3.A.8 The Mitochondrial Protein Translocase (MPT) Family
Outer and inner membranes of mitochondria and hydrogenosomes are equipped with characteristic sets of membrane proteins required for energy conversion, metabolite and protein transport, membrane fusion and fission, and signal transduction (Becker et al. 2009; Rada et al., 2011). The mitochondrial protein translocase (MPT), which brings nuclearly encoded preproteins into mitochondria, is very complex with numerous identified protein constituents that comprise at least five translocons, two in the outer membrane: Tom (Tom40/70/22/20) and Sam (Sam50/Sam35/Mas37) [SAM=sorting and assembly machinery], and three in the inner membrane, Tim23 (Tim23/17/44/50/Hsp70/Pam16/Pam18/Mge1) [PAM=presequence-associated import motor]; Tim22 (Tim22/54/18) and Oxa1p (TC #2.A.9) (Wiedemann et al., 2004; Dukanovic and Rapaport, 2011). These systems function in conjunction with several chaperone proteins (Rehling et al., 2004). The outer membrane translocase (Tom) consists of import receptors and the proteins of the Tom channel complex. Non-identical integral outer membrane receptor proteins are called Tom70, Tom22 and Tom20. Of these receptor proteins, only Tom22 is essential for protein import. The receptor complex delivers the substrate proteins to the outer membrane channel consisting of 5 hydrophobic proteins, Tom40, Tom38, Tom7, Tom6 and Tom5. Tom40 is the core oligomeric subunit of this channel. It forms a β-stranded, cation-selective, high conductance pore that specifically binds to and transports mitochondrial-targeting peptides. The inner pore diameter has been estimated to be about 22 Å. The small Tom proteins may function in regulatory capacities and are not essential (Rehling et al., 2003, 2004). The membrane-embedded C-terminal segment of TOM40 constitutes the pore and recognizes the preprotein substrate (Suzuki et al., 2004). Endo et al. (2011) have reviewed high-resolution structures of the components of the mitochondrial protein import machineries that afford structural and mechanistic insight into how the mitochondrial import system works. Harner et al. (2011) have demonstrated lateral release of proteins from the TOM complex into the outer mitochondrial membrane. Mia40 is an intermembrane protein that acts during import into the mitochondrial intermembrane space (Hofmann et al. 2005).
Most mitochondrial proteins, and all outer membrane proteins, are synthesized in the cytosol, imported into mitochondria via the TOM40 (translocase of the mitochondrial outer membrane 40) complex, and follow several distinct sorting pathways to reach their destination submitochondrial compartments. The phosphate carrier (PiC) requires the Tim9-Tim10 complex and the TIM22 complex to be inserted into the inner membrane.PiC can be sorted to either the TIM22 pathway or the TIM23 pathway for its import into yeast mitochondria (Yamano et al. 2005). Precursors of β-barrel and α-helical proteins are transported into the outer membrane via distinct import routes. The translocase of the outer membrane (TOM complex) transports beta-barrel precursors across the outer membrane, and the sorting and assembly machinery (SAM complex) inserts them into the target membrane. The mitochondrial import (MIM) complex (TC# 3.A.29) constitutes the major integration site for alpha-helical embedded proteins. The import of some MIM-substrates involves TOM receptors, while others are imported in a TOM-independent manner. Thus, TOM, SAM and MIM complexes dynamically interact to import a large set of different proteins and to coordinate their assembly into protein complexes (Gupta and Becker 2020).
The translocase of the outer mitochondrial membrane (TOM) complex is the general entry site into the organelle for newly synthesized proteins. Mim1 (mitochondrial import protein 1) is a mitochondrial outer membrane protein composed of an N-terminal cytosolic domain, a central putative transmembrane segment (TMS) and a C-terminal domain facing the intermembrane space (Popov-Celeketić et al., 2008). Mim1 is required for the integration of the import receptor Tom20 into the outer membrane. The TMS of Mim1 is the minimal functional domain of the protein. It forms homo-oligomeric structures via its TMS which contains two helix-dimerization GXXXG motifs. Mim1 with mutated GXXXG motifs does not form oligomeric structures and is inactive. Thus, the homo-oligomerization of Mim1 allows it to fulfill its function in promoting the integration of Tom20 into the mitochondrial outer membrane (Popov-Celeketić et al., 2008).
