Mitochondrial Targeting Sequence Research Articles

The human mitochondrial enzyme BCO2 exhibits catalytic activity toward carotenoids and apocarotenoids

The enzyme β-carotene oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish, and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, because of a 4-amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expressed in ARPE-19 cells. Removal of the MTS increased solubility of BCO2a when expressed in Escherichia coli and rendered the recombinant protein enzymatically active. The expression of the enzymatically active recombinant human BCO2a was further improved by codon optimization and its fusion with maltose-binding protein. Introduction of the 4-amino acid insertion into mouse Bco2 did not impede the chimeric enzyme's catalytic proficiency. We further showed that the chimeric BCO2 displayed broad substrate specificity and converted carotenoids into two ionones and a central C14-apocarotendial by oxidative cleavage reactions at C9,C10 and C9',C10'. Thus, our study demonstrates that human BCO2 is a catalytically competent enzyme. Consequently, information on BCO2 becomes broadly applicable in human biology with important implications for the physiology of the eyes and other tissues.

Elimination of Mutant mtDNA by an Optimized mpTALEN Restores Differentiation Capacities of Heteroplasmic MELAS-iPSCs

Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling.

Phosphorylation of STAT3 by axonal Cdk5 promotes axonal regeneration by modulating mitochondrial activity

Cyclin-dependent kinase 5 (Cdk5) is involved in neural organization and synaptic functions in developing and adult brains, yet its role in axonal regeneration is not known well. Here, we characterize Cdk5 function for axonal regeneration after peripheral nerve injury. Levels of Cdk5 and p25 were elevated in sciatic nerve axons after injury. Cdk5 activity was concomitantly induced from injured nerve and increased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) on the serine 727 residue. Pharmacological and genetic blockades of Cdk5 activity phosphorylating STAT3 resulted in the inhibition of axonal regeneration as evidenced by reduction of retrograde labeling of dorsal root ganglion (DRG) sensory neurons and spinal motor neurons and also of neurite outgrowth of preconditioned DRG neurons in culture. Cdk5 and STAT3 were found in mitochondrial membranes of the injured sciatic nerve. Cdk5-GFP, which was translocated into the mitochondria by the mitochondrial target sequence (MTS), induced STAT3 phosphorylation in transfected DRG neurons and was sufficient to induce neurite outgrowth. In the mitochondria, Cdk5 activity was positively correlated with increased mitochondrial membrane potential as measured by fluorescence intensity of JC-1 aggregates. Our data suggest that Cdk5 may play a role in modulating mitochondrial activity through STAT3 phosphorylation, thereby promoting axonal regeneration.

Optimization of 1,2,4-butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance.

1,2,4-Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2-ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2-keto-3-deoxy-xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2-ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.

Somatic Mitochondrial DNA Point Mutations Used as Biomarkers to Demonstrate Genomic Heterogeneity in Primary Prostate Cancer.

Primary prostate tumor heterogeneity is poorly understood, leaving research efforts with challenges regarding the initiation and advancement of the disease. The growth of tumor cells is accompanied by mutations in nuclear and in mitochondrial genomes. Thus, mitochondrial DNA mutations may be used as tumor cell markers. By the use of laser capture microdissection coupled with assays for mitochondrial point mutation detection, mtDNA mutations were used to trace mutated cells at a histological level. Point mutations in mtDNA were determined in 12 primary prostate cancers. The tumors represent different pathology-prognostic grade groups. Known mutational hotspots of the mtDNA were scanned for heteroplasmy. All specimens with mtDNA heteroplasmy were subsequently subsampled by laser capture microdissection. From a total number of 1728 microsamples, mitochondrial DNA target sequences were amplified and base substitutions detected by cycling temperature capillary electrophoresis. Real-time PCR was used as a quantitative assay to determine the relative mtDNA copy number of 12 tumors studied, represented by two samples from each (N = 24); a high degree (75%) demonstrated tumor specimen heterogeneity. A grid of 96 spots isolated by laser capture microdissection demonstrated interfocal sample heterogeneity and increased the limit of detection. The spots demonstrated a wide range of mutant fractions from 0 to 100% mutant copies. The mitochondrial DNA copy number in the samples was determined by real-time PCR. No correlation between copy number and pathology-prognostic grade groups was observed. Somatic mitochondrial DNA point mutations represent traceable biomarkers demonstrating heterogeneity in primary prostate cancer. Mutations can be detected in areas before changes in tissue histopathology are evident to the pathologist.

