Mitochondrial Targeting Presequence Research Articles

Cross-Species Functional Conservation and Possible Origin of the N-Terminal Specificity Domain of Mitochondrial Presequences.

Plants have two endosymbiotic organelles, chloroplast and mitochondrion. Although they have their own genomes, proteome assembly in these organelles depends on the import of proteins encoded by the nuclear genome. Previously, we elucidated the general design principles of chloroplast and mitochondrial targeting signals, transit peptide, and presequence, respectively, which are highly diverse in primary structure. Both targeting signals are composed of N-terminal specificity domain and C-terminal translocation domain. Especially, the N-terminal specificity domain of mitochondrial presequences contains multiple arginine residues and hydrophobic sequence motif. In this study we investigated whether the design principles of plant mitochondrial presequences can be applied to those in other eukaryotic species. We provide evidence that both presequences and import mechanisms are remarkably conserved throughout the species. In addition, we present evidence that the N-terminal specificity domain of presequence might have evolved from the bacterial TAT (twin-arginine translocation) signal sequence.

Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line.

The cholesterol hydroxylase/lyase (CHL) system, consisting of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR), initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The foot-and-mouth disease virus 2A-based method allows to express multiple proteins from a single transcript. We developed a 2A-based multicistronic system for the coexpression of three bovine CHL system proteins as the self-processing polyprotein pCoxIV-P450scc-2A-Adx-2A-AdR-GFP (pCoxIV-CHL-GFP), with a cleavable N-terminal mitochondrial targeting presequence. HEK293T cells transfected with plasmid, containing complementary DNA (cDNA) for pCoxIV-CHL-GFP, efficiently performed the expression of P450scc-2A, targeted to mitochondria, and Adx-2A, AdR-GFP and the fusion protein Adx-2A-AdR-GFP, which were predominantly localized in the cytosol. Despite the spatial separation of expressed P450scc and redox partners, the transfected HEK293T cells were able to convert the steroid substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T cells were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. HEK293T cells transfected with a vector directing the synthesis of only P450scc-2A demonstrated cytochrome P450scc activity comparable to that of cells expressing all three CHL system components, and to that of nature steroidogenic cells. Thus, the P450scc activity detected in cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3β-hydroxysteroid dehydrogenase. Therefore, HEK293T cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.

Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions.

TIMM50 is an essential component of the TIM23 complex, the mitochondrial inner membrane machinery that imports cytosolic proteins containing a mitochondrial targeting presequence into the mitochondrial inner compartment. Whole exome sequencing (WES) identified compound heterozygous pathogenic mutations in TIMM50 in an infant patient with rapidly progressive, severe encephalopathy. Patient fibroblasts presented low levels of TIMM50 and other components of the TIM23 complex, lower mitochondrial membrane potential, and impaired TIM23‐dependent protein import. As a consequence, steady‐state levels of several components of mitochondrial respiratory chain were decreased, resulting in decreased respiration and increased ROS production. Growth of patient fibroblasts in galactose shifted energy production metabolism toward oxidative phosphorylation (OxPhos), producing an apparent improvement in most of the above features but also increased apoptosis. Complementation of patient fibroblasts with TIMM50 improved or restored these features to control levels. Moreover, RNASEH1 and ISCU mutant fibroblasts only shared a few of these features with TIMM50 mutant fibroblasts. Our results indicate that mutations in TIMM50 cause multiple mitochondrial bioenergetic dysfunction and that functional TIMM50 is essential for cell survival in OxPhos‐dependent conditions.

Targeting the Amyloid Precursor Protein (APP) to Mitochondria of White Adipose Tissues Triggers Mitochondrial Dysfunction and Obesity

