Functional crosstalk between the TIM22 complex and YME1 machinery maintains mitochondrial proteostasis and integrity.

TIM22 pathway cargos are essential for sustaining mitochondrial homeostasis as an excess of these proteins leads to proteostatic stress and cell death. Yme1 is an inner membrane metalloprotease that regulates protein quality control with chaperone-like and proteolytic activities. Although the mitochondrial translocase and protease machinery are critical for organelle health, their functional association remains unexplored. The present study unravels a novel genetic connection between the TIM22 complex and YME1 machinery in Saccharomyces cerevisiae that is required for maintaining mitochondrial health. Our genetic analyses indicate that impairment in the TIM22 complex rescues the respiratory growth defects of cells without Yme1. Furthermore, Yme1 is essential for the stability of the TIM22 complex and regulates the proteostasis of TIM22 pathway substrates. Moreover, impairment in the TIM22 complex suppressed the mitochondrial structural and functional defects of Yme1-devoid cells. In summary, excessive levels of TIM22 pathway substrates could be one of the reasons for respiratory growth defects of cells lacking Yme1, and compromising the TIM22 complex can compensate for the imbalance in mitochondrial proteostasis caused by the loss of Yme1.

Acylglycerol Kinase Mutated in Sengers Syndrome Is a Subunit of the TIM22 Protein Translocase in Mitochondria.

Mutations in mitochondrial acylglycerol kinase (AGK) cause Sengers syndrome, which is characterized by cataracts, hypertrophic cardiomyopathy, and skeletal myopathy. AGK generates phosphatidic acid and lysophosphatidic acid, bioactive phospholipids involved in lipid signaling and the regulation of tumor progression. However, the molecular mechanisms of the mitochondrial pathology remain enigmatic. Determining its mitochondrial interactome, we have identified AGK as a constituent of the TIM22 complex in the mitochondrial inner membrane. AGK assembles with TIMM22 and TIMM29 and supports the import of a subset of multi-spanning membrane proteins. The function of AGK as a subunit of the TIM22 complex does not depend on its kinase activity. However, enzymatically active AGK is required to maintain mitochondrial cristae morphogenesis and the apoptotic resistance of cells. The dual function of AGK as lipid kinase and constituent of the TIM22 complex reveals that disturbances in both phospholipid metabolism and mitochondrial protein biogenesis contribute to the pathogenesis of Sengers syndrome.

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis.

BACKGROUND: Hydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown. RESULTS: We report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes. CONCLUSIONS: The hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.

Conserved regions of budding yeast Tim22 have a role in structural organization of the carrier translocase.

Mitochondrial biogenesis requires efficient sorting of various proteins into different mitochondrial sub-compartments, mediated by dedicated protein machinery present in the outer and inner membrane. Among them, the TIM22 complex enables the integration of complex membrane proteins with internal targeting signals into the inner membrane. Although the Tim22 protein forms the core of the complex, the dynamic recruitment of subunits to the channel is still enigmatic. In this study, we highlight that the intermembrane space (IMS) and transmembrane 4 (TM4) regions of Tim22 are critically required for interactions with the membrane-embedded subunits, including Tim54, Tim18, and Sdh3, and thereby maintain the functional architecture of the TIM22 translocase. Furthermore, we find that the TM1 and TM2 regions of Tim22 are important for association with Tim18, whereas TM3 is exclusively required for the interaction with Sdh3. Moreover, impairment of TIM22 complex assembly influences its translocase activity, the mitochondrial network, and the viability of cells lacking mitochondrial DNA. Overall, our findings provide compelling evidence highlighting the significance of conserved regions of Tim22 that are important for the maintenance of the TIM22 complex and mitochondrial integrity.

The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments.

BACKGROUND: The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. RESULTS: Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and 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. CONCLUSIONS: The carrier pathway can import paired and non-paired transmembrane helices 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.

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria.

