The majority of the mitochondrial proteome, required to fulfil its diverse range of functions, is cytosolically synthesised and translocated via specialised machinery. The dedicated translocases, receptors, and associated proteins have been characterised in great detail in yeast over the last several decades, yet many of the mechanisms that regulate these processes in higher eukaryotes are still unknown. In this review, we highlight the current knowledge of mitochondrial protein import in plants. Despite the fact that the mechanisms of mitochondrial protein import have remained conserved across species, many unique features have arisen in plants to encompass the developmental, tissue-specific, and stress-responsive regulation in planta. An understanding of unique features and mechanisms in plants provides us with a unique insight into the regulation of mitochondrial biogenesis in higher eukaryotes.

Introduction

Mitochondria represent an interesting case for the study of protein-targeting mechanisms as the majority of its proteome is nuclear-encoded and synthesised in the cytosol. These proteins need to be maintained in an import-competent state to be targeted to the mitochondria usually by cytosolic chaperones that direct the precursor to the translocase of the outer membrane (TOM) complex. Proteins are then directed to their specific subcompartments, the outer membrane (OM), inner membrane (IM), intermembrane space (IMS), or matrix utilising specialist machinery and pathways (Figure 1). In the following paragraphs, we will examine the mechanisms and machinery involved in protein transport across and into the plant mitochondrial subcompartments. We will discuss the current research in characterising these import components, the consequences of the modulation of these proteins, and focus on factors that have been identified to regulate these processes and pathways. Furthermore, we will discuss the various identified proteolytic pathways that deal with targeting peptide accumulation and amino acid and examine the protein export and assembly of mitochondrially encoded proteins. Plant-specific features will be reviewed to highlight the unique mechanisms and features that have evolved to deal with the complexities of a sessile organism (Table 1).

The four major protein import pathways in plant mitochondria.

Figure 1.
The four major protein import pathways in plant mitochondria.

The coloured lines indicate the four protein import pathways. All precursor proteins cross the TOM complex in the outer membrane. The orange line demonstrates the MIA pathway for proteins carrying twin cysteine residues that are assembled by the MIA complex. The pink line demonstrates the general import pathway for proteins directed to the matrix, via passage across the TIM17:23 (translocase of the inner membrane) complex upon which the cleavable targeting sequence is removed. The yellow line demonstrates the carrier protein pathway for assembly of carrier proteins, usually containing internal signals, via tiny TIM-mediated transport across the IMS to the TIM22 complex for insertion into the inner membrane. The green line demonstrates the SAM pathway for assembly β-barrel proteins on the outer membrane. OM, outer membrane; IM, inner membrane; IMS, intermembrane space.

Figure 1.
The four major protein import pathways in plant mitochondria.

The coloured lines indicate the four protein import pathways. All precursor proteins cross the TOM complex in the outer membrane. The orange line demonstrates the MIA pathway for proteins carrying twin cysteine residues that are assembled by the MIA complex. The pink line demonstrates the general import pathway for proteins directed to the matrix, via passage across the TIM17:23 (translocase of the inner membrane) complex upon which the cleavable targeting sequence is removed. The yellow line demonstrates the carrier protein pathway for assembly of carrier proteins, usually containing internal signals, via tiny TIM-mediated transport across the IMS to the TIM22 complex for insertion into the inner membrane. The green line demonstrates the SAM pathway for assembly β-barrel proteins on the outer membrane. OM, outer membrane; IM, inner membrane; IMS, intermembrane space.

Table 1
Key features of the plant mitochondrial protein import apparatus
Import component Details Ref. 
Tom70/Tim54/Tim18/OM14/Hot13 Lack plant orthologues [1,2
Tom20 Convergent evolution [3,4
Tom9 Orthologue to yeast TOM22, lacking receptor domain [5
Om64 Plant-specific outer membrane receptor [6,7
Tim17-1 ABA-responsive, germination-specific import component [8
Tim17-2 C-terminal extension links the inner and outer membrane [9
Tim21 Interacts with respiratory Complex III [10
Tim23-2 and B14.7 Dual-located within Complex I and TIM17:23 [10
PreP/OOP/M17/M17.10 Dual-located and conserved presequence peptidolytic processes with chloroplasts [1116
MPP/bc1 Dual function in processing and respiration in the inner membrane [17,18
Import component Details Ref. 
Tom70/Tim54/Tim18/OM14/Hot13 Lack plant orthologues [1,2
Tom20 Convergent evolution [3,4
Tom9 Orthologue to yeast TOM22, lacking receptor domain [5
Om64 Plant-specific outer membrane receptor [6,7
Tim17-1 ABA-responsive, germination-specific import component [8
Tim17-2 C-terminal extension links the inner and outer membrane [9
Tim21 Interacts with respiratory Complex III [10
Tim23-2 and B14.7 Dual-located within Complex I and TIM17:23 [10
PreP/OOP/M17/M17.10 Dual-located and conserved presequence peptidolytic processes with chloroplasts [1116
MPP/bc1 Dual function in processing and respiration in the inner membrane [17,18

A list summarising the key plant-specific features that have been identified for the protein import apparatus in plant mitochondria.

Precursor protein sorting

The majority of mitochondrial precursor proteins contain either cleavable peptide extensions known as presequences or internal signals that serve as sorting signals to the mitochondria [19,20]. These signals are pivotal for the correct sorting of precursor proteins and contain information for recognition by the outer membrane receptors and for the directed translocation of the preprotein across the membranes via four distinct protein import pathways as illustrated in Figure 1.