The role of mitochondrial dysfunction in white adipose tissues (WAT) under different metabolic conditions is not well understood. Here, we report that under high fat diet (HFD) conditions, endogenous production of APP is significantly induced and, instead of being secreted, the protein is highly enriched in WAT mitochondria. To better understand the role of APP, we established an inducible model by directing the full-length APP with a mitochondrial targeting presequence quantitatively to mitochondria, selectively in adipocytes. We validated that APP mRNA and protein levels were significantly elevated in subcutaneous WAT, and the protein was markedly accumulated in isolated mitochondria from sWAT. EM imaging suggested widespread loss of mitochondrial cristae. Respiratory measurements on a Seahorse instrument revealed a rapid impairment in mitochondrial functions in sWAT, concomitant with an increase in adipocyte size within a few days. APP transgenic mice displayed significantly enhanced fat mass, along with impaired glucose tolerance and reduced insulin sensitivity under a long term HFD challenge. By H and E staining, we observed widespread inflammatory cell infiltration in WAT, and consistent with the adipose tissue dysfunction, we also noticed massive hepatic steatosis. Adiponectin levels in plasma and sWAT were significantly reduced. We specifically eliminated endogenous APP in WAT through a conditioned knock-out mouse model (Appflox/flox) to demonstrate that adipocyte-specific deletion of APP can ameliorate high fat diet-induced mitochondrial dysfunction. In summary, accumulated APP in WAT mitochondria significantly induces mitochondrial dysfunction in adipocytes, leading to unhealthy adipose tissue expansion and eventually resulting in obesity and systemic insulin resistance. Disclosure Y.A. An: None. P.E. Scherer: None.

Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria

BackgroundThere is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.ResultsHere we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (nif) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, nifU, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration.ConclusionsWe present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.

AAE13 encodes a dual-localized malonyl-CoA synthetase that is crucial for mitochondrial fatty acid biosynthesis.

Malonyl-CoA is a key intermediate in a number of metabolic processes associated with its role as a substrate in acylation and condensation reactions. These types of reactions occur in plastids, the cytosol and mitochondria, and although carboxylation of acetyl-CoA is the known mechanism for generating the distinct plastidial and cytosolic pools, the metabolic origin of the mitochondrial malonyl-CoA pool is still unclear. In this study we demonstrate that malonyl-CoA synthetase encoded by the Arabidopsis AAE13 (AT3G16170) gene is localized in both the cytosol and the mitochondria. These isoforms are translated from two types of transcripts, one that contains and one that does not contain a mitochondrial-targeting pre-sequence. Whereas the cytosolic AAE13 protein is not essential, due to the presence of a redundant malonyl-CoA generating system provided by a cytosolic acetyl-CoA carboxylase, the mitochondrial AAE13 protein is essential for plant growth. Phenotypes of the aae13-1 mutant are transgenically reversed only if the mitochondrial pre-sequence is present in the ectopically expressed AAE13 proteins. The aae13-1 mutant exhibits typical metabolic phenotypes associated with a deficiency in the mitochondrial fatty acid synthase system, namely depleted lipoylation of the H subunit of the photorespiratory enzyme glycine decarboxylase, increased accumulation of glycine and glycolate and reduced levels of sucrose. Most of these metabolic alterations, and associated morphological changes, are reversed when the aae13-1 mutant is grown in a non-photorespiratory condition (i.e. a 1% CO2 atmosphere), demonstrating that they are a consequence of the deficiency in photorespiration due to the inability to generate lipoic acid from mitochondrially synthesized fatty acids.

MutS HOMOLOG1 silencing mediates ORF220 substoichiometric shifting and causes male sterility in Brassica juncea.

Cytoplasmic male sterility (CMS) has consistently been associated with the expression of mitochondrial open reading frames (ORFs) that arise from genomic rearrangements. Spontaneous fertility reversion in CMS has been observed in several cases, but a clear understanding of fertility reversion controlled by nuclear genetic influences has been lacking. Here, we identified spontaneous fertile revertant lines for Brassica juncea CMS cytoplasm in which the mitochondrial genome has undergone substoichiometric shifting (SSS) to suppress ORF220 copy number. We placed ORF220, with or without a mitochondrial targeting presequence, under the control of the CaMV35S and AP3 promoters in Arabidopsis to confirm that ORF220 causes male sterility when mitochondrially localized. We found that copy number of the ORF220 gene was altered under conditions that suppress MSH1, a nuclear gene that controls illegitimate recombination in plant mitochondria. MSH1-RNAi lines with increased ORF220 copy number were male sterile compared with wild type. We found that a wide range of genes involved in anther development were up- and down-regulated in revertant and MSH1-RNAi lines, respectively. The system that we have developed offers valuable future insight into the interplay of MSH1 and SSS in CMS induction and fertility reversion as a mediator of nuclear-mitochondrial crosstalk.