Two protein translocases transport precursor proteins into or across the inner mitochondrial membrane. The presequence translocase (TIM23 complex) sorts precursor proteins with a cleavable presequence either into the matrix or into the inner membrane. The carrier translocase (TIM22 complex) inserts multispanning proteins into the inner membrane. Both protein import pathways depend on the presence of a membrane potential, which is generated by the activity of the respiratory chain. The non-bilayer-forming phospholipids cardiolipin and phosphatidylethanolamine are required for the activity of the respiratory chain and therefore to maintain the membrane potential for protein import. Depletion of cardiolipin further affects the stability of the TIM23 complex. The role of bilayer-forming phospholipids like phosphatidylcholine (PC) in protein transport into the inner membrane and the matrix is unknown. Here, we report that import of presequence-containing precursors and carrier proteins is impaired in PC-deficient mitochondria. Surprisingly, depletion of PC does not affect stability and activity of respiratory supercomplexes, and the membrane potential is maintained. Instead, the dynamic TIM23 complex is destabilized when the PC levels are reduced, whereas the TIM22 complex remains intact. Our analysis further revealed that initial precursor binding to the TIM23 complex is impaired in PC-deficient mitochondria. We conclude that reduced PC levels differentially affect the TIM22 and TIM23 complexes in mitochondrial protein transport.

The emerging picture of the mitochondrial protein import complexes of Amoebozoa supergroup.

BACKGROUND: The existence of mitochondria-related organelles (MROs) is proposed for eukaryotic organisms. The Amoebozoa includes some organisms that are known to have mitosomes but also organisms that have aerobic mitochondria. However, the mitochondrial protein apparatus of this supergroup remains largely unsampled, except for the mitochondrial outer membrane import complexes studied recently. Therefore, in this study we investigated the mitochondrial inner membrane and intermembrane space complexes, using the available genome and transcriptome sequences. RESULTS: When compared with the canonical cognate complexes described for the yeast Saccharomyces cerevisiae, amoebozoans with aerobic mitochondria, display lower differences in the number of subunits predicted for these complexes than the mitochondrial outer membrane complexes, although the predicted subunits appear to display different levels of diversity in regard to phylogenetic position and isoform numbers. For the putative mitosome-bearing amoebozoans, the number of predicted subunits suggests the complex elimination distinctly more pronounced than in the case of the outer membrane ones. CONCLUSION: The results concern the problem of mitochondrial and mitosome protein import machinery structural variability and the reduction of their complexity within the currently defined supergroup of Amoebozoa. This results are crucial for better understanding of the Amoebozoa taxa of both biomedical and evolutionary importance.

Protein insertion into the inner membrane of mitochondria: routes and mechanisms.

The inner membrane of mitochondria contains hundreds of different integral membrane proteins. These proteins transport molecules into and out of the matrix, they carry out multifold catalytic reactions and they promote the biogenesis or degradation of mitochondrial constituents. Most inner membrane proteins are encoded by nuclear genes and synthesized in the cytosol from where they are imported into mitochondria by translocases in the outer and inner membrane. Three different import routes direct proteins into the inner membrane and allow them to acquire their appropriate membrane topology. First, mitochondrial import intermediates can be arrested at the level of the TIM23 inner membrane translocase by a stop-transfer sequence to reach the inner membrane by lateral insertion. Second, proteins can be fully translocated through the TIM23 complex into the matrix from where they insert into the inner membrane in an export-like reaction. Carriers and other polytopic membrane proteins embark on a third insertion pathway: these hydrophobic proteins employ the specialized TIM22 translocase to insert from the intermembrane space (IMS) into the inner membrane. This review article describes these three targeting routes and provides an overview of the machinery that promotes the topogenesis of mitochondrial inner membrane proteins.

The mitochondrial carrier SFXN1 is critical for complex III integrity and cellular metabolism.

