Mitochondrial protein-import machinery: correlating structure with function

Most mitochondrial proteins are synthesized in the cytosol, translocated into the organelle and directed along specific sorting pathways. Over the past 20 years, >30 proteins have been identified as having key roles in mitochondrial protein import. Recently, the elucidation of the structures of several import components has provided fresh insight into the import process. Here, we review the different pathways involved in sorting proteins into mitochondrial subcompartments. Along the way, we highlight the available structural information about the protein-import machinery and discuss how these structures correlate with previously ascribed functions. Future challenges for the cell biologists will be to use this structural information to test specific hypotheses addressing the molecular mechanisms of mitochondrial protein import.     

Mitochondrial Fusion Is Essential for Steroid Biosynthesis

Although the contribution of mitochondrial dynamics (a balance in fusion/fission events and changes in mitochondria subcellular distribution) to key biological process has been reported, the contribution of changes in mitochondrial fusion to achieve efficient steroid production has never been explored. The mitochondria are central during steroid synthesis and different enzymes are localized between the mitochondria and the endoplasmic reticulum to produce the final steroid hormone, thus suggesting that mitochondrial fusion might be relevant for this process. In the present study, we showed that the hormonal stimulation triggers mitochondrial fusion into tubular-shaped structures and we demonstrated that mitochondrial fusion does not only correlate-with but also is an essential step of steroid production, being both events depend on PKA activity. We also demonstrated that the hormone-stimulated relocalization of ERK1/2 in the mitochondrion, a critical step during steroidogenesis, depends on mitochondrial fusion. Additionally, we showed that the SHP2 phosphatase, which is required for full steroidogenesis, simultaneously modulates mitochondrial fusion and ERK1/2 localization in the mitochondrion. Strikingly, we found that mitofusin 2 (Mfn2) expression, a central protein for mitochondrial fusion, is upregulated immediately after hormone stimulation. Moreover, Mfn2 knockdown is sufficient to impair steroid biosynthesis. Together, our findings unveil an essential role for mitochondrial fusion during steroidogenesis. These discoveries highlight the importance of organelles’ reorganization in specialized cells, prompting the exploration of the impact that organelle dynamics has on biological processes that include, but are not limited to, steroid synthesis.     

Mutation in MRPS34 Compromises Protein Synthesis and Causes Mitochondrial Dysfunction

The evolutionary divergence of mitochondrial ribosomes from their bacterial and cytoplasmic ancestors has resulted in reduced RNA content and the acquisition of mitochondria-specific proteins. The mitochondrial ribosomal protein of the small subunit 34 (MRPS34) is a mitochondria-specific ribosomal protein found only in chordates, whose function we investigated in mice carrying a homozygous mutation in the nuclear gene encoding this protein. The Mrps34 mutation causes a significant decrease of this protein, which we show is required for the stability of the 12S rRNA, the small ribosomal subunit and actively translating ribosomes. The synthesis of all 13 mitochondrially-encoded polypeptides is compromised in the mutant mice, resulting in reduced levels of mitochondrial proteins and complexes, which leads to decreased oxygen consumption and respiratory complex activity. The Mrps34 mutation causes tissue-specific molecular changes that result in heterogeneous pathology involving alterations in fractional shortening of the heart and pronounced liver dysfunction that is exacerbated with age. The defects in mitochondrial protein synthesis in the mutant mice are caused by destabilization of the small ribosomal subunit that affects the stability of the mitochondrial ribosome with age.     

Exposure to arginine analog canavanine induces aberrant mitochondrial translation products,mitoribosome stalling,and instability of the mitochondrial proteome

Impairment of mitochondrial protein homeostasis disrupts mitochondrial function and causes human diseases and aging, but the molecular mechanisms of protein synthesis and quality control in mammalian mitochondria are not fully understood. Here we demonstrate in human cells that misincorporation of an arginine analog, canavanine, during mitochondrial protein synthesis, induced aberrant translation products and destabilized the mtDNA-encoded proteome, leading to loss of mitochondrial respiratory chain complexes. Furthermore, in the presence of a high concentration of canavanine, mitoribosome stalling could be demonstrated. The stalling did not, however, occur at arginine codons, but downstream of those codons. In particular, two adjacent arginines induced the most prominent downstream stalling effect, with the distance between the arginine codons and the stalling peak corresponding roughly to the length of the ribosomal exit tunnel. These results suggest that misincorporated canavanine disrupted the proper folding of the hydrophobic nascent polypeptides within the exit tunnel or while being inserted into the inner mitochondrial membrane. The canavanine treatment provides a model system for studying the consequences of mitoribosome stalling and the responses to misfolded proteins exiting the mitochondrial ribosome.     

