 10, 46). Tom70 mainly functions as the receptor for preproteins with internal targeting sequences, such as carrier precursors (47–52). Presequence-carrying precursors cross the Tom40 channel as linear polypeptide chains (44, 45, 53–57), and interact with the tail of the Tom22 receptor in the IMS (57). Tom22 has presequence binding sites on both the cytoplasmic and IMS sides of the OM. The exact roles of the three small subunits, Tom5, Tom6, and Tom7, have not been well-clarified. It has been proposed that they are not essential for TOM functions but are involved in the assembly and stability of the TOM complex (58–61).

The TIM23 complex translocates cleavable preproteins into mitochondrial matrix or IM. Tim50, Tim23, Tim17, and Tim21 compose the main elements of the TIM23 complex (12, 13, 16, 62–65). Tim50 functions as a presequence receptor that binds preproteins emerging in the IMS (63) and a channel blocker that closes out the TIM23 channel in the absence of preproteins (25, 66–69). Tim23 and associated partner Tim17 form the channel (16, 25, 28, 64, 70, 71). Tim21, Tim50, and Tim23 expose domains to the IMS that transiently connect the TOM and TIM23 complexes to facilitate the preprotein transfer (16, 67, 72–74). Additionally, Tim21 also physically links the TIM23 complex to the respiratory chain III-IV supercomplex [bc1 complex and cytochrome c oxidase (COX)] (65, 75, 76). Tim21 thus plays a dual role in TOM-TIM23 transfer and the recruitment of respiratory chain complexes. A small membrane protein, Mgr2, functions as a lateral gatekeeper for preproteins that are sorted into the IM (35, 76). The Δψ across the IM is crucial for translocation of the presequences through the Tim23 channel, which is negative at the matrix side and positive at the IMS side of the IM, whereas presequences are mostly positively charged. Two roles have been assigned to Δψ: activation of TIM23 channel (25) and an electrophoretic effect that drives the import of presequences (77, 78).

PAM. The Δψ is a prerequisite for translocation of the presequence across the TIM23 channel. Nevertheless, it is not sufficient to import the entire protein into the matrix. PAM is necessary for the translocation of matrix proteins. The core of PAM is formed by the molecular chaperone mitochondrial 70 kDa heat shock protein (mtHsp70) (17, 18) and its co-chaperones (Tim44, Mge1, Pam16, Pam17, and Pam18) (16, 21, 79, 80). mtHsp70 binds the unfolded polypeptide chain and drives its translocation into the matrix in an ATP-dependent manner (17, 18). The peripheral membrane protein Tim44 is a docking site for mtHsp70 at the TIM23 complex (21). Mge1 (also known as mitochondrial GrpE) stimulates the release of ADP from mtHsp70 (81). Pam16, Pam17, and Pam18 are three membrane-associated co-chaperones. Pam18 (also termed Tim14) is a J-type co-chaperone that stimulates the ATPase activity of mtHsp70 (82, 83). The J-related Pam16 (Tim16) forms a complex with Pam18 and functions as a negative regulator (82–86). Pam17 mediates the organization of the TIM23–PAM interaction (79, 87).

MPP. Once arriving in the matrix, the presequences of both IM-sorted and matrix-targeted precursors are removed by a heterodimeric enzyme, MPP (3, 29–31, 88). Additional proteases, the intermediate cleaving peptidase (Icp55) (7, 89) and the octapeptidyl aminopeptidase (Oct1, also termed mitochondrial intermediate peptidase, MIP) (90, 91), can remove destabilizing N-terminal amino acid residues of the imported proteins. mtHSP70 and other chaperones, like the HSP60–HSP10 chaperonin complex, further assist proteins folding into their active forms (92). The clipped presequence peptides undergo subsequent degradation by the matrix peptidasome, termed presequence protease (PreP) or Cym1 (93, 94).

OXA translocase is vital for exporting proteins from the mitochondrial matrix into the IM. OXA has three different roles. (1) Proteins encoded by the mitochondrial genome are exported into the IM by Oxa1 (42, 95–97). (2) Some presequence-carrying proteins imported into the matrix via the TOM-TIM23 machinery are exported into the IM via Oxa1. This import-export pathway is termed conservative sorting of nuclear-encoded IM proteins (38–41, 98–100). (3) Oxa1 is also vital for the assembly of the carrier translocase TIM22 (38, 101).

The carrier pathway is the second mitochondrial protein import pathway to be discovered, and is responsible for importing precursor proteins without a cleavable presequence, yet with different kinds of internal targeting signals (3, 102–104). The carrier precursors are accompanied by cytosolic chaperones, such as the HSP70 and HSP90 classes in the cytosol, directly delivered to the Tom70 receptor of the TOM complex (47, 105, 106), and then bound by small TIM chaperones in the IMS (107–110) and eventually integrated into the IM by the carrier translocase of the IM (TIM22) complex in a Δψ-dependent manner (Figure 1) (109, 111–116).

