Comparative proteomics as a new tool for exploring human mitochondrial tRNA disorders.

More than 70 different point mutations in human mitochondrial tRNA genes are correlated with severe disorders, including fatal cardiopathies, encephalopathies, myopathies, and others. So far, investigation of the molecular impact(s) of mutations has focused on the affected tRNA itself by seeking structural and/or functional perturbations capable of interfering with synthesis of the 13 mitochondrion-encoded subunits of respiratory chain complexes. Here, a proteomic approach was used to investigate whether such mutations would affect the pattern of mitochondrial proteins at a broader level. Analysis of several hundred mitochondrial proteins from sibling cybrid cell lines by two-dimensional electrophoresis, an approach that takes into account all regulatory steps of mitochondrial and nuclear gene expression, indeed reveals a number of up- and downregulated proteins when healthy and single-point-mutation-carrying mitochondria representative of either MELAS or MERRF syndrome were compared. Assignment by mass spectrometry of the two proteins which exhibit obvious large quantitative decreases in the levels of both pathologic mitochondria identified nuclear-encoded subunits of cytochrome c oxidase, a respiratory chain complex. This clearly shows a linkage between the effects of mutations in mitochondrial tRNA genes and the steady-state level of nuclear-encoded proteins in mitochondria. It opens new routes toward a large-scale exploration of potential proteic partners involved in the genotype-phenotype correlation of mitochondrial disorders.     

A survey of the plant mitochondrial proteome in relation to development

To expand the functional analysis of plant mitochondria, we have undertaken the building of the proteome of pea mitochondria purified from leaves (green and etiolated), roots and seeds. In the first stage, we focused our proteomic exploration on the soluble protein complement of the green leaf mitochondria. We used traditional two-dimensional polyacrylamide gel electrophoresis, in combination with size exclusion chromatography as a third dimension, to identify the major proteins and further resolve their macromolecular complexity. The two-dimensional map of soluble proteins of green leaf mitochondria revealed 433 spots (with Coomassie blue staining) and around 73% of the proteins (in mass) were identified using three different approaches: Edman degradation, matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry. Quite a lot of the polypeptides were present in multiforms which indicated the presence of isoforms or the occurrence of post-translational modifications. Among these proteins, we uncovered an abundant family that was identified as aldehyde dehydrogenases, representing approximately 7.5% of the soluble proteins. The comparative analysis of soluble mitochondrial proteomes led to the identification of a number of proteins which were specifically present in root or in seed mitochondria, thus revealing the impact of tissue differentiation at the mitochondrial level.     

Diseases caused by mutations in mitochondrial DNA

Mitochondrial diseases associated with mutations within mitochondrial genome are a subgroup of metabolic disorders since their common consequence is reduced metabolic efficiency caused by impaired oxidative phophorylation and shortage of ATP. Although the vast majority of mitochondrial proteins (approximately 1500) is encoded by nuclear genome, mtDNA encodes 11 subunits of respiratory chain complexes, 2 subunits of ATP synthase, 22 tRNAs and 2 rRNAs. Up to now, more than 250 pathogenic mutations have been described within mtDNA. The most common are point mutations in genes encoding mitochondrial tRNAs such as 3243A-->G and 8344T-->G that cause, respectively, MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) or MIDD (maternally-inherited diabetes and deafness) and MERRF (myoclonic epilepsy with ragged red fibres) syndromes. There have been also found mutations in genes encoding subunits of ATP synthase such as 8993T-->G substitution associated with NARP (neuropathy, ataxia and retinitis pigmentosa) syndrome. It is worth to note that mitochondrial dysfunction can also be caused by mutations within nuclear genes coding for mitochondrial proteins.     

Treatment of human cells derived from MERRF syndrome by peptide-mediated mitochondrial delivery

Background aimsThe feasibility of delivering mitochondria using the cell-penetrating peptide Pep-1 for the treatment of MERRF (myoclonic epilepsy with ragged red fibers) syndrome, which is caused by point mutations in the transfer RNA genes of mitochondrial DNA, is examined further using cellular models derived from patients with MERRF syndrome.MethodsHomogenesis of mitochondria (wild-type mitochondria) isolated from normal donor cells with about 83.5% preserved activity were delivered into MERRF fibroblasts by Pep-1 conjugation (Pep-1-Mito).ResultsDelivered doses of 52.5 μg and 105 μg Pep-1-Mito had better delivered efficiency and mitochondrial biogenesis after 15 days of treatment. The recovery of mitochondrial function in deficient cells receiving 3 days of treatment with peptide-mediated mitochondrial delivery was comprehensively demonstrated by restoration of oxidative phosphorylation subunits (complex I, III and IV), mitochondrial membrane potential, adenosine triphosphate synthesis and reduction of reactive oxygen species production. The benefits of enhanced mitochondrial regulation depended on the function of foreign mitochondria and not the existence of mitochondrial DNA and can be maintained for at least 21 days with dramatically elongated mitochondrial morphology. In contrast to delivery of wild-type mitochondria, the specific regulation of Pep-1-Mito during MERRF syndrome progression in cells treated with mutant mitochondria was reflected by the opposite performance, with increase in reactive oxygen species production and matrix metalloproteinase activity.ConclusionsThe present study further illustrates the feasibility of mitochondrial intervention therapy using the novel approach of peptide-mediated mitochondrial delivery and the benefit resulting from mitochondria-organelle manipulation.     