In vivo functional analysis of a nuclear restorer PPR protein.

BackgroundNuclear restorers of cytoplasmic male fertility (CMS) act to suppress the male sterile phenotype by down-regulating the expression of novel CMS-specifying mitochondrial genes. One such restorer gene is Rfo, which restores fertility to the radish Ogura or ogu CMS. Rfo, like most characterized restorers, encodes a pentatricopeptide repeat (PPR) protein, a family of eukaryotic proteins characterized by tandem repeats of a 35 amino acid motif. While over 400 PPR genes are found in characterized plant genomes and the importance of this gene family in organelle gene expression is widely recognized, few detailed in vivo assessments of primary structure-function relationships in this protein family have been conducted.ResultsIn contrast to earlier studies, which identified 16 or 17 PPR domains in the Rfo protein, we now find, using a more recently developed predictive tool, that Rfo has 18 repeat domains with the additional domain N-terminal to the others. Comparison of transcript sequences from pooled rfo/rfo plants with pooled Rfo/Rfo plants of a mapping population led to the identification of a non-restoring rfo allele with a 12 bp deletion in the fourth domain. Introduction into ogu CMS plants of a genetic construct in which this deletion had been introduced into Rfo led to a partial loss in the capacity to produce viable pollen, as assessed by vital staining, pollen germination and the capacity for seed production following pollination of CMS plants. The degree of viable pollen production among different transgenic plants roughly correlated with the copy number of the introduced gene and with the reduction of the levels of the ORF138 CMS-associated protein. All other constructs tested, including one in which only the C-terminal PPR repeat was deleted and another in which this repeat was replaced by the corresponding domain of the related, non-restoring gene, PPR-A, failed to result in any measure of fertility restoration.ConclusionsThe identification of the additional PPR domain in Rfo indicates that the protein, apart from its N-terminal mitochondrial targeting presequence, consists almost entirely of PPR repeats. The newly identified rfo allele carries the same 4 amino acid deletion as that found in the neighboring, related, non-restoring PPR gene, PPR-A. Introduction of this four amino acid deletion into a central domain the Rfo protein, however, only partially reduces its restoration capacity, even though this alteration might be expected to alter the spacing between the adjoining repeats. All other tested alterations, generated by deleting specific PPR repeats or exchanging repeats with corresponding domains of PPR-A, led to a complete loss of restorer function. Overall we demonstrate that introduction of targeted alterations of Rfo into ogu CMS plants provides a sensitive in vivo readout for analysis of the relationship between primary structure and biological function in this important family of plant proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0313-4) contains supplementary material, which is available to authorized users.

Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide

The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-β peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation is due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP.

Complete sequences of organelle genomes from the medicinal plant Rhazya stricta (Apocynaceae) and contrasting patterns of mitochondrial genome evolution across asterids.

BackgroundRhazya stricta is native to arid regions in South Asia and the Middle East and is used extensively in folk medicine to treat a wide range of diseases. In addition to generating genomic resources for this medicinally important plant, analyses of the complete plastid and mitochondrial genomes and a nuclear transcriptome from Rhazya provide insights into inter-compartmental transfers between genomes and the patterns of evolution among eight asterid mitochondrial genomes.ResultsThe 154,841 bp plastid genome is highly conserved with gene content and order identical to the ancestral organization of angiosperms. The 548,608 bp mitochondrial genome exhibits a number of phenomena including the presence of recombinogenic repeats that generate a multipartite organization, transferred DNA from the plastid and nuclear genomes, and bidirectional DNA transfers between the mitochondrion and the nucleus. The mitochondrial genes sdh3 and rps14 have been transferred to the nucleus and have acquired targeting presequences. In the case of rps14, two copies are present in the nucleus; only one has a mitochondrial targeting presequence and may be functional. Phylogenetic analyses of both nuclear and mitochondrial copies of rps14 across angiosperms suggests Rhazya has experienced a single transfer of this gene to the nucleus, followed by a duplication event. Furthermore, the phylogenetic distribution of gene losses and the high level of sequence divergence in targeting presequences suggest multiple, independent transfers of both sdh3 and rps14 across asterids. Comparative analyses of mitochondrial genomes of eight sequenced asterids indicates a complicated evolutionary history in this large angiosperm clade with considerable diversity in genome organization and size, repeat, gene and intron content, and amount of foreign DNA from the plastid and nuclear genomes.ConclusionsOrganelle genomes of Rhazya stricta provide valuable information for improving the understanding of mitochondrial genome evolution among angiosperms. The genomic data have enabled a rigorous examination of the gene transfer events. Rhazya is unique among the eight sequenced asterids in the types of events that have shaped the evolution of its mitochondrial genome. Furthermore, the organelle genomes of R. stricta provide valuable genomic resources for utilizing this important medicinal plant in biotechnology applications.Electronic supplementary materialThe online version of this article (doi: 10.1186/1471-2164-15-405) contains supplementary material, which is available to authorized users.