In the model plant Arabidopsis thaliana (Arabidopsis), the majority of presequences are less than 40 amino acids length [21]. They are typically rich in positively charged polar residues such as serine and arginine which are required as a recognition site for cleavage [2123]. Arginine residues in position −2 (P2) or −3 (P3) from the scissile bond site of precursor proteins often serve as a binding site for MPP [21,23]. Most presequences have a high abundance of positively charged residues and have a strong tendency to form amphiphilic α-helices, which turns out to be a prerequisite for the binding to protein import machinery at the outer membrane [21,24]. The physicochemical properties of presequences, such as secondary structure and overall polarity, rather than its length and primary structure, determine correct sorting and the subsequent post-import process of a mitochondria-destined precursor protein [21]. Owing to the presence of an additional endosymbiotic organelle, the chloroplast, that also requires importing the majority of its proteome, targeting signals thus need to be specific. This is further complicated by the fact that several hundred proteins have been shown to be dual-targeted to both the mitochondrial and chloroplast with over 400 proteins being predicted to be dual-targeted and 250 proteins being experimentally verified to be dual-targeted across organelles or intracellular locations [25,26]. Various researchers have focussed on identifying key residues that are responsible for maintaining targeting specificity [21,27]. These studies have identified that multiple sequence motifs or modules are required to co-operate synergistically allowing presequences to maintain import specificity despite their diversity. Several reports have additionally investigated the molecular mechanisms of dual-targeting specificity between the mitochondria and chloroplast [2832]. The targeting ability of the presequence belonging to the dual-localised Thr–tRNA synthetase (ThrRS–dTP) was investigated by a variety of deletion constructs coupled to GFP. These results showed that the 23 amino acid long N-terminal portion was essential for import, but the minimum length of peptide essential to drive dual-targeting specificity was 60 amino acid long [29]. While specific residues have not been identified, positive residues within the N-terminal of dual-targeted proteins might, in fact, function as an ‘avoidance signal’ for the chloroplast import [30]. Investigations into the dual-targeting properties of pMDHAR1 and pMDHAR2 (monodehydroascorbate reductase) revealed that only a four amino acid change was sufficient to confer dual-targeting ability [33].

Transport across the outer mitochondrial membrane

The first point of contact for nuclear-encoded mitochondrial proteins occurs at the outer membrane, namely the TOM complex and the sorting and assembly machinery (SAM) complex (Figure 2). The TOM complex is certainly the most fascinating translocase, involved in the translocation of a wide range of proteins with varying signals; it has also been shown to interact with every subsequent protein transport complex from which the precursor protein may be directed. In yeast, the TOM complex consists of the core translocation pore Tom40, the membrane-anchored and cytosolically facing receptor proteins; Tom20, Tom22, and Tom70. Small accessory proteins Tom5, 6, and 7 are also associated with and involved in complex stability (Figure 2).

The outer membrane and intermembrane space protein translocase in plant mitochondria.

Figure 2.
The outer membrane and intermembrane space protein translocase in plant mitochondria.

All proteins targeted to the mitochondria are translocated through the TOM40 complex. Plants lack a Tom70 and contain a truncated Tom22, named Tom9. Outer membrane B-barrel proteins are inserted via the SAM complex with the assistance of the small TIMs, while IMS proteins are assembled via the Mia40–Erv1 complex in the intermembrane space. OM, outer membrane; IM, inner membrane; IMS, intermembrane space. Plant-specific features/components are indicated with a leaf, and yeast-specific featured are indicated with yeast in pale yellow.

Figure 2.
The outer membrane and intermembrane space protein translocase in plant mitochondria.

All proteins targeted to the mitochondria are translocated through the TOM40 complex. Plants lack a Tom70 and contain a truncated Tom22, named Tom9. Outer membrane B-barrel proteins are inserted via the SAM complex with the assistance of the small TIMs, while IMS proteins are assembled via the Mia40–Erv1 complex in the intermembrane space. OM, outer membrane; IM, inner membrane; IMS, intermembrane space. Plant-specific features/components are indicated with a leaf, and yeast-specific featured are indicated with yeast in pale yellow.

The initial recognition of the targeting presequence occurs via the Tom20 receptor, an N-terminal-anchored protein that exhibits substrate specificity within its C-terminal domain. Plant Tom20 was first identified by biochemical characterisation in potato [34] and is one of the few plant protein import receptors that have evolved by independent means. Plant Tom20s are not orthologous to yeast, human, or fungal Tom20. In addition, they are structurally reversed, anchored to the outer membrane via the C-terminal domain, rather than an N-terminal anchor as observed in other species [3]. Plant Tom20s also contain unique bidentate-binding site. Nevertheless, plant Tom20 appears to be functionally the same as the non-plant counterparts [4]. Subsequent crystallographic and NMR studies of plant Tom20 presequence interactions confirm its dynamic affinity and recognition of presequences specific for both mitochondrial and dual-targeted presequences [31,35,36].

In Arabidopsis, four genes encode Tom20 proteins. Reverse genetic approaches have shown that the removal of three isoforms of Tom20 from Arabidopsis results in a significant decrease in protein uptake ability of specific precursor proteins such as AOX which is imported via the general import pathway [37], Tom40 and the carrier protein PiC but not the FAd-subunit of ATP synthase or the dual-targeted protein Glutathione Reductase [7]. Interestingly, no phenotype was observed with any single T-DNA insertion line tom20-2, tom20-3, or tom20-4 and only a slight delayed growth phenotype was observed in the triple tom20-2::20-3::20-4 knockout line, suggesting that there exists overlap in the substrate specificity across the four isoforms and possibly the requirement for additional outer membrane receptors [7].