Signal-anchored proteins are a class of mitochondrial outer membrane proteins that expose a hydrophilic domain to the cytosol and are anchored to the membrane by a single N-terminal TMS. Like other mitochondrial proteins, signal-anchored proteins are synthesized on cytosolic ribosomes and are subsequently imported into the organelle. Ahting et al. 2005 studied the mechanisms by which precursors of these proteins are recognized by the mitochondria and are inserted into the outer membrane. The import of signal-anchored proteins is independent of the known import receptors, Tom20 and Tom70, but requires the major Tom component, Tom40. In contrast to precursors destined to internal compartments of mitochondria and those of outer membrane beta-barrel proteins, precursors of signal-anchored proteins appear not to be inserted via the general import pore (Ahting et al. 2005).
The TOM complex can apparently function independently of the two TIM complexes, transporting proteins from the cytoplasm to the intermembrane space (Herrmann, 2003). Conserved, intraprotein, hydrophilic targeting sequences, different from matrix targeting sequences, are involved. Targeting translocation across the outer membrane may be independent of ATP and the pmf, but subsequent transport steps require ATP. About 30% of mitochondrial proteins lack matrix targeting sequences. They are present in the outer membrane, the intermembrane space and in the inner membrane. These proteins may be imported initially via the TOM complex, but their transport across or into the inner membrane is achieved by two independent systems, each with a different subset of targeted proteins. The Tom complex distinguishes between β-barrel proteins and other types of preproteins, thus playing an active role in the transfer of preproteins to subsequent translocases for insertion into the correct mitochondrial subcompartment (Sherman et al., 2006). Tom40 forms the pore and contains the 'gating machinery' of the complex. However, for proper functioning, additional proteins (Tom22, Tom7, Tom6 and Tom5) are required that act as modulators of pore dynamics by significantly reducing the energy barrier between different conformational states (Poynor et al., 2008; Walther and Rapaport, 2009).
The high-resolution cryo-EM structures of the core TOM complex from Saccharomyces cerevisiae in dimeric and tetrameric forms have been determined (Tucker and Park 2019). Dimeric TOM consists of two copies each of five proteins arranged in two-fold symmetry: pore-forming beta-barrel protein Tom40 and four auxiliary alpha-helical transmembrane proteins. The pore of each Tom40 has an overall negatively charged inner surface due to multiple functionally important acidic patches. The tetrameric complex is a dimer of dimeric TOM, which may be capable of forming higher-order oligomers. Yeast mitochondrial pyruvate carrier, MPC, proteins with 3 TMSs and a matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9.10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. Thus, the carrier pathway can import paired and non-paired TMSs and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins (Rampelt et al. 2020).
Mia40p and Erv1p are probably chaperone-like redox components of a translocation pathway for the import of cysteine-rich proteins into the mitochondrial intermembrane space. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC-motif. The second and the third bond belong to the twin Cx9C-motif and bridge the cysteine residues of two Cx9C-segments. In contrast to the stabilizing disulfide bonds of the twin Cx9C-motif, the first disulfide bond is easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond, as well as fully reduced Mia40C is oxidized by Erv1p in vitro, and mixed disulfides of Mia40C and Erv1p form (Grumbt et al., 2007). Thus, these proteins probably facilitate disulfide bond formation.
The two inner membrane channel-forming complexes are both multicomponent. One, the Tim23 complex, consists of at least 2 integral membrane Tim proteins: Tim23 and Tim17 as well as peripheral membrane motor proteins, Tim44, Pam16 and Pam18. In this complex, other motor components (Tim14 and Mge1) are required (Mokranjac et al., 2003; Rehling et al., 2004; Truscott et al., 2003). Tim23, together with Tim17, exhibits channel activity. Tim23 has 4 TMSs in its C-terminal half. This region forms part of the channel, particularly TMS2 (Walsh and Koehler, 2008; Alder et al., 2008). The hydrophilic N-terminus regulates gate opening (with Tim50). The channel accomodates the unfolded, extended polypeptide chain (Truscott et al., 2001). Two ATP-dependent matrix chaperone proteins (mhsp70 and mGrpE) have been suggested to function together with Tim44 to drive active uptake. Tim44 (with or without the chaperone proteins) may function as an ATP-driven import motor that pulls the precursor polypeptide chain through the Tim23 channel into the matrix. The Tim23 complex imports presequence-containing matrix proteins as well as presequence-containing inner membrane monotonic (1TMS) proteins. The latter can laterally diffuse out of the channel into the lipid bilayer (Herrmann, 2003). Tim14, Tim16 and Tim23 interact to form the fully active complex (Mokranjac et al. 2007).
