1 Department of Biology and 2 Department of Kinesiology and Health Science, York University, Toronto, Ontario, Canada M3J 1P3; and 3 Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 862, Japan
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ABSTRACT |
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Mitochondrial biogenesis is accompanied by an increased expression of components of the protein import machinery, as well as increased import of proteins destined for the matrix. We evaluated the role of the outer membrane receptor Tom20 by varying its expression and measuring changes in the import of malate dehydrogenase (MDH) in differentiating C2C12 muscle cells. Cells transfected with Tom20 had levels that were twofold higher than in control cells. Labeling of cells followed by immunoprecipitation of MDH revealed equivalent increases in MDH import. This parallelism between import rate and Tom20 levels was also evident as a result of thyroid hormone treatment. Using antisense oligodeoxynucleotides, we inhibited Tom20 expression by 40%, resulting in 40-60% reductions in MDH import. In vitro assays also revealed that import into the matrix was more sensitive to Tom20 inhibition than import into the outer membrane. These data indicate a close relationship between induced changes in Tom20 and the import of a matrix protein, suggesting that Tom20 is involved in determining the kinetics of import. However, this relationship was dissociated during normal differentiation, since the expression of Tom20 remained relatively constant, whereas imported MDH increased 12-fold. Thus Tom20 is important in determining import during organelle biogenesis, but other mechanisms (e.g., intramitochondrial protein degradation or nuclear transcription) likely also play a role in establishing the final mitochondrial phenotype during normal muscle differentiation.
mitochondrial biogenesis; antisense oligodeoxynucleotides; thyroid hormone; translocases of the outer membrane
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INTRODUCTION |
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THE MITOCHONDRION POSSESSES a 16-kb genome that encodes a small portion of its constituent proteins. The remaining proteins are encoded by nuclear genes and synthesized on free ribosomes in the cytosol (2). These nuclear-encoded preproteins are translocated into the mitochondria by an import machinery that is well characterized in Neurospora crassa and Saccharomyces cerivisiae (20), the components of which are beginning to be described in mammals (1, 10, 11, 17, 26, 31, 37, 42). The major complexes comprising the import machinery include translocases of the outer membrane (Toms) and translocases of the inner membrane (Tims) (28, 32, 35). Tom20, Tom22, Tom37, and Tom70 are referred to as import receptors, and they are involved in the initial recognition of preproteins at the surface of the organelle (28, 32, 35). These form dynamic protein oligomers. The Tom20-Tom22 and Tom37-Tom70 heterodimers bind preproteins with their cytosolic domains and initiate import into the mitochondria (21). Certain preproteins are targeted primarily to the Tom37-Tom70 heterodimer through the action of an ATP-requiring cytosolic chaperone termed the mitochondrial import stimulation factor (MSF) (11), whereas others are directed to Tom20-Tom22 via a heat shock protein 70 (HSP70)-preprotein complex in a process that does not require ATP (23). The Tom37-Tom70 subunits mainly recognize preproteins with hydrophobic internal presequences (5), which are then transferred to the Tom20-Tom22 subunits and translocated into the mitochondria. The Tom20-Tom22 heterodimer has a highly acidic cytosolic domain (3) that preferentially binds preproteins possessing basic, cleavable amino-terminal presequences (5), but also those containing a variety of other targeting signals (36), thus implicating this heterodimer in the import of most mitochondrial proteins (28).
Previous studies have shown that when striated muscles such as heart and skeletal muscle undergo mitochondrial biogenesis, there is an increased expression of Tom20 as well as other components of the import machinery, coincident with an increased import of malate dehydrogenase (MDH), a protein destined for the matrix space (6, 39). However, these studies could not conclude that the induction of Tom20 was responsible for the accelerated import observed. To address this, we used muscle cell differentiation-induced mitochondrial biogenesis (27, 38). In this model, increases in nuclear-encoded mitochondrial enzymes have been reported to be four- to sixfold, whereas ultrastructural indexes of mitochondrial inner membrane density increase about twofold (27). Whether this increase in mitochondrial protein is due to transcription or posttranscriptional mechanisms remains unknown. To begin an evaluation of the role of protein import in determining mitochondrial phenotype during differentiation, we manipulated Tom20 levels using expression constructs and antisense oligodeoxynucleotides (ODNs) to assess whether specific changes in Tom20 expression could be associated with changes in the import of a typical matrix protein. Although many studies have examined protein import in vitro (see Refs. 20 and 28 for reviews) relatively few (12, 15, 25, 29, 34, 41, 44) have documented rates of import in intact mammalian cells. In view of the role of Tom20 at the intersection of the MSF-HSP70 chaperone-mediated import pathway, we hypothesized that Tom20 could function as a point of regulation in the import process. This may be particularly relevant to investigate in muscle cells, in view of reported mitochondrial myopathies that are associated with an import malfunction (14, 16). Relevant to this is the recent finding that deletions of the outer membrane import receptor, Tom70, led to mitochondrial DNA deletions and rearrangements, potentially leading to a respiratory chain deficiency (18).