Interestingly, no plant orthologues to Tom70 have been identified, either by biochemical or by homology-based approaches [1]. Yeast Tom70 functions as a receptor specifically for proteins containing internal targeting signals, so we could safely assume that an equivalent receptor must exist. A paralogue to the chloroplastic Toc64 import receptor has been identified in plants, named Om64, that utilises TPR domains to bind to Hsp70 and Hsp90 [6,38] (Figure 2). The depletion of Om64 resulted in a decrease in the import of FAd-subunit of ATP synthase, known to diverge from the general import pathway [39], but no difference was observed with any of the dual-targeted proteins tested [7]. It is noteworthy to mention that the generation of a quadruple knockout of tom20-2::20-3::20-4::om64 is lethal [40].

It would be tempting to speculate that an additional plant-specific outer membrane receptor exists. A recent study by Li et al. [41] characterised a novel plant-specific mitochondrial β-barrel protein named Om47 that belongs to the Porin3 superfamily of proteins that also encompasses Tom40, Sam50, and the chloroplastic Toc75 protein transporter. However, characterisation of T-DNA insertional lines showed no defect in protein uptake ability. Furthermore, as Om47 was able to complement the yeast vdac deletion mutant, it was concluded that Om47 might be involved in metabolite transport [41].

The sorting and assembly machinery

In yeast, the SAM protein complex located on the OM is responsible for mediating insertion of outer membrane β-barrel proteins consisting of Sam50, Sam37 (also known as metaxin), and Sam35 (Figure 2). The subunits, Sam35 and Sam37, face the cytosol, while Sam50 is an integral embedded inside the OM [42,43]. An additional subunit Mdm10 has also been identified to interact with the SAM complex and plays a role in the import of Tom40 [4446].

Plant orthologues of both Sam50 and Sam37/metaxin have been identified by sequence homology; however, only Sam37/metaxin has been characterised to any extent [7]. Loss-of-function mutants for SAM37/metaxin exhibited delayed growth phenotypes and altered starch metabolism. Its role in protein import was confirmed by a substantial decrease in the import uptake rates of all precursor proteins tested including the β-barrel proteins TOM40 and VDAC/porin [7]. It seems likely that the decrease in import rates of the non-β barrel proteins was rather affected by a consequential lack of the TOM40 complex, yet surprisingly the steady-state abundance of Tom40 protein was comparable to the Arabidopsis wild-type ecotype Col-0. This mechanism of TOM40 complex assembly would require further investigation. While T-DNA insertions for the two genes encoding Sam50 (At3g11070 and At5g05520) have yet to be characterised, Sam50 has been confirmed to resolve at 460 and 1000 kDa on a BN-PAGE gel along its interacting subunit Sam37 and a DNA J-homologue encoded by At2g35720 that raises the possibility that this protein may play a role in protein assembly and insertion [47]. An additional protein associates with the SAM complex named Mim1 (mitochondrial import 1 protein) involved in the insertion of Tom40 and the tail-anchored receptors Tom70 and Tom20 [48,49], though this protein appears to be yeast-specific (Figure 2).

Recent studies in yeast have uncovered multiple examples of dynamic interactions between SAM and TOM complexes, in addition to interactions with additional structures and complexes of the inner mitochondrial membrane. As mentioned above, the SAM complex contains a subunit named Mdm10 that it shares with the ER–mitochondria encounter structure (ERMES) complex, an outer membrane complex responsible for docking the mitochondria and the ER allowing metabolite exchange [50,51]. Tom7 has also been shown to interact with Mdm10, promoting the segregation of Mdm10 from the SAM complex [44,45]. So far, it appears that plants lack direct orthologues to Mdm10 or any other ERMES subunits [1].

The SAM complex has also been shown to interact with another inner membrane complex named mitochondrial contact site and cristae organising system (MICOS) or mitochondrial inner membrane organising system (MINOS). MICOS subunits were initially identified in yeast for maintaining inner membrane contact sites [52]. Composed of six subunits, homology-based searches revealed that five subunits, such as Mic10, Mic12, Mic25, DNAJC11, and Mic60, are present in plants [53]. The core subunit Mic60 was recently found to interact directly with both the TOM and the SAM complex, with a role in recruiting Mia40 to the TOM complex as to promote efficient translocation of proteins into the IMS [54]. Plant Mic60 has also been characterised, and it is suggested that it similarly connects the inner and outer membranes as it was found to bind to Tom40 via pull-down assays and proposed to play a role in lipid transfer [55].

Import of intermembrane space proteins

MIA–ERV1

The intermembrane space proteome is the smallest subcompartment of mitochondria and only consists of tens to hundreds of proteins (37 in yeast and 127 in human mitochondria) [56,57], yet a distinct import pathway has nevertheless evolved (Figure 2). Import of proteins can go through the MIA (mitochondrial intermembrane space import and assembly)-dependent (Figure 2) or via an MIA-independent pathway. The MIA-dependent precursors are distinguishable as they acquire disulfide bonds during their import and assembly by the essential MIA–Erv1 machinery. MIA-dependent precursors typically contain twin CX9C or CX3C motifs that are reduced during translocation across the OM, and upon import into the IMS, Mia40 catalyses the oxidative folding of the precursor, with electrons transferred to Erv1 [58,59]. Mia40–Erv1 has also been shown to interact with a yeast-specific protein named Hot13, a zinc-binding protein [60]. Characterisation of the Arabidopsis Mia40–Erv1 import pathway found that it contains several unique features, firstly unlike yeast, Mia40 is a non-essential protein and is soluble in the IMS protein, lacking the inner membrane docking domain [61]. Moreover, Erv1 has the ability to directly oxidise proteins independently of Mia40 [62]. This finding proposes that plants acquired Mia40 subsequently to Erv1 to increase the efficiency of, rather than to directly mediate protein oxidation [62]. Interestingly, Mia40 itself is dual-targeted to both the mitochondria and peroxisomes [61,63], though its exact role in peroxisomes is still to be elucidated.

