A mitochondrial iron-responsive pathway regulated by DELE1.

The heme-regulated kinase HRI is activated under heme/iron deficient conditions; however, the underlying molecular mechanism is incompletely understood. Here, we show that iron-deficiency-induced HRI activation requires the mitochondrial protein DELE1. Notably, mitochondrial import of DELE1 and its subsequent protein stability are regulated by iron availability. Under steady-state conditions, DELE1 is degraded by the mitochondrial matrix-resident protease LONP1 soon after mitochondrial import. Upon iron chelation, DELE1 import is arrested, thereby stabilizing DELE1 on the mitochondrial surface to activate the HRI-mediated integrated stress response (ISR). Ablation of this DELE1-HRI-ISR pathway in an erythroid cell model enhances cell death under iron-limited conditions, suggesting a cell-protective role for this pathway in iron-demanding cell lineages. Our findings highlight mitochondrial import regulation of DELE1 as the core component of a previously unrecognized mitochondrial iron responsive pathway that elicits stress signaling following perturbation of iron homeostasis.

Dynamic Regulation of Mitochondrial Import by the Ubiquitin System.

Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.

Tom20 gates PINK1 activity and mediates its tethering of the TOM and TIM23 translocases upon mitochondrial stress.

Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early-onset Parkinson's disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by triggering mitophagy mediated by the ubiquitin (Ub) ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane forming a high-molecular-weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates Ub, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 N-terminal-C-terminal extension module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.

Aggregation-prone Tau impairs mitochondrial import, which affects organelle morphology and neuronal complexity.

Mitochondrial protein import is essential for organellar biogenesis, and thereby for the sufficient supply of cytosolic ATP - which is particularly important for cells with high energy demands like neurons. This study explores the prospect of import machinery perturbation as a cause of neurodegeneration instigated by the accumulation of aggregating proteins linked to disease. We found that the aggregation-prone Tau variant (TauP301L) reduces the levels of components of the import machinery of the outer (TOM20, encoded by TOMM20) and inner membrane (TIM23, encoded by TIMM23) while associating with TOM40 (TOMM40). Intriguingly, this interaction affects mitochondrial morphology, but not protein import or respiratory function; raising the prospect of an intrinsic rescue mechanism. Indeed, TauP301L induced the formation of tunnelling nanotubes (TNTs), potentially for the recruitment of healthy mitochondria from neighbouring cells and/or the disposal of mitochondria incapacitated by aggregated Tau. Consistent with this, inhibition of TNT formation (and rescue) reveals Tau-induced import impairment. In primary neuronal cultures, TauP301L induced morphological changes characteristic of neurodegeneration. Interestingly, these effects were mirrored in cells where the import sites were blocked artificially. Our results reveal a link between aggregation-prone Tau and defective mitochondrial import relevant to disease.

The TOM complex from an evolutionary perspective and the functions of TOMM70.

In humans, up to 1,500 mitochondrial precursor proteins are synthesized at cytosolic ribosomes and must be imported into the organelle. This is not only essential for mitochondrial but also for many cytosolic functions. The majority of mitochondrial precursor proteins are imported over the translocase of the outer membrane (TOM). In recent years, high-resolution structure analyses from different organisms shed light on the composition and arrangement of the TOM complex. Although significant similarities have been found, differences were also observed, which have been favored during evolution and could reflect the manifold functions of TOM with cellular signaling and its response to altered metabolic situations. A key component within these regulatory mechanisms is TOMM70, which is involved in protein import, forms contacts to the ER and the nucleus, but is also involved in cellular defense mechanisms during infections.

The Interaction and Effect of a Small MitoBlock Library as Inhibitor of ALR Protein-Protein Interaction Pathway.

MIA40 and ALR of the MIA pathway mediate the import of protein precursors that form disulfides into the mitochondrial intermembrane space. This import pathway is suggested to be a linear pathway in which MIA40 first binds to the precursor via a disulfide linkage and oxidizes it. Subsequently, ALR re-oxidizes MIA40 and then ALR transfers electrons to terminal electron acceptors. However, the precise mechanism by which ALR and MIA40 coordinate translocation is unknown. With a collection of small molecule modulators (MB-5 to MB-9 and MB-13) that inhibit ALR activity, we characterized the import mechanism in mitochondria. NMR studies show that most of the compounds bind to a similar region in ALR. Mechanistic studies with small molecules demonstrate that treatment with compound MB-6 locks the precursor in a state bound to MIA40, blocking re-oxidation of MIA40 by ALR. Thus, small molecules that target a similar region in ALR alter the dynamics of the MIA import pathway differently, resulting in a set of probes that are useful for studying the catalysis of the redox-regulated import pathway in model systems.