Mitochondrial carriers (MCs) mediate the passage of small molecules across the inner mitochondrial membrane (IMM), enabling regulated crosstalk between compartmentalized reactions. Despite MCs representing the largest family of solute carriers in mammals, most have not been subjected to a comprehensive investigation, limiting our understanding of their metabolic contributions. Here, we functionally characterize SFXN1, a member of the non-canonical, sideroflexin family. We find that SFXN1, an integral IMM protein with an uneven number of transmembrane domains, is a TIM22 complex substrate. SFXN1 deficiency leads to mitochondrial respiratory chain impairments, most detrimental to complex III (CIII) biogenesis, activity, and assembly, compromising coenzyme Q levels. The CIII dysfunction is independent of one-carbon metabolism, the known primary role for SFXN1 as a mitochondrial serine transporter. Instead, SFXN1 supports CIII function by participating in heme and α-ketoglutarate metabolism. Our findings highlight the multiple ways that SFXN1-based amino acid transport impacts mitochondrial and cellular metabolic efficiency.

Role of the Mitochondrial Protein Import Machinery and Protein Processing in Heart Disease.

Mitochondria are essential organelles for cellular energy production, metabolic homeostasis, calcium homeostasis, cell proliferation, and apoptosis. About 99% of mammalian mitochondrial proteins are encoded by the nuclear genome, synthesized as precursors in the cytosol, and imported into mitochondria by mitochondrial protein import machinery. Mitochondrial protein import systems function not only as independent units for protein translocation, but also are deeply integrated into a functional network of mitochondrial bioenergetics, protein quality control, mitochondrial dynamics and morphology, and interaction with other organelles. Mitochondrial protein import deficiency is linked to various diseases, including cardiovascular disease. In this review, we describe an emerging class of protein or genetic variations of components of the mitochondrial import machinery involved in heart disease. The major protein import pathways, including the presequence pathway (TIM23 pathway), the carrier pathway (TIM22 pathway), and the mitochondrial intermembrane space import and assembly machinery, related translocases, proteinases, and chaperones, are discussed here. This review highlights the importance of mitochondrial import machinery in heart disease, which deserves considerable attention, and further studies are urgently needed. Ultimately, this knowledge may be critical for the development of therapeutic strategies in heart disease.

Independent accretion of TIM22 complex subunits in the animal and fungal lineages.

Background: The mitochondrial protein import complexes arose early in eukaryogenesis. Most of the components of the protein import pathways predate the last eukaryotic common ancestor. For example, the carrier-insertase TIM22 complex comprises the widely conserved Tim22 channel core. However, the auxiliary components of fungal and animal TIM22 complexes are exceptions to this ancient conservation. Methods: Using comparative genomics and phylogenetic approaches, we identified precisely when each TIM22 accretion occurred. Results: In animals, we demonstrate that Tim29 and Tim10b arose early in the holozoan lineage. Tim29 predates the metazoan lineage being present in the animal sister lineages, choanoflagellate and filastereans, whereas the erroneously named Tim10b arose from a duplication of Tim9 at the base of metazoans. In fungi, we show that Tim54 has representatives present in every holomycotan lineage including microsporidians and fonticulids, whereas Tim18 and Tim12 appeared much later in fungal evolution. Specifically, Tim18 and Tim12 arose from duplications of Sdh3 and Tim10, respectively, early in the Saccharomycotina. Surprisingly, we show that Tim54 is distantly related to AGK suggesting that AGK and Tim54 are extremely divergent orthologues and the origin of AGK/Tim54 interaction with Tim22 predates the divergence of animals and fungi. Conclusions: We argue that the evolutionary history of the TIM22 complex is best understood as the neutral structural divergence of an otherwise strongly functionally conserved protein complex. This view suggests that many of the differences in structure/subunit composition of multi-protein complexes are non-adaptive. Instead, most of the phylogenetic variation of functionally conserved molecular machines, which have been under stable selective pressures for vast phylogenetic spans, such as the TIM22 complex, is most likely the outcome of the interplay of random genetic drift and mutation pressure.