Transport across the inner membrane

Proteins destined to the inner membrane or matrix undergo translocation across the inner membrane via two protein complexes the TIM17:23 complex and the TIM22 complex which constitute the general import pathway and the carrier import pathway, respectively (Figure 3).

The inner membrane protein translocases in plant mitochondria.

Figure 3.
The inner membrane protein translocases in plant mitochondria.

The inner membrane contains the TIM17:23 complex for the translocation of the majority of mitochondrial precursor proteins containing cleavable presequences and the TIM22 complex for the insertion of carrier-type proteins into the inner membrane. Mitochondrially encoded proteins synthesised in the matrix are inserted in the inner membrane via OXA translocase. OM, outer membrane; IM, inner membrane; IMS, intermembrane space. Plant-specific features/components are indicated with a leaf, and yeast-specific featured are indicated with yeast in pale yellow.

Figure 3.
The inner membrane protein translocases in plant mitochondria.

The inner membrane contains the TIM17:23 complex for the translocation of the majority of mitochondrial precursor proteins containing cleavable presequences and the TIM22 complex for the insertion of carrier-type proteins into the inner membrane. Mitochondrially encoded proteins synthesised in the matrix are inserted in the inner membrane via OXA translocase. OM, outer membrane; IM, inner membrane; IMS, intermembrane space. Plant-specific features/components are indicated with a leaf, and yeast-specific featured are indicated with yeast in pale yellow.

TIM17:23

The TIM17:23 complex is one of the best-characterised inner membrane translocases. It is composed of 11 subunits, exists in multiple states and has been shown to dynamically interact with the TOM complex and various respiratory chain complexes of the inner membrane. This complex is built around the core translocases Tim17 and Tim23 that constitute the channel, and associated proteins, Tim50, Tim21, Tim44, Mgr2, Pam16, Pam17, Pam18, Tim15, Mge1, and Hsp70 responsible for the recognition of presequences, recruiting motors, translocation motoring, and complex stability (Figure 3). The TIM17:23 translocase acts as a voltage-sensitive channel specific for proteins containing N-terminal targeting signals [64,65]. This translocase is highly dynamic, existing as two distinct forms: the TIM17:23SORT complex required for the membrane potential-dependent and lateral insertion of preproteins into the inner mitochondrial membrane, and the TIM17:23MOTOR, which in association with the PAM complex, responsible for ATP-dependent insertion of proteins occurs into the matrix [66]. In plants, as in yeast, the rate of protein import is principally regulated by the abundance of this translocase [10,67].

Numerous plant-specific features have been identified with this complex, a common feature of higher plants, both Tim17 and Tim23 are encoded by multiple genes and belong to the preprotein and amino acid transporter (PRAT) family identified by the conserved PRAT or Tim17/23/22 domain [6870]. This highly conserved family of proteins are thought to have originated from a single eubacterial ancestral LivH amino acid permease [71], although its homology to LivH has been questioned [72]. Collectively known for their role in the transport of peptides/metabolites across the membranes of mitochondria and chloroplasts, evolutionary and functional significance of PRAT family is evidenced by their large-scale conservation across numerous eukaryotic species [72].

Like its yeast counterpart, Tim17 is essential for viability [73] and while all three Tim17s are predicted to contain the four transmembrane spanning and the conserved PRAT domain required for its function [69,72], only Tim17-2 and Tim17-3 contain the twin cysteine residues shown to be essential for voltage gating and import in yeast [74,75]. Of the three Arabidopsis Tim17 proteins, Tim17-1 and Tim17-2 also exhibit unique features when compared with yeast, predominantly containing C-terminal extensions of up to 143 amino acid (aa) long (Tim17-2) when compared with the yeast and mammalian. This C-terminal extension appears to be of particular importance, as has been shown to link the inner and outer membranes with a portion exposed on the outer membrane and has substrate-binding capabilities (Figure 3). Removal of this extension resulted in the ability to complement a yeast Tim17 deletion strain, not obtainable by Tim17-3, indicative of a plant-specific feature for the Tim17 C-terminal extensions [9,68,76]. The ability to link the TIM complex with the TOM is a feature that was also observed in yeast but with regard to Tim23 that contains an N-terminal extension that has the ability to bind presequences and tether to the TOM complex [67].

Tim17-1 appears to play a specific role in germination. Mitochondrial biogenesis is especially active during germination, being the primary source of ATP production prior to the establishment of photosynthesis as such the expression of the protein import apparatus is most abundant at these early stages in both rice and Arabidopsis [7779]. Tim17-1 is one homologue that is only expressed in dry seed and is highly stress responsive [8,80]. It was determined that the presence of Tim17-1 regulates the initiation of mitochondrial biogenesis and the rate of germination. Furthermore, its expression was under the regulation of an ABA-responsive promoter and thus is the first mitochondrial protein import component identified to be under hormonal regulation [8].