Chaperone-guided transport of carrier precursors (including chaperones in the cytosol and IMS). The carrier import pathway uses the same mitochondrial entry gate as the presequence pathway, the TOM complex. However, the mechanisms of translocation differ significantly. The involvement of cytosolic (47, 105, 106) and mitochondrial IMS chaperones, which is crucial to prevent aggregation of the hydrophobic carrier precursors in the aqueous environment, is the main feature distinguishing this from the presequence pathway. Chaperones of the Hsp70 and Hsp90 classes directly participate in delivering the precursors to Tom70 (47, 105, 106). The receptor Tom70 possesses two distinct binding sites, one for the precursor and another for a chaperone (47, 49, 117), ATP is needed to release the precursor proteins from the cytosolic chaperones (47, 48). Upon binding to Tom70, the carrier precursors are transferred to the central receptor Tom22, followed by insertion into the Tom40 channel in a loop conformation (118, 119), and transferred to small TIM chaperones in the IMS (107–110). These small TIM heterohexameric chaperone complexes, like the Tim9-Tim10 complex (120, 121) and the homologous Tim8-Tim13 complex (122), bind to the precursor proteins and transfer them through the aqueous IMS to IM.

Insertion of carrier precursors into the IM. The TIM22 complex consists of the receptor-like protein Tim54, the channel-forming protein Tim22, the Tim9-Tim10-Tim12 chaperone complex, and the Tim18-Sdh3 module. The majority of Tim54 domain is exposed to the IMS and probably functions as the binding site for the Tim9-Tim10-Tim12 complex (123, 124). The Tim9-Tim10-Tim12 complex is a modified form of the IMS chaperone, docking onto the TIM22 complex (123, 125). Carrier precursors are inserted into the Tim22 channel in a Δψ-dependent manner (115). The Tim18-Sdh3 module is involved in the assembly of the TIM22 complex (126, 127). The carrier precursors are first bound to the Tim9–Tim10–Tim12 chaperone complex on the surface of the translocase. Upon activation of the Tim22 channel (Δψ-dependent), the precursors are inserted into the translocase, probably in a loop structure. Finally, the proteins are laterally released into the lipid phase of the IM.

Many IMS proteins contain internal targeting signals and characteristic cysteine motifs. In the cytosol, the precursors are kept in a reduced and unfolded state (128). Upon import by the TOM complex (60), they are oxidized by the MIA machinery, and stay in the IMS in an oxidized state (Figure 1). The MIA system consists of two main components: the oxidoreductase Mia40 and the sulfhydryl oxidase Erv1 (129–133).

Mia40 serves as a receptor and protein disulfide carrier. Most IMS proteins are synthesized without cleavable presequences but contain cysteine motifs. Unlike presequence-carrying precursors and carrier precursors, none of the Tom receptors is necessary for the import of MIA substrates (60, 61). Instead, upon passage through the Tom40 channel (60), Mia40 functions as a receptor on the IMS side of the Tom40 channel (133–138). It recognizes an internal signal of the precursor proteins, typically consisting of a hydrophobic element flanked by a cysteine residue (133, 139, 140). Mia40 binds to precursors via hydrophobic interaction and catalyzes the formation of disulfide bonds in imported proteins (133, 138). The disulfide bonds facilitate the conformational stabilization and assembly of many IMS proteins.

Erv1 cooperates with Mia40 in a disulfide relay. Mia40 does not form disulfide bonds de novo. Disulfide bonds are generated by Erv1 and transferred to Mia40 by the formation of transient intermolecular disulfide bonds (141). Mia40 then transfers the disulfides onto the imported protein. Upon transfer of disulfide bonds to proteins, cysteines of Mia40 become reduced and are re-oxidized by Erv1. Electrons originating from the oxidation of imported proteins flow in the opposite direction. They flow from Mia40 to Erv1 and then to O₂ or cytochrome c of the respiratory chain (141–144). In addition to most IMS proteins, some IM and matrix proteins are also MIA-system–dependent (28, 71, 113, 114, 145).

All the mitochondrial OM proteins are imported from the cytosol. The membrane contains two different types of integral protein: β-barrel proteins, which are integrated into the membrane by multiple transmembrane β-strands, and α-helical proteins, which are membrane-anchored by one or more α-helical transmembrane segments.

