Contractile activity-induced adaptations in the mitochondrial protein import system

Mark Takahashi, Alan Chesley, Damien Freyssenet, and David A. Hood

Department of Biology and Department of Kinesiology and Health Science, York University, Toronto, Ontario, Canada M3J 1P3

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We previously demonstrated that subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subfractions import proteins at different rates. This study was undertaken to investigate 1) whether protein import is altered by chronic contractile activity, which induces mitochondrial biogenesis, and 2) whether these two subfractions adapt similarly. Using electrical stimulation (10 Hz, 3 h/day for 7 and 14 days) to induce contractile activity, we observed that malate dehydrogenase import into the matrix of the SS and IMF mitochondia isolated from stimulated muscle was significantly increased by 1.4- to 1.7-fold, although the pattern of increase differed for each subfraction. This acceleration of import may be mitochondrial compartment specific, since the import of Bcl-2 into the outer membrane was not affected. Contractile activity also modified the mitochondrial content of proteins comprising the import machinery, as evident from increases in the levels of the intramitochondrial chaperone mtHSP70 as well as the outer membrane import receptor Tom20 in SS and IMF mitochondria. Addition of cytosol isolated from stimulated or control muscles to the import reaction resulted in similar twofold increases in the ability of mitochondria to import malate dehydrogenase, despite elevations in the concentration of mitochondrial import-stimulating factor within the cytosol of chronically stimulated muscle. These results suggest that chronic contractile activity modifies the extra- and intramitochondrial environments in a fashion that favors the acceleration of precursor protein import into the matrix of the organelle. This increase in protein import is likely an important adaptation in the overall process of mitochondrial biogenesis.

mitochondrial subfractions; mitochondrial biogenesis; exercise; gene expression; molecular chaperones

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

MITOCHONDRIAL BIOGENESIS appears to begin with an increase in the quantity of phospholipids comprising the mitochondrial membranes to expand the membrane surface area (7, 29). This is followed by an increase in the quantity of mitochondrial proteins embedded in the inner and outer membrane systems, as well as within the matrix. The incorporation of matrix and membrane proteins into the organelle is the subject of intense study in eukaryotes such as yeast and fungi (20, 25) but is less well understood in mammalian tissues. Skeletal muscle provides a useful model in this regard, since mitochondrial biogenesis can be elicited by the imposition of various physiological demands. For example, chronic contractile activity leads to large changes in mitochondrial content (6, 8). Although a number of studies have focused on the modifications observed in gene expression that occur during such conditions (9, 17, 19, 32, 33), no study has investigated the potentially adaptive response of the protein import system during mitochondrial biogenesis.

Another feature of skeletal muscle is that it contains mitochondria that are physically and biochemically distinct (3, 10, 11, 14, 21). On the basis of their locations within the muscle, they have been termed subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. These two subfractions differ with respect to respiratory rate and nuclear-encoded protein composition, with two- to threefold differences in the rate of precursor protein import (28). Because earlier studies have illustrated that the two subfractions adapt differentially to changes in the level of contractile activity (10, 11, 14, 16), we wanted to determine whether adaptations in the protein import pathway occurred in parallel in these heterogeneous mitochondrial subfractions. In addition, we examined potential alterations in the expression of selected components of the mitochondrial protein import machinery to address some potential underlying causes involved in the adaptation of the import process.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals and surgery. Male Sprague-Dawley rats (Charles River, St. Constant, PQ, Canada) weighing 389 ± 8 g (n = 42) were housed individually and given food and water ad libitum. The procedure as outlined previously (29) was followed for implantation of electrodes and stimulation of animals. Briefly, animals were anesthetized with pentobarbital sodium (60 mg/kg). Under aseptic conditions, two stimulating electrodes (Medwire, Leico Industries, New York, NY) were passed subcutaneously from the thigh and exteriorized at the back of the neck. At the thigh the electrodes were sutured on either side of the common peroneal nerve, and the overlying tissues were sutured closed. Sterile ampicillin (Penbritin, Ayerst, Montreal, PQ, Canada) was injected to minimize infection. A portable stimulator unit (31) was placed in a plastic housing and attached to the exteriorized wires at the neck. The whole assembly was then secured to the animal's back with cloth tape. Care was taken to ensure that the procedure did not restrict animal movement, cause discomfort, or restrict breathing. Stimulation was adjusted at the time of electrode implantation to result in palpable contractions of the tibialis anterior and extensor digitorum longus muscles. The contralateral limb was used as a nonstimulated internal control in all animals.