To drive matrix translocation, the TIM23 complex recruits the presequence translocase-associated motor (PAM) with the matrix heat shock protein 70 (mtHsp70) as the central subunit. Activity and localization of mtHsp70 are regulated by four membrane-associated cochaperones: the adaptor protein Tim44, the stimulatory J-complex Pam18/Pam16, and Pam17. It has been proposed that Tim44 serves as a molecular platform to localize mtHsp70 and the J-complex at the TIM23 complex. Pam17 interacts directly with the channel protein Tim23. Thus, the motor PAM is composed of functional modules that bind to different sites on the translocase. Tim44 may not be merely a scaffold for binding of motor subunits; it may additionally play a role in the recruitment of PAM modules to the inner membrane translocase (Hutu et al., 2008).
The motor-free form of the presequence translocase integrates preproteins into the membrane. The reconstituted presequence translocase responds to targeting peptides and mediates voltage-driven preprotein translocation, lateral release and insertion into the lipid phase. The minimal system for preprotein integration into the mitochondrial inner membrane is the presequence translocase, a cardiolipin-rich membrane and a membrane potential (van der Laan et al., 2007). The MPT system have been implicated in Alzheimer disease (Hong et al. 2010).
The TIM23 complex mediates translocation of proteins across, and their lateral insertion into, the mitochondrial inner membrane. Translocation of proteins requires both the membrane-embedded core of the complex and its ATP-dependent import motor. Insertion of some proteins, however, occurs in the absence of ATP, questioning the need for the import motor during lateral insertion. Popov-Čeleketić et al. (2011) showed that the import motor associates with laterally inserted proteins even when its ATPase activity is not required. A role for the import motor in lateral insertion is suggested. Thus, the import motor is involved in ATP-dependent translocation and ATP-independent lateral insertion. The TIM23 channel undergoes structural changes in response to the energized state of the membrane, the pmf (Malhotra et al. 2013).
The second inner membrane channel-forming complex consists of at least three integral membrane proteins, Tim22, Tim54 and Tim18. The Tim22 complex has been purified and shown to insert inner membrane proteins by a twin-pore translocase that uses the pmf as the exclusive energy source and a half of sites mechanism (Rehling et al., 2003, 2004). The polytopic substrate proteins of the Tim22 complex (including Tim22 and Tim23) lack typical presequences and instead have internal targeting signals. Tim22 is a dynamic ligand-gated, high conductance, slightly cation-selective channel, solely active in the presence of its cargo protein (Peixto et al., 2007). The Tim22 carrier translocase is built via a modular process and each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein (Wagner et al., 2008).
The Tom and Tim proteins have homologues in yeast, fungi, animals, plants, and other eukaryotes. Some constituents are homologous between these eukaryotes, but others are non homologous (Lister et al., 2007). Tim23, Tim17 and Tim22 within a single organism are additionally homologous to each other. Tim9 and Tim10 form an intermembrane hexameric complex with the two subunits arranged alternatively in a ring structure. This complex plays a chaperone role during import of mitochondrial membrane proteins (Ivanova et al., 2008).
Some of the inner membrane proteins are translocated into the matrix via the Tim23 complex and then, following cleavage of its first signal sequence, they are targeted to the Oxa1 complex (TC #2.A.9). Proteins that are translated within the mitochondrial matrix are directly inserted into the inner membrane by the Oxa1 complex. Another group of inner membrane proteins is directly targeted to the inner membrane in a pathway requiring intermembrane protein complexes as well as the integral inner membrane Tim22 complex. While the proteins that use the Tim22 complex are of eukaryotic origin (e.g., MC family members (TC #2.A.29)), those that use the Oxa1 complex are usually of bacterial origin (Chacinska et al., 2002; Herrmann, 2003).