In Arabidopsis, three genes encode for Tim23 that share 70–92% sequence identity, with deletion of at least two genes, results in a lethal phenotype suggests that they share functionality [81]. Tim23-2 is the most abundantly expressed gene and has the most interesting characteristics [68]. It was shown to be dually located within the TIM17:23 translocase and existing as a monomer with respiratory Complex I [10]. Remarkably, the overexpression of Tim23 correlates to a decrease in Complex I and vice versa. This association raises the possibility that such a physical interaction may be a mechanism for regulating the abundance of both mitochondrial respiration components and protein import apparatus as a loss of complex I resulted in an up-regulation of the TIM17:23 complex and consequently protein uptake ability [82]. Another PRAT family member was also found to associate with the TIM17:23 complex, named B14.7 [10]. Originally identified as a Complex I subunit in bovine [83], and in Arabidopsis by multiple independent studies [84], B14.7 has the ability to interact with the TIM17:23 complex, though its exact role in either complex is unknown.

TIM17:23 subunits have also been demonstrated to interact with other respiratory chain complexes. Tim21, a protein associated with TIM17:23 and involved in tethering to the TOM translocase [85], was revealed to interact with Complexes III and IV [86]. Proposed as a mechanism to allow for the efficient insertion of respiratory components [87], a similar interaction has been identified in Arabidopsis with Tim21 interacting with respiratory Complex III [10]. Plants contain additional Tim21 proteins that are related to Tim21 but are plant-specific, named Tim21-like, and while the exact functions of these proteins are yet to be elucidated, evidence points to their role as respiratory complex assembly factors [88].

PAM subunits

The presequence translocase-associated motor (PAM) is associated with the TIM17:23 translocase and it is responsible for the folding of matrix-targeted proteins and provides a source of energy to drive the translocation of precursors across the inner membrane. There are seven essential subunits associated with the PAM subcomplex in yeast: Hsp70, Tim44, Tim15, Pam18, Pam16, Pam17, and Mge1. Tim44 recruits Hsp70 to the TIM23 channel [89], interacting with the matrix domains of Tim23 and Tim17 [90]. Tim15 stabilises the PAM complex and prevents aggregation of key residues [91]. Pam18 stimulates the ATPase activity of Hsp70 [92], and Pam16 tethers Pam18 to the PAM complex [93]. Pam17 was shown to stabilise this complex, while Mge-1 catalyses the exchange of ADP for ATP [94]. All PAM proteins belong to the DnaJ and DnaJ-like family of proteins [95]. In Arabidopsis, multiple putative orthologues to Pam16 and 18 have been identified by homology, but only Pam16 has so far been experimentally characterised [96,97]. The phenotypes of single pam16-1 or pam16-2 mutants were investigated with pam16-1 displaying limited growth, with an increased pathogenic response attributed to the accumulation of reactive oxygen species [97]. The double knockout pam16-1::16-2 was lethal, confirming a similarly essential role for plant Pam16 as observed in yeast [98]. While the single mutant of pam16-2 did not exhibit the same pathogenic responses as pam16-1, it did exhibit reduced growth, suggesting that the two genes may have acquired specialised functions [97].

Mge1 stimulates the exchange of ADP for ATP by Hsp70 leading to a conformational change from the closed to open state so that a new preprotein can bind [94]. In Arabidopsis, two putative orthologues exist for Mge1: Mge1 and Mge2. It was shown that Arabidopsis Mge1 could functionally replace Mge1/GrpE in yeast under heat stress conditions suggesting similar functionality, and a T-DNA insertional deletion line of mge2 could only exhibit a clear phenotype when grown under long-term heat stress conditions [99]. GFP-tagged localisation assays have confirmed that Mge2 is distinctly mitochondrial (SUBA4, http://suba.live) [100]; surprisingly, Mge1 has been shown to be dual-targeted to mitochondria and chloroplasts [80]. As chloroplasts also contain their own Mge1-like proteins termed Cge1/2 that play a similar role in nucleotide exchange with the chloroplastic chaperonins [101], it would be interesting to determine if plants have acquired a distinct role for Mge1 that is required in both organelles.

The yeast Tim15 also known as Hsp70 Escort Protein 1 (Hep1) or Zim17 is a peripheral inner membrane protein facing the matrix and a member of the zinc ribbon protein family [91,102104]. Validated as a member of the TIM17:23 complex in yeast, the absence of which depleted the import of matrix-targeted proteins [105]. Tim15 is proposed to supplement Pam18 by providing the zinc finger for loading the substrate on Hsp70. The zinc finger also has a role in maintaining the tertiary structure of J-proteins and Hsp70. In Arabidopsis, there are three putative orthologues [1], and GFP-tagged localisation assays can so far confirm one of these (Tim15-2) as mitochondrial [102].

TIM22

The TIM22 complex responsible for the import of carrier-type proteins is the least-characterised protein import complex of the inner membrane (Figure 3). In yeast, it is composed of the Tim22 translocase channel and its accessory subunits Tim18, Tim12, Tim54, and the succinate dehydrogenase complex subunit, Sdh3. Sdh3 was shown to be a genuine subunit of the TIM22 complex interacting with Tim18 and involved in the assembly of the Tim22 complex [106].