Proteasomal degradation induced by DPP9‐mediated processing competes with mitochondrial protein import

Plasticity of the proteome is critical to adapt to varying conditions. Control of mitochondrial protein import contributes to this plasticity. Here, we identified a pathway that regulates mitochondrial protein import by regulated N‐terminal processing. We demonstrate that dipeptidyl peptidases 8/9 (DPP8/9) mediate the N‐terminal processing of adenylate kinase 2 (AK2) en route to mitochondria. We show that AK2 is a substrate of the mitochondrial disulfide relay, thus lacking an N‐terminal mitochondrial targeting sequence and undergoing comparatively slow import. DPP9‐mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Besides AK2, we identify more than 100 mitochondrial proteins with putative DPP8/9 recognition sites and demonstrate that DPP8/9 influence the cellular levels of a number of these proteins. Collectively, we provide in this study a conceptual framework on how regulated cytosolic processing controls levels of mitochondrial proteins as well as their dual localization to mitochondria and other compartments.

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

BackgroundThe 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.ResultsWe 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.ConclusionsOur 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.

Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

ObjectiveCholesterol plays a pivotal role in mitochondrial steroidogenesis, membrane structure, and respiration. Mitochondrial membranes are intrinsically low in cholesterol content and therefore must be replenished with cholesterol from other subcellular membranes. However, the molecular mechanisms underlying mitochondrial cholesterol transport remains poorly understood. The Aster-B gene encodes a cholesterol binding protein recently implicated in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (ER). In this study, we investigated the function and underlying mechanism of Aster-B in mediating mitochondrial cholesterol transport. MethodsCRISPR/Cas9 gene editing was carried out to generate cell lines deficient in Aster-B expression. The effect of Aster-B deficiency on mitochondrial cholesterol transport was examined by both confocal imaging analysis and biochemical assays. Deletion mutational analysis was also carried out to identify the function of a putative mitochondrial targeting sequence (MTS) at the N-terminus of Aster-B for its role in targeting Aster-B to mitochondria and in mediating mitochondrial cholesterol trafficking. ResultsAblation of Aster-B impaired cholesterol transport from the ER to mitochondria, leading to a significant decrease in mitochondrial cholesterol content. Aster-B is also required for mitochondrial transport of fatty acids derived from hydrolysis of cholesterol esters. A putative MTS at the N-terminus of Aster-B mediates the mitochondrial cholesterol uptake. Deletion of the MTS or ablation of Arf1 GTPase which is required for mitochondrial translocation of ER proteins prevented mitochondrial cholesterol transport, leading to mitochondrial dysfunction. ConclusionsWe identified Aster-B as a key regulator of cholesterol transport from the ER to mitochondria. Aster-B also coordinates mitochondrial cholesterol trafficking with uptake of fatty acids derived from cholesterol esters, implicating the Aster-B protein as a novel regulator of steroidogenesis.

Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast.

Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded.