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METHODS |
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Materials. The pCAGGs expression vector used for human Tom20 (hTom20) overexpression was originally described by Niwa et al. (30). Primary antibodies against metaxin and MDH were supplied by Dr. P. Bornstein (University of Washington, Seattle, WA) and Dr. K. Freeman (McMaster University, Hamilton, Ontario, Canada), respectively. Secondary antibodies were purchased from Sigma (St. Louis, MO). The mitochondrial transcription factor A (TFAM) construct was provided by Dr. Rudolf Wiesner (University of Cologne, Germany). The Bcl-2 antibody was obtained from Santa Cruz Biotechnology, (Santa Cruz, CA). Lipofectamine and 3,3,5'-triiodothyronine (T3) were obtained from GIBCO BRL (Burlington, Canada) and Sigma, respectively.
ODN synthesis.
Two different regions of the rat Tom20 cDNA (GenBank accession no.
Q62760) were used in the synthesis of phosphorothioated antisense ODNs
(Dalton Chemical Laboratories, Toronto, Canada). These regions either
overlapped the start codon from position 4 to +12
(5'-CCGGCCCACCATCTTC-3', designated ODN 12) or were located further
downstream from +62 to +77 (5'-CGGTCAAAGTAGATGC-3', designated ODN 77).
A sense ODN corresponding to positions
4 to +12
(5'-GAAGATGGTGGGCCGG-3', designated 12S) was synthesized as a control.
Cell culture and preparation of cell extracts.
C2C12 myoblasts were cultured in 100-mm dishes in 10 ml of growth
medium [DMEM including 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin] at 37°C under an atmosphere of 5% CO2-95% air. At a confluence of 60-70%, the cells
were washed twice with Dulbecco's phosphate-buffered saline (DPBS),
switched to a low serum medium (DMEM containing 5% heat-inactivated
horse serum and 1% penicillin-streptomycin), and allowed to
differentiate for the indicated times, as done previously
(9). For T3 treatment, cells cultured as
described above were incubated with either 10 mM NaOH (vehicle) or
T3 (100 nM) and allowed to differentiate for 4 and 8 days.
The myotubes were solubilized with 2× Laemmli buffer (62.5 mM
Tris · HCl, pH 6.8, 20% glycerol, 2% SDS, 5%
-mercaptoethanol), including protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 1 µM dithiothreitol, 10 µg/ml
leupeptin and pepstatin), and scraped from the plates to prepare cell
extracts. The protein concentrations were determined using the Bradford
assay (4).
Transfection. For the transfection of Tom20, C2C12 cells were cultured as described above, then were transiently transfected with 20 µg of control plasmid or Tom20 expression construct (pCAGGS-hTom20) using a poly-L-ornithine-DMSO shock method (33). Cells were then switched to low serum conditions and allowed to differentiate for the indicated times. For transfection of the ODNs, C2C12 cells were cultured in 24-well plates as described above. After 24 h, the cells were rinsed with 1 ml of DMEM, and each well was incubated with 0.3 ml DMEM containing Lipofectamine at a final concentration of 10 µg/ml, and ODNs at a concentration of 200 nM for 5 h at 37°C. The medium containing Lipofectamine was then replaced with growth medium lacking bacterial antibiotics and, after 24 h, the cells were switched to low serum conditions and allowed to differentiate for the indicated times. The cells were then scraped and the resulting extracts were subjected to 8% SDS-PAGE and analyzed by immunoblotting.