Stimulation protocol and tissue sampling. When animals had recovered from surgery for >= 1 wk, they were stimulated (10 Hz, 0.1-ms duration) for 3 h/day for 7 (n = 32) or 14 (n = 13) days, as done previously (30). After the indicated number of days, stimulation was terminated, and 21 h later the animals were anesthetized with pentobarbital sodium (60 mg/kg) and the tibialis anterior and extensor digitorum longus muscles were quickly removed from the stimulated and the contralateral limbs. Muscles were immediately placed into ice-cold buffer for mitochondrial isolation.

Mitochondrial isolation. IMF and SS mitochondrial subfractions were isolated by differential centrifugation after a brief Polytron homogenization of rat tibialis anterior muscle, as described previously in detail (3, 28). IMF and SS mitochondria were resuspended in a buffer consisting of 10 mM HEPES, 0.25 M sucrose, 2.5 mM potassium phosphate dibasic, 10 mM succinate, 0.21 mM ADP, and 1 mM dithiothreitol (pH 7.4), and protein concentrations were measured (2).

Isolation of cytosolic fraction. After the final centrifugation to isolate the SS mitochondria, the supernate was removed and retained. This supernate, obtained from the chronically stimulated and control muscles, was centrifuged at 100,000 g for 1 h at 4°C. The supernate was concentrated in an ultrafiltration cell (Amicon, Beverly, MA; mol wt cutoff 10 kDa) to a volume of <1 ml, and this was referred to as the cytosolic fraction.

In vitro synthesis of precursor proteins. Full-length cDNAs encoding malate dehydrogenase (MDH, pGMDH) and Bcl-2 (pBCL-2) were provided by Dr. A. Strauss (Washington University School of Medicine) and Dr. G. C. Shore (McGill University), respectively. pBCL-2 DNA was isolated using miniprep plasmid DNA isolations followed by RNase treatment (1.0 µg RNase/2.5 µg DNA) for 1 h at room temperature. pBCL-2 was linearized by Pst 1, treated with proteinase K (0.1 mg/ml final concentration) for 1 h at 37°C, and recovered by phenol extraction and ethanol precipitation. pGMDH DNA was isolated using CsCl gradient centrifugation and linearized using BamH I. Linearized plasmids were resuspended in Tris-EDTA (pH 7.8) to a final concentration of 0.8 mg/ml. Transcription reactions were carried out with SP6 and T3 RNA polymerase for 90 min at 40°C for MDH and 37°C for Bcl-2 mRNA synthesis, respectively (5, 28). Synthesized MDH and Bcl-2 mRNAs were isolated by phenol extraction followed by ethanol precipitation, and final concentrations were adjusted to 2 mg/ml. In vitro translation was performed at 30°C using a cell-free rabbit reticulocyte lysate (Promega) in the presence of [35S]methionine (Amersham; 20 µCi/reaction).

Import of precursor proteins into isolated mitochondrial subfractions. Isolated IMF and SS mitochondria and the reticulocyte lysate containing the translated radiolabeled precursor proteins were allowed to equilibrate separately at 30°C for 10 min. To initiate the protein import reaction, reticulocyte lysate was added to the mitochondrial samples. Final import reactions consisted of 25 µg of mitochondria, 12 µl of reticulocyte lysate, and 0, 1, 5, or 35 µg of cytosolic fraction incubated at 30°C for 10 min (MDH) or for 0, 1, and 5 min (Bcl-2). Mitochondria were then recovered by centrifugation (18,000 g) through 600 µl of 20% (wt/vol) sucrose in 0.1 M potassium chloride, 2 mM magnesium chloride, and 20 mM HEPES (pH 7.4) at 4°C for 10 min. The supernate was removed, and the pellet was resuspended in 0.6 M sorbitol and 20 mM HEPES-KOH (pH 7.4). Mitochondria that were used for Bcl-2 import were additionally treated with 0.1 M NaCO3 (pH 11.5) for 30 min on ice after the import incubation. Mitochondria were then reisolated by centrifugation, and the samples were resuspended as described above. Equal volumes of sample buffer (10% vol/vol glycine, 80 mM SDS, 62.5 mM Tris · HCl, pH 6.8, 5% vol/vol 2-mercaptoethanol) and dye (5% vol/vol) were added. Samples were denatured (5 min) and electrophoresed through an 8% SDS-polyacrylamide gel at low voltage overnight. After electrophoresis, gels were treated for 5 min in boiling 5% TCA, 5 min in distilled water, 5 min in 10 mM Tris base (pH 9.0), and 30 min in 1 M sodium salicylate. Treated gels were subsequently dried and quantified by electronic autoradiography (Instantimager, Packard) or exposed to film (beta -MAX, Amersham) at -80°C.

