3.A.8.1.1
Mitochondrial protein translocase (MPT) (Chacinska et al., 2005; Mokranjac et al., 2005; Bihlmaier et al., 2007). The crystal structure of the intermembrane space domain of yeast Tim50 has been solved to 1.83 Å resolution (Qian et al., 2011). A protruding beta-hairpin of Tim50 is crucial for interaction with Tim23, providing a molecular basis for the cooperation of Tim50 and Tim23 in preprotein translocation to the protein-conducting channel of the mitochondrial inner membrane (Qian et al., 2011).  TIM23-mediates insertion of transmembrane α-helices into the mitochondrial inner membrane (Botelho et al., 2011). The TIM23 channel undergoes structural changes in response to the energized state of the membrane, the pmf (Malhotra et al. 2013).  TMS1 in TIM23 is required for homodimerization while it and TMS2 are involved in pre-protein binding in the channel (Pareek et al. 2013).  The Tom40 outer membrane channel may be a 19 β-stranded barrel, possibly homologous to the VDAC porins (TC# 1.B.8) (Lackey et al. 2014).  Tim23 and Tim17 interact with each other as well as Tim44 and Pam17, respectively.  These last two proteins may serve regulatory functions (Ting et al. 2014).  Tom20, 22, 40 and 70 recognize presequences in various mitochondrially targetted proteins (Melin et al. 2015; Melin et al. 2014).  In the 4 TMS TIM17 protein, mutations in TMSs1 and 2 impair the interaction of Tim17 with Tim23, whereas mutations in TM3 compromise binding of the import motor (Demishtein-Zohary et al. 2017); further, residues in the matrix-facing region of Tim17 involved in binding of the import motor were identified. TIM22, forms an intramolecular disulfide bond in yeast and humans.  If not oxidized, they do not properly integrate into the membrane complex, and the lack of Tim17 oxidation disrupts the TIM23 translocase complex (Wrobel et al. 2016). Mgr2 (TC# 1.A.111.1.3) and Pam18 are involved in precursormembrane protein quality control (Schendzielorz et al. 2018). Tom7 and OMA1 play reciprocal roles during mitochondrial import and activation of the PTEN-induced kinase 1, PINK1, in humans (Sekine et al. 2019).  Organellar beta-barrel proteins are unique as most of them do not contain typical targeting information in the form of an N-terminal cleavable targeting signal. Instead, targeting and surface recognition of mitochondrial beta-barrel proteins in yeast, humans and plants depends on the hydrophobicity of the last beta-hairpin of the beta-barrel. Klinger et al. 2019 demonstrated that hydrophobicity is not sufficient for the discrimination of targeting to chloroplasts or mitochondria. Using atVDAC1 (TC# 1.B.8.1.15) and psOEP24 (1.B.28.1.1) they showed that the presence of a hydrophilic amino acid at the C-terminus of the penultimate beta-strand is required for mitochondrial targeting. A mutation of the chloroplast beta-barrel protein psOEP24 which mimics such a profile is efficiently targeted to mitochondria (Klinger et al. 2019). 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. Negatively charged residues in the N-terminus of Tim17 are critical for the preprotein-induced gating of the TIM23 translocase, possibly by recognizing the positive charges in the leader sequence of the substrate proteins (Meier et al. 2005). Tom22, Tom40 and Tom7 have been observed in primorial eukaryotes, arguing that these subunits were core to the complex early in evolution (Maćasev et al. 2004). The assembly of the Tim22 complex have been studied () Kumar et al. 2020. The intermembrane space (IMS) and TMS4 regions of Tim22 are required for interactions with membrane-embedded subunits, including Tim54, Tim18, and Sdh3, and thereby maintain the functional architecture of the TIM22 translocase. The TMS1 and TMS2 regions of Tim22 are important for association with Tim18, whereas TMS3 is exclusively required for the interaction with Sdh3 (Kumar et al. 2020). TIM8.13 and TIM9.10 are chaparone proteins that form complexes, 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 in TIM9.10. Consequently, TIM9.10 outcompetes TIM8.13 in binding hydrophobic clients, while TIM8.13 is tuned to a few clients with both hydrophilic and hydrophobic parts (Sučec et al. 2020). The TOM complex is a multisubunit membrane protein complex consisting of a beta-barrel protein Tom40 and six alpha-helical transmembrane (TM) proteins, receptor subunits Tom20, Tom22, and Tom70, and regulatory subunits Tom5, Tom6, and Tom7 (Araiso et al. 2020). CryoEM structures showed a symmetric dimer containing five different subunits including Tom22. Different translocation paths within the Tom40 import channel seem to be present for different classes of translocating precursor proteins (Araiso et al. 2020). The Mgr2 subunit of the TIM23 complex regulates membrane insertion of marginal stop-transfer signals in the mitochondrial inner membrane (Lee et al. 2020). The architecture of the human TIM22 complex has been examined by chemical crosslinking (Valpadashi et al. 2021).  The TIM22 complex mediates the import of sideroflexins and is required for efficient mitochondrial one-carbon metabolism (Jackson et al. 2021). About 60% of more than 1,000 different mitochondrial proteins are synthesised with amino-terminal targeting signals, termed presequences, which form positively charged amphiphilic α-helices. TIM23 sorts the presequence proteins into the inner membrane or matrix. Various views including regulatory and coupling functions have been reported on the essential TIM23 subunit Tim17. The interaction of Tim17 with matrix-targeted and inner membrane-sorted preproteins occurs during translocation in the native membrane environment. Fielden et al. 2023 showed that Tim17 contains conserved negative charges close to the intermembrane space side of the bilayer, which are essential to initiate presequence protein translocation along a distinct transmembrane cavity of Tim17 for both classes of preproteins. The amphiphilic character of mitochondrial presequences directly matches this Tim17-dependent translocation mechanism. This mechanism permits direct lateral release of transmembrane segments of inner membrane-sorted precursors into the inner membrane (Fielden et al. 2023). Tom20 acts as a dynamic gatekeeper, guiding preproteins into the pores of the TOM complex. Ornelas et al. 2023 analyzed the interactions of Tom20 with other TOM subunits, presented insight into the structure of the TOM holo complex, and suggested a translocation mechanism.  Distinct structural motifs are necessary for targeting and import of Tim17 in Trypanosoma brucei mitochondrion (Darden et al. 2024). Inactivation of TIM complex components leads to a decrease in the level of DNA import into Arabidopsis mitochondria (Tarasenko et al. 2023). Intermembrane proteins involved in uniport and assembly of other intermembrane proteins include Mia40 or TIM40 (403aas with 1 TMS near the N-terminus; P36046) and Erv1 (Mitochondrial FAD-linked sulfhydryl oxidase, P27882) (Chacinska et al., 2004; Grumbt et al., 2007).  Frameshift mutations in the Timm8a1 (Timm8A, Ddp1, Tim8a) gene in mouse leads to an abnormal mitochondrial structure in the brain, correlating with hearing and memory impairment (Song et al. 2021).  Sengers syndrome caused by biallelic TIMM29 variants and RNAi silencing in Drosophila orthologue recapitulates the human phenotype (Shalata et al. 2025).

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