Bi-Allelic UQCRFS1 Variants Are Associated with Mitochondrial Complex III Deficiency, Cardiomyopathy, and Alopecia Totalis.

Isolated complex III (CIII) deficiencies are among the least frequently diagnosed mitochondrial disorders. Clinical symptoms range from isolated myopathy to severe multi-systemic disorders with early death and disability. To date, we know of pathogenic variants in genes encoding five out of 10 subunits and five out of 13 assembly factors of CIII. Here we describe rare bi-allelic variants in the gene of a catalytic subunit of CIII, UQCRFS1, which encodes the Rieske iron-sulfur protein, in two unrelated individuals. Affected children presented with low CIII activity in fibroblasts, lactic acidosis, fetal bradycardia, hypertrophic cardiomyopathy, and alopecia totalis. Studies in proband-derived fibroblasts showed a deleterious effect of the variants on UQCRFS1 protein abundance, mitochondrial import, CIII assembly, and cellular respiration. Complementation studies via lentiviral transduction and overexpression of wild-type UQCRFS1 restored mitochondrial function and rescued the cellular phenotype, confirming UQCRFS1 variants as causative for CIII deficiency. We demonstrate that mutations in UQCRFS1 can cause mitochondrial disease, and our results thereby expand the clinical and mutational spectrum of CIII deficiencies.

CHCHD4 (MIA40) and the mitochondrial disulfide relay system.

Mitochondria are pivotal for normal cellular physiology, as they perform a crucial role in diverse cellular functions and processes, including respiration and the regulation of bioenergetic and biosynthetic pathways, as well as regulating cellular signalling and transcriptional networks. In this way, mitochondria are central to the cell's homeostatic machinery, and as such mitochondrial dysfunction underlies the pathology of a diverse range of diseases including mitochondrial disease and cancer. Mitochondrial import pathways and targeting mechanisms provide the means to transport into mitochondria the hundreds of nuclear-encoded mitochondrial proteins that are critical for the organelle's many functions. One such import pathway is the highly evolutionarily conserved disulfide relay system (DRS) within the mitochondrial intermembrane space (IMS), whereby proteins undergo a form of oxidation-dependent protein import. A central component of the DRS is the oxidoreductase coiled-coil-helix-coiled-coil-helix (CHCH) domain-containing protein 4 (CHCHD4, also known as MIA40), the human homologue of yeast Mia40. Here, we summarise the recent advances made to our understanding of the role of CHCHD4 and the DRS in physiology and disease, with a specific focus on the emerging importance of CHCHD4 in regulating the cellular response to low oxygen (hypoxia) and metabolism in cancer.

The C-terminal region of the oxidoreductase MIA40 stabilizes its cytosolic precursor during mitochondrial import.

BACKGROUND: The mitochondrial intermembrane space (IMS) is home to proteins fulfilling numerous essential cellular processes, particularly in metabolism and mitochondrial function. All IMS proteins are nuclear encoded and synthesized in the cytosol and must therefore be correctly targeted and transported to the IMS, either through mitochondrial targeting sequences or conserved cysteines and the mitochondrial disulfide relay system. The mitochondrial oxidoreductase MIA40, which catalyzes disulfide formation in the IMS, is imported by the combined action of the protein AIFM1 and MIA40 itself. Here, we characterized the function of the conserved highly negatively charged C-terminal region of human MIA40. RESULTS: We demonstrate that the C-terminal region is critical during posttranslational mitochondrial import of MIA40, but is dispensable for MIA40 redox function in vitro and in intact cells. The C-terminal negatively charged region of MIA40 slowed import into mitochondria, which occurred with a half-time as slow as 90 min. During this time, the MIA40 precursor persisted in the cytosol in an unfolded state, and the C-terminal negatively charged region served in protecting MIA40 from proteasomal degradation. This stabilizing property of the MIA40 C-terminal region could also be conferred to a different mitochondrial precursor protein, COX19. CONCLUSIONS: Our data suggest that the MIA40 precursor contains the stabilizing information to allow for postranslational import of sufficient amounts of MIA40 for full functionality of the essential disulfide relay. We thereby provide for the first time mechanistic insights into the determinants controlling cytosolic surveillance of IMS precursor proteins.