Metabolic labeling of cells and immunoprecipitation. Culture and transfection of C2C12 cells were performed as described above, except that 150-mm dishes and 40 µg of plasmid DNA were used for transfection. After 6 days, C2C12 myotubes were washed twice with DPBS and then incubated at 37°C for 1.5 h in 6 ml of cysteine-methionine-free medium. The myotubes were then radiolabeled with 100 µCi of [35S]methionine-[35S]cysteine (ICN, Montreal, Canada) for either 0.5 or 1 h. The myotubes were washed with cold DPBS containing 100 µg/ml cycloheximide and permeabilized with TENT buffer (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100). The resulting cell lysate was then centrifuged at 10,000 g for 10 min at 4°C. MDH was immunoprecipitated with 7 µl of MDH antiserum and 100 µl of a 10% suspension of protein A-Sepharose (Amersham-Pharmacia Biotech, Baie D'Urfé, Canada). The protein samples were subjected to 10% SDS-PAGE and fluorography, dried, and quantified by electronic autoradiography.
Immunoblotting. Cell extracts were subjected to 8% (for MDH and metaxin), 10% (for Bcl-2), or 12% (for Tom20) SDS-PAGE as described above and then electrophoretically transferred to nitrocellulose membranes (Hybond-C, Amersham). The membranes were blocked with 5% skim milk-PBS and incubated separately with anti-Tom20 (1:1,000), anti-metaxin (1:1,000), anti-MDH (1:1,000), or anti-Bcl-2 (1:100) antibodies. The Tom20 signal was detected using anti-rabbit IgG coupled to alkaline phosphatase (1:1,000). MDH, metaxin, and Bcl-2 signals were detected using anti-rabbit, peroxidase-coupled IgG (1:1,000) using the ECL chemiluminescence detection system (Amersham-Pharmacia Biotech). The blots were then quantified by laser densitometry (39).
Protein import in vitro. To evaluate the functional role of Tom20 in mediating the import of matrix and outer membrane proteins into mitochondria in vitro, import reactions were performed with isolated rat muscle intermyofibrillar mitochondria. The isolation of mitochondria and the import of MDH and Bcl-2 were performed as done previously (39, 40). For the import of TFAM, the construct was linearized with Xho I, transcribed with T7 RNA polymerase, and the mRNA was translated in a rabbit reticulocyte lysate. Import was performed for 15 min (30°C). In all cases, the import reactions were performed in the presence or absence of Tom20 antibody (200 ng/ml).
Cytochrome-c oxidase enzyme activity.
C2C12 myotubes were scraped from 100-mm plates at 0, 2, 4, 6, and 8 days of differentiation in ice-cold DPBS, pelleted, and resuspended in
200 µl of extraction buffer (100 mM NaKPO4, 2 mM EDTA, pH
7.2). Cells were sonicated (4 × 10 s) and subjected to one
freeze-thaw cycle. After a clearing spin (5 min, 4°C), the supernate
was used in the enzyme assay. Approximately 350-400 µg of
cellular protein were incubated with a reduced cytochrome c
solution (1 mg/ml), and cytochrome-c oxidase enzyme activity (nmol · min1 · mg protein
1)
was determined spectrophotometrically (
= 550 nm), as done previously (13).
Statistics. Statistical analysis was done using Student's paired t-test. The data are expressed as means ± SE, and values of P < 0.05 were used to indicate statistical significance.
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RESULTS |
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Protein expression in C2C12 cells during cellular differentiation.
When C2C12 myoblasts were switched to low serum conditions, they fused
and formed myotubes that contracted asynchronously. During this
differentiation period (0-8 days), total cellular protein
increased progressively from 467 ± 75 to 1,913 ± 151 µg/plate (n = 6). As an indicator of mitochondrial
biogenesis, cytochrome-c oxidase enzyme activity was used,
since it contains multiple protein subunits derived from both the
nuclear and the mitochondrial genomes (19). Enzyme
activity increased approximately threefold from 78.2 ± 8.1 to
237.9 ± 14.1 nmol · min1 · mg
protein
1 (n = 6-7 samples/day)
between 0 and 6 days and remained constant at 8 days of
differentiation. In contrast, Tom20 was expressed at a constant level
per microgram of protein at all indicated time points during
differentiation (Fig.
