Structural Basis of Membrane Protein Chaperoning through the Mitochondrial Intermembrane Space.

The exchange of metabolites between the mitochondrial matrix and the cytosol depends on ß-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and ß-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and ß-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment.

Intermembrane space-localized TbTim15 is an essential subunit of the single mitochondrial inner membrane protein translocase of trypanosomes.

All mitochondria import >95% of their proteins from the cytosol. This process is mediated by protein translocases in the mitochondrial membranes, whose subunits are generally highly conserved. Most eukaryotes have two inner membrane protein translocases (TIMs) that are specialized to import either presequence-containing or mitochondrial carrier proteins. In contrast, the parasitic protozoan Trypanosoma brucei has a single TIM complex consisting of one conserved and five unique subunits. Here, we identify candidates for new subunits of the TIM or the presequence translocase-associated motor (PAM) using a protein-protein interaction network of previously characterized TIM and PAM subunits. This analysis reveals that the trypanosomal TIM complex contains an additional trypanosomatid-specific subunit, designated TbTim15. TbTim15 is associated with the TIM complex, lacks transmembrane domains, and localizes to the intermembrane space. TbTim15 is essential for procyclic and bloodstream forms of trypanosomes. It contains two twin CX9C motifs and mediates import of both presequence-containing and mitochondrial carrier proteins. While the precise function of TbTim15 in mitochondrial protein import is unknown, our results are consistent with the notion that it may function as an import receptor for the non-canonical trypanosomal TIM complex.

Evolution of mitochondrial protein import - lessons from trypanosomes.

The evolution of mitochondrial protein import and the systems that mediate it marks the boundary between the endosymbiotic ancestor of mitochondria and a true organelle that is under the control of the nucleus. Protein import has been studied in great detail in Saccharomyces cerevisiae. More recently, it has also been extensively investigated in the parasitic protozoan Trypanosoma brucei, making it arguably the second best studied system. A comparative analysis of the protein import complexes of yeast and trypanosomes is provided. Together with data from other systems, this allows to reconstruct the ancestral features of import complexes that were present in the last eukaryotic common ancestor (LECA) and to identify which subunits were added later in evolution. How these data can be translated into plausible scenarios is discussed, providing insights into the evolution of (i) outer membrane protein import receptors, (ii) proteins involved in biogenesis of α-helically anchored outer membrane proteins, and (iii) of the intermembrane space import and assembly system. Finally, it is shown that the unusual presequence-associated import motor of trypanosomes suggests a scenario of how the two ancestral inner membrane protein translocases present in LECA evolved into the single bifunctional one found in extant trypanosomes.

Mitochondrial protein import in trypanosomes: Expect the unexpected.

Mitochondria have many different functions, the most important one of which is oxidative phosphorylation. They originated from an endosymbiotic event between a bacterium and an archaeal host cell. It was the evolution of a protein import system that marked the boundary between the endosymbiotic ancestor of the mitochondrion and a true organelle that is under the control of the nucleus. In present day mitochondria more than 95% of all proteins are imported from the cytosol in a proces mediated by hetero-oligomeric protein complexes in the outer and inner mitochondrial membranes. In this review we compare mitochondrial protein import in the best studied model system yeast and the parasitic protozoan Trypanosoma brucei. The 2 organisms are phylogenetically only remotely related. Despite the fact that mitochondrial protein import has the same function in both species, only very few subunits of their import machineries are conserved. Moreover, while yeast has 2 inner membrane protein translocases, one specialized for presequence-containing and one for mitochondrial carrier proteins, T. brucei has a single inner membrane translocase only, that mediates import of both types of substrates. The evolutionary implications of these findings are discussed.

Origin and Evolutionary Alteration of the Mitochondrial Import System in Eukaryotic Lineages.

Protein transport systems are fundamentally important for maintaining mitochondrial function. Nevertheless, mitochondrial protein translocases such as the kinetoplastid ATOM complex have recently been shown to vary in eukaryotic lineages. Various evolutionary hypotheses have been formulated to explain this diversity. To resolve any contradiction, estimating the primitive state and clarifying changes from that state are necessary. Here, we present more likely primitive models of mitochondrial translocases, specifically the translocase of the outer membrane (TOM) and translocase of the inner membrane (TIM) complexes, using scrutinized phylogenetic profiles. We then analyzed the translocases' evolution in eukaryotic lineages. Based on those results, we propose a novel evolutionary scenario for diversification of the mitochondrial transport system. Our results indicate that presequence transport machinery was mostly established in the last eukaryotic common ancestor, and that primitive translocases already had a pathway for transporting presequence-containing proteins. Moreover, secondary changes including convergent and migrational gains of a presequence receptor in TOM and TIM complexes, respectively, likely resulted from constrained evolution. The nature of a targeting signal can constrain alteration to the protein transport complex.

Tim62, a Novel Mitochondrial Protein in Trypanosoma brucei, Is Essential for Assembly and Stability of the TbTim17 Protein Complex.