In Arabidopsis, two identical genes (At3g10110/At1g18320) encode for Tim22 that is also essential for viability as deletion of one gene resulted in a sterile and developmentally delayed plant, establishing the likelihood that the other gene is not expressed [81]. It has the ability to complement the yeast Tim22 deletion strain, so it is likely to play the same role in protein import [69]. While the TIM22 complex remains to be biochemically characterised in plants, it is likely to vary significantly to yeast, as no orthologues for Tim12, Tim54 and Tim18 can be identified in plants or other species [1]. Human TIM22 complex has recently been experimentally characterised and novel interacting partners identified by immunoprecipitation: Tim29, which is involved in complex assembly and stability and AGK, a lipid kinase that associates with TIM22 and TIM29 to facilitate import of precursor proteins [107109], although neither of these mammalian TIM22 constituents have direct plant orthologues when searched by homology in plant genome databases (Phytozome 9.0) [110]. An interesting feature of the plant Tim22 is that it has been identified by proteomic studies to co-migrate within respiratory Complex I resolving at 1000 kDa [47,111,112]. This localisation would need to be confirmed via additional methods as several studies did not identify this protein as a Complex I subunit [113]. The ever-increasing examples of protein import components interacting with respiratory chain complexes and/or subunits of dual location/function (see reviews [81,87]) support the feasibility that plant TIM22 may associate with respiratory Complex I. While the presence of the carrier import pathway in plant mitochondria has been long established [76,114,115], the specific role of Arabidopsis Tim22 in this pathway has not been elucidated. The proteins encoded by At3g10110/At1g18320 were able to complement a yeast Tim22 deletion mutant [69]. Žárský and Doležal [72] have recently phylogenetically analysed the Tim17 family from over 5000 protein sequences from many species. They assigned the proteins encoded by At4g26670 and At5g55510, to the TIM22 clade, though these proteins have been shown to be located in the chloroplast [69] and play a role in protein import (named as HP20) [116].

Sorting and processing

Upon successful import into the organelle, presequences will be cleaved off, proteins folded, and sorted into their respective location (Figure 4). Processing is initially carried out by the mitochondrial processing peptidase (MPP) [21,23] to generate free peptides. MPP is a heterodimeric enzyme comprising α and β subunits which belong to the metalloendopeptidase M16B pitrilysin family as classified by MEROPS [117,118]. In plants, MPP is unique in that it is incorporated into cytochrome bc1 complex of electron transport chain and found to have both processing and electron transfer capabilities [17,18] (Figure 4). The negatively charged pocket of the MPP catalytic site is capable of binding the positively charged arginine residue at the P2 or P3 from the precursor cleavage site [21]. Its catalytic action occurs in a single processing step at a conserved Arginine residue (−2R) position although a two-step processing might be observed, primarily when the presequence contains two MPP recognition motifs [119121]. Presequences can also be subsequently cleaved by distinct peptidases such as intermediate cleavage peptidase 55 kDa (Icp55) [122], Oct1 [123], and the inner membrane peptidase (IMP) [124,125]. Icp55 removes a single amino acid from mitochondrial proteins at position -3 from a conserved arginine (-3R) of the cleavage site, which required for protein stability [122]. But, IMP cleaves off hydrophobic targeting signal required for the protein to be imported into mitochondrial IMS [126,127].

Processing and sorting machinery.

Figure 4.
Processing and sorting machinery.

The processing machinery involved in presequence cleavage and degradation following import. Upon import through the TIM17:23 complex, the membrane-bound MPP/bc1 (mitochondrial processing peptidase/cytochrome bc1) removes the targeting peptide. A series of step-wise cleavage events occur to degrade the peptide into single amino acids that can be recycled or exported out of the mitochondria. OM, outer membrane; IM, inner membrane; IMS, intermembrane space.

Figure 4.
Processing and sorting machinery.

The processing machinery involved in presequence cleavage and degradation following import. Upon import through the TIM17:23 complex, the membrane-bound MPP/bc1 (mitochondrial processing peptidase/cytochrome bc1) removes the targeting peptide. A series of step-wise cleavage events occur to degrade the peptide into single amino acids that can be recycled or exported out of the mitochondria. OM, outer membrane; IM, inner membrane; IMS, intermembrane space.

Following the release of the targeting peptide from the preprotein by MPP, the cleaved peptide is further processed into smaller fragments by the presequence of peptidases (PreP) and organellar oligopeptidase (OOP) to prevent the accumulation of free peptides and its toxic effects to membrane integrity and can even inhibit protein import [128131] (Figure 4). PreP is a metalloendopeptidase belonging to M16C pitrilysin oligopeptidase family [11]. PreP has been first identified in potato (Solanum tuberosum) mitochondria and shown experimentally to digest the presequence of F1β subunit of ATP synthase [12]. Further analysis revealed that PreP is also located in the chloroplasts and could degrade the transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) [132]. In Arabidopsis, PreP is also dually localised in the chloroplastic stroma and the mitochondrial matrix [132,133]. PreP orthologues have been identified in the mitochondrial matrix of yeast and human [13] and a mutation in human PreP results in the accumulation of amyloid β-proteins, responsible for protofibril plaque formation in the onset of Alzheimer's disease [14,134].

Similar to PreP, OOP was also found dually localised in both mitochondria and chloroplasts of Arabidopsis [11], but determined to degrade targeting peptides into shorter fragments of three to five aa residues in a parallel fashion with PreP as their substrates appear to overlap in length [11]. PreP can digest peptides of 10–65 residues in length, whereas OOP degrades shorter peptides ranging from 8 to 23 aa [11,133]. From these results, it was hypothesised that PreP produced shorter peptides that can act as substrates for OOP and while OOP is capable of processing short targeting peptides independently of PreP, they constitute only ∼10% of the total substrate pool based on their length [11].