Functional Dissection of the Dictyostelium discoideum Dynamin B Mitochondrial Targeting Sequence

Most mitochondrial proteins are nuclear encoded and synthesized in the cytosol with an N-terminal mitochondrial targeting sequence or presequence for subsequent import into mitochondria. Here, we describe the proteolytic processing and inner membrane potential-dependent translocation of a dynamin family member by the Dictyostelium discoideum mitochondrial import system. Our results show that the unusual D. discoideum dynamin B presequence is removed through a processing mechanism that is common for mitochondrial matrix proteins. We identified a minimal segment of the dynamin B presequence containing seven lysine residues. This 47-residue region is, in combination with consensus matrix protease cleavage sites, necessary and sufficient for mitochondrial targeting. The correct positioning of these lysine residues plays a critical role for the proper processing and mitochondrial import of dynamin B in D. discoideum. Fluorescent proteins tagged with the dynamin B presequence or presequence regions supporting mitochondrial import in D. discoideum are imported with similar efficiency into the mitochondrial matrix of mammalian cells, indicating that the basic mechanisms underlying mitochondrial protein import are highly conserved from amoebozoa to mammalia.

Euglena gracilis and Trypanosomatids Possess Common Patterns in Predicted Mitochondrial Targeting Presequences

Euglena gracilis possessing chloroplasts of secondary green algal origin and parasitic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania major belong to the protist phylum Euglenozoa. Euglenozoa might be among the earliest eukaryotic branches bearing ancestral traits reminiscent of the last eukaryotic common ancestor (LECA) or missing features present in other eukaryotes. LECA most likely possessed mitochondria of endosymbiotic α-proteobacterial origin. In this study, we searched for the presence of homologs of mitochondria-targeted proteins from other organisms in the currently available EST dataset of E. gracilis. The common motifs in predicted N-terminal presequences and corresponding homologs from T. brucei, T. cruzi and L. major (if found) were analyzed. Other trypanosomatid mitochondrial protein precursor (e.g., those involved in RNA editing) were also included in the analysis. Mitochondrial presequences of E. gracilis and these trypanosomatids seem to be highly variable in sequence length (5-118 aa), but apparently share statistically significant similarities. In most cases, the common (M/L)RR motif is present at the N-terminus and it is probably responsible for recognition via import apparatus of mitochondrial outer membrane. Interestingly, this motif is present inside the predicted presequence region in some cases. In most presequences, this motif is followed by a hydrophobic region rich in alanine, leucine, and valine. In conclusion, either RR motif or arginine-rich region within hydrophobic aa-s present at the N-terminus of a preprotein can be sufficient signals for mitochondrial import irrespective of presequence length in Euglenozoa.