Clements et al. (2009) have identified in alpha-proteobacteria the component parts of a mitochondrial protein transport machine. In bacteria, the components are found in the inner membrane, topologically equivalent to the mitochondrial proteins. Although the bacterial proteins function in simple assemblies, relatively few mutations might be required to convert them to function as a protein transport machine. This analysis of protein transport provides a blueprint for the evolution of cellular machinery in general (Clements et al., 2009).
The membrane-embedded TIM23(SORT) complex mediates the membrane potential-dependent membrane insertion of precursor proteins with a stop-transfer sequence downstream of the mitochondrial targeting signal. In contrast, translocation of precursor proteins into the matrix requires the recruitment of the presequence translocase-associated motor (PAM) to the TIM23 complex. This ATP-driven import motor consists of mitochondrial Hsp70 and several membrane-associated co-chaperones. These two structurally and functionally distinct forms of the TIM23 complex (TIM23(SORT) and TIM23(MOTOR)) are in a dynamic equilibrium with each other (van der Laan et al., 2010; Chacinska et al., 2010).
The mitochondrial outer membrane contains two translocase machineries for precursor proteins-the translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The TOM complex functions as the main mitochondrial entry gate for nuclear-encoded proteins, whereas the SAM complex was identified according to its function in the biogenesis of beta-barrel proteins of the outer membrane (Dukanovic and Rapaport, 2011). The SAM complex, together with the TOB complex (Endo and Yamano, 2009) is required for the assembly of precursors of the TOM complex, including not only the beta-barrel protein Tom40 but also a subset of alpha-helical subunits. Thornton et al. (2010) reported that the SAM core complex can associate with different partner proteins to form two large SAM complexes with different functions in the biogenesis of alpha-helical Tom proteins. They found that a subcomplex of TOM, Tom5-Tom40, associated with the SAM core complex to form a new large SAM complex. This SAM-Tom5/Tom40 complex binds the alpha-helical precursor of Tom6 after the precursor has been inserted into the outer membrane in an Mim1 (mitochondrial import protein 1)-dependent manner. The second large SAM complex, SAM-Mdm10 (mitochondrial distribution and morphology protein), binds the alpha-helical precursor of Tom22 and promotes its membrane integration. Thornton et al. (2010) and Endo and Yamano (2009) suggested that the modular composition of the SAM complex provides a flexible platform to integrate the sorting pathways of different precursor proteins and to promote their assembly into oligomeric complexes. Tom6 and Tom40 interact, yielding a more stable Tom40 β-barrel. Sam37 appears to be required for growth at higher temperatures because it enhances the biogenesis of Tom40. This requirement may be overruled by improved stability of newly synthesized Tom40 molecules (Dukanovic et al., 2009). Tom5 may play a two-stage role in the interaction of Tom40 with the SAM complex (Becker et al., 2010).
Opposing mechanisms of preprotein insertion into the membrane have been debated: stop-transfer with arrest in the inner membrane versus conservative sorting via the matrix. Bohnert et al. (2010) dissected the membrane insertion of a multispanning ABC transporter. The N-terminal membrane domain was laterally released from the presequence translocase of the inner membrane (TIM23 complex) by a stop-transfer mechanism, whereas the subsequent domain was imported via the matrix heat-shock protein 70 (mtHsp70) motor and exported by the oxidase assembly (OXA) translocase. These observations lead to an unexpected solution to the controversial debate about mitochondrial preprotein sorting. Stop-transfer and conservative sorting are not mutually exclusive pathways but represent sorting mechanisms that cooperate in the membrane integration of a protein with complex topology. Bohnert et al. (2010) concluded that the multispanning protein is inserted in a modular manner by the coordinated action of two inner-membrane preprotein translocases.
Proteins of the mitochondrial outer membrane are synthesized as precursors on cytosolic ribosomes and sorted via internal targeting sequences to mitochondria. Two different types of integral outer membrane proteins exist: proteins with a transmembrane beta-barrel and proteins embedded by a single or multiple alpha-helices. The import pathways of these two types of membrane proteins differ fundamentally. Precursors of beta-barrel proteins are first imported across the outer membrane via the TOM complex which is coupled to the sorting and assembly machinery (SAM complex), which catalyzes folding and membrane insertion of these precursors. The mitochondrial import machinery (MIM complex) promotes import of proteins with multiple alpha-helical membrane spans. Depending on the topology, precursors of proteins with a single alpha-helical membrane anchor are imported via one of several distinct routes (Ellenrieder et al. 2015).
