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PubMed2002

Import of nuclear encoded RNAs into yeast and human mitochondria: experimental approaches and possible biomedical applications.

Mitochondria import from the cytoplasm the vast majority of proteins and some RNAs. Although there exists extended knowledge concerning the mechanisms of protein import, the import of RNA is poorly understood. It was almost exclusively studied on the model of tRNA import, in several protozoans, plants and yeast. Mammalian mitochondria, which do not import tRNAs naturally, are hypothesized to import other small RNA molecules from the cytoplasm. We studied tRNA import in the yeast system, both in vitro and in vivo, and applied similar approaches to study 5S rRNA import into human mitochondria. Despite the obvious divergence of RNA import systems suggested for different species, we find that in yeast and human cells this pathway involves similar mechanisms exploiting cytosolic proteins to target the RNA to the organelle and requiring the integrity of pre-protein import apparatus. The import pathway might be of interest from a biomedical point of view, to target into mitochondria RNAs that could suppress pathological mutations in mitochondrial DNA. Yeast represents a good model to elaborate such a gene therapy approach. We have described here the various approaches and protocols to study RNA import into mitochondria of yeast and human cells in vitro and in vivo.

Humans↗

PubMed1994

In vivo expression and mitochondrial import of normal and mutated tRNA(thr) in Leishmania.

Evidence suggests that mitochondria of protozoans and plants contain nuclear-encoded tRNAs. In trypanosomatids, the entire set of tRNAs in the mitochondria are presumably imported from the nucleus, but the mechanism of tRNA import is not presently understood. In this study, we have employed a plasmid-encoded nuclear tRNA gene as a means of investigating tRNA expression and mitochondrial import in vivo in Leishmania tarentolae. Using a Leishmania plasmid, we cloned a 1-kb or 250-bp restriction fragment carrying the nuclear tRNA(thr) gene and three in vitro mutagenized derivatives: Tac6 (an insertion of 6 nucleotides at the anticodon loop), Td4 (a 4-nt insert at the D-loop) and Tv4 (a 4-nt insert at the variable arm). Leishmania cells stably transfected with these plasmids were then examined for tRNA expression and import by Northern analysis. The results show that the plasmid-encoded wild type tRNA(thr) gene produced a significantly elevated level of expression in the cytosol. Similarly, the Tac6-transfected cells exhibited a large abundance of the mutant RNA relative to the normal tRNA (chromosome-encoded gene transcripts) in the cytosol. Furthermore, the mutant Tac6 RNA was found imported into mitochondria, although the proportion of the mutant vs. normal tRNA in mitochondria was greatly reduced as compared to that in the cytosol. We suggest that the mitochondrial import machinery is capable of discriminating against the mutant RNA in favor of the normal tRNA for import. In another example, we found that the Tv4 gene showed expression, albeit somewhat reduced, but its import into mitochondria was completely blocked. Unexpectedly, the 4-base addition mutation (Td4) at the D-loop showed neither expression nor import. While these results clearly signify the importance of various segments within the tRNA gene for in vivo expression, our data underscore the significance of the variable loop for mitochondrial import. It is our belief that this plasmid-encoded tRNA gene expression system in Leishmania may be useful in gaining further insights on tRNA import.

Animals↗

PubMed2002

Zinc-dependent intermembrane space proteins stimulate import of carrier proteins into plant mitochondria.

Mitochondrial inner membrane carrier proteins are imported into mitochondria from yeast, fungi and mammals by specific machinery, some components of which are distinct from those utilized by other proteins. Import of two different carriers into plant mitochondria showed that one contains a cleavable presequence which was processed during import, while the other imported in a valinomycin-sensitive manner without processing. Mild osmotic shock of mitochondria released intermembrane space (IMS) components and impaired carrier protein import. Adding back the released IMS proteins as a concentrate in the presence of micromolar ZnCl2 stimulated carrier import into IMS-depleted mitochondria, but did not stimulate import of a non-carrier control precursor protein, the alternative oxidase. Anion-exchange separation of IMS components before addition to IMS-depleted mitochondria revealed a correlation between several 9-10 kDa proteins and stimulation of carrier import. MS/MS sequencing of these proteins identified them as plant homologues of the yeast zinc-finger carrier import components Tim9 and Tim10. Stimulation of import was dependent on either Zn2+ or Cd2+ and inhibited by both N-ethylmalamide (NEM) and a divalent cation chelator, consistent with a functional requirement for a zinc finger protein. This represents direct functional evidence for a distinct carrier import pathway in plant mitochondria, and provides a tool for determining the potential function of other IMS proteins associated with protein import.