Direct Interaction of Mitochondrial Targeting Presequences with Purified Components of the TIM23 Protein Complex

Precursor proteins that are imported from the cytosol into the matrix of mitochondria carry positively charged amphipathic presequences and cross the inner membrane with the help of vital components of the TIM23 complex. It is currently unclear which subunits of the TIM23 complex recognize and directly bind to presequences. Here we analyzed the binding of presequence peptides to purified components of the TIM23 complex. The interaction of three different presequences with purified soluble domains of yeast Tim50 (Tim50IMS), Tim23 (Tim23IMS), and full-length Tim44 was examined. Using chemical cross-linking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50IMS and Tim44 to interact directly with the yeast Hsp60 presequence. We also analyzed their interaction with presequences derived from precursors of yeast mitochondrial 70-kDa heat shock protein (mHsp70) and of bovine cytochrome P450SCC. Moreover, we characterized the nature of the interactions and determined their KDs. On the basis of our results, we suggest a mechanism of translocation where stronger interactions of the presequences on the trans side of the channel support the import of precursor proteins through TIM23 into the matrix.

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.

Extreme mitochondrial evolution in the ctenophore Mnemiopsis leidyi: Insight from mtDNA and the nuclear genome

Recent advances in sequencing technology have led to a rapid accumulation of mitochondrial DNA (mtDNA) sequences, which now represent the wide spectrum of animal diversity. However, one animal phylum—Ctenophora—has, to date, remained completely unsampled. Ctenophores, a small group of marine animals, are of interest due to their unusual biology, controversial phylogenetic position, and devastating impact as invasive species. Using data from the Mnemiopsis leidyi genome sequencing project, we Polymerase Chain Reaction (PCR) amplified and analyzed its complete mitochondrial (mt-) genome. At just over 10 kb, the mt-genome of M. leidyi is the smallest animal mtDNA ever reported and is among the most derived. It has lost at least 25 genes, including atp6 and all tRNA genes. We show that atp6 has been relocated to the nuclear genome and has acquired introns and a mitochondrial targeting presequence, while tRNA genes have been genuinely lost, along with nuclear-encoded mt-aminoacyl tRNA synthetases. The mt-genome of M. leidyi also displays extremely high rates of sequence evolution, which likely led to the degeneration of both protein and rRNA genes. In particular, encoded rRNA molecules possess little similarity with their homologs in other organisms and have highly reduced secondary structures. At the same time, nuclear encoded mt-ribosomal proteins have undergone expansions, likely to compensate for the reductions in mt-rRNA. The unusual features identified in M. leidyi mtDNA make this organism an interesting system for the study of various aspects of mitochondrial biology, particularly protein and tRNA import and mt-ribosome structures, and add to its value as an emerging model species. Furthermore, the fast-evolving M. leidyi mtDNA should be a convenient molecular marker for species- and population-level studies.

Comparative Analysis of Structural Diversity and Sequence Evolution in Plant Mitochondrial Genes Transferred to the Nucleus

The transfer of functional mitochondrial genes to the nucleus is an ongoing process during plant evolution that has made a major impact on cytonuclear interactions and mitochondrial genome evolution. Analysis of evolutionarily recent transfers in plants provides insights into the evolutionary dynamics of the process and how transferred genes become functional in the nucleus. Here, we report 42 new transferred genes in various angiosperms, including 9 separate transfers of the succinate dehydrogenase gene sdh3. We performed comparative analyses of gene structures and sequence evolution of 77 genes transferred to the nucleus in various angiosperms, including multiple transfers of 10 genes in different lineages. Many genes contain mitochondrial targeting presequences, and potentially 5' cis-regulatory elements, that were acquired from pre-existing nuclear genes for mitochondrial proteins to create chimeric gene structures. In eight separate cases, the presequence was acquired from either the hsp70 chaperonin gene or the hsp22 chaperonin gene. The most common location of introns is in the presequence, and the least common is in the region transferred from the mitochondrion. Several genes have an intron between the presequence and the core region, or an intron in the 5'UTR (untranslated region) or 3'UTR, suggesting presequence and/or regulatory element acquisition by exon shuffling. Both synonymous and nonsynonymous substitution rates have increased considerably in the transferred genes compared with their mitochondrial counterparts, and the degree of rate acceleration varies by gene, species, and evolutionary timing of transfer. Pairwise and branchwise K(a)/K(s) analysis identified four genes with evidence for positive selection, but positive selection is generally uncommon in transferred genes. This study provides a detailed portrayal of structural and sequence evolution in mitochondrial genes transferred to the nucleus, revealing the frequency of different mechanisms for how presequences and introns are acquired and showing how the sequences of transferred genes evolve after movement between cellular genomes.