A novel mitochondrial targeted hybrid peptide modified HPMA copolymers for breast cancer metastasis suppression

Primary tumor metastasis remains to be a tough obstacle for clinical breast cancer treatment. Since evidences have shown that mitochondria play a crucial role in tumor metastasis, we designed a mitochondrial targeted drug delivery system (P-D-R8MTS) based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers to simultaneously inhibit breast cancer progression and metastasis. A novel mitochondrial targeted hybrid peptide R8MTS, which consists of a cell penetrating peptide octaarginine (R8) and a mitochondrial targeting sequence ALD5MTS, was used as targeting ligand and attached to doxorubicin (DOX) as model drug (DOX-R8MTS). After entering into the tumor cells, DOX-R8MTS was pH-responsibly released from HPMA copolymer backbone in acidic lysosome and efficiently targeted to mitochondria, resulting in enhanced reactive oxygen species (ROS) generation and apoptosis initiation. By destroying mitochondria, P-D-R8MTS not only inhibited cell proliferation but also suppressed migration and invasion of breast cancer 4T1 and MDA-MB-231 cells in vitro. Moreover, P-D-R8MTS exhibited superior inhibition of tumor growth and showed no apparent lung metastatic nodules on 4T1-bearing mice in vivo, which was partially via down-regulation of typical proteins associated with tumor metastasis and invasion: matrix metalloproteinases-2 (MMP-2), vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β). Collectively, our work provided a prospectively potential strategy for metastatic cancer treatment through mitochondrial targeted drug delivery.

Targeting mitochondrial and cytosolic substrates of TRIT1 isopentenyltransferase: Specificity determinants and tRNA-i6A37 profiles.

The tRNA isopentenyltransferases (IPTases), which add an isopentenyl group to N6 of A37 (i6A37) of certain tRNAs, are among a minority of enzymes that modify cytosolic and mitochondrial tRNAs. Pathogenic mutations to the human IPTase, TRIT1, that decrease i6A37 levels, cause mitochondrial insufficiency that leads to neurodevelopmental disease. We show that TRIT1 encodes an amino-terminal mitochondrial targeting sequence (MTS) that directs mitochondrial import and modification of mitochondrial-tRNAs. Full understanding of IPTase function must consider the tRNAs selected for modification, which vary among species, and in their cytosol and mitochondria. Selection is principally via recognition of the tRNA A36-A37-A38 sequence. An exception is unmodified tRNATrpCCA-A37-A38 in Saccharomyces cerevisiae, whereas tRNATrpCCA is readily modified in Schizosaccharomyces pombe, indicating variable IPTase recognition systems and suggesting that additional exceptions may account for some of the tRNA-i6A37 paucity in higher eukaryotes. Yet TRIT1 had not been characterized for restrictive type substrate-specific recognition. We used i6A37-dependent tRNA-mediated suppression and i6A37-sensitive northern blotting to examine IPTase activities in S. pombe and S. cerevisiae lacking endogenous IPTases on a diversity of tRNA-A36-A37-A38 substrates. Point mutations to the TRIT1 MTS that decrease human mitochondrial import, decrease modification of mitochondrial but not cytosolic tRNAs in both yeasts. TRIT1 exhibits clear substrate-specific restriction against a cytosolic-tRNATrpCCA-A37-A38. Additional data suggest that position 32 of tRNATrpCCA is a conditional determinant for substrate-specific i6A37 modification by the restrictive IPTases, Mod5 and TRIT1. The cumulative biochemical and phylogenetic sequence analyses provide new insights into IPTase activities and determinants of tRNA-i6A37 profiles in cytosol and mitochondria.

A mitochondrial recruitment assay to characterize a family of DENN‐domain proteins, Rab GDP/GTP exchange factors.