Biological Transport↗

PubMed2000

Hydrogen peroxide inhibition of nuclear protein import is mediated by the mitogen-activated protein kinase, ERK2.

H(2)O(2) alters gene expression in many cell types. Alterations in nuclear import of transcription factors or similar key proteins may be responsible for these changes. To investigate this possibility, a cytosolic nuclear import cocktail was treated with varying ¿H(2)O(2) and used in import assays. H(2)O(2) caused a dose- and time-dependent inhibition of import at concentrations as low as 100 microM. Catalase reversed this effect. H(2)O(2) treatment of permeablized cells did not affect import, suggesting that H(2)O(2) was acting on a cytosolic factor. Treatment of import cocktail with two different free radical generating systems had no effect, but treatment of permeablized cells inhibited import, suggesting H(2)O(2) works via a distinct process from hydroxyl or superoxide radicals. Pretreatment of import cocktail with genistein reversed the effect of H(2)O(2) on import. Western blotting revealed that H(2)O(2) activated ERK2. The specific MEK1/2 inhibitor, PD98059, completely blocked the effects of H(2)O(2) on import. Activated ERK2 mimicked H(2)O(2)'s effect on import. Immunocytochemistry revealed that H(2)O(2) treatment of whole cells increased cytosolic Ran/TC4 levels, an effect reversible by catalase or PD98059. These data demonstrate that H(2)O(2) inhibits nuclear protein import and that this effect is mediated by mitogen-activated protein (MAP) kinase activation, possibly by altering Ran/TC4 function.

Animals↗

PubMed2001

An optimization method for importance factors and beam weights based on genetic algorithms for radiotherapy treatment planning.

We propose a new method for selecting importance factors (for regions of interest like organs at risk) used to plan conformal radiotherapy. Importance factors, also known as weighting factors or penalty factors, are essential in determining the relative importance of multiple objectives or the penalty ratios of constraints incorporated into cost functions, especially in dealing with dose optimization in radiotherapy treatment planning. Researchers usually choose importance factors on the basis of a trial-and-error process to reach a balance between all the objectives. In this study, we used a genetic algorithm and adopted a real-number encoding method to represent both beam weights and importance factors in each chromosome. The algorithm starts by optimizing the beam weights for a fixed number of iterations then modifying the importance factors for another fixed number of iterations. During the first phase, the genetic operators, such as crossover and mutation, are carried out only on beam weights, and importance factors for each chromosome are not changed or 'frozen'. In the second phase, the situation is reversed: the beam weights are 'frozen' and the importance factors are changed after crossover and mutation. Through alternation of these two phases, both beam weights and importance factors are adjusted according to a fitness function that describes the conformity of dose distribution in planning target volume and dose-tolerance constraints in organs at risk. Those chromosomes with better fitness are passed into the next generation, showing that they have a better combination of beam weights and importance factors. Although the ranges of the importance factors should be set in advance by using this algorithm, it is much more convenient than selecting specific numbers for importance factors. Three clinical examples are presented and compared with manual plans to verify this method. Three-dimensional standard displays and dose-volume histograms are shown to demonstrate that this method is feasible, automatic and convenient.

Abdominal Neoplasms↗

PubMed1987

The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information.

The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein.

Amino Acid Sequence↗

PubMed1998

Thyroid hormone modifies mitochondrial phenotype by increasing protein import without altering degradation.

