1 School of Kinesiology and Health Science and 3 Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada; and 2 Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611
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ABSTRACT |
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Thyroid hormone [3,5,3'-triiodo-L-thyronine (T3)] induces phenotypic alterations in cardiac mitochondria, in part by influencing protein import and the expression of the import motor mitochondrial heat shock protein (mtHsp70). Here we examined the adaptability of translocases of the inner membrane (Tim) proteins, as well as the outer membrane receptor Tom34, to T3. Administration of T3 to rats for 5 days increased cardiac Tim23 and Tim44 mRNA levels by 55 and 50%, respectively, but had no effect on Tim17. T3 treatment also induced a 45% increase in Tom34 mRNA, with no accompanying changes at the protein level, suggesting regulation at the posttranscriptional level. In H9c2 cardiac cells, Tim17 mRNA was elevated by 114% by 9 days of differentiation, whereas Tim23 and Tim44 declined by 25 and 29%, respectively. To determine the functional consequences of these T3-induced changes, malate dehydrogenase (MDH) import rates were measured in H9c2 cells stably overexpressing Tim44 and mtHsp70, either alone or in combination. MDH import remained unaltered in cells overexpressing Tim44 or in cells overexpressing both Tim44 and mtHsp70. However, when mtHsp70 was overexpressed alone, a 13% (P < 0.05) increase in MDH import rate was observed. These findings indicate that import machinery components are differentially regulated in response to stimuli that induce mitochondrial biogenesis, like T3 and differentiation. In addition, the induction of an import machinery component in response to T3 may not necessarily result in functional changes in protein import during mitochondrial biogenesis. Finally, mtHsp70 may play a regulatory role in the import process that is independent of its interaction with Tim44.
mitochondrial biogenesis; heart; translocases of the inner membrane; mitochondrial heat shock protein 70; muscle differentiation
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INTRODUCTION |
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MITOCHONDRIAL BIOGENESIS is a process that involves the expansion of the mitochondrial reticulum via the synthesis of lipids, and the insertion of nascent proteins in the lipid bilayer. This complex process has been well characterized in striated muscle, and it involves the coordinated expression of mitochondrial proteins encoded by both the nuclear and mitochondrial genomes (8). It is well established that the induction of mitochondrial biogenesis in striated muscle can be enhanced in response to various physiological stimuli. One stimulus that has been shown to have a profound effect on mitochondrial function is thyroid hormone (3,3,5'-triiodo-L-thyronine, or T3; see Refs. 24 and 27). Studies in vivo have demonstrated that T3 elicits a number of adaptations in the mitochondrial compartment, including changes in mitochondrial composition and enzyme activities (20, 27). Such alterations in mitochondrial phenotype during conditions of T3-induced mitochondrial biogenesis are mediated, in part, via adaptations in the mitochondrial protein import pathway.
The majority of proteins required for mitochondrial structure and function are encoded on nuclear genes and synthesized as precursor proteins on cytosolic polysomes (19). These precursors harbor mitochondrial-targeting sequences, which allow cytosolic chaperones to identify the precursor as a mitochondrial protein and escort it to its proper cellular location (13). Once at the mitochondrion, the precursor interacts with import receptors located in both the outer and inner mitochondrial membranes. These import molecules, collectively referred to as translocases of the outer and inner membrane (Toms and Tims, respectively), help guide the precursor in the mitochondrion, whereupon its targeting signal is proteolytically cleaved to form a mature mitochondrial protein (2).
Previous work in our laboratory has demonstrated that the kinetics of protein import in mitochondria are influenced by T3. The outer membrane receptor Tom20 and the matrix chaperone mitochondrial heat shock protein 70 (mtHsp70) are upregulated in response to T3 (4, 22). In addition to increased expression levels of these import machinery components, T3 also stimulates the import of precursor proteins in the mitochondrial matrix. The increase in Tom20 expression was accompanied by a parallel increase in the rate of import of the mitochondrial enzymes malate dehydrogenase (MDH) and ornithine carboxytransferase (4). Similarly, the increase in mtHsp70 levels within the matrix of the mitochondrion was attributable to an accelerated rate of import of this chaperone in the mitochondrion (22).