Intracellular membrane trafficking controls the levels, localization and functional activity of a myriad of proteins and is thus critical for cellular function. Rab GTPases are molecular switches that regulate membrane trafficking by toggling between GTP‐bound (active; membrane‐bound) and GDP‐bound (inactive; cytosolic) states. These GTPases are controlled largely by guanine nucleotide exchange factors (GEFs) that mediate the exchange of GDP for GTP. Mammalian cells possess 61 Rabs but many of their GEFs are unknown. One of the largest family of Rab GEFs contain an evolutionarily conserved protein module called the DENN domain. There are 18 DENN domain proteins but many of their corresponding Rab GTPase partner(s) are either not known or are controversial. This is due to the fact that all studies concerning DENN domains have used biochemical and in‐vitro assays, which do not reflect physiological conditions. Here we describe a cell‐based GEF assay as a method to identify GTPase substrates of DENN domain proteins. By overexpressing DENN domains fused to a mitochondrial targeting sequence we can drive the recruitment of physiological GTPase substrates to the mitochondrial membrane, which can then be detected by immunofluorescence analysis. We validated our assay with a known DENN/Rab pair, DENND1A and Rab35. Using a panel of GFP‐tagged Rabs we have resolved controversies over previously reported DENN/Rab pairs and have identified new Rab substrates for DENN domains. Identifying the full complement of DENN/Rab pairs will provide new insights into membrane trafficking and cellular function.Support or Funding Information1) Canada First Research Excellence Fund, awarded to McGill University for Healthy Brains for Healthy Lives. 2) ALS Canada

Mitochondrial Translocation of DJ-1 Is Mediated by Grp75: Implication in Cardioprotection of Resveratrol Against Hypoxia/Reoxygenation-Induced Oxidative Stress.

Resveratrol (Res) was recently reported to ameliorate hypoxia/reoxygenation (H/R)-caused oxidative stress in H9c2 cardiomyocytes through promoting the mitochondrial translocation of DJ-1 protein and subsequently preserving the activity of mitochondrial complex I. However, it is noteworthy that DJ-1 possesses no mitochondria-targeting sequence. Therefore, how Res induces DJ-1 mitochondrial translocation is an important and interesting question for further exploration. Glucose-regulated protein 75 (Grp75), whose N-terminus contains a 51-amino acid long mitochondrial-targeting signal peptide, is a cytoprotective chaperone that partakes in mitochondrial import of several proteins. Here, the contribution of Grp75 to mitochondrial import of DJ-1 by Res was investigated in a cellular model of H/R. Our results showed that Res upregulated the expression of DJ-1 protein, enhanced the interaction of DJ-1 and Grp75, and promoted DJ-1 translocation to mitochondria from cytosol in H9c2 cardiomyocytes undergoing H/R. Importantly, knockdown of Grp75 markedly reduced the interaction of DJ-1 with Grp75 and subsequent DJ-1 mitochondrial translocation induced by Res. Furthermore, Res pretreatment promoted the association of DJ-1 with ND1 and NDUFA4 subunits of complex I, preserved the activity of complex I, decreased mitochondria-derived reactive oxygen species production, and eventually ameliorated H/R-caused oxidative stress damage. Intriguingly, these effects were largely prevented also by small interfering RNA targeting Grp75. Overall, these results suggested that Grp75 interacts with DJ-1 to facilitate its translocation from cytosol to mitochondria, which is required for Res-mediated preservation of mitochondria complex I and cardioprotection from H/R-caused oxidative stress injury.

CHCHD2 harboring Parkinson's disease-linked T61I mutation precipitates inside mitochondria and induces precipitation of wild-type CHCHD2.

The T61I mutation in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a protein residing in the mitochondrial intermembrane space (IMS), causes an autosomal dominant form of Parkinson's disease (PD), but the underlying pathogenic mechanisms are not well understood. Here, we compared the subcellular localization and solubility of wild-type (WT) and T61I mutant CHCHD2 in human cells. We found that mitochondrial targeting of both WT and T61I CHCHD2 depended on the four cysteine residues in the C-terminal coiled-coil-helix-coiled-coil-helix (CHCH) domain but not on the N-terminal predicted mitochondrial targeting sequence. The T61I mutation did not interfere with mitochondrial targeting of the mutant protein but induced its precipitation in the IMS. Moreover, T61I CHCHD2 induced increased mitochondrial production of reactive oxygen species and apoptosis, which was prevented by treatment with anti-oxidants. Retention of T61I CHCHD2 in the cytosol through mutation of the cysteine residues in the CHCH domain prevented its precipitation as well as its apoptosis-inducing effect. Importantly, T61I CHCHD2 potently impaired the solubility of WT CHCHD2. In conclusion, our data show that the T61I mutation renders mutant CHCHD2 insoluble inside mitochondria, suggesting loss of function of the mutant protein. In addition, T61I CHCHD2 exerts a dominant-negative effect on the solubility of WT CHCHD2, explaining the dominant inheritance of this form of PD.