The mitochondrial phenotype within cardiac muscle cells is dramatically altered by thyroid hormone. We report here that this can be accounted for, in part, by modifications in the rate of mitochondrial protein import. The import of matrix-localized precursor proteins malate dehydrogenase (MDH) and ornithine carbamoyltransferase was augmented, whereas the insertion of the outer membrane protein Bcl-2 was unaffected by thyroid hormone treatment. Coincident with increases in the import of these matrix-localized precursors were thyroid hormone-induced elevations in the outer membrane receptor Tom20 and the matrix heat-shock protein mthsp70. The phospholipid cardiolipin was not involved in mediating the thyroid hormone-induced increase in import, as judged from adriamycin inhibition studies. When the import reaction was supplemented with rat heart cytosol, we found that 1) MDH import was stimulated, but Bcl-2 import was inhibited and 2) thyroid hormone did not influence the effect of the cytosol on import rates. Thus distinct requirements exist for the mitochondrial import of precursor proteins, destined for different organellar compartments. Although import of these matrix-localized proteins was augmented by thyroid hormone treatment, the proteolysis of matrix proteins was unaffected as indicated by the degradation of cytob2(167)RIC-dihydrofolate reductase, a chimeric protein missorted to the matrix. Thus our data indicate that at least some thyroid hormone-induced modifications of the mitochondrial phenotype occur due to the compartment-specific upregulation of precursor protein import rates, likely mediated via changes in the expression of protein import machinery components.

14-3-3 Proteins↗

PubMed2006

Improving the use of research evidence in guideline development: 6. Determining which outcomes are important.

BACKGROUND: The World Health Organization (WHO), like many other organisations around the world, has recognised the need to use more rigorous processes to ensure that health care recommendations are informed by the best available research evidence. This is the sixth of a series of 16 reviews that have been prepared as background for advice from the WHO Advisory Committee on Health Research to WHO on how to achieve this. OBJECTIVES: We reviewed the literature on determining which outcomes are important for the development of guidelines. METHODS: We searched five databases of methodological studies for existing systematic reviews and relevant methodological research. We did not conduct a complete systematic review ourselves. Our conclusions are based on the available evidence, consideration of what WHO and other organisations are doing and logical arguments. KEY QUESTIONS AND ANSWERS: We did not find a systematic review that addresses any of the following key questions and we found limited relevant research evidence. What methods should WHO use to identify important outcomes? Methods of outcome identification should be transparent and explicit. The consultation process should start with identification of all relevant outcomes associated with an intervention. Those affected, including consumers, should be involved in the selection of outcomes. A question driven approach (what is important?) is preferable to a data driven approach (what data are at hand?) to identify important outcomes. What type of outcomes should WHO consider and how should cultural diversity be taken account of in the selection of outcomes? Desirable (benefits, less burden and savings) and undesirable effects should be considered in all guidelines. Undesirable effects include harms (including the possibility of unanticipated adverse effects), greater burden (e.g. having to go to the doctor) and costs (including opportunity costs). Important outcomes (e.g. mortality, morbidity, quality of life) should be preferred over surrogate, indirect outcomes (e.g. cholesterol levels, lung function) that may or may not correlate with patient important outcomes. Ethical considerations should be part of the evaluation of important outcomes (e.g. impacts on autonomy). If the importance of outcomes is likely to vary across cultures, stakeholders from diverse cultures should be consulted and involved in the selection of outcomes. How should the importance of outcomes be ranked? Outcomes should be ranked by relative importance, separated into benefits and downsides. Information from research on values and preferences should inform the ranking of outcomes whenever possible. If the importance of outcomes is likely to vary across cultures, ranking of outcomes should be done in specific settings. If evidence is lacking for an important outcome, this should be acknowledged, rather than ignoring the outcome.

Journal Article↗

PubMed1995

[Cryptosporidia and other endoparasites in heifers imported into the Czech Republic].