Although the important adaptive role of the mitochondrial protein import pathway in response to T3 is well documented, there is limited information regarding the relative importance of individual import machinery components in the overall import process in mammalian cells. One study has examined the role of the outer membrane component Tom20 for mitochondrial protein import in C2C12 muscle cells (7). Overexpression of Tom20 resulted in an increase in MDH import rate in the mitochondrial matrix, an observation that paralleled the extent to which Tom20 was overexpressed. In the same study, inhibition of Tom20 expression using antisense oligonucleotides reduced the import rate of MDH. These findings suggest that this receptor plays a vital role in skeletal muscle mitochondrial protein import. However, studies have yet to examine the role of inner membrane machinery components in import and their adaptability during conditions of altered mitochondrial biogenesis. Thus the purpose of this study was to evaluate the mRNA expression of Tim44, Tim23, and Tim17 in response to T3-induced mitochondrial biogenesis in cardiac tissue. In addition, we wished to determine the functional consequences of changes in the expression of selected import machinery components by overexpressing these in H9c2 cardiac cells and evaluating their impact on protein import rates. Finally, we determined the effect of altered thyroid status on cardiac levels of Tom34, a newly identified component of the mammalian import system that is imperative for protein import (3).
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MATERIALS AND METHODS |
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Materials.
The Tom34 antibody and the cDNA encoding human Tom34 were generously
provided by Dr. M. Mori (Kumamoto University School of Medicine,
Kumamoto, Japan). Geneticin, Lipofectamine, and Lipofectamine Plus
reagents were purchased from Invitrogen Life Technologies (Burlington,
Ontario, Canada). T3 and an Anti-Flag M2 monoclonal antibody for Tim44 detection (TIM-Flag) were obtained from
Sigma-Aldrich (St. Louis, MO). Antibodies for thyroid
receptor-1 (TR-
1) and mtHsp70 were
purchased from Santa Cruz (Santa Cruz, CA) and Stressgen Biotechnologies (Victoria, BC, Canada), respectively.
Animal care and treatments. Male Sprague-Dawley rats received daily injections of either T3 (0.4 mg/kg body wt) dissolved in vehicle [100% propylene glycol-0.9% NaCl (1.5:1, vol/vol)], or vehicle solution alone, for a period of 5 days. On the 6th day, animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt), and the hearts were removed and quick-frozen in liquid nitrogen. Heart weights were recorded and compared with body weight to assess the extent of cardiac hypertrophy. The tissues were then pulverized and stored in liquid nitrogen for subsequent mRNA and protein analyses.
Culturing and treatment of H9c2 cardiac cells.
H9c2 cardiac myocytes were cultured in 100-mm dishes in 10 ml DMEM
supplemented with 10% FBS and 1% penicillin/streptomycin (P/S). At a
confluence of 70%, the cells were switched to low serum conditions
(DMEM containing 5% horse serum and 1% P/S) and were differentiated
in the presence of either T3 (100 nM) or vehicle for
periods of 3, 6, and 9 days for mRNA determination and 3 and 9 days for
TR-1 measurements (see below).
Stable overexpression of selected import components in H9c2 cells. H9c2 myocytes were cultured as described above. At 75-80% confluence, the cells were transfected with either a Tim44 [pcDNA 3.1/TIM-Flag (26)] or mtHsp70 (pcDNA 3.1/mtHsp70) expression construct or both. The DNA (3 µg) was diluted in 0.75 ml of serum- and antibiotic-free DMEM and precomplexed with Lipofectamine Plus reagent for 15 min at room temperature. A separate mixture containing Lipofectamine diluted in 0.75 ml of serum- and antibiotic-free DMEM was then prepared and added to the DNA-containing mixture (30 µg Lipofectamine/ml, final concentration). The combined volumes were incubated for an additional 15 min at room temperature. During this time, the plates were washed three times with Dulbecco's PBS (DPBS), and 5 ml of DMEM (no serum or antibiotics) were added to each plate. At the appropriate time, the DNA-Lipofectamine-Lipofectamine Plus solution was added to each plate, and the plates were incubated at 37°C for 3 h. The transfection medium was then discarded and replaced with 10 ml growth medium (DMEM supplemented with 10% FBS, no P/S).