Quantitative global studies reveal differential translational control by start codon context across the fungal kingdom.

Eukaryotic protein synthesis generally initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but the quantitative importance of this context in different species is unclear. We tested this concept in two pathogenic Cryptococcus yeast species by genome-wide mapping of translation and of mRNA 5′ and 3′ ends. We observed thousands of AUG-initiated upstream open reading frames (uORFs) that are a major contributor to translation repression. uORF use depends on the Kozak sequence context of its start codon, and uORFs with strong contexts promote nonsense-mediated mRNA decay. Transcript leaders in Cryptococcus and other fungi are substantially longer and more AUG-dense than in Saccharomyces. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Analysis of other fungal species shows that such dual-localization is also predicted to be common in the ascomycete mould, Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that quantitatively programs both the expression and the structures of proteins in diverse fungi.

Mitochondrial import, health and mtDNA copy number variability using type II and type V CRISPR effectors

Current methodologies for targeting the mitochondrial genome for research and/or therapy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. A molecular toolbox for CRISPR-mediated mitochondrial genome editing is desirable, as this could enable targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes. Such 'MitoCRISPR' systems described to date lack reproducibility and independent corroboration. We have explored the requirements for MitoCRISPR in human cells by CRISPR nuclease engineering, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of guide (g)RNA modifications for mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and effects on mitochondrial dynamics/function of different CRISPR nucleases, with Lachnospiraceae bacterium ND2006 (Lb) Cas12a being better targeted and tolerated than Cas9 variants. We also provide evidence of Cas9 gRNA association with mitochondria in HeLa cells and isolated yeast mitochondria, even in the absence of a targeting RNA aptamer. Our data link mitochondrial-targeted LbCas12a/crRNA with increased mtDNA copy number dependent upon DNA binding and cleavage activity. We discuss reproducibility issues and the future steps necessary for MitoCRISPR.

Molecular Mechanism Underlying Hypoxic Preconditioning-Promoted Mitochondrial Translocation of DJ-1 in Hypoxia/Reoxygenation H9c2 Cells.

DJ-1 was recently reported to be involved in the cardioprotection of hypoxic preconditioning (HPC) against hypoxia/reoxygenation (H/R)-induced oxidative stress damage, by preserving mitochondrial complex I activity and, subsequently, inhibiting mitochondrial reactive oxygen species (ROS) generation. However, the molecular mechanism by which HPC enables mitochondrial translocation of DJ-1, which has no mitochondria-targeting sequence, to preserve mitochondrial complex I, is largely unknown. In this study, co-immunoprecipitation data showed that DJ-1 was associated with glucose-regulated protein 75 (Grp75), and this association was significantly enhanced after HPC. Immunofluorescence imaging and Western blot analysis showed that HPC substantially enhanced the translocation of DJ-1 from cytosol to mitochondria in H9c2 cells subjected to H/R, which was mimicked by DJ-1 overexpression induced by pFlag-DJ-1 transfection. Importantly, knockdown of Grp75 markedly reduced the mitochondrial translocation of DJ-1 induced by HPC and pFlag-DJ-1 transfection. Moreover, HPC promoted the association of DJ-1 with mitochondrial complex I subunits ND1 and NDUFA4, improved complex I activity, and inhibited mitochondria-derived ROS production and subsequent oxidative stress damage after H/R, which was also mimicked by pFlag-DJ-1 transfection. Intriguingly, these effects of HPC and pFlag-DJ-1 transfection were also prevented by Grp75 knockdown. In conclusion, these results indicated that HPC promotes the translocation of DJ-1 from cytosol to mitochondria in a Grp75-dependent manner and Grp75 is required for DJ-1-mediated protection of HPC on H/R-induced mitochondrial complex I defect and subsequent oxidative stress damage.