Role of mitochondrial import-stimulating factor in protein import. To establish the role of the mitochondrial import-stimulating factor (MSF) in MDH import, the procedure as outlined by Alam et al. (1) was followed. Briefly, 200 µg of cytosolic protein were preincubated with 1 mg/ml of anti-MSF antibody (from Drs. M. Sakaguchi, K. Mihara, and T. Omura) specific for the large (MSF-L) or the small subunit (MSF-S1) for 10 min at room temperature. Appropriate quantities of the cytosolic fraction were then added to the mitochondria and allowed to equilibrate at 30°C for 10 min before the addition of the lysate to initiate the protein import reaction.

RNA isolation and hybridization. Total RNA was isolated as previously described (4). Briefly, tissue powders (100-200 mg) were homogenized in 1.25 ml of denaturing solution (4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% sarkosyl, and 0.1 M beta -mercaptoethanol), 1.25 ml of phenol, and 0.125 ml of 2 M sodium acetate solution (pH 4.0). Phase separation was achieved by the addition of 0.4 ml of chloroform, and the mixture was centrifuged for 15 min at 12,000 g (4°C). The aqueous phase was then quantitatively removed, 1 ml of isopropanol was added, and total RNA was precipitated for 10 min at room temperature. The RNA was centrifuged for 10 min at 12,000 g (4°C), and the supernate was discarded. The pellet was washed with 1 ml of 75% ethanol and transferred to an Eppendorf tube. Total RNA was centrifuged at 10,000 g (4°C) for 5 min, the supernate was discarded, and the pellet was dried in a vacuum desiccator. The RNA was then resuspended in 75-300 µl of sterile H2O to a concentration of ~1 µg/µl, determined by measuring the absorbance at 260 nm.

To quantify specific mRNA levels, total RNA was applied to a nylon membrane (Hybond N, Amersham) using a vacuum slot-blotting apparatus, or samples were run on agarose-formaldehyde gels and then transferred to a nylon membrane by capillary action overnight (4, 9). Blots were air dried, and the RNA was fixed to the membrane by exposure to ultraviolet light for 15 min. Deoxy-[32P]CTP-labeled cDNA probes specific for human Tom20 (from Dr. N. Nomura), rat MDH (from Dr. A. Strauss), human mtHSP70 (from Dr. R. Morimoto), and rat MSF-L (from Drs. M. Sakaguchi, K. Mihara, and T. Omura) were hybridized overnight with the blots at 42°C (4, 9, 22). On the next day, blots were washed three times for 10 min each in 2× SSC (1× SSC = 0.15 M NaCl-0.015 M sodium citrate) and 0.1% SDS at room temperature. Subsequently, all blots were washed stringently in 0.1× SSC and 0.1% SDS. Wash conditions were as follows: twice for 15 min each at 55°C for Tom20, twice for 15 min each at 50°C for mtHSP70, and 15 min at 55°C and 15 min at 60°C for MDH and MSF-L. Blots were then quantified electronically (Instantimager) or exposed to autoradiography film (Hyperfilm, Amersham) at -70°C. After hybridization, blots were stripped and rehybridized with a radiolabeled cDNA encoding 18S rRNA (4), the level of which does not change during the course of 7 or 14 days of chronic stimulation for 3 h/day (M. Takahashi and D. A. Hood, unpublished observations). Thus all mRNA values were corrected for loading on the basis of the 18S rRNA content.

Western blot analyses of mtHSP70, MDH, and MSF. Frozen mitochondrial and cytosolic samples were used for the quantification of specific proteins involved in the protein import pathway. Equal amounts of mitochondrial or cytosolic protein were added to each lane of an SDS-polyacrylamide gel. After overnight electrophoresis, gels were electroblotted (semidry electroblotter, Enprotech) for 1.5 h onto nitrocellulose membranes (Hybond C, Amersham) and probed with monoclonal antibodies directed against mtHSP70 (1:1,000; StressGen Biotechnologies, Victoria, BC, Canada) or sera containing antibodies directed against MSF-L (diluted 1:2,500), Tom20 (1:1,000; from Dr. M. Mori), or mitochondrial MDH (1:1,000; from Dr. K. Freeman). Sheep anti-mouse IgG (1:1,000) conjugated to alkaline phosphatase was used for the detection of mtHSP70, and goat anti-rabbit IgG (1:1,000) conjugated to alkaline phosphatase was used for detection of MSF-L, Tom20, and MDH. Blots were incubated for ~5 min with substrates 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. The color products were quantified by laser densitometry.

