Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish

Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. The import of some, but not all, nuclear-encoded mitochondrial proteins begins with translation of messenger RNAs (mRNAs) on the surface of mitochondria. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a-/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to employ localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.

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Biallelic Mutations in MTPAP Associated with a Lethal Encephalopathy

Neuropediatrics ◽

10.1055/s-0039-3400979 ◽

2019 ◽

Vol 51 (03) ◽

pp. 178-184 ◽

Cited By ~ 1

Author(s):

Lien Van Eyck ◽

Francesco Bruni ◽

Anne Ronan ◽

Tracy A. Briggs ◽

Tony Roscioli ◽

...

Keyword(s):

De Novo ◽

Mitochondrial Gene ◽

Mitochondrial Protein ◽

Missense Variant ◽

Mitochondrial Proteins ◽

Compound Heterozygous ◽

Altered Expression ◽

Mitochondrial Transcripts ◽

Biallelic Mutations ◽

First Year Of Life

Abstract Background A homozygous founder mutation in MTPAP/TENT6, encoding mitochondrial poly(A) polymerase (MTPAP), was first reported in six individuals of Old Order Amish descent demonstrating an early-onset, progressive spastic ataxia with optic atrophy and learning difficulties. MTPAP contributes to the regulation of mitochondrial gene expression through the polyadenylation of mitochondrially encoded mRNAs. Mitochondrial mRNAs with severely truncated poly(A) tails were observed in affected individuals, and mitochondrial protein expression was altered. Objective To determine the genetic basis of a perinatal encephalopathy associated with stereotyped neuroimaging and infantile death in three patients from two unrelated families. Methods Whole-exome sequencing was performed in two unrelated patients and the unaffected parents of one of these individuals. Variants and familial segregation were confirmed by Sanger sequencing. Polyadenylation of mitochondrial transcripts and de novo synthesis of mitochondrial proteins were assessed in patient's fibroblasts. Results Compound heterozygous p.Ile428Thr and p.Arg523Trp substitutions in MTPAP were recorded in two affected siblings from one family, and a homozygous p.Ile385Phe missense variant identified in a further affected child from a second sibship. Mitochondrial poly(A) tail analysis demonstrated shorter posttranscriptional additions to the mitochondrial transcripts, as well as an altered expression of mitochondrial proteins in the fibroblasts of the two siblings compared with healthy controls. Conclusion Mutations in MTPAP likely cause an autosomal recessive perinatal encephalopathy with lethality in the first year of life.

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LPS-Induced Endotoxemia Evokes Epigenetic Alterations in Mitochondrial DNA That Impacts Inflammatory Response

Cells ◽

10.3390/cells9102282 ◽

2020 ◽

Vol 9 (10) ◽

pp. 2282

Author(s):

Björn Koos ◽

Eva Lotta Moderegger ◽

Katharina Rump ◽

Hartmuth Nowak ◽

Katrin Willemsen ◽

...

Keyword(s):

Immune Response ◽

Mitochondrial Dna ◽

Dna Methyltransferase ◽

Protein Import ◽

Mononuclear Cells ◽

Mitochondrial Protein ◽

Mitochondrial Fraction ◽

Protein Abundance ◽

Peripheral Blood Mononuclear ◽

Cellular Expression

Mitochondrial DNA (mtDNA) plays a vital role as a damage-associated molecular pattern in sepsis being able to shape the immune response. Since pathogen recognition receptors of innate immune cells are activated by demethylated DNA only, we set out to investigate the amount of DNA methyltransferase 1 (DNMT1) in mitochondria and the extent of mtDNA methylation in a human endotoxin model. Peripheral blood mononuclear cells of 20 healthy individuals were isolated from whole blood and stimulated with lipopolysaccharide (LPS) for 48 h. Subsequently, DNMT1 protein abundance was assessed in whole cells and a mitochondrial fraction. At the same time, methylation levels of mtDNA were quantified, and cytokine expression in the supernatant was measured. Despite increased cellular expression of DNMT1 after LPS stimulation, the degree of mtDNA methylation slightly decreased. Strikingly the mitochondrial protein abundance of DNMT1 was reduced by 50% in line with the lower degree of mtDNA methylation. Although only modest alterations were seen in the degree of mtDNA methylation, these strongly correlated with IL-6 and IL-10 expression. Our data may hint at a protein import problem for DNMT1 into the mitochondria under LPS stimulation and suggest a role of demethylated mtDNA in the regulation of the inflammatory immune response.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21113820 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3820 ◽

Cited By ~ 1

Author(s):

Jia Xin Tang ◽

Kyle Thompson ◽

Robert W. Taylor ◽

Monika Oláhová

Keyword(s):

Gene Expression ◽

Protein Synthesis ◽

Protein Import ◽

Acyl Carrier Protein ◽

Early Stage ◽

Mitochondrial Gene ◽

Mitochondrial Protein ◽

Fine Tuning ◽

Mitochondrial Translation ◽

Oxphos Complex

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.