Visualization of Sirtuin 4 Distribution between Mitochondria and the Nucleus, Based on Bimolecular Fluorescence Self-Complementation.

Mitochondrial sirtuins (Sirts) control important cellular processes related to stress. Despite their regulatory importance, however, the dynamics and subcellular distributions of Sirts remain debatable. Here, we investigate the subcellular localization of sirtuin 4 (Sirt4), a sirtuin variant with a mitochondrial targeting sequence (MTS), by expressing Sirt4 fused to the superfolder green fluorescent protein (Sirt4-sfGFP) in HeLa and pancreatic β-cells. Super resolution fluorescence microscopy revealed the trapping of Sirt4-sfGFP to the outer mitochondrial membrane (OMM), possibly due to slow mitochondrial import kinetics. In many cells, Sirt4-sfGFP was also present within the cytosol and nucleus. Moreover, the expression of Sirt4-sfGFP induced mitochondrial swelling in HeLa cells. In order to bypass these effects, we applied the self-complementing split fluorescent protein (FP) technology and developed mito-STAR (mitochondrial sirtuin 4 tripartite abundance reporter), a tripartite probe for the visualization of Sirt4 distribution between mitochondria and the nucleus in single cells. The application of mito-STAR proved the importation of Sirt4 into the mitochondrial matrix and demonstrated its localization in the nucleus under mitochondrial stress conditions. Moreover, our findings highlight that the self-complementation of split FP is a powerful technique to study protein import efficiency in distinct cellular organelles.