One day after transfection, the cells from each plate were trypsinized and dispersed in two separate dishes. Each group of two plates was labeled for identification purposes. An additional day later, geneticin was added to the growth medium at a concentration of 400 µg/ml. To generate stable cell lines expressing the protein(s) of interest, the cells were propagated for a period of 2 wk, with the growth medium and resistance antibiotic being replaced every 2 days. At the end of the selection period, one plate from each group was trypsinized, resuspended in freezing medium (90% FBS-10% DMSO), and stored in liquid nitrogen. Protein extracts were prepared from the remaining plates for purposes of verifying protein overexpression.RNA isolation and analysis. Total RNA was isolated from cells and frozen tissue powders using TRIzol Reagent (Invitrogen), according to the manufacturer's instructions. Total RNA (10-30 µg) was electrophoresed on formaldehyde gels and transferred to nylon membranes for Northern blot analyses. In some cases, total RNA (20-40 µg) was also applied directly to nylon membranes using a slot-blot apparatus. All membranes were exposed to ultraviolet light to fix the RNA to the membranes. Hybridization was carried out overnight at 42°C with 32P-labeled cDNA probes encoding Tim44, Tim23, Tim17, or Tom34, as done previously (5, 9, 25). To correct for any loading differences between samples, the membranes were hybridized with radiolabeled 18S rRNA probes. All signals were quantified using electronic autoradiography.
Immunoblotting.
Cellular protein was isolated from H9c2 cells and frozen tissue
powders, as done previously (5, 25). Protein extracts (10-50 µg) were subjected to SDS-PAGE and transferred to
nitrocellulose membranes. The membranes were subsequently incubated
overnight with the appropriate primary antibody (1:1,000 for Tom34,
mtHsp70, and TR-1; 1:400 for TIM-Flag) followed by a 1-h
incubation with an anti-mouse (TIM-Flag and mtHsp70) or anti-rabbit
(Tom34 and TR-
1) peroxidase-conjugated secondary
antibody (1:1,000). Signals were detected using the enhanced
chemiluminescence system (Amersham Biosciences, Baie d'Urfé,
Canada), and the band intensities were quantified using Sigma Gel software.
Imunoprecipitation in intact cells. Frozen stocks of stable transfectants were thawed and cultured as described above upon verification of overexpression of the protein(s) of interest from parallel plates. The cells were switched to low-serum medium at 70% confluence and allowed to differentiate for a period of 6 days, at which point the immunoprecipitation was performed. Cells were washed three times with DPBS and then incubated for 90 min at 37°C in 3 ml DMEM devoid of methionine and cysteine. After this incubation period, 100 µCi [35S]methionine were added to each plate, and the cultures were incubated at 37°C for 2 h. Upon removal of the medium, 5 ml PBS containing cycloheximide (100 µg/ml PBS) were added to each plate, and the plates were incubated at 4°C to halt protein synthesis. The PBS was subsequently discarded, and 900 µl of 50 mM Tris (pH 7.4), 5 mM EDTA, 150 mM NaCl, and 0.5% Triton X-100 (TENT buffer) containing 200 µg phenylmethylsulfonyl fluoride (PMSF)/ml TENT were added to each plate. The plates were incubated with end-over-end rocking at 4°C for 30 min, and the cells were then scraped and transferred to Eppendorf tubes. The samples were spun for 10 min at 4°C to precipitate insoluble material, and the supernatants were transferred to new tubes. An equal quantity of protein from each sample, as determined by a Bradford assay, was incubated with 7 µl MDH antibody at 4°C for 1 h. To recover the immune complex, 100 µl of a 10% solution of protein A-Sepharose beads were added to each sample, and the samples were incubated under conditions identical to those used to immunprecipitate MDH. After a brief spin, the precipitated MDH-antibody-protein A-Sepharose complex was washed three times with TENT buffer containing PMSF. Laemmli buffer was then added to each sample, and the beads were dissociated by incubating the samples at 95°C for 5 min. The samples were spun again for 5 min, and the supernatants were subjected to electrophoresis on 10% polyacrylamide gels. The proteins were fixed in the gels by boiling in 5% TCA for 5 min. After the gels were washed in 10 mM Tris base (pH 9.0) for 5 min and 1 M sodium salicylate for 30 min, the gels were dried at 80°C for 1 h. The gels were then subjected to electronic autoradiography or exposed on film, and the bands representing the precursor, intermediate, and mature forms of MDH were quantified using Instant Imager or Sigma Gel software. The calculation of percent import was based on the intensity of mature MDH relative to the sum of the band intensities of the precursor, intermediate, and mature forms times 100.