Statistics. Specific mRNA and Western blot data were analyzed using paired Student's t-tests (alpha  = 0.05). Two-way ANOVA with repeated measures was carried out on the import data as a function of the cytosol protein concentration. Values are means ± SE.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Mitochondrial biogenesis. Chronic stimulation of the tibialis anterior muscle for 7 and 14 days resulted in significant increases in the extractable content of mitochondrial protein (Table 1). The SS and IMF mitochondrial content from 7-day-stimulated muscles was increased by 1.3 ± 0.1-fold (Table 1; n = 12) above the control muscle. After 14 days of stimulation, the SS and IMF protein content within the stimulated muscles remained elevated to 1.3 ± 0.1- and 1.5 ± 0.2-fold (P <=  0.05, n = 12) above the control muscles, respectively.

                              
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Table 1.   Total mitochondrial protein content in contralateral control muscle and muscle chronically stimulated for 7 and 14 days

Total RNA content in stimulated muscle was increased (P <=  0.05, n = 7) by ~1.4-fold after 7 and 14 days of stimulation (Table 2) compared with the control muscles, similar to our previous findings with this model (9). The mRNA level encoding the mitochondrial matrix enzyme MDH was significantly increased by 1.3 ± 0.1-fold (n = 7) in stimulated muscle compared with the control muscle after 7 days of stimulation (Fig. 1A, Table 2). However, MDH mRNA levels were no longer increased by 14 days of chronic stimulation. MDH protein levels within the two mitochondrial subfractions were more extensively altered by chronic contractile activity. By 7 days the MDH contents were 1.9 ± 0.2- and 1.6 ± 0.2-fold greater in IMF and SS mitochondria isolated from stimulated muscle. After 14 days of stimulation the MDH content was 2.9 ± 1.1- and 1.6 ± 0.2-fold greater in SS and IMF mitochondria, respectively, than in the control muscle (Fig. 1B, Table 3; P <=  0.05, n = 7).

                              
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Table 2.   Total RNA in contralateral control muscle and muscle chronically stimulated for 7 and 14 days and specific mRNA content after 7 and 14 days of chronic stimulation


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Fig. 1.   Changes in gene expression of specific intra- and extramitochondrial proteins resulting from chronic contractile activity. A: autoradiograms of Northern blots and slot blots probed with malate dehydrogenase (MDH), large mitochondrial import-stimulating factor subunit (MSF-L), Tom20, and mtHSP70 cDNA. Blots contained total RNA isolated from 7-day chronically stimulated (S) and contralateral control (C) muscles as follows: 12 µg/lane (MDH), 4 µg/lane (MSF-L), 20 µg/slot (Tom20), and 8 µg/slot (mtHSP70). B: immunoblots of MDH, Tom20, and mtHSP70 protein content in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. Blots contained total mitochondrial protein isolated from 7-day chronically stimulated and contralateral control muscles as follows: 30 µg/lane (MDH), 150 µg/lane (Tom20), and 30 µg/lane (mtHSP70). MSF-L protein content was quantified using 300 µg/lane of cytosolic fraction isolated from stimulated and control muscles.

                              
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Table 3.   Protein content of specific cytosolic and mitochondrial proteins after 7 and 14 days of chronic stimulation

Protein import into SS and IMF mitochondrial subfractions. MDH import into mitochondrial subfractions isolated from control muscles was 2.8 ± 0.4-fold greater in the IMF than in the SS mitochondria and closely matched our previous findings (28) (Fig. 2A, lane 1 vs. lane 7). Chronic contractile activity resulted in significant increases in the ability of both mitochondrial subfractions to import MDH. After 7 days of chronic stimulation, MDH import was 1.4 ± 0.03- and 1.7 ± 0.2-fold (n = 5-6) greater in SS and IMF mitochondria from stimulated muscle (Fig. 2B), respectively, than in mitochondria from control muscle (Fig. 2A