Totally 887 heifers of Holstein-Friesian breed mostly in late pregnancy imported to the Czech Republic from France (597), Germany (89), Denmark (181) and Holland (20) were examined coprologically from September 1993 to March 1995 in the parasitological laboratory of the National Veterinary Institute (NVI). Prague. Feces were sampled individually, rectally, always on days 1-3 following importation from heifers housed in particular quarantine sheds. In compliance with presently valid veterinary regulations, all animals were examined for liver fluke disease (fascioliasis) and lungworm. Moreover, 634 heifers were submitted to qualitative coprological examination aimed at revealing the presence of cysts and oocysts of protozoa, eggs of taenias and nematodes of gastrointestinal tract. The method according to Pavlásek (1991), especially designed for proving oocysts of the genus Cryptosporidium, was applied in all fecal specimens delivered to the SVI from animals in quarantine (N = 887). From trematodes, 12 heifers imported from France were positive for eggs of Fasciola hepatica and in other two animals eggs of the genus Paramphistomum were found. None of the imported heifers showed lungworm disease. Summary of data on occurrence of endoparasites gained during qualitative examination of samples of feces taken from heifers imported from France, Germany and Denmark is presented in Tab. I. Parasitologically, 91.2 to 100% of imported animals were positive. Taeniasis (the genus Moniezia) was detected in 2.8% of heifers imported from France and in 9.8% animals from Denmark. Protozoal parasites were found in 58.8% (Denmark) and 92.8% (Germany) heifers. Coccidial oocysts most frequently observed represented the genus Eimeria (E. bovis, E. auburnensis and E. zuernii). Gastrointestinal nematodes of nine genera were found in 72.5 to 80.8% of heifers. The most frequent findings were genera Ostertagia, Haemonchus and Trichostrongylus. Oocysts, morphologically identical with Cryptosporidium muris Tyzzer (1907), 1910, were detected in 4.5% of heifers imported into the Czech Republic from France and in 7.9% of those from Germany. In view of the fact the imported heifers were sampled always on days 1-3 of their quarantine following their importation it is quite impossible, considering the development of the protozoon, they could become infected just in the territory of the Czech Republic. Therefore, with the highest probability, our findings of C. muris-like oocysts in heifers are of priority importance for France and Germany because in the literature these countries do not report cattle as a host of this protozoon. We have found out 57.9% out of 19 animals positive for C. muris on one farm of a private cattle keeper. On the basis of a long-term monitoring of three dairy cows and one bull, the duration of the patent period is longer than 18 months, while we do not know precise onset of shedding oocysts of the protozoon in these naturally infected animals. Furthermore, the paper discusses the need of future studies of C. muris from the point of view of spread, pathogenicity, specificity and host spectrum. The author proposes and recommends obligatory examinations of imported animals with special attention paid to presence of coccidia of the genus Cryptosporidium in order to maintain, with respect to their zoonotic character, these protozoal infections under proper control. At present the parasitological laboratory of the NVI in Prague has a bank of oocyst isolates of the C. muris type from cattle (Bos taurus), from desert hamsters (Phodopus roborovskii Satunin, 1903) and camels (Camelus bactrianus). Experimental infections is permanently kept in laboratory mice following successful transmission from desert hamsters.

Animals↗

PubMed2001

Rheumatology patient and physician concordance with respect to important health and symptom status outcomes.

OBJECTIVE: In chronic diseases such as arthritis, patients' priorities and values are of prime importance in the measurement of outcome. Relatively few indices take this into account. We sought to identify individual rheumatology patients' choices for important health and symptom status outcomes and assess physicians' ability to detect them. METHODS: A cross-sectional, observational study design was used to examine rheumatology patients' choices for important health and symptom status outcomes. A descriptive summary of patients' choices of "most important" health and symptom status outcomes was compiled. The sensitivity and specificity of physicians' ability to detect their patients' choices for important health or symptom status outcomes were calculated. RESULTS: The sample consisted of 79 rheumatology patients, 30 men and 49 women, with a mean age of 52.6 years. The feeling of being in control was the mental health outcome rated most important by the largest number of the patients (42%); cognitive function (making decisions, solving problems) (22%), feelings of being happy (18%), and feelings of being satisfied (16%) were also rated as being "most important" by many patients. Feelings of hope were rated "most important" by the fewest patients (2%). The physicians' sensitivity to detecting patients' choices of most important outcomes was highest for social functioning and lowest for mental functioning. The specificity to detect patients' choices of less important outcomes was highest for physical functioning and lowest for symptom status. CONCLUSION: In this study involving 4 rheumatologists, patients were found to differ in their choices of important health and symptom status outcomes. Physicians often fail to identify these choices.

Adult↗

PubMed2005

Outcomes generated by patients with rheumatoid arthritis: how important are they?