Cytochrome c oxidase activity. The activity of the cytochrome c oxidase (COX) holoenzyme was determined as described previously (9). Briefly, enzyme extractions were prepared from H9c2 cells at 3, 6, and 9 days of differentiation. A volume of extract (100 µl) was then mixed with a test solution (900 µl) containing reduced cytochrome c, and the rate of cytochrome c oxidation was immediately recorded spectrophotometrically at 550 nm.
Statistics. A Student's unpaired t-test was used to evaluate immunoprecipitation and in vivo data. All time- and treatment-dependent data in cells were analyzed with a two-way ANOVA, whereas one-way ANOVA and Tukey's post hoc test were used for time-dependent data only. Significance was set at the P < 0.05 level.
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RESULTS |
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T3 induces cardiac hypertrophy.
Table 1 summarizes basic anatomic
modifications brought about by the in vivo administration of
T3. The hearts of animals injected with T3
weighed 43% more than those from vehicle-treated animals
(P < 0.01). To determine whether this difference was
because of changes in body weight or the result of T3
treatment, cardiac hypertrophy was assessed by examining the
heart-to-body weight ratios of the animals. This value was 58% higher
(P < 0.01) in T3-treated animals,
reflecting the effectiveness of the treatment.
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Effect of T3 on cardiac Tim23, Tim44, and Tim17 mRNA
levels.
To assess the effect of T3 on the mRNA expression of Tim
machinery components in cardiac muscle, Northern blots were performed (Fig. 1). T3 treatment
resulted in a 55% increase in Tim23 mRNA levels compared with
vehicle-treated animals (P < 0.01). A similar increase
(50%) in Tim44 mRNA levels occurred over the 5-day period (P < 0.02). However, there was no effect of
T3 on Tim17 mRNA levels in cardiac muscle.
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Effect of T3 on cardiac Tom34 mRNA and protein levels.
We also examined the effect of T3 on the mRNA expression of
the outer membrane receptor Tom34 in cardiac muscle (Fig.
2A). Slot-blot analysis
revealed that Tom34 mRNA was elevated by 45% in the hearts of
T3- compared with vehicle-treated animals
(P < 0.01). Surprisingly, this increase in Tom34
transcript levels was not accompanied by any concomitant changes in the
protein expression of this import component (Fig. 2B),
whether examined in whole cell extracts (top) or in isolated
mitochondria (bottom).
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Effect of T3 and differentiation on Tim23, Tim44, and
Tim17 levels in H9c2 cardiac cells.
To determine whether the changes observed in the mRNA expression of the
Tim components were replicable in an in vitro environment, we measured
the mRNA levels of Tim23, Tim44, and Tim17 in vehicle- and
T3-treated H9c2 cardiac cells. In contrast to the findings seen in vivo, T3 had no detectable effect on the mRNA
expression of the Tim components at any of the time points examined
(Fig. 3A). In addition, no
effect of T3 was observed on COX activity (data not shown),
suggesting that T3 does not induce mitochondrial biogenesis
in H9c2 cells. To determine whether this lack of response could be
because of low levels of expression of the T3 receptors (TR) in H9c2 cells, we assessed the levels of TR-1 by
Western blotting. TR-
1 levels were detectable, and there
was no difference between 3- and 9-day differentiated cardiac cells. In
addition, T3 treatment had no effect on the expression of
TR-
1 (Fig. 3A). This lack of responsiveness
of H9c2 cells to T3, in contrast to our observations in the
intact adult rat, is likely developmentally related, since H9c2 cells
are derived from embryonic heart cells. As discussed previously, the
response of the heart to thyroid hormone is known to be developmentally
dependent (25).
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Functional consequences of T3-induced changes for
protein import.