Sorting and Assembly Machinery for β-Barrel Proteins. The precursors of β-barrel proteins initially pass through the TOM complex at the OM (146), then bind to small TIM chaperones in the IMS (110), like the carrier precursors, to avoid aggregation (Figure 1). Subsequent insertion of the β-barrel proteins into the OM is performed by the SAM complex, which consists of a membrane-integrated protein, Sam50 (Tob55), and two peripheral membrane proteins exposed to the cytosol, Sam35 and Sam37 (147–149). Folding of the β-barrel proteins occurs at Sam50-Sam35, followed by lateral release into the lipid phase of the OM (148, 150, 151). Sam37 directly interacts with Tom22, coupling the TOM and SAM complexes into a transient supercomplex that promotes the efficient transfer of precursor proteins (54, 152).

OM Insertion of α-Helical Proteins. The main α-helical proteins are classified as signal-anchored proteins (containing an α-helical transmembrane segment at the N-terminus), tail-anchored proteins (containing an α-helical transmembrane segment at the C-terminus), and polytopic (multi-spanning) OM proteins. α-helical OM proteins are imported via distinct routes that do not involve the Tom40 channel, in contrast to most mitochondrial proteins. The insertion of some signal-anchored and polytopic OM proteins is performed by the MIM complex (153–156), which consists of multiple copies of Mim1 and one copy of Mim2, both of which are small single-spanning OM proteins (Figure 1) (157, 158). Tom70 is required for the insertion of some polytopic proteins into MIM (155, 156). In the case of tail-anchored proteins and some multi-spanning proteins, import is aided by the lipid composition of the membrane, and no proteinaceous machinery has been identified (159–162). However, the exact mechanism for sorting and insertion of α-helical outer membrane proteins is only partially understood, and further studies are urgently needed.

Tom70 mainly serves as the receptor for hydrophobic precursors without a cleavable presequence, such as carrier precursors. Tom70 protein was downregulated in hypertrophic heart of animals and humans, which was associated with increased oxidative stress. Furthermore, upregulation of Tom70 provided cardiomyocytes with full resistance to diverse pro-hypertrophic insults (218). Tom70 expression was also reduced by I/R insult in cardiomyocytes (219–222). Supplementation of Tom70 significantly attenuated I/R injury by promoting translocation of PKCε (216, 217) [to increase the expression of KATP channel pore-forming subunit Kir6.2 (223), augment mitochondrial respiratory capacity, and modulate cardiac glucose metabolism (224)], MICU1 (to reduce mitochondrial Ca²⁺ overload), (222) and PINK1 (associated with mitophagy) (221, 225) into mitochondria. Increased expression of PGC-1α/Tom70 was also involved in melatonin-induced cardiac protection against post-myocardial infarction, which was associated with inhibited mitochondrial impairment and reduced ROS generation (219, 220). In the hearts of diabetic db/db mice, Tom70 expression was suppressed. Reconstitution of Tom70 protected against diabetic cardiomyopathy through its antioxidant and antiapoptotic properties (226). Moreover, in aging hearts of diabetic db/db mice, only the expression of mitochondrial membrane proteins like Tom70 and VDAC, but not respiratory enzymes, could be increased by short-term exercise (227). Further, phosphoproteome mapping in a rat model of heart failure revealed phosphorylation of several import machinery proteins (Tom70, HSP90, and Tim8a), suggesting that the modification of mitochondrial protein import was involved in heart failure (228).

AGK (acylglycerol kinase). AGK is a mitochondrial lipid kinase that was recently identified as a subunit of the TIM22 complex. It plays an indispensable role in the import and assembly of mitochondrial carrier proteins in the IM (299). It has been shown that loss-of-function mutations in the AGK gene cause Sengers syndrome (281–286), an autosomal recessive mitochondrial disorder characterized by hypertrophic cardiomyopathy, congenital cataracts, skeletal myopathy, exercise intolerance, and lactic acidosis. Loss of AGK led to destabilized TIM22 complex; defects in the biogenesis of carrier substrates (such as adenine nucleotide translocator); lower complex I, III, and IV activities; perturbed tricarboxylic acid (TCA) cycle; and higher citrate synthase activity (300).

Tim8a (Tim8a/DDP1 and Tim8b/DDP2 are human homologs of yeast Tim8) is a small TIM chaperone in the IMS. Tim8a expression in ischemic rat heart was downregulated by treatment with GSK inhibitor SB216763, which showed a protective effect against I/R stress (258), suggesting a potential role of Tim8a in ischemic heart disease.

FAD-linked sulfhydryl oxidase ALR is the human homolog of yeast Erv1. The interaction between Mia40 and Erv1/ALR facilitates the import of the small Tim proteins and cysteine-rich proteins. Inhibition of ALR activity by MitoBloCK-6 or a translation initiation codon (ATG) morpholino targeted to ALR in zebrafish embryos led to retarded cardiac development and impaired cardiac function (287).