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Plant mitochondrial protein import: mechanisms and control

Functional Plant Biology ◽

10.1071/pp99064 ◽

1999 ◽

Vol 26 (8) ◽

pp. 725 ◽

Cited By ~ 1

Author(s):

James Whelan

Keyword(s):

Protein Import ◽

Current Knowledge ◽

Mitochondrial Protein ◽

Mitochondrial Proteins ◽

Mitochondrial Protein Import ◽

Future Directions ◽

Receptor Structure ◽

Processing Peptidase ◽

Targeting Signals ◽

And Control

The characterisation of components of the plant mitochondrial import apparatus along with the availability of over one hundred nuclear-encoded mitochondrial proteins allows the study of plant mitochondrial protein import in homologous systems. From these studies it has emerged that although similarities in the import process exist with other organisms, significance differences exist, such as receptor structure, location of processing peptidase and targeting signals. These differences mean that previous studies carried out in heterologous systems must be re-evaluated. Further studies into protein import in plants need to be directed at understanding the mechanism of import and how this process may be controlled. In this review the latter points will be dealt with in terms of summarising our current knowledge and possible future directions.

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Mitochondrial protein import: An unexpected disulfide bond

The Journal of Cell Biology ◽

10.1083/jcb.201607117 ◽

2016 ◽

Vol 214 (4) ◽

pp. 363-365 ◽

Cited By ~ 4

Author(s):

Dejana Mokranjac

Keyword(s):

Disulfide Bond ◽

Protein Import ◽

Protein Translocation ◽

Mitochondrial Protein ◽

Channel Gating ◽

Mitochondrial Proteins ◽

Mitochondrial Protein Import ◽

Cell Biol ◽

Molecular Nature

Most mitochondrial proteins are imported through the TIM23 translocation channel, the structure and molecular nature of which are still unclear. In this issue, Ramesh et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201602074) show that the TIM23 subunit Tim17 contains a disulfide bond that is crucial for protein translocation and channel gating.

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The intermembrane space protein Mix23 is a novel stress-induced mitochondrial import factor

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014247 ◽

2020 ◽

Vol 295 (43) ◽

pp. 14686-14697 ◽

Cited By ~ 2

Author(s):

Eva Zöller ◽

Janina Laborenz ◽

Lena Krämer ◽

Felix Boos ◽

Markus Räschle ◽

...

Keyword(s):

Protein Import ◽

Mitochondrial Protein ◽

Mitochondrial Proteins ◽

Temperature Sensitive ◽

Intermembrane Space ◽

Mitochondrial Import ◽

Induced Stress ◽

Evolutionarily Conserved ◽

Precursor Proteins ◽

Biogenesis Of Mitochondria

The biogenesis of mitochondria requires the import of hundreds of precursor proteins. These proteins are transported post-translationally with the help of chaperones, meaning that the overproduction of mitochondrial proteins or the limited availability of chaperones can lead to the accumulation of cytosolic precursor proteins. This imposes a severe challenge to cytosolic proteostasis and triggers a specific transcription program called the mitoprotein-induced stress response, which activates the proteasome system. This coincides with the repression of mitochondrial proteins, including many proteins of the intermembrane space. In contrast, herein we report that the so-far-uncharacterized intermembrane space protein Mix23 is considerably up-regulated when mitochondrial import is perturbed. Mix23 is evolutionarily conserved and a homolog of the human protein CCDC58. We found that, like the subunits of the proteasome, Mix23 is under control of the transcription factor Rpn4. It is imported into mitochondria by the mitochondrial disulfide relay. Mix23 is critical for the efficient import of proteins into the mitochondrial matrix, particularly if the function of the translocase of the inner membrane 23 is compromised such as in temperature-sensitive mutants of Tim17. Our observations identify Mix23 as a novel regulator or stabilizer of the mitochondrial protein import machinery that is specifically up-regulated upon mitoprotein-induced stress conditions.

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From cytosol to mitochondria: the beginning of a protein journey

Biological Chemistry ◽

10.1515/hsz-2020-0110 ◽

2020 ◽

Vol 401 (6-7) ◽

pp. 645-661 ◽

Cited By ~ 5

Author(s):

Maria Clara Avendaño-Monsalve ◽

José Carlos Ponce-Rojas ◽

Soledad Funes

Keyword(s):

Mitochondrial Biogenesis ◽

Stress Responses ◽

Protein Import ◽

Protein Targeting ◽

Mitochondrial Protein ◽

Past Research ◽

Membrane Receptors ◽

Mitochondrial Proteins ◽

Mitochondrial Protein Import ◽

The Past

AbstractMitochondrial protein import is one of the key processes during mitochondrial biogenesis that involves a series of events necessary for recognition and delivery of nucleus-encoded/cytosol-synthesized mitochondrial proteins into the organelle. The past research efforts have mainly unraveled how membrane translocases ensure the correct protein sorting within the different mitochondrial subcompartments. However, early steps of recognition and delivery remain relatively uncharacterized. In this review, we discuss our current understanding about the signals on mitochondrial proteins, as well as in the mRNAs encoding them, which with the help of cytosolic chaperones and membrane receptors support protein targeting to the organelle in order to avoid improper localization. In addition, we discuss recent findings that illustrate how mistargeting of mitochondrial proteins triggers stress responses, aiming to restore cellular homeostasis.