To determine the functional consequences of increased in vivo mRNA
expression of protein import machinery components, we overexpressed selected components in H9c2 cells and measured MDH import rates in
intact cells. H9c2 cells transfected with a Tim44 mammalian expression
construct had levels of Tim44 that were 2.7-fold higher than
pcDNA-transfected control cells (Fig.
4A). Despite this high extent
of overexpression, immunoprecipitation studies revealed no difference
in MDH import rates between control- and Tim44-transfected samples
(Fig. 4A; n = 2).
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DISCUSSION |
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It is well established that T3 has profound effects on mitochondrial morphology and phenotype in cardiac muscle. These adaptations may result from T3-mediated changes at the transcriptional or posttranscriptional level (18, 21) or via an acceleration of posttranslational import in mitochondria (4, 22). This latter change may, in turn, be because of T3-induced changes in the expression of components of the protein import machinery. This machinery consists of a large number of components, only a few of which have been characterized in mammalian cells. One goal of our ongoing work is to characterize the biochemical and functional roles of these import components, their inducibility by physiological stimuli, and their roles in the etiology of mitochondrial disorders. Protein import is an essential step in the overall process of mitochondrial biogenesis, and defects in mitochondrial protein import have been implicated in neurodegenerative disease and muscle myopathies (10, 11, 12). We have previously shown that T3 induces Tom20 (4) and mtHsp70 (22), coincident with increased import rates in mitochondria. The purpose of this study was to investigate the adaptability of other import machinery components in response to T3 and to determine whether some of these changes were of functional significance for protein import during conditions of mitochondrial biogenesis.
Our results show, for the first time, that T3 treatment can
mediate changes in the mRNA expression of various components of the Tim
machinery in vivo. Because T3 stimulates the import of precursors destined for the mitochondrial matrix (4, 22), and interactions of precursor proteins with Tim proteins are required for import in this compartment, these adaptations could play a key role in mediating changes in the mitochondrial phenotype
during T3-induced organelle biogenesis. However, the
responses we observed were not regulated coordinately. Cardiac mRNA
levels of Tim44 and Tim23 were upregulated in response to
T3, similar to the increase previously observed in mtHsp70
mRNA (22). In contrast, Tim17 mRNA levels remained
unaltered. These changes, or lack thereof, may be because of the
presence or absence of functional thyroid response elements (TREs) in
the promoters of the genes encoding these import proteins. Currently,
the promoter regions of the genes encoding the Tim proteins have yet to
be characterized. However, transcriptional mechanisms may account for
the T3-induced increase in mtHsp70 mRNA, since the murine
mtHsp70 gene has been shown to contain TRE-related sites within its
upstream regulatory region (15). The increases in Tim23
and Tim44 mRNA levels could also be in part because of
T3-induced increases in mRNA stability, as observed with
the cardiac - and
-myosin heavy chain subunit mRNAs in response
to T3 (18, 23). These possibilities remain to
be investigated.
We also examined the effect of T3 treatment on the in vivo expression of the outer membrane receptor Tom34. This import component is unique in that yeast or fungal homologues have not yet been identified (17). The important role played by this molecule in mitochondrial protein import is underscored by the finding that its overexpression in COS-7 cells stimulates precursor import in vitro, whereas inhibition of its expression attenuates this process (3). Our preliminary results in skeletal muscle myocytes confirm this finding (A. M. Joseph and D. A. Hood). Interestingly, the intracellular localization of Tom34 is not exclusively restricted to the mitochondrion. In fact, the majority of the protein is found in the cytosolic fraction of rat tissues, suggesting that it may also be involved in protein targeting to the mitochondrion (3). Our results show that Tom34 mRNA levels were increased by 45% in response to T3 treatment. However, the observed changes in Tom34 mRNA expression were not accompanied by any concomitant increases in the protein level of this molecule, either in cardiac mitochondria or in whole tissue extracts. This indicates that posttranscriptional mechanisms are likely involved in regulating the expression of Tom34 in cardiac muscle. Specifically, detectable increases in the steady-state level of this protein may require a larger mRNA induction, or a latency period of longer duration may be necessary for T3-induced increases in Tom34 transcript concentration to be observed at the protein level.