1A). Levels of the
35-kDa precursor MDH (pMDH) increased as a function of differentiation time, as did levels of mature, imported MDH (14-fold) and metaxin (7-fold; Fig. 1, B and C). Bcl-2 protein levels
declined to ~40% of that found at day 0 by days
6-8 (Fig. 1D). These data reveal that the
expression of Tom20, metaxin, mature MDH, and Bcl-2 is differentially
regulated during mitochondrial biogenesis induced during muscle cell
differentiation. Further, they indicate that dramatic increases in the
level of the mature form of MDH can occur during normal differentiation
even in the absence of changes in Tom20 concentration.
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Effect of T3.
We have previously shown that T3 treatment results in an
upregulation of both the import rate as well as the expression of components of the import machinery in cardiac mitochondria when administered in vivo (6). Here we used T3
treatment to investigate the inducibility of the protein import system
as a whole in skeletal muscle cells. T3 treatment resulted
in a significant increase in Tom20 and mature MDH levels compared with
vehicle-treated cells at days 4 and 8 of
differentiation (Fig. 2, A and
C). In contrast, metaxin (Fig. 2B), pMDH (Fig.
2C), and Bcl-2 (Fig. 2D) protein levels were not
significantly altered. These results indicate that T3
treatment of cultured skeletal muscle cells can bring about specific
alterations in mitochondrial composition, in particular related to the
mitochondrial import pathway.
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Tom20 expression and MDH import.
To evaluate whether changes in only one component (Tom20) of the
import machinery could influence the rate of mitochondrial protein
import in intact cells, we forced the overexpression of Tom20, and we
inhibited Tom20 expression in normal differentiating cells using an
antisense ODN approach. Evidence for the overexpression of Tom20 in
C2C12 cells is shown in Fig.
3A. Increases
(P < 0.05) in Tom20 protein levels of 2.4 ± 0.1- and 1.8 ± 0.4-fold compared with control are evident at 4 and 8 days of differentiation, respectively (Fig. 3A). The amount
of newly synthesized MDH translocated into the mitochondria of the
C2C12 myotubes overexpressing Tom20 was also assessed under the same
conditions. The results show that increases in MDH import of 1.9 ± 0.3- and 1.6 ± 0.3-fold (P < 0.05) above
control cells at 30 and 60 min of metabolic labeling, respectively,
were observed in cells in which Tom20 was overexpressed (Fig.
3B). No consistent effect of Tom20 overexpression on pMDH levels was detected. These data also appear to indicate that
overexpression of human Tom20 adequately complemented the mouse
mitochondrial import machinery.
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Effect of Tom20 inhibition on import into the matrix and outer
membrane.
To evaluate the importance of Tom20 in the import of proteins into the
matrix and outer membrane, the import of the matrix proteins MDH and
TFAM (Fig. 5, A and
C) was compared with that of the outer membrane protein
Bcl-2 (Fig. 5B) in response to inhibition of function with
Tom20 antibody. TFAM import was most dramatically reduced, to only
35 ± 9% of control (P < 0.05), whereas MDH
import was reduced to 59 ± 3% of control (P < 0.05). In contrast, Bcl-2 import into the outer membrane was unaffected
(102 ± 9% of control) at this concentration of Tom20 antibody
(Fig. 5D).
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DISCUSSION |
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Tom20 is part of a hetero-oligomeric protein import receptor that mediates the import of cytosolic preproteins into the mitochondria. In yeast, this import receptor functions as a Tom20-Tom22 heterodimer, and it appears to be involved in the import of all preproteins known to use the general import machinery (22, 28). A lack of Tom20 causes cell growth arrest as well as a reduction in the mitochondrial capacity for oxidative phosphorylation and protein synthesis (28). In contrast to Tom20 deficiency, Tom20 overexpression can be achieved in skeletal muscle by a program of increased contractile activity (39). This treatment induces the expression of other members of the mitochondrial protein import machinery as well (e.g., MSF, mitochondrial HSP70), and the result is a coincident increase in the capacity for precursor protein import, as measured in vitro in isolated mitochondria. This is likely an important contributing process to the overall increase in mitochondrial biogenesis observed (39). A similar parallel relationship between Tom20 levels and in vitro measured protein import capacity has been shown in cardiac mitochondria as a result of T3 administration in vivo (6). These results suggest that changes in the expression of mitochondrial import machinery components are important in accelerating the translocation of precursor proteins into the mitochondria. The extension of those in vitro import assays was to evaluate the functional role of Tom20 in an intact cellular environment. In most in vitro studies the preprotein is synthesized in a reticulocyte lysate and imported into isolated mitochondria. However, this situation does not reflect changes that may occur in the cytosol of an intact cell during the differentiation process or in response to physiological perturbations. Relatively few studies have examined mitochondrial protein import in vivo (12, 15, 25, 29, 34, 41, 44), and none have done so under the conditions of enhanced mitochondrial biogenesis. Given that a rapid increase in mitochondrial biogenesis occurs in intact muscle cells during differentiation (27, 38), we used this model to study protein import. Under these conditions, we wanted to determine whether specific changes in the expression of Tom20 alone could alter the import of a matrix-destined protein. Our results indicate that the import of MDH paralleled that of Tom20 during both forced overexpression, as well as during conditions of reduced expression, of the import receptor. In addition, this proportionality was also evident when a mature MDH level was measured under conditions of mitochondrial biogenesis induced by T3 treatment. These effects on MDH import were independent of changes in precursor MDH (pMDH) levels, and they indicate that the rate of protein import can be directly modified by changes in the expression of the single mitochondrial outer membrane import receptor Tom20. This not only confirms its role in the import process, but suggests its involvement in determining the kinetics of the process.