Studies on rhodanese synthesis in bovine adrenocortical cells

The synthesis of adrenodoxin, a mitochondrial iron-sulfur protein required for adrenocortical steroidogenesis, is known to be regulated chronically by ACTH. Rhodanese, also a mitochondrial enzyme, is thought to be required for synthesis of iron-sulfur centers, such as those contained in adrenodoxin. In this study it has been found that rhodanese synthesis and activity are not regulated by ACTH, under the same conditions whereby ACTH induces adrenodoxin synthesis. In addition, unlike adrenodoxin, rhodanese is found to be synthesized in the mature form rather than as a higher molecular weight precursor protein.     

Proteomic analysis of the mouse liver mitochondrial inner membrane

Mitochondria play a crucial role in cellular homeostasis, which justifies the increasing interest in mapping the different components of these organelles. Here we have focused our study on the identification of proteins of the mitochondrial inner membrane (MIM). This membrane is of particular interest because, besides the well known components of the respiratory chain complexes, it contains several ion channels and many carrier proteins that certainly play a key role in mitochondrial function and, therefore, deserve to be identified at the molecular level. To achieve this goal we have used a novel approach combining the use of highly purified mouse liver mitochondrial inner membranes, extraction of membrane proteins with organic acid, and two-dimensional liquid chromatography coupled to tandem mass spectrometry. This procedure allowed us to identify 182 proteins that are involved in several biochemical processes, such as the electron transport machinery, the protein import machinery, protein synthesis, lipid metabolism, and ion or substrate transport. The full range of isoelectric point (3.9-12.5), molecular mass (6-527 kDa), and hydrophobicity values (up to 16 transmembrane predicted domains) were represented. In addition, of the 182 proteins found, 20 were unknown or had never previously been associated with the MIM. Overexpression of some of these proteins in mammalian cells confirmed their mitochondrial localization and resulted in severe remodeling of the mitochondrial network. This study provides the first proteome of the MIM and provides a basis for a more detailed study of the newly characterized proteins of this membrane.     

Proteomic profiling of aging in the mouse heart: Altered expression of mitochondrial proteins

Using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, we have used a systems biology approach to study the molecular basis of aging of the mouse heart. We have identified 8 protein spots whose expression is up-regulated due to aging and 36 protein spots whose expression is down-regulated due to aging (p0.05 as judged by Wilcoxon Rank Sum test). Among the up-regulated proteins, we have characterized 5 protein spots and 2 of them, containing 3 different enzymes, are mitochondrial proteins. Among the down-regulated proteins, we have characterized 27 protein spots and 16 of them are mitochondrial proteins. Mitochondrial damage is believed to be a key factor in the aging process. Our current study provides molecular evidence at the level of the proteome for the alteration of structural and functional parameters of the mitochondria that contribute to impaired activity of the mouse heart due to aging.     

The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria

AAA proteases comprise a conserved family of membrane bound ATP-dependent proteases that ensures the quality control of mitochondrial inner-membrane proteins. Inactivation of AAA proteases causes pleiotropic phenotypes in various organisms, including respiratory deficiencies, mitochondrial morphology defects, and axonal degeneration in hereditary spastic paraplegia (HSP). The molecular basis of these defects, however, remained unclear. Here, we describe a regulatory role of an AAA protease for mitochondrial protein synthesis in yeast. The mitochondrial ribosomal protein MrpL32 is processed by the m-AAA protease, allowing its association with preassembled ribosomal particles and completion of ribosome assembly in close proximity to the inner membrane. Maturation of MrpL32 and mitochondrial protein synthesis are also impaired in a HSP mouse model lacking the m-AAA protease subunit paraplegin, demonstrating functional conservation. Our findings therefore rationalize mitochondrial defects associated with m-AAA protease mutants in yeast and shed new light on the mechanism of axonal degeneration in HSP.     

Effect of chloramphenicol,antimycin A and hydroxamate on the morphogenetic development of the dimorphic ascomyceteEndomycopsis capsularis

Mitochondrial protein synthesis, primary (antimycin-sensitive) respiration and secondary (antimycin-insensitive, salicyl-hydroxamate-sensitive) respiration, have been characterized in the dimorphic yeastEndomycopsis capsularis. The inhibition by chloramphenicol (CAP) of the morphogenetic development from the yeast-like form to the mycelial structure in this yeast could represent the intervention in the morphogenetic process of mitochondrial protein synthesis, since chloramphenicol blocks in vivo and in vitro mitochondrial protein synthesis. In fact, other functions such as primary and secondary respiration, do not seem to play a role in the morphogenetic development since their inhibition by antimycin A (AA) or by salicyl-hydroxamic acid (SHAM) does not affect the process. In addition, mitochondrial protein synthesis has been shown to be uninhibited by the two respiratory inhibitors.     