The kinetics of protein import in mitochondria may differ depending on the conditions in which mitochondrial biogenesis is induced. For instance, protein levels of the outer membrane receptor Tom20 are increased in response to T3 (4) but remain unaltered during myocyte differentiation (7), another condition in which mitochondrial content is increased (7, 16). However, increases in mature levels of MDH within the mitochondrion are seen in both conditions, and, in the case of T3, this is because of an accelerated rate of import of MDH in the matrix (4). If increases in precursor import contribute to the differentiation-induced increase in mature MDH content, this would suggest that Tom20 may not play as integral a role in mediating changes in mitochondrial content and composition during myocyte differentiation. In our study, we wanted to determine whether differentiation of H9c2 cells could induce a pattern of expression of the Tim proteins that is distinct from that seen with T3 treatment. We found that differentiation results in a decline in Tim44 and Tim23 mRNA levels (Fig. 3, A and B). At the same time, an increase in the Tim17 mRNA level was observed. These results suggest that Tim17 may play a more important role in protein import than Tim44 or Tim23 during differentiation-induced biogenesis, whereas the opposite may be true during conditions of elevated thyroid hormone levels.
To determine the function of single components of the protein import machinery, selective expression of each, along with functional protein import measurements, is required. To date, only one study in myocytes has attempted to determine whether changes in the expression of import components during conditions of altered mitochondrial biogenesis could have functional consequences for protein import (7). When Tom20 was overexpressed in C2C12 skeletal muscle cells, parallel increases in protein import rates were observed. Conversely, inhibition of Tom20 expression using antisense oligonucleotides led to a similar decline in import rates. In this study, we wished to evaluate the role of Tim44 in import by stably overexpressing this protein and assessing the effects on MDH import via immunoprecipitation from intact cells. Because the function of Tim44 is integrally linked to mtHsp70 (1, 14), we also overexpressed this protein either alone or in combination with Tim44. When Tim44 was overexpressed on its own, no increase in MDH import was observed compared with control cells. On the other hand, mtHsp70 overexpression resulted in a modest increase in MDH import. However, this increase did not parallel the extent of overexpression. Surprisingly, no changes in MDH import rate were seen when Tim44 and mtHsp70 were overexpressed together. These results suggest that at least a fraction of the role that mtHsp70 plays in pulling the precursor into the mitochondrial matrix can be performed independently of its interaction with Tim44. A recent study in yeast, using a temperature-sensitive Tim44 mutant that is impaired in its interaction with mtHsp70, found that precursor proteins with tightly folded domains were imported in the mitochondrial matrix with reduced efficiency (1). A subsequent study determined that those proteins that were imported in mitochondria with this mutation remained uncharacteristically bound to mtHsp70 for extended periods, resulting in a delay in their folding into the mature form within the matrix (6). Thus the interaction of mtHsp70 with Tim44 is likely very important but may not account for all mtHsp70 function during the import process.
In conclusion, proteins of the mitochondrial import apparatus are differentially regulated in response to conditions of altered mitochondrial biogenesis, such as myocyte differentiation and T3 treatment. More importantly, the inducibility of an import machinery component to a stimulus evoking mitochondrial biogenesis does not necessarily reflect the magnitude of its role in mediating precursor protein import during this time. Future studies must focus on measuring the adaptability of other protein import components, specifically those of the mitochondrial inner membrane, and evaluating their functional importance in the import process during conditions of mitochondrial biogenesis in mammalian cells. Such work will have clinical significance for the treatment of mitochondrial diseases that arise because of inherent defects in the mitochondrial protein import system (10, 11, 12).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. M. Mori and colleagues (Kumamoto University School of Medicine, Kumamoto, Japan) for supplying the human (h) Tom34 antibody and cDNA encoding hTom34.
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FOOTNOTES |
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This work was supported by the Canadian Institutes of Health Research. D. A. Hood is the holder of a Canada Research Chair in Cell Physiology.
Address for reprint requests and other correspondence: D. A. Hood, Kinesiology and Health Science, York Univ., Toronto, Ontario, M3J 1P3, Canada (E-mail: dhood{at}yorku.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
September 17, 2002;10.1152/ajpendo.00294.2002
Received 8 July 2002; accepted in final form 4 September 2002.
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