Tim23 forms the core of voltage gated import channel, while Tim17 maintains the stoichiometry of the translocase. Matta et al. 2016 demonstrated that Tim17 harbours conserved G/AXXXG/A motifs within its transmembrane regions and plays an imperative role in translocase assembly through interaction with Tim23. Tim17 transmembrane regions regulate the dynamic assembly of translocase to form either the TIM23 (PAM)-complex or the TIM23 (SORT)-complex by recruiting PAM-machinery or Tim21, respectively. The TIM23 complex) facilitates anterograde precursor transport into the matrix and lateral release of precursors with a stop-transfer signal into the membrane (sorting). Sorting requires precursor exit from the translocation channelvia the lateral gate of TIM23. Schendzielorz et al. 2018 showed that the import motor J-protein, Pam18, essential for matrix import, controls lateral protein release into the lipid bilayer. Pam18 obstructs lateral precursor transport, while Mgr2, implicated in precursor quality control, is displaced from the translocase. During motor-dependent matrix protein transport, the transmembrane segment of Pam18 closes the lateral gate to promote anterograde polypeptide movement.
The structure of the TOM core complex has been determined by cryoelectron microscopy (cryo-EM) (Bausewein et al. 2017). The complex is a 148 kDa symmetrical dimer of ten membrane protein subunits that create a shallow funnel on the cytoplasmic membrane surface. In the core of the dimer, the beta-barrels of the Tom40 pore form two identical preprotein conduits. Each Tom40 pore is surrounded by the TMSs of the α-helical subunits, Tom5, Tom6, and Tom7. Tom22, the central preprotein receptor, connects the two Tom40 pores at the dimer interface. This structure offers detailed insight into the molecular architecture of the mitochondrial preprotein import machinery (Bausewein et al. 2017). Araiso et al. 2019 presented the high-resolution architecture of the translocator consisting of two Tom40 beta-barrel channels and alpha-helical transmembrane subunits. Each Tom40 beta-barrel is surrounded by small Tom subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis revealed its dual role in early and late steps in biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites: Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the midst of the dimer, whereas Tom5 and the Tom40 N-extension guide presequence-less preproteins to the exit at the dimer periphery (Araiso et al. 2019).
In mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane. Tom70 represents the major receptor for these precursors. After transport across the outer membrane, the hydrophobic carriers engage with the small TIM protein complex composed of Tim9 and Tim10 for transport across the intermembrane space (IMS) toward the TIM22 complex. Tim22 is the pore-forming core unit of the complex. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Gomkale et al. 2020 used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, subunits of the mitochondrial pyruvate carrier (MPC) (TC#s 2.A.105.1.1-3) were identified as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels.
Doan et al. 2020 reported that the yeast MIM complex promotes the insertion of proteins with N-terminal (signal-anchored) or C-terminal (tail-anchored) membrane anchors. The MIM complex exists in three dynamic populations. MIM interacts with TOM to accept precursor proteins from the receptor Tom70. Free MIM complexes insert single-spanning proteins that are imported in a Tom70-independent manner. Finally, coupling of MIM and SAM promotes early assembly steps of TOM subunits. Thus, the MIM complex is a major and versatile protein translocase of the mitochondrial outer membrane (Doan et al. 2020). It consists of Mim1, Mim2 and porin (see 1.B.33.3.1).
TIM8.13 and TIM9.10 are two chaparone proteins that target different integral membrane proteins, the all-transmembrane mitochondrial carriers, Ggc1 and Tim23, the latter which has an additional disordered hydrophilic domain. Sučec et al. 2020 determined the structures of Tim23/TIM8.13 and Tim23/TIM9.10 complexes. TIM8.13 uses transient salt bridges to interact with the hydrophilic part of its client, but its interactions to the transmembrane part are weaker than for TIM9.10. Consequently, TIM9.10 outcompetes TIM8.13 when binding hydrophobic clients, while TIM8.13 is tuned to clients with both hydrophilic and hydrophobic parts (Sučec et al. 2020).
The generalized transport reaction catalyzed by the MPT is:
protein (cell cytoplasm) protein (mitochondrial matrix or membrane)