Functional consequences of mitochondrial proteome heterogeneity

Potential functional consequences of the differences in protein distribution between the mitochondria of the rat liver, heart, brain, and kidney, as determined in the companion paper in this issue (Johnson DT, French S, Blair PV, You JS, Bemis KG, Wang M, Harris RA, and Balaban RS. The tissue heterogeneity of the mammalian mitochondrial proteome. Am J Physiol Cell Physiol292: C689–C697, 2006), were analyzed using a canonical metabolic pathway approach as well as a functional domain homology analysis. These data were inserted into the Kyoto Encyclopedia of Genes and Genomes pathway framework to give global and metabolic pathway-specific information on the impact of the differential protein distribution on mitochondrial function. Custom pathway analysis was also performed using pathways limited to the mitochondrion. With the use of this approach, several well-known functional differences between these mitochondrial populations were confirmed. These included GABA metabolism in the brain, urea synthesis in the liver, and the domination of oxidative phosphorylation in the heart. By comparing relative protein amounts of mitochondria across tissues, a greater understanding of functional emphasis is possible as well as the nuclear "programming" required to enhance a given function within the mitochondria. For proteins determined to be mitochondrial and lacking a defined role functional domain BLAST analyses were performed. Several proteins associated with DNA structural modification and a novel CoA transferase were identified. A protein was also identified capable of catalyzing the first three steps of de novo pyrimidine synthesis. This analysis demonstrates that the distribution of nuclear encoded proteins significantly modifies the overall functional emphasis of the mitochondria to meet tissue-specific needs. These studies demonstrate the existence of mitochondrial biochemical functions that at present are poorly defined. oxidative phosphorylation; liquid chromatography; mass spectrometry; electrophoresis; histone; liver; heart; kidney; brain     

Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis

The mitochondrial respiratory chain consists of multi-subunit protein complexes embedded in the inner membrane. Although the majority of subunits are encoded by nuclear genes and are imported into mitochondria, 13 subunits in humans are encoded by mitochondrial DNA. The coordinated assembly of subunits encoded from two genomes is a poorly understood process, with assembly pathway defects being a major determinant in mitochondrial disease. In this study, we monitored the assembly of human respiratory complexes using radiolabeled, mitochondrially encoded subunits in conjunction with Blue Native polyacrylamide gel electrophoresis. The efficiency of assembly was found to differ markedly between complexes, and intermediate complexes containing newly synthesized mitochondrial DNA-encoded subunits could be observed for complexes I, III, and IV. In particular, we detected human cytochrome b as a monomer and as a component of a novel approximately 120 kDa intermediate complex at early chase times before being totally assembled into mature complex III. Furthermore, we show that this approach is highly suited for the rapid detection of respiratory complex assembly defects in fibroblasts from patients with mitochondrial disease and, thus, has potential diagnostic applications.     

Shotgun proteomics for the characterization of subunit composition of mitochondrial complex I

Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.     

Partial purification of mitochondrial ribosomes from broad bean and identification of proteins encoded by the mitochondrial genome

An approach towards the identification at the protein level of the ribosomal proteins encoded by the mitochondrial genome of broad bean (Vicia faba) has been developed. After Triton X-100 treatment of isolated mitochondria, a fraction enriched in mitochondrial ribosomes was obtained by successive centrifugation, first onto a sucrose cushion, and then in a sucrose gradient. Mitochondrial translation products were labelled in isolated mitochondria with [³⁵S]methionine and added to the enriched mitochondrial ribosomal proteins before separation by two-dimensional gel electrophoresis. Six spots, identified both by Coomassie blue staining and autoradiography, were analysed by protein micro-sequencing. Two of these were shown to correspond to ribosomal proteins S10 and S12. We conclude that these two proteins are encoded by the mitochondrial genome of broad bean and that the method described here can be used to identify other proteins encoded by the mitochondrial genome. Received: 4 September 1996 / Accepted: 30 November 1996     