However, we also present some evidence for a dissociation between the expression of Tom20 and the level of imported MDH. This result was apparent at days 4, 6, and 8 of normal differentiation, in which the level of imported MDH far exceeded the change in Tom20 level during the same period. In this case, levels of pMDH were also increased along with the imported form of the protein, suggesting that events upstream of import (e.g., transcription) may be involved in regulating the import process, independent of Tom20 levels. In addition, the large accumulation of MDH in the mitochondrial matrix during normal differentiation could be a result, in part, of a reduced rate of intramitochondrial protein degradation (i.e., increased protein stability) as differentiation proceeds. These interesting possibilities remain to be evaluated.
It is established that Tom20 is involved in mediating the import of a variety of proteins destined for different mitochondrial compartments (28). Antibody inhibition studies have shown that the import of matrix and outer membrane proteins can be reduced by applying high concentrations of Tom20 antibody (24). However, using low amounts of antibody, our data suggest that the sensitivity of the import process directed toward the matrix in response to Tom20 inhibition is greater than that of import into the outer membrane, at least with respect to the proteins used in the present study. These data indicate that Tom20 does mediate MDH import into mitochondria, and they support the contention that the interaction of Tom20 with preproteins differs (5, 41). It is of interest to relate these results with those of Dekker et al. (7), in which it was reported that only one in four Tom complexes formed a stable supercomplex, with membrane-spanning preproteins linking all the Tim core complexes. If these overabundant Tom20 complexes that are not involved in interacting with Tim proteins are uniquely involved in the import of precursor proteins into the outer membrane, then this suggests that import into the outer membrane may follow different import kinetics and regulatory mechanisms than import into the matrix, even though Tom20 is involved in both processes. Our data certainly suggest that Tom20 plays a regulatory role in the import of MDH into the matrix under most conditions.
We also investigated the expression pattern of two mitochondrial outer membrane proteins, metaxin and Bcl-2, during muscle cell differentiation. The gradual decline in Bcl-2 concentration is similar to the pattern observed in differentiating ML-1 cells (43) and is consistent with the idea that Bcl-2 is involved at an early myogenic stage in promoting muscle cell proliferation (8). In contrast to the decline in the levels of Bcl-2, the expression of metaxin, an import receptor subunit that is a homologue of Tom37, was increased during differentiation. At this time, the role of metaxin and its relationship to Tom20 and the import process itself is not defined. Nonetheless, the expression pattern of mitochondrial proteins observed during muscle differentiation indicates that proteins destined for the same mitochondrial compartment (e.g., the outer membrane) are subjected to different regulatory influences.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Heart and Stroke Foundation of Canada (Ontario). The antibodies against metaxin and MDH were kindly supplied by Dr. P. Bornstein (University of Washington, Seattle, WA) and Dr. K. Freeman (McMaster University, Hamilton, Canada), respectively. The cDNA encoding TFAM was provided by Dr. Rudolf Wiesner (University of Cologne, Germany).
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. A. Hood, Dept. of Biology, York Univ., 4700 Keele St., Toronto, Ontario, Canada, M3J 1P3 (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.
Received 17 November 1999; accepted in final form 4 May 2000.
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