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Identification of Paramecium mitochondrial proteins using antibodies raised against fused mitochondrial gene products

Gene ◽

10.1016/0378-1119(86)90392-6 ◽

1986 ◽

Vol 49 (1) ◽

pp. 129-138 ◽

Cited By ~ 6

Author(s):

Ravi Mahalingam ◽

Jeffrey J. Seilhamer ◽

Arthur E. Pritchard ◽

Donald J. Cummings

Keyword(s):

Mitochondrial Gene ◽

Mitochondrial Proteins ◽

Gene Products

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Translational inhibitors as potential therapeutic tool of human neuroblastoma through mitochondrial gene expression

European Psychiatry ◽

10.1016/j.eurpsy.2017.01.517 ◽

2017 ◽

Vol 41 (S1) ◽

pp. S464-S464

Author(s):

S. Hina

Keyword(s):

Gene Expression ◽

Oxidative Phosphorylation ◽

Neuroendocrine Tumour ◽

Mitochondrial Gene ◽

Mitochondrial Protein ◽

Genetic Material ◽

Neuronal Cell ◽

Mitochondrial Proteins ◽

Mitochondrial Gene Expression ◽

Human Neuroblastoma

Neuroblastoma is a solid neuroendocrine tumour and most common type of cancer of infancy. It is a complex heterogeneous disease and many factors such as molecular, cellular and genetic features are involved in its development. Mitochondria play a pivotal role in neuronal cell survival or death. Neurons are highly reliant on aerobic oxidative phosphorylation (OXPHOS) for their energy needs. Defective activities of mitochondrial complexes I, II, III and IV have been identified in many neurological and neurodegenerative diseases. Human mitochondria with its own genetic material meet the needs required for the assembly of subunits of the oxidative phosphorylation (OXPHOS) complexes. A number of translational inhibitors are known that could potentially effect translation of mitochondrial protein synthesis. Among these puromycin, homoharringtonine and cyclohexamide were selected for the present study. The effect of these translational inhibitors on mitochondrial gene expression for the treatment of neuroblastoma are not well established. Therefore, in this study, we have investigated the effects of these translational inhibitors on the expression of human mitochondrial gene expression in SH-SY5Y neuroblastoma cells.We observed a significant effect on the level of mitochondrial transcripts upon exposure to these translation inhibitors in SH-SY5Y cells, however, the effects on expression of mitochondrial proteins were minimal. This suggests that translational inhibitors might not directly affect the abundance of mitochondrial proteins. Translational inhibitors induce significant effect on mitochondrial gene expression that can be lead to the new-targeted therapy for treating neuroblastoma.

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Mitochondrial diseases caused by dysfunctional mitochondrial protein import

Biochemical Society Transactions ◽

10.1042/bst20180239 ◽

2018 ◽

Vol 46 (5) ◽

pp. 1225-1238 ◽

Cited By ~ 8

Author(s):

Thomas Daniel Jackson ◽

Catherine Sarah Palmer ◽

Diana Stojanovski

Keyword(s):

Mitochondrial Disease ◽

Genetic Disease ◽

Molecular Basis ◽

Protein Import ◽

Mitochondrial Protein ◽

Mitochondrial Diseases ◽

Mitochondrial Proteins ◽

Eukaryotic Cells ◽

Mitochondrial Protein Import ◽

Mitochondrial Proteome

Mitochondria are essential organelles which perform complex and varied functions within eukaryotic cells. Maintenance of mitochondrial health and functionality is thus a key cellular priority and relies on the organelle's extensive proteome. The mitochondrial proteome is largely encoded by nuclear genes, and mitochondrial proteins must be sorted to the correct mitochondrial sub-compartment post-translationally. This essential process is carried out by multimeric and dynamic translocation and sorting machineries, which can be found in all four mitochondrial compartments. Interestingly, advances in the diagnosis of genetic disease have revealed that mutations in various components of the human import machinery can cause mitochondrial disease, a heterogenous and often severe collection of disorders associated with energy generation defects and a multisystem presentation often affecting the cardiovascular and nervous systems. Here, we review our current understanding of mitochondrial protein import systems in human cells and the molecular basis of mitochondrial diseases caused by defects in these pathways.

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