Proteomic Analysis of Mitochondrial Membrane Protein FAM210B in Erythroid Cells

(Background) In the context of erythroid differentiation, the importance of transcription factor GATA-1 has been unequivocally demonstrated through cell-based ex vivoassays, knockout mouse models, and rare patients with anemia. GATA-1regulates the expression of erythroid-related genes such as globins, and those involved in heme biosynthesis and an unrecognized function. A novel FAM210B (C20orf108) gene was recently identified as a novel GATA-1 target gene (Kondo et al. Int J Hematol. 2016). FAM210B gene is abundantly expressed in the later stage of erythroid differentiation and encodes a protein containing an N-terminal mitochondrial-targeting sequence, which was considered as a mitochondrial membrane protein. Although the FAM210B protein has been suggested to regulate mitochondrial metabolism (Sun et al. Cell Death Dis. 2017), its detailed function remains to be elucidated. (Method) Endogenous FAM210B protein was deleted by CRISPR/Cas9 based on human iPS-derivederythroid progenitor (HiDEP) cells (Kurita et al. PLoS ONE 2103). To induce erythroid differentiation, HiDEP cells were co-cultured with OP9 stromal cells (ATCC) with IMDM medium supplemented with FBS, erythropoietin, dexamethasone, MTG, insulin-transferrin-selenium, ascorbic acid, and sodium ferrous citrate (Saito and Suzuki et al. MCB2019). Affinity purification of the FAM210B complex was conducted in K562 erythroid cell line (ATCC) stably expressing His/Biotin-tagged FAM210B. MitoXpress Xtra (Agilent) and Human Oligo chip 25K (Toray) were used to evaluate oxygen consumption rate and transcription profiling, respectively. (Results) Clonal lines with HiDEP cells were established, which harbored deletion within coding exon 3 of FAM210B. Quantitative real-time polymerase chain reaction (RT-PCR) analysis confirmed a strong decrease in FAM210B expression. Microarray analysis revealed >1.5-fold up- and down-regulation of 104 and 53 genes caused by the FAM210B knockout, respectively. The down-regulated gene ensemble included TTC19, which is involved in mitochondrial respiratory chain complex III assembly, and genes encoding mitochondrial proteins (AARS2, YWHAE, CLIC1), whereas, the expressions for ALAS2, HBG, HBA, and HMOX1 were not significantly affected by FAM210B depletion. Intriguingly, when wild-type and FAM210B-depleted HiDEP cells were further induced to undergo erythroid differentiation, erythroid differentiation was more pronounced by FAM210B depletion. This was reflected by enhanced nuclear condensation and hemoglobinization, as well as upregulations for ALAS2, HBG, HBA, and HMOX1 in the FAM210B-depleted erythroblasts. Recent report suggests that FAM210B functions as an adaptor protein in the mitochondria (Yien et al. JBC 2018). Thus, FAM210B-interacting protein was purified from K562 cells stably expressing His/Biotin-tagged FAM210B.Mass spectrometry analysis based on the His/Biotin-purified material showed that FAM210B may interact with multiple mitochondrial proteins, including multiple subunits of mitochondrial adenosine triphosphate synthases and mitochondrial heat shock proteins. Further functional analysis showed that oxygen consumption rates might be decreased in FAM210B-depleted HiDEP cells, indicating that FAM210B was possibly involved in mitochondrial energy metabolism in erythroblasts. At present, we are conducting detailed biological analyses to characterize the role of FAM210B during erythroid differentiation. (Conclusion) Further characterization of FAM210B provides new insights in the study of erythroid differentiation, and this may lead to better understanding of the pathophysiology of certain anemias. Disclosures Fukuhara: Mundi: Honoraria; Janssen Pharma: Honoraria; Mochida: Honoraria; Gilead: Research Funding; Bayer: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Kyowa-Hakko Kirin: Honoraria; AbbVie: Research Funding; Solasia Pharma: Research Funding; Celgene Corporation: Honoraria, Research Funding; Nippon Shinkyaku: Honoraria; Zenyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria. Onishi:MSD: Honoraria, Research Funding; Kyowa-Hakko Kirin: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Celgene: Honoraria; Pfizer Japan Inc.: Honoraria; Astellas Pharma Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Nippon Shinyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Yokoyama:Astellas: Other: Travel expenses.

Diet and Adaptive Evolution of Alanine-Glyoxylate Aminotransferase Mitochondrial Targeting in Birds.

Adaptations to different diets represent a hallmark of animal diversity. The diets of birds are highly variable, making them an excellent model system for studying adaptive evolution driven by dietary changes. To test whether molecular adaptations to diet have occurred during the evolution of birds, we examined a dietary enzyme alanine-glyoxylate aminotransferase (AGT), which tends to target mitochondria in carnivorous mammals, peroxisomes in herbivorous mammals, and both mitochondria and peroxisomes in omnivorous mammals. A total of 31 bird species were examined in this study, which included representatives of most major avian lineages. Of these, 29 have an intact mitochondrial targeting sequence (MTS) of AGT. This finding is in stark contrast to mammals, which showed a number of independent losses of the MTS. Our cell-based functional assays revealed that the efficiency of AGT mitochondrial targeting was greatly reduced in unrelated lineages of granivorous birds, yet it tended to be high in insectivorous and carnivorous lineages. Furthermore, we found that proportions of animal tissue in avian diets were positively correlated with mitochondrial targeting efficiencies that were experimentally determined, but not with those that were computationally predicted. Adaptive evolution of AGT mitochondrial targeting in birds was further supported by the detection of positive selection on MTS regions. Our study contributes to the understanding of how diet drives molecular adaptations in animals, and suggests that caution must be taken when computationally predicting protein subcellular targeting.