Apoptosis-Inducing Factor (AIF) was initially characterized as a pro-apoptotic factor. It translocates from the mitochondrial IMS to the nucleus in the presence of apoptotic insults. It is also critical for the mitochondrial import and maturation of CHCHD4 (in human)/Mia40 (in yeast) (301–303). Mutations of the AIF-encoding gene AIFM1 led to early prenatal ventriculomegaly (288) and childhood cardiomyopathy (289), accompanied by respiratory chain complex I and IV deficiency. Global loss of AIF in mice during embryogenesis resulted in embryonic growth retardation and death during mid-gestation. Muscle-specific loss of AIF in mice led to severe DCM and skeletal muscle atrophy, associated with a significant defect in respiratory chain complex I activity (290). The Harlequin (Hq) mice, a genetic model with an 80% reduction in mitochondrial AIF, displayed more severe ischemic damage than wild-type hearts after acute I/R injury (291, 292).

Cardiolipin (CL) is a unique phospholipid that is localized and synthesized in mitochondrial IM. CL plays a central role in many biological processes, such as mitochondrial biogenesis, protein import, morphology and mitophagy, oxidative phosphorylation, and apoptosis (304–306). Defective remodeling of CL due to genetic mutations of TAZ-1 causes Barth syndrome, a rare, X-linked recessive, infantile-onset debilitating disorder characterized by early-onset cardiomyopathy, skeletal myopathy, growth delay, and neutropenia (304–309). The molecular mechanisms were partially mediated by impaired mitochondrial import machinery. CL was verified to play a central role in the structural integrity and functions of mitochondrial translocases, such as TOM Complex (44, 310), TIM22 Complex, and TIM23 Complex (26, 311, 312).

Connexin 43 (Cx43) is the predominant protein forming gap junctions and non-junctional hemichannels in ventricular cardiomyocytes and is also localized at the IM of cardiomyocyte mitochondria (313–315). The translocation of Cx43 to the IM was TOM-HSP90–dependent and was enhanced by ischemic preconditioning (IP). The beneficial role of mitochondrial Cx43 in I/R stress was associated with its regulation of mitochondrial potassium influx and ROS production (257, 313–315). The cardioprotection of IP was abolished by genetic ablation of Cx43, blockade of mitochondrial Cx43 import, or age-related loss of mitochondrial Cx43 (257, 316–318).

PINK1 is imported into the mitochondrial matrix in healthy conditions, followed by retrotranslocation into cytosol and degradation by the proteasome. Perturbation of this process causes mitophagy, which plays a vital role in the quality control of mitochondria in heart disease. Given that it has been well-studied and summarized in many other reviews (319, 320), we do not discuss it here in detail.

NDUFB10, an accessory subunit of complex I, is a substrate of the MIA machinery (CHCHD4/ Mia40) for oxidation-dependent protein import into the mitochondrial IMS. Mutation of cysteine 107 of NDUFB10 impaired its mitochondrial import via CHCHD4 and resulted in complex I assembly defect, led to fatal infantile lactic acidosis and cardiomyopathy in a single-case report of an infant (321).

In addition, some other proteins also showed a protective effect against I/R or H/R stress through enhancing their translocation from the cytosol to the mitochondria; these include α-crystallin B (cryAB, the major small heat shock protein in cardiomyocytes) via VDAC-Tom20 (322) and DJ-1 via mtHSP70 (246, 323–325).

According to their subcellular spatial arrangement in cardiomyocytes, mitochondria are classified into three groups: subsarcolemmal mitochondria (SSM) existing below the cell membrane, interfibrillar mitochondria (IFM) residing in rows between the myofibrils, and perinuclear mitochondria located at the nuclear poles. Mitochondrial subpopulations vary in structure and function and appear to be influenced disparately in different cardiac pathologies, including I/R, heart failure, aging, exercise, and diabetes mellitus. According to recent studies, the mitochondrial import machinery in IFM of T1DM hearts (247, 326) and SSM of T2DM hearts (248, 327) were more susceptible to inefficiency. Further, many studies reported the downregulation of mitochondrial import-machinery components in heart disease, such as heart failure, DCM, ischemic cardiomyopathy, diabetic cardiomyopathy, etc. Supplements of corresponding components could partially recover cardiac function.

However, some studies pointed out an enhancement of mitochondrial protein import in aging animals. Craig et al. found mitochondria from senescent animals exhibited a higher import rate of precursors into the matrix than mitochondria from young animals (328). Later studies showed that, although expression of some key import machinery components was upregulated in the aging heart, import efficiency was compromised (238). The mechanism and significance need to be determined in future studies.

In addition, the import rate of matrix-localized proteins was found to be increased in heart of hyperthyroid animals or by T3 treatment (329–332), which was partially mediated by elevated levels of the OM receptor Tom20 and mtHSP70. Meanwhile, the proteolysis of matrix proteins was unaffected.