Rim2, a pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria

Mitochondria transport and utilize iron for the synthesis of haem and Fe-S clusters. Although many proteins are known to be involved in these processes, additional proteins are likely to participate. To test this hypothesis, in the present study we used a genetic screen looking for yeast mutants that are synthetically lethal with the mitochondrial iron carriers Mrs3 and Mrs4. Several genes were identified, including an isolate mutated for Yfh1, the yeast frataxin homologue. All such triple mutants were complemented by increased expression of Rim2, another mitochondrial carrier protein. Rim2 overexpression was able to enhance haem and Fe-S cluster synthesis in wild-type or Δmrs3/Δmrs4 backgrounds. Conversely Rim2 depletion impaired haem and Fe-S cluster synthesis in wild-type or Δmrs3/Δmrs4 backgrounds, indicating a unique requirement for this mitochondrial transporter for these processes. Rim2 was previously shown to mediate pyrimidine exchange in and out of vesicles. In the present study we found that isolated mitochondria lacking Rim2 exhibited concordant iron defects and pyrimidine transport defects, although the connection between these two functions is not explained. When organellar membranes were ruptured to bypass iron transport, haem synthesis from added iron and porphyrin was still markedly deficient in Rim2-depleted mitochondrial lysate. The results indicate that Rim2 is a pyrimidine exchanger with an additional unique function in promoting mitochondrial iron utilization.     

Synthesis of the mitochondrial inner membrane in cultured Xenopus laevis oocytes.

The purpose of this study was to investigate the contribution of mitochondrial and cytoplasmic protein synthesis to the biogenesis of cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase EC 1.9.3.1) and rutamycin-sensitive adenosine triphosphatase (ATP phosphohydrolase EC 3.6.1.3) in cultured oocytes of the toad, Xenopus laevis. X. laevis cytochrome oxidase was purified over 23-fold with respect to specific activity and over 29-fold with respect to specific heme a content from oocyte submitochondrial particles. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate separated the enzyme into six subunits with molecular weights of 44,000, 33,000, 23,000, 17,000, 12,000 and 9,500. the synthesis of the three larger subunits is sensitive to chloramphenicol (an inhibitor of mitochondrial protein synthesis), indicating that these subunits are made on mitochondrial ribosomes; the synthesis of the three smaller subunits is sensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and therefore occurs on cytoplasmic ribosomes. X. laevis rutamycin-sensitive ATPase, purified over 19-fold from oocyte submitochondrial pparticles, consists of 10 subunits with molecular weights of 56,000, 53,000, 41,000, 32,000, 29,000, 24,000, 21,000, 17,500 (2), and 11,500 on sodium dodecyl sulfate-polyacrylamide gels. The 29,000, 21,000, and one of the 17,500-dalton polypeptides are synthesized in the presence of cycloheximide and are, therefore, products of mitochondrial protein synthesis; the synthesis of the remaining seven subunits occurs in the presence of chloramphenicol, indicating that these subunits are made on cytoplasmic ribosomes. The synthesis of protein by mitochondria in cultured oocytes appears to be dependent upon cytoplasmic protein synthesis. In the presence of cycloheximide, the mitoribosomal synthesis of the subunits of cytochrome oxidase and rutamycin-sensitive ATPase is detectable only after a prior inhibition of mitochondrial protein synthesis by chloramphenicol. Oocyte mitochondrial ribosomes synthesize at least nine polypeptides after chloramphenicol treatment, three of which are components of neither cytochrome oxidase nor rutamycin-sensitive ATPase.     

P43-dependent mitochondrial activity regulates myoblast differentiation and slow myosin isoform expression by control of Calcineurin expression

We have previously shown that mitochondrial protein synthesis regulates myoblast differentiation, partly through the control of c-Myc expression, a cellular oncogene regulating myogenin expression and myoblast withdrawal from the cell cycle. In this study we provide evidence of the involvement of Calcineurin in this regulation. In C2C12 myoblasts, inhibition of mitochondrial protein synthesis by chloramphenicol decreases Calcineurin expression. Conversely, stimulation of this process by overexpressing the T3 mitochondrial receptor (p43) increases Calcineurin expression. Moreover, expression of a constitutively active Calcineurin (ΔCN) stimulates myoblast differentiation, whereas a Calcineurin antisense has the opposite effect. Lastly, ΔCN expression or stimulation of mitochondrial protein synthesis specifically increases slow myosin heavy chain expression. In conclusion, these data clearly suggest that, partly via Calcineurin expression, mitochondrial protein synthesis is involved in muscle development through the control of myoblast differentiation and probably the acquisition of the contractile and metabolic phenotype of muscle fibres.     