Trypanosoma brucei, the causative agent of human African trypanosomiasis, possesses non-canonical mitochondrial protein import machinery. Previously, we characterized the essential translocase of the mitochondrial inner membrane (TIM) consisting of Tim17 in T. brucei. TbTim17 is associated with TbTim62. Here we show that TbTim62, a novel protein, is localized in the mitochondrial inner membrane, and its import into mitochondria depends on TbTim17. Knockdown (KD) of TbTim62 decreased the steady-state levels of TbTim17 post-transcriptionally. Further analysis showed that import of TbTim17 into mitochondria was not inhibited, but its half-life was reduced >4-fold due to TbTim62 KD. Blue-native gel electrophoresis revealed that TbTim62 is present primarily in ∼150-kDa and also in ∼1100-kDa protein complexes, whereas TbTim17 is present in multiple complexes within the range of ∼300 to ∼1100 kDa. TbTim62 KD reduced the levels of both TbTim62 as well as TbTim17 protein complexes. Interestingly, TbTim17 was accumulated as lower molecular mass complexes in TbTim62 KD mitochondria. Furthermore, depletion of TbTim62 hampered the assembly of the ectopically expressed TbTim17-2X-myc into TbTim17 protein complex. Co-immunoprecipitation analysis revealed that association of TbTim17 with mHSP70 was markedly reduced in TbTim62 KD mitochondria. All together our results demonstrate that TbTim62, a unique mitochondrial protein in T. brucei, is required for the formation of a stable TbTim17 protein complex. TbTim62 KD destabilizes this complex, and unassembled TbTim17 degrades. Therefore, TbTim62 acts as a novel regulatory factor to maintain the levels of TIM in T. brucei mitochondria.

FUNDC1 interacts with GPx4 to govern hepatic ferroptosis and fibrotic injury through a mitophagy-dependent manner.

INTRODUCTION: Liver fibrosis is a life-threatening pathological anomaly which usually evolves into advanced liver cirrhosis and hepatocellular carcinoma although limited therapeutic option is readily available. FUN14 domain containing 1 (FUNDC1) is a mitophagy receptor with little information in liver fibrosis. OBJECTIVE: This study was designed to examine the role for FUNDC1 in carbon tetrachloride (CCl4)-induced liver injury. METHODS: GEO database analysis and subsequent validation of biological processes including western blot, immunofluorescence, and co-immunoprecipitation were applied to clarify the regulatory role of FUNDC1 on mitophagy and ferroptosis. RESULTS: Our data revealed elevated FUNDC1 levels in liver tissues of patients with liver fibrotic injury and CCl4-challenged mice. FUNDC1 deletion protected against CCl4-induced hepatic anomalies in mice. Moreover, FUNDC1 deletion ameliorated CCl4-induced ferroptosis in vivo and in vitro. Mechanically, FUNDC1 interacted with glutathione peroxidase (GPx4), a selenoenzyme to neutralize lipid hydroperoxides and ferroptosis, via its 96-133 amino acid domain to facilitate GPx4 recruitment into mitochondria from cytoplasm. GPx4 entered mitochondria through mitochondrial protein import system-the translocase of outer membrane/translocase of inner membrane (TOM/TIM) complex, prior to degradation of GPx4 mainly through mitophagy along with ROS-induced damaged mitochondria, resulting in hepatocyte ferroptosis. CONCLUSION: Taken together, our data favored that FUNDC1 promoted hepatocyte injury through GPx4 binding to facilitate its mitochondrial translocation through TOM/TIM complex, where GPx4 was degraded by mitophagy to trigger ferroptosis. Targeting FUNDC1 may be a promising therapeutic approach for liver fibrosis.

LPGAT1 controls MEGDEL syndrome by coupling phosphatidylglycerol remodeling with mitochondrial transport.

Phosphatidylglycerol (PG) is a mitochondrial phospholipid required for mitochondrial cristae structure and cardiolipin synthesis. PG must be remodeled to its mature form at the endoplasmic reticulum (ER) after mitochondrial biosynthesis to achieve its biological functions. Defective PG remodeling causes MEGDEL (non-alcohol fatty liver disease and 3-methylglutaconic aciduria with deafness, encephalopathy, and Leigh-like) syndrome through poorly defined mechanisms. Here, we identify LPGAT1, an acyltransferase that catalyzes PG remodeling, as a candidate gene for MEGDEL syndrome. We show that PG remodeling by LPGAT1 at the ER is closely coordinated with mitochondrial transport through interaction with the prohibitin/TIMM14 mitochondrial import motor. Accordingly, ablation of LPGAT1 or TIMM14 not only causes aberrant fatty acyl compositions but also ER retention of newly remodeled PG, leading to profound loss in mitochondrial crista structure and respiration. Consequently, genetic deletion of the LPGAT1 in mice leads to cardinal features of MEGDEL syndrome, including 3-methylglutaconic aciduria, deafness, dilated cardiomyopathy, and premature death, which are highly reminiscent of those caused by TIMM14 mutations in humans.

Minimal peptide length required to span the mitochondrial protein translocases in Trypanosoma brucei.

Mitochondrial protein import depends on heterooligomeric translocases in the outer and inner membranes. Using import substrates consisting of various lengths of the N-terminal part of mitochondrial dihydrolipoamide dehydrogenase (LDH) fused to dihydrofolate reductase we present an in vivo analysis showing that in Trypanosoma brucei at least 96 aa of mature LDH are required to efficiently produce an import intermediate that spans both translocases. This is different to yeast, where around 50 aa are sufficient to achieve the same task and likely reflects the different arrangement and architecture of the trypanosomal mitochondrial translocases. Furthermore, we show that formation of the stuck import intermediate leads to a strong growth inhibition suggesting that, depending on the length of the LDH, the import channels in the translocases are quantitatively blocked.