BACKGROUND: It has been shown previously that patients with rheumatoid arthritis (RA) can generate a wide range of outcomes that they consider important in treatment. It is not known if these outcomes are generally important in the wider RA patient community. OBJECTIVES: (1) To examine whether recent patient-generated outcomes are generalizable within a wider RA population; (2) to assess the relative importance of each outcome; and(3) to explore whether any important outcomes have been omitted. METHODS: A questionnaire, listing 23 outcomes previously generated by RA patients, was distributed through three rheumatology centres in the UK. Patients gave an importance score to each outcome (0-3), selected their top three most important outcomes, and then listed any outcomes of personal importance that were missing. RESULTS: 323 questionnaires were returned (65%). All outcomes were deemed important. Independence, pain, and mobility were most frequently selected by patients in their top three outcomes but were not chosen by 61-66% of patients. The next most commonly chosen outcomes related to feeling well and fatigue. Factor analysis revealed six reasonably distinct groupings: general well-being (11.9% explained variance), day-to-day functioning(10.6%), emotional and psychological well-being (10.6%), social role and confidence (10%), physical symptoms (9.5%) and medication issues (7.9%). CONCLUSION: Outcomes generated by patients as important in RA, are generalizable and inclusive. The most important (independence, pain and mobility) are routinely treated and measured. The next most important (feeling well, fatigue) are infrequently addressed and deserve urgent consideration for measurement, treatment and research.

Journal Article↗

PubMed1996

Mechanisms of tRNA import into yeast mitochondria: an overview.

Mitochondrial import of tRNA is now considered as a quasi-universal phenomenon. In the yeast Saccharomyces cerevisiae, one of the three lysine isoacceptors, the tRNA(Lys)1 with the anticodon CUU (tRNA-K1), is encoded by the nuclear genome and distributed between the cytoplasmic (> 95%) and mitochondrial (< 5%) compartments. In vivo and in vitro import assays were developed to study the mechanisms of tRNA-K1 mitochondrial import. Transmembrane translocation of the tRNA requires the intactness of at least two of the components of the mitochondrial import machinery of pre-proteins, MOM19 and MIM44, as well as energy of ATP hydrolysis and an electrochemical potential across the inner membrane. The import of tRNA-K1 involves formation of an RNP complex on the mitochondrial outer membrane. tRNA-K1 import is also dependent upon cytosolic protein factors, one of which was identified as the precursor of the mitochondrial lysyl-tRNA synthetase (MSK). Although essential for tRNA-K1 import in vitro and in vivo, pre-MSK is however not sufficient to direct the import in vitro, which suggests the need of additional cytosolic factor(s). The tRNA can be imported in its mature form and nucleoside modification is not essential. Aminoacylation of the imported tRNA by the cytoplasmic lysyl-tRNA synthetase is a prerequisite for import. Possible mechanisms of intracellular partitioning and mitochondrial membrane translocation of tRNA-K1 are discussed.

Amino Acyl-tRNA Synthetases↗

PubMed2003

Environmental stresses inhibit and stimulate different protein import pathways in plant mitochondria.

The impact of various environmental stresses (drought, chilling or herbicide treatment) on the capacity of plant mitochondria to import precursor proteins was investigated. Drought treatment stimulated import and processing of various precursor proteins via the general import pathway. The stimulatory effect of drought on the general import pathway was due to an increased rate of import, was accompanied by an increased rate of processing, and could be attributed to the presequence of the precursor protein. Interestingly, drought decreased the import of the F(A)d subunit of ATP synthase suggesting a bypass of the point of stimulation during import of this precursor. Both chilling and herbicide treatment of plants, on the other hand, caused inhibition of import with all precursors tested. No decrease in processing of imported proteins was observed by these stress treatments. Western analysis of several mitochondrial proteins indicated that the steady-state level of several mitochondrial components, including the TOM20 receptor and the core subunits of the cytochrome bc(1) complex responsible for processing, remained largely unchanged. Thus environmental stresses differentially affect import of precursor proteins in a complicated manner dependent on the import pathway utilised.

Chloroplasts↗

PubMed2000

Selective importation of RNA into isolated mitochondria from Leishmania tarentolae.

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNA(Ile)(UAU) and tRNA(Gln)(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA(Asp) was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than approximately 17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNA(Ile) as compared to the level of importation of the mainly cytosol-localized tRNA(Gln). Furthermore, exchanging the D-arm between the tRNA(Ile) and the tRNA(Gln) resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.

Adenosine Triphosphate↗

PubMed2003

Protein structure and import into the peroxisomal matrix.