Mitochondrial malate dehydrogenases in Brassica napus: altered protein patterns in different nuclear mitochondrial combinations

Two-dimensional analyses of mitochondrial proteins of Brassica napus revealed a set of differences in patterns of mitochondrial matrix proteins isolated from different nuclear backgrounds. One of these varying proteins was identified as mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37) by homology analyses of the partial amino acid sequence. Immunological detection identified additional mMDH subunits and detected different patterns of mMDH subunits in two distinct mitochondria types although they were isolated from plants with the same nuclear genotype. These differences are also reflected in isozym patterns, whereas Southern analyses showed no alteration in genome structure. Therefore mitochondria type-specific mMDH modifications are possible.     

Modifications of the lipoamide-containing mitochondrial subproteome in a yeast mutant defective in cysteine desulfurase

Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulfurase-defective (nfs1-14, carrying a hypomorphic allele of NFS1) yeast strains, using two-dimensional gel electrophoresis coupled to mass spectrometry analyses, revealed large changes in the amounts of various proteins. Protein spots that were specifically increased in the nfs1-14 mutant included subunits of lipoamide-containing enzyme complexes: Kgd2, Lat1, and Gcv3, subunits of the mitochondrial alpha-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and glycine cleavage system complexes, respectively. Moreover the increased protein spots corresponded to lipoamide-deficient forms in the nfs1-14 mutant. The increased proteins migrated as separate, cathode-shifted spots, consistent with gain of a lysine charge due to lack of lipoamide addition. Lack of lipoylation of these proteins was further validated using an antibody specific for lipoamide-containing proteins. In addition, this antibody revealed a fourth lipoamide-containing protein, probably corresponding to the E2 component of the branched-chain keto acid dehydrogenase complex. Like the lipoamide-containing forms of Kgd2, Lat1, and Gcv3, this protein also showed decreased lipoic acid reactivity in the nfs1-14 mutant. Cysteine desulfurases, such as yeast NFS1, are required for sulfur addition to iron-sulfur clusters and other sulfur-requiring processes. The results demonstrate that Nfs1 protein is required for the proper post-translational modification of the lipoamide-containing mitochondrial subproteome in yeast and pave the road toward a thorough understanding of its precise role in lipoic acid synthesis.     

The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii

Two genes encoding cytochrome c oxidase subunits, Cox2a and Cox2b, are present in the nuclear genomes of apicomplexan parasites and show sequence similarity to corresponding genes in chlorophycean algae. We explored the presence of COX2A and COX2B subunits in the cytochrome c oxidase of Toxoplasma gondii. Antibodies were raised against a synthetic peptide containing a 14-residue fragment of the COX2A polypeptide and against a hexa-histidine-tagged recombinant COX2B protein. Two distinct immunochemical stainings localized the COX2A and COX2B proteins in the parasite's mitochondria. A mitochondria-enriched fraction exhibited cyanide-sensitive oxygen uptake in the presence of succinate. T. gondii mitochondria were solubilized and subjected to Blue Native Electrophoresis followed by second dimension electrophoresis. Selected protein spots from the 2D gels were subjected to mass spectrometry analysis and polypeptides of mitochondrial complexes III, IV and V were identified. Subunits COX2A and COX2B were detected immunochemically and found to co-migrate with complex IV; therefore, they are subunits of the parasite's cytochrome c oxidase. The apparent molecular mass of the T. gondii mature COX2A subunit differs from that of the chlorophycean alga Polytomella sp. The data suggest that during its biogenesis, the mitochondrial targeting sequence of the apicomplexan COX2A precursor protein may be processed differently than the one from its algal counterpart.     

Specific changes in total and mitochondrial proteomes are associated with higher levels of heterosis in maize hybrids

The phenomenon of hybrid vigor (heterosis) has long been harnessed by plant breeders to improve world food production. However, the changes that are essential for heterotic responses and the mechanisms responsible for heterosis remain undefined. Large increases in biomass and yield in high-heterosis hybrids suggest that alterations in bioenergetic processes may contribute to heterosis. Progeny from crosses between various inbred lines vary in the extent of vigor observed. Field-grown maize F(1) hybrids that consistently exhibited either low or high heterosis across a variety of environments were examined for changes in proteins that may be correlated with increased plant vigor and yield. Unpollinated ears at the time of flowering (ear shoots) were selected for the studies because they are metabolically active, rich in mitochondria, and the sizes of the ears are diagnostic of yield heterosis. Total protein and mitochondrial proteomes were compared among low- and higher-heterosis hybrids. Two-dimensional difference gel electrophoresis was used to identify allelic and/or isoform differences linked to heterosis. Identification of differentially regulated spots by mass spectrometry revealed proteins involved in stress responses as well as primary carbon and protein metabolism. Many of these proteins were identified in multiple spots, but analysis of their abundances by label-free mass spectrometry suggested that most of the expression differences were due to isoform variation rather than overall protein amount. Thus, our proteomics studies suggest that expression of specific alleles and/or post-translational modification of specific proteins correlate with higher levels of heterosis.     