Presequence proteases, such as PreP and OOP, can only degrade presequences to short peptide fragments of 3–5 aa. Therefore, an additional step is required for individual amino acid release which is carried out by aminopeptidases [135]. Aminopeptidases are found in various cellular locations, with nearly half of 28 putative Arabidopsis aminopeptidases are confirmed/predicted to be located within the mitochondria and chloroplasts [136]. Specific aminopeptidases belonging to the M1, M17.10, M17.20, and M18 families have been shown to complete the peptide processing pathway by recovering single amino acids from short peptides provided from earlier stages of processing by PreP and OOP [15,16], highlighting a series of conserved enzymatic reactions from the presequence cleavage, short peptide generation, and amino acid recovery in both the mitochondria and chloroplasts (Figure 4).

Protein export and assembly

Although almost all mitochondrial proteins are nuclear-encoded and synthesised in the cytosol, a few of them are mitochondrially encoded, translated, and incorporated into the inner membrane. Most of these are hydrophobic proteins involved in respiration or energy generation [137139], and the hydrophobic nature of these proteins is a characteristic that has been proposed to prevent their transfer to the nuclear genome [140].

Protein synthesis within mitochondria occurs via inner membrane-bound mitochondrial ribosomes [141] situated in the cristae which appear to facilitate the co-translational insertion of synthesised proteins into the inner membrane or the matrix [142144]. One of the first and most widely studied translocator complexes involved in this process is the cytochrome oxidase assembly (OXA) translocase [142,145,146]. Oxa1 is the major component embedded within the inner membrane [146149] responsible for inserting proteins from the matrix to the inner membrane [138,139]. Oxa orthologues can be found in its prokaryotic ancestor bacteria (named YidC proteins) as well as the chloroplasts (Alb3 proteins) [150].

In Arabidopsis, at least eight different genes are encoding Oxa1 and Oxa2 [151], with four Oxa homologues exclusively found in mitochondria and localised to the inner membrane [151,152]. Oxa1a and Oxa1b possess similar structural features like their yeast counterpart Oxa1, including the matrix-facing ribosome-binding coils [151]. Remarkably, in contrast with Oxa1, Oxa2a and Oxa2b contain four tetratricopeptide repeat (TPR) motifs near the C-terminus, which is specific to Oxa2 and found exclusively in plants [153].

Distinct structural features of two Oxa homologues in plant mitochondria explain their difference in molecular functions. Oxa1 proteins contain coiled-coil domain responsible for binding with mitoribosomes. To perform this function, positively charged Oxa1 C-terminal coils electrostatically interact with the negatively charged 21S RNA of the large ribosomal subunit, which is located near the exit tunnel of the synthesised polypeptide [144,154]. As a result, Oxa1 is capable of contacting the translated protein directly and promotes its co-translational insertion into the IM [155,156]. On the other hand, the specific functions of Oxa2 TPR domains are yet to be experimentally discovered and are difficult to bioinformatically predict as Oxa2 is the only known group of Oxa-like proteins that possess TPR motifs [153]. TPR domains usually act in protein–protein interactions in various biological processes. For example, Tom70 has TPR domains capable of binding cytoplasmic chaperones Hsc70/Hsp70 and Hsp90 [157]. However, it is unlikely that the TPR domain of Oxa2 assists in chaperone-binding as the mitochondrial matrix chaperone Hsp70 lacks the conserved TPR-binding motif [158]. TPR domains in Oxa2 may have a role similar to TPR motifs in its yeast counterpart Cox18, which interacts with another TPR domain of mitochondrial splicing system-related protein Mss2p that is responsible for the biogenesis and assembly of cytochrome c oxidase (COX) complex [159].

Recently, another protein insertion pathway has been identified in plant mitochondria: the twin-arginine translocation (Tat) pathway [160]. This pathway is distinct from the Oxa1p pathway in that it is involved in the transport of fully folded proteins and is similarly conserved across bacterial, mitochondria, and chloroplast thylakoid membranes. Shown to be involved in the assembly of a Complex III subunit, Rieske Fe/S protein (Rip) that is nuclear-encoded and imported via the general import pathway where it is folded at the C-terminus and Fe/S clusters inserted within the matrix [161]. Rip is subsequently chaperoned back into the inner membrane with a fully folded C-terminus delivered to the intermembrane space with the Tat machinery. While the extent of this pathway in plant mitochondria is still to be investigated, it does open up a new area of interest regarding complexome assembly, regulation, and mitochondrial biogenesis [162].

Controlling protein import rates by regulating transcript and protein abundance

Characterisation of individual subunits using both forward and reverse genetic approaches has revealed that the subunits Tim23, Tim17, Tim44, Tom40, Tim21, B14.7, Pam16, and Tim50 are all essential for plant viability [81,97,163,164]. Furthermore, protein import ability can be up-regulated or down-regulated by regulating protein abundance, in the case of Tim23 and metaxin [7,10]. Therefore, controlling protein abundance appears to be a direct mechanism of regulating protein uptake ability and thus mitochondrial biogenesis.