MSW, a yeast gene coding for mitochondrial tryptophanyl-tRNA synthetase

E569 and E606 are noncomplementing pet mutants of Saccharomyces cerevisiae. Both strains are defective in mitochondrial protein synthesis and as a result exhibit a pleiotropic deficiency in respiratory components that are translated on mitochondrial ribosomes. The wild type gene MSW capable of complementing the protein synthesis defect has been cloned by transformation of one of the mutants with a genomic library of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein with a molecular weight of 42,414 which is 37 and 39% identical to the tryptophanyl-tRNA synthetases of Escherichia coli and Bacillus stearothermophilus, respectively. A strain containing an insertion in the chromosomal copy of MSW was constructed by in situ gene replacement. This mutant fails to charge mitochondrial tryptophanyl-tRNA providing further evidence that MSW is the structural gene for mitochondrial tryptophanyl tRNA synthetase. The existence of another gene coding for the cytoplasmic tryptophanyl-tRNA synthetase is inferred from the observation that mutations in MSW are not lethal but only result in a respiratory deficiency.     

Mitochondrial free radical generation, oxidative stress, and aging

Mitochondria have been described as "the powerhouses of the cell" because they link the energy-releasing activities of electron transport and proton pumping with the energy conserving process of oxidative phosphorylation, to harness the value of foods in the form of ATP. Such energetic processes are not without dangers, however, and the electron transport chain has proved to be somewhat "leaky." Such side reactions of the mitochondrial electron transport chain with molecular oxygen directly generate the superoxide anion radical (O2*-), which dismutates to form hydrogen peroxide (H2O2), which can further react to form the hydroxyl radical (HO*). In addition to these toxic electron transport chain reactions of the inner mitochondrial membrane, the mitochondrial outer membrane enzyme monoamine oxidase catalyzes the oxidative deamination of biogenic amines and is a quantitatively large source of H2O2 that contributes to an increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol. In this article we review the mitochondrial rates of production and steady state levels of these reactive oxygen species. Reactive oxygen species generated by mitochondria, or from other sites within or outside the cell, cause damage to mitochondrial components and initiate degradative processes. Such toxic reactions contribute significantly to the aging process and form the central dogma of "The Free Radical Theory of Aging." In this article we review current understandings of mitochondrial DNA, RNA, and protein modifications by oxidative stress and the enzymatic removal of oxidatively damaged products by nucleases and proteases. The possible contributions of mitochondrial oxidative polynucleotide and protein turnover to apoptosis and aging are explored.     

Dominant optic atrophy,OPA1, and mitochondrial quality control: understanding mitochondrial network dynamics

Mitochondrial quality control is fundamental to all neurodegenerative diseases, including the most prominent ones, Alzheimer’s Disease and Parkinsonism. It is accomplished by mitochondrial network dynamics – continuous fission and fusion of mitochondria. Mitochondrial fission is facilitated by DRP1, while MFN1 and MFN2 on the mitochondrial outer membrane and OPA1 on the mitochondrial inner membrane are essential for mitochondrial fusion. Mitochondrial network dynamics are regulated in highly sophisticated ways by various different posttranslational modifications, such as phosphorylation, ubiquitination, and proteolytic processing of their key-proteins. By this, mitochondria process a wide range of different intracellular and extracellular parameters in order to adapt mitochondrial function to actual energetic and metabolic demands of the host cell, attenuate mitochondrial damage, recycle dysfunctional mitochondria via the mitochondrial autophagy pathway, or arrange for the recycling of the complete host cell by apoptosis. Most of the genes coding for proteins involved in this process have been associated with neurodegenerative diseases. Mutations in one of these genes are associated with a neurodegenerative disease that originally was described to affect retinal ganglion cells only. Since more and more evidence shows that other cell types are affected as well, we would like to discuss the pathology of dominant optic atrophy, which is caused by heterozygous sequence variants in OPA1, in the light of the current view on OPA1 protein function in mitochondrial quality control, in particular on its function in mitochondrial fusion and cytochrome C release. We think OPA1 is a good example to understand the molecular basis for mitochondrial network dynamics.     

The yeast GTPase Mtg2p is required for mitochondrial translation and partially suppresses an rRNA methyltransferase mutant, mrm2

The assembly of ribosomes involves the coordinated processing and modification of rRNAs with the temporal association of ribosomal proteins. This process is regulated by assembly factors such as helicases, modifying enzymes, and GTPases. In contrast to the assembly of cytoplasmic ribosomes, there is a paucity of information concerning the role of assembly proteins in the biogenesis of mitochondrial ribosomes. In this study, we demonstrate that the Saccharomyces cerevisiae GTPase Mtg2p (Yhr168wp) is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to petite cells. In addition, cells expressing a temperature-sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lowered levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. We propose that Mtg2p is involved in mitochondrial ribosome biogenesis. Consistent with this role, we show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner.     

Optimization of in vitro protein synthesis by isolated mouse adrenal mitochondria

The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)