Proteins destined for the peroxisomal matrix are synthesized in the cytosol, and imported post-translationally. It has been previously demonstrated that stably folded proteins are substrates for peroxisomal import. Mammalian peroxisomes do not contain endogenous chaperone molecules. Therefore, it is possible that proteins are required to fold into their stable, tertiary conformation in order to be imported into the peroxisome. These investigations were undertaken to determine whether proteins rendered incapable of folding were also substrates for import into peroxisomes. Reduction of albumin resulted in a less compact tertiary structure as measured by analytical centrifugation. Microinjection of unfolded albumin molecules bearing the PTS1 targeting signal resulted in their import into peroxisomes. Kinetic analysis indicated that native and unfolded molecules were imported into peroxisomes at comparable rates. While import was unaffected by treatment with cycloheximide, hsc70 molecules were observed to be imported along with the unfolded albumin molecules. These results indicate that proteins, which are incapable of assuming their native conformation, are substrates for peroxisomal import. When combined with previous observations demonstrating the import of stably folded proteins, these results support the model that tertiary structure has no effect on protein import into the peroxisomal matrix.

Cycloheximide↗

PubMed2004

A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria.

The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDH(sp)-DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDH(sp)-DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.

Aldehyde Dehydrogenase↗

PubMed2003

The anticodon and the D-domain sequences are essential determinants for plant cytosolic tRNA(Val) import into mitochondria.

In higher plants, one-third to one-half of the mitochondrial tRNAs are encoded in the nucleus and are imported into mitochondria. This process appears to be highly specific for some tRNAs, but the factors that interact with tRNAs before and/or during import, as well as the signals present on the tRNAs, still need to be identified. The rare experiments performed so far suggest that, besides the probable implication of aminoacyl-tRNA synthetases, at least one additional import factor and/or structural features shared by imported tRNAs must be involved in plant mitochondrial tRNA import. To look for determinants that direct tRNA import into higher plant mitochondria, we have transformed BY2 tobacco cells with Arabidopsis thaliana cytosolic tRNA(Val)(AAC) carrying various mutations. The nucleotide replacements introduced in this naturally imported tRNA correspond to the anticodon and/or D-domain of the non-imported cytosolic tRNA(Met-e). Unlike the wild-type tRNA(Val)(AAC), a mutant tRNA(Val) carrying a methionine CAU anticodon that switches the aminoacylation of this tRNA from valine to methionine is not present in the mitochondrial fraction. Furthermore, mutant tRNAs(Val) carrying the D-domain of the tRNA(Met-e), although still efficiently recognized by the valyl-tRNA synthetase, are not imported any more into mitochondria. These data demonstrate that in plants, besides identity elements required for the recognition by the cognate aminoacyl-tRNA synthetase, tRNA molecules contain other determinants that are essential for mitochondrial import selectivity. Indeed, this suggests that the tRNA import mechanism occurring in plant mitochondria may be different from what has been described so far in yeast or in protozoa.

Acylation↗

PubMed2002

Import of yeast mitochondrial transcription factor (Mtf1p) via a nonconventional pathway.

The yeast mitochondrial (mt) transcription factor Mtf1p is imported into the mitochondria from the cytoplasm without a conventional mt-targeting presequence. To understand its import the mt translocation of wild type and mutant Mtf1p constructs was investigated in vitro under various assay conditions. We report here that Mtf1p, unlike most mt matrix proteins hitherto studied, is translocated into the mitochondria independent of membrane potential, ATP hydrolysis, and membrane receptor. This unusual import of Mtf1p was also observed on ice (3 degrees C). Sub-mitochondrial fractionation demonstrated that Mtf1p was translocated in vitro to one or more of the same mt sites as the endogenous protein that includes the matrix. To identify the mt-targeting sequence of Mtf1p, various N-terminal, C-terminal, or internally deleted Mtf1p derivatives were generated. The full-length and C-terminal deletions but not the N-terminal truncated Mtf1p were imported into mitochondria, indicating the importance of its N-terminal sequence for mt targeting. However, the internal deletion of Mtf1p revealed that the first 150-amino acid N-terminal sequence alone was not sufficient for mt targeting of Mtf1p, suggesting that an extended rather than a short N-terminal sequence is required for import. We favor a model in which Mtf1p adopts an import-competent conformation during translation. Consistent with this model are three findings: most of the protein sequence appears to be required for optimal import, urea denaturation eliminates its import competence, and the import-competent form of the protein is more resistant to tryptic hydrolysis than is the denatured protein. This represents a novel mechanism for mitochondrial protein import.

Adenosine Triphosphate↗