Maintenance and turnover of the protein transport components

ATP-dependent proteases have long been implicated in maintaining mitochondrial protein integrity and homeostasis [165,166]. The plant i-AAA protease, Ftsh4, has recently been implicated in specifically maintaining protein import component abundance [167]. By co-immunoprecipitation of a proteolytic inactive form of Ftsh4, binding partners and proteolytic substrates were identified and they revealed the interaction of Ftsh4 with six individual import components across a range of protein import pathways [167]. These proteins included the inner membrane protein import components TIM17-2 and PAM18s, along with the outer membrane Sam50-2 and the matrix located Oxa1p-like protein. Characterisation of the Ftsh4 knockout mutant lines further reveals that mutant plants exhibited an increased capacity for import, specifically through the TIM17:23 channel and that this was most likely due to an accumulation of Tim17-2 abundance [167]. Therefore, the abundance of the rate-defining Tim17-2 can be attributed to Ftsh4 activity.

Undertaking an omics approach to plant mitochondrial protein stability has revealed the regulatory role of Tim17 and partner proteins in import. Li et al. [168] carried out a systematic analysis of the turnover of over nearly 500 protein mitochondrial proteins using a 15N labelling approach. Protein turnover rates of mitochondrial proteins can be determined and classified as relatively stable, intermediate or fast following defined criteria [168]. Twenty-eight protein import components were identified from hydroponically grown Arabidopsis mitochondria and classified according to their turnover rates (Table 2). Interestingly, a component of the rate-defining TIM17:23 complex Tim17-2 and its partner protein Tim50 along with the outer membrane receptors Tom20 and Tom9-2 exhibit fast turnover rates, with slow turnover rates being observed for Tom40 and Hsp10 and Hsp60 (Table 2). As fast turnover rates suggest high potential on protein abundance changes during growth and developmental transitions which require a high energy demand for fast protein synthesis and degradation, we hereby postulate that the ‘fast’ proteins most probably play a regulatory role compared with the ‘slow’ house-keeping components.

Table 2
Protein turnover rates

The classifications of relatively stable, intermediate, and fast turnover rates are based on the defined criteria as outlined [169].

A table listing protein turnover rates for 28 mitochondrial protein import components as identified in the study by Li et al. [170].

 
 

Phosphorylation of presequences and receptors

Phosphorylation of proteins, at both the presequence and the transport component, can influence protein uptake ability resulting in either an up- or down-regulation in import ability. In yeast mitochondria, multiple receptors and presequence proteins such as Tom40, Tom22, Tom70, and Mim1 [171,172] and similarly in chloroplasts Toc34 and Toc159 [173,174] have been shown to be phosphorylated. While this area of research is still in its infancy in plant mitochondria, more examples of this type of regulation are emerging. Om64 has recently been shown to be phosphorylated, as observed within its chloroplastic paralogue Toc64, Om64 is phosphorylated within its TPR domain at a single serine residue [175]. This phosphorylation, in turn, reduces the binding ability of Hsp90 and consequently resulted in the impairment of the import of Om64 substrate protein pFAD [175].

Multiple reports have identified that the transit peptides of chloroplastic proteins can be reversibly phosphorylated to regulate protein import ability. STY kinases have been implicated in the specific phosphorylation of several chloroplastic proteins [176,177] and not the mitochondrial F1β subunit of ATP synthase. So far, there is one report that identified the phosphorylation of a mitochondrial protein pMORF3 (multiple organellar editing factor) which was shown to be phosphorylated at threonine/serine residues by STY kinases. This phosphorylation inhibited protein import ability most likely via its interactions with 14:3:3 proteins and Hsp70 [178]. While there have been no further reports of presequence phosphorylation regulating protein uptake in plants, this is likely to change as phosphorylation of both the presequence and the receptor provides an additional level of control [172]. As the dual-targeted purple acid phosphatase (AtPAP2) was implicated in their dephosphorylation [179], it raises an intriguing question of whether the targeting signals within dual-targeting proteins are phosphorylated. This could be an underexplored mechanism that regulates protein import rates and co-ordinates specificity in multiple plant organelles.

Summary

Over the years, molecular, biochemical, and reverse genetic approaches have advanced our understanding of the mechanisms of protein transport into plant mitochondria. While conservation across species is observed, plants have developed unique and specialised mechanisms to deal with their sessile nature and complex intracellular networks.

Abbreviations

     
  • aa

    amino acid

  •  
  • ERMES

    ER–mitochondria encounter structure

  •  
  • IM

    inner membrane

  •  
  • IMP

    inner membrane peptidase

  •  
  • IMS

    intermembrane space

  •  
  • MIA

    mitochondrial intermembrane space import and assembly

  •  
  • MICOS

    mitochondrial contact site and cristae organising system

  •  
  • Mim1

    mitochondrial import 1 protein

  •  
  • MINOS

    mitochondrial inner membrane organising system

  •  
  • MPP

    mitochondrial processing peptidase

  •  
  • OM

    outer membrane

  •  
  • OOP

    organellar oligopeptidase

  •  
  • OXA

    oxidase assembly

  •  
  • PAM

    presequence translocase-associated motor

  •  
  • PRAT

    preprotein and amino acid transporter

  •  
  • PreP

    presequence of peptidases

  •  
  • Rip

    Rieske Fe/S protein

  •  
  • SAM

    sorting and assembly machinery

  •  
  • Tat

    twin-arginine translocation

  •  
  • TOM

    translocase of the outer membrane

  •  
  • TIM

    translocase of the inner membrane

  •  
  • TPR

    tetratricopeptide repeat

Funding

M.W.M. is supported by Australian Research Council (ARC) Future Fellowship [FT130100112]. A.S.G is supported by the Indonesian Endowment Fund for Education (LPDP) scholarship.

Acknowledgments

The authors thank Aneta Ivanova and Lei Li for their helpful comments.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contributed equally to this work.