A male sterility‐associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants

In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST-ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.

A 24-residue presequence localizes human factor B to mitochondria

We reported previously that the human factor B precursor is a 215-amino acid polypeptide, the first 40 amino acid residues of which function as a mitochondrial targeting presequence [G.I. Belogrudov, Y. Hatefi, J. Biol. Chem. 277 (2002) 6097–6103]. Confocal microscopy of live HEK293 cells, transiently transfected with factor B constructs tagged at the C-terminus with green fluorescent protein (GFP) revealed that either a 40- or 25-residue presequence localized factor B to mitochondria. Indirect immunofluorescent labeling of fixed, permeabilized HEK293 cells that were transiently transfected with a construct lacking a presequence, showed diffuse, intracellular staining that was consistent with targeting of ectopically expressed factor B to cellular compartments distinct from the mitochondria. Mutants in which either Met −25 or both Met −25/Met −24 residues of the presequence were deleted exhibited decreased or undetectable levels, respectively, of the GFP-tagged factor B. The factor B presequence alone was shown to target a reporter polypeptide GFP to mitochondria. Our studies, therefore, demonstrate that a 24-residue presequence is sufficient to localize factor B to mitochondria, and suggest that the human factor B precursor is a 199-amino acid polypeptide.

Evolutionary transfers of mitochondrial genes to the nucleus in the Populus lineage and coexpression of nuclear and mitochondrial Sdh4 genes

The transfer of mitochondrial genes to the nucleus is an ongoing evolutionary process in flowering plants. Evolutionarily recent gene transfers provide insights into the evolutionary dynamics of the process and the way in which transferred genes become functional in the nucleus. Genes that are present in the mitochondrion of some angiosperms but have been transferred to the nucleus in the Populus lineage were identified by searches of Populus sequence databases. Sequence analyses and expression experiments were used to characterize the transferred genes. Two succinate dehydrogenase genes and six mitochondrial ribosomal protein genes have been transferred to the nucleus in the Populus lineage and have become expressed. Three transferred genes have gained an N-terminal mitochondrial targeting presequence from other pre-existing genes and two of the transferred genes do not contain an N-terminal targeting presequence. Intact copies of the succinate dehydrogenase gene Sdh4 are present in both the mitochondrion and the nucleus. Both copies of Sdh4 are expressed in multiple organs of two Populus species and RNA editing occurs in the mitochondrial copy. These results provide a genome-wide perspective on mitochondrial genes that were transferred to the nucleus and became expressed, functional genes during the evolutionary history of Populus.

One Single In-frame AUG Codon Is Responsible for a Diversity of Subcellular Localizations of Glutaredoxin 2 in Saccharomyces cerevisiae

Glutaredoxins belong to a family of small proteins with glutathione-dependent disulfide oxidoreductase activity involved in cellular defense against oxidative stress. The product of the yeast GRX2 gene is a protein that is localized both in the cytosol and mitochondria. To throw light onto the mechanism responsible for the dual subcellular distribution of Grx2 we analyzed mutant constructs containing different targeting information. By altering amino acid residues around the two in-frame translation initiation start sites of the GRX2 gene, we could demonstrate that the cytosolic isoform of Grx2 was synthesized from the second AUG, lacking an N-terminal extension. Translation from the first AUG resulted in a long isoform carrying a mitochondrial targeting presequence. The mitochondrial targeting properties of the presequence and the influence of the mature part of Grx2 were analyzed by the characterization of the import kinetics of specific fusion proteins. Import of the mitochondrial isoform is relatively inefficient and results in the accumulation of a substantial amount of unprocessed form in the mitochondrial outer membrane. Substitution of Met(35), the second translation start site, to Val resulted in an exclusive targeting to the mitochondrial matrix. Our results show that a plethora of Grx2 subcellular localizations could spread its antioxidant functions all over the cell, but one single A to G [corrected] mutation converts Grx2 into a typical protein of the mitochondrial matrix. The "A" denotes adenine, rather than alanine, and the "G" refers to guanine, not glycine [corrected]