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.
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).
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).
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).
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).
The generalized transport reaction catalyzed by the MPT is:
protein (cell cytoplasm) protein (mitochondrial matrix or membrane)