Mysteries of mitochondrial protein synthesis

Mitochondria possess an endogenous system of translation, in which all constituent components are unique. An electrophoretic analysis of mitochondrial translation products revealed that the content of polypeptides in mitochondria is two times as high as that of mitochondrial DNA genes. The electrophoretically determined molecular mass of proteins synthesized in mitochondria is much less than that calculated from gene sequencing data. The average amino acid composition of the proteins synthesized in mitochondria differs significantly from that encoded by the nucleotide sequence of corresponding mitochondrial genes. These enigmas of mitochondrial protein synthesis await further solution.     

Conservative and compensatory evolution in oxidative phosphorylation complexes of angiosperms with highly divergent rates of mitochondrial genome evolution

Interactions between nuclear and mitochondrial gene products are critical for eukaryotic cell function. Nuclear genes encoding mitochondrial‐targeted proteins (N‐mt genes) experience elevated rates of evolution, which has often been interpreted as evidence of nuclear compensation in response to elevated mitochondrial mutation rates. However, N‐mt genes may be under relaxed functional constraints, which could also explain observed increases in their evolutionary rate. To disentangle these hypotheses, we examined patterns of sequence and structural evolution in nuclear‐ and mitochondrial‐encoded oxidative phosphorylation proteins from species in the angiosperm genus Silene with vastly different mitochondrial mutation rates. We found correlated increases in N‐mt gene evolution in species with fast‐evolving mitochondrial DNA. Structural modeling revealed an overrepresentation of N‐mt substitutions at positions that directly contact mutated residues in mitochondrial‐encoded proteins, despite overall patterns of conservative structural evolution. These findings support the hypothesis that selection for compensatory changes in response to mitochondrial mutations contributes to the elevated rate of evolution in N‐mt genes. We discuss these results in light of theories implicating mitochondrial mutation rates and mitonuclear coevolution as drivers of speciation and suggest comparative and experimental approaches that could take advantage of heterogeneity in rates of mtDNA evolution across eukaryotes to evaluate such theories.     

Deletion of mtDNA disrupts mitochondrial function and structure, but not biogenesis

The role of nuclear DNA (nDNA)-encoded proteins in the regulation of mitochondrial fission and fusion has been documented, yet the role of mitochondrial DNA (mtDNA) and encoded proteins in mitochondrial biogenesis remains unknown. Long-term treatment of a lymphoblastoid cell line Molt-4 with ethidium bromide generated mtDNA-deficient rho0 mutants. Depletion of mtDNA in rho0 cells produced functional and morphological changes in mitochondria without affecting the nuclear genome and encoded proteins. Indeed, the gene encoding subunit II of mitochondrial cytochrome c oxidase (COX II), a prototypical mitochondrial gene, was reduced in rho0 mutants blunting the activity of mitochondrial cytochrome coxidase. Yet, the amount of the nuclear beta-actin gene and the activity of citrate synthase, a mitochondrial matrix enzyme encoded by nDNA, remained unaffected in rho0 cells. Loss of mtDNA in rho0 cells was associated with significant distortion of mitochondrial structure, decreased electron density of the matrix and disorganized inner and outer membranes, resulting in the appearance of 'ghost-like' mitochondria. However, the number of mitochondria-like structures was not significantly different between mtDNA-deficient and parental cells. Thus, we conclude that cells lacking mtDNA still generate mitochondrial scaffolds, albeit with aberrant function.     

Characterization of the Drosophila melanogaster mitochondrial proteome

We have combined high-resolution two-dimensional (2-D) gel electrophoresis with mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of Drosophila melanogaster mitochondria. First, we purified mitochondria from third instar Drosophila larvae and constructed a high-resolution 2-D gel database containing 231 silver-stained polypeptides. Next, we carried out preparative 2-D PAGE to isolate some of the polypeptides and characterize them by MALDI-TOF analysis. Using this strategy, we identified 66 mitochondrial spots in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis. In addition, we generated antibodies against two of the mitochondrial proteins as tools for characterizing the organelle.     

Comparative profiling of the mammalian mitochondrial proteome: multiple aconitase-2 isoforms including N-formylkynurenine modifications as part of a protein biomarker signature for reactive oxidative species

The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.     

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.