How does protein degradation regulate TOM machinery-dependent mitochondrial import?

Mitochondrial dysregulation is a pivotal hallmark of aging-related disorders. Although there is a considerable understanding of the molecular counteracting responses toward damaged mitochondria, the molecular underpinnings connecting the abnormal aggregation of mitochondrial precursor protein fragments and abrogation of mitochondrial import machinery are far from clear. Recently, proteasomal-dependent degradation was unveiled as a pivotal fine-tuner of TOM machinery-dependent mitochondrial import. Herein, the role of proteasomal-mediated degradation in regulating fidelity of TOM-dependent import is briefly discussed and analyzed. The insights obtained from the characterization of this process may be applied to targeting mitochondrial import dysfunction in some neurodegenerative disorders.

Protein import by the mitochondrial disulfide relay in higher eukaryotes.

The proteome of the mitochondrial intermembrane space (IMS) contains more than 100 proteins, all of which are synthesized on cytosolic ribosomes and consequently need to be imported by dedicated machineries. The mitochondrial disulfide relay is the major import machinery for soluble proteins in the IMS. Its major component, the oxidoreductase MIA40, interacts with incoming substrates, retains them in the IMS, and oxidatively folds them. After this reaction, MIA40 is reoxidized by the sulfhydryl oxidase augmenter of liver regeneration, which couples disulfide formation by this machinery to the activity of the respiratory chain. In this review, we will discuss the import of IMS proteins with a focus on recent findings showing the diversity of disulfide relay substrates, describing the cytosolic control of this import system and highlighting the physiological relevance of the disulfide relay machinery in higher eukaryotes.

Mitochondrial OXPHOS Biogenesis: Co-Regulation of Protein Synthesis, Import, and Assembly Pathways.

The assembly of mitochondrial oxidative phosphorylation (OXPHOS) complexes is an intricate process, which-given their dual-genetic control-requires tight co-regulation of two evolutionarily distinct gene expression machineries. Moreover, fine-tuning protein synthesis to the nascent assembly of OXPHOS complexes requires regulatory mechanisms such as translational plasticity and translational activators that can coordinate mitochondrial translation with the import of nuclear-encoded mitochondrial proteins. The intricacy of OXPHOS complex biogenesis is further evidenced by the requirement of many tightly orchestrated steps and ancillary factors. Early-stage ancillary chaperones have essential roles in coordinating OXPHOS assembly, whilst late-stage assembly factors-also known as the LYRM (leucine-tyrosine-arginine motif) proteins-together with the mitochondrial acyl carrier protein (ACP)-regulate the incorporation and activation of late-incorporating OXPHOS subunits and/or co-factors. In this review, we describe recent discoveries providing insights into the mechanisms required for optimal OXPHOS biogenesis, including the coordination of mitochondrial gene expression with the availability of nuclear-encoded factors entering via mitochondrial protein import systems.

Decoding the Divergent Subcellular Location of Two Highly Similar Paralogous LEA Proteins.

Many mitochondrial proteins are synthesized as precursors in the cytosol with an N-terminal mitochondrial targeting sequence (MTS) which is cleaved off upon import. Although much is known about import mechanisms and MTS structural features, the variability of MTS still hampers robust sub-cellular software predictions. Here, we took advantage of two paralogous late embryogenesis abundant proteins (LEA) from Arabidopsis with different subcellular locations to investigate structural determinants of mitochondrial import and gain insight into the evolution of the LEA genes. LEA38 and LEA2 are short proteins of the LEA_3 family, which are very similar along their whole sequence, but LEA38 is targeted to mitochondria while LEA2 is cytosolic. Differences in the N-terminal protein sequences were used to generate a series of mutated LEA2 which were expressed as GFP-fusion proteins in leaf protoplasts. By combining three types of mutation (substitution, charge inversion, and segment replacement), we were able to redirect the mutated LEA2 to mitochondria. Analysis of the effect of the mutations and determination of the LEA38 MTS cleavage site highlighted important structural features within and beyond the MTS. Overall, these results provide an explanation for the likely loss of mitochondrial location after duplication of the ancestral gene.

Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import.

To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease. We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked.

Dual-targeted proteins tend to be more evolutionarily conserved.

In eukaryotic cells, identical proteins can be located in more than a single subcellular compartment, a phenomenon termed dual targeting. We hypothesized that dual-targeted proteins should be more evolutionary conserved than exclusive mitochondrial proteins, due to separate selective pressures administered by the different compartments to maintain the functions associated with the protein sequences. We employed codon usage bias, propensity for gene loss, phylogenetic relationships, conservation analysis at the DNA level, and gene expression, to test our hypothesis. Our findings indicate that, indeed, dual-targeted proteins are significantly more conserved than their exclusively targeted counterparts. We then used this trait of gene conservation, together with previously identified traits of dual-targeted proteins (such as protein net charge and mitochondrial targeting sequence strength) to 1) create, for the first time (due to addition of conservation parameters), a tool for the prediction of dual-targeted mitochondrial proteins based on protein and mRNA sequences, and 2) show that molecular mechanisms involving one versus two translation products are not correlated with specific dual-targeting parameters. Finally, we discuss what evolutionary pressure maintains protein dual targeting in eukaryotes and deduce, as we initially hypothesized, that it is the discrete functions of these proteins in the different subcellular compartments, regardless of their dual-targeting mechanism.

Mitochondrially-imported RNA in drug discovery.

The import of nuclear transcribed RNAs into mitochondria is an emerging area that presents a tremendous opportunity to develop human metabolic therapeutics. However, our knowledge base is quite limited. Much remains to be discovered regarding specific RNA localization and mechanisms of import. To identify novel RNAs imported into mitochondria, all RNAs within the mitochondria were characterized using next generation sequencing technology. Several nuclear transcribed RNAs were found within mitochondrial RNA (mtRNA) samples, including nuclear ribosomal RNAs, gamma satellite RNA and VL30 retroelement RNA. The presence of these RNAs within mitochondria coupled with RNA sequencing data from other laboratories investigating mtRNA processing, lead us to hypothesize that nuclease treatment of mitoplasts is insufficient for removing contaminating cytoplasmic RNAs. In contrast to traditional methodology, mitochondrial import was evaluated by qRT-PCR after stepwise removal of the outer mitochondrial membrane and subsequent lysis of mitochondria. This allowed identification of RNAs lost from the mitochondria with the same kinetics as mitochondrial DNA-transcribed RNAs. This approach provided an improved evaluation of nuclear RNA enrichment within mitochondrial membranes to characterize nuclease protection and mitochondrial import and identify false-positive detection errors. qRT-PCR results confirmed the presence of VL30 retroelement RNA within mitochondria and question the hypothesis that the RNA component of RNase P is imported. These results illustrate a reliable approach for evaluating the presence of RNAs within mitochondria and open new avenues of investigation relating to mtRNA biology and in targeting mitochondrial based therapeutics.

Evolving dual targeting of a prokaryotic protein in yeast.

Dual targeting is an important and abundant phenomenon. Indeed, we estimate that more than a third of the yeast mitochondrial proteome is dual localized. The enzyme fumarase is a highly conserved protein in all organisms with respect to its sequence, structure, and enzymatic activity. In eukaryotes, it is dual localized to the cytosol and mitochondria. In Saccharomyces cerevisiae, the dual localization of fumarase is achieved by the reverse translocation mechanism; all fumarase molecules harbor a mitochondrial targeting sequence (MTS), are targeted to mitochondria, begin their translocation, and are processed by mitochondrial processing peptidase in the matrix. A subset of these processed fumarase molecules in transit is then fully imported into the matrix, whereas the majority moves back into the cytosol by reverse translocation. The proposed driving force for fumarase distribution is protein folding during import. Here, we asked how reverse translocation could have evolved on a prokaryotic protein that had already acquired expression from the nuclear genome and a targeting sequence. To address this question, we used, as a model, the Escherichia coli FumC Class II fumarase, which is homologous to eukaryotic fumarases (∼58% identity and ∼74% similarity to the yeast Fum1). Starting with an exclusively mitochondrial targeted FumC (attached to a strong MTS), we show that two randomly acquired mutations within the prokaryotic FumC sequence are sufficient to cause substantial dual targeting by reverse translocation. In fact, the unmutated MTS-FumC also has some ability to be dual targeted but only at low temperatures. Our results suggest that in this case, evolution of dual targeting by reverse translocation is based on naturally occurring and fortuitously conserved features of fumarase folding.

Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol.

The vast majority of mitochondrial precursor proteins is synthesized in the cytosol and subsequently imported into the organelle with the help of targeting signals that are present within these proteins. Disruptions in mitochondrial import will result in the accumulation of the organellar precursors in the cytosol of the cell. If mislocalized proteins exceed their critical concentrations, they become prone to aggregation. Under certain circumstances, protein aggregation becomes an irreversible process, which eventually endangers cellular health. Impairment in mitochondrial biogenesis and its effect on cellular protein homeostasis were recently linked to neurodegeneration, therefore placing this process in the center of attention. In this chapter, we are presenting a set of techniques that allows to monitor and study mitochondrial precursor protein aggregates upon mitochondrial dysfunction in the cytosol of both yeast and human cells.

Structural determinants of mitochondrial STAT3 targeting and function.

Signal transducer and activator of transcription (STAT) 3 has been found within mitochondria in addition to its canonical role of shuttling between cytoplasm and nucleus during cytokine signaling. Mitochondrial STAT3 has been implicated in modulation of cellular metabolism, largely through effects on the respiratory electron transport chain. However, the structural requirements underlying mitochondrial targeting and function have remained unclear. Here, we show that mitochondrial STAT3 partitions between mitochondrial compartments defined by differential detergent solubility, suggesting that mitochondrial STAT3 is membrane associated. The majority of STAT3 was found in an SDS soluble fraction copurifying with respiratory chain proteins, including numerous components of the complex I NADH dehydrogenase, while a minor component was found with proteins of the mitochondrial translation machinery. Mitochondrial targeting of STAT3 required the amino-terminal domain, and an internal linker domain motif also directed mitochondrial translocation. However, neither the phosphorylation of serine 727 nor the presence of mitochondrial DNA was required for the mitochondrial localization of STAT3. Two cysteine residues in the STAT3 SH2 domain, which have been previously suggested to be targets for protein palmitoylation, were also not required for mitochondrial translocation, but were required for its function as an enhancer of complex I activity. These structural determinants of STAT3 mitochondrial targeting and function provide potential therapeutic targets for disrupting the activity of mitochondrial STAT3 in diseases such as cancer.

Isolation of yeast mitochondria by affinity purification using magnetic beads.

Isolated mitochondria have been widely utilized in various model organisms to investigate the diverse functions of the organelle. Techniques such as differential centrifugation, density gradient ultracentrifugation and antibody-coated magnetic beads are employed for isolation of the organelle from whole cells. However, mitochondria isolated using differential centrifugation are often contaminated with other organelles; isolation using density gradient ultracentrifugation can reduce contamination but is time-intensive and requires large amounts of starting materials; and mitochondria isolated using antibody-coated magnetic beads are irreversibly bound to the beads. Here, we provide a step-by-step protocol for the isolation of highly pure mitochondria from Saccharomyces cerevisiae using a magnetic bead affinity purification method that overcomes these limitations. This protocol describes how to isolate mitochondria, tagged by insertion of 6 histidines (6xHis) into the chromosomal copy of the TOM70 (Translocase of outer membrane 70) gene using Ni-NTA (nickel(II) nitrilotriacetic acid) paramagnetic beads, and the subsequent release of mitochondria from the beads using a buffer containing imidazole. We provide examples of expected results, highlighting the purity, integrity and import activity of isolated mitochondria. These affinity-purified mitochondria are intact and functional, containing less contamination with cytosol and other organelles compared to mitochondria isolated by other methods. Our method is adaptable and can be applied to other model organisms that can be genetically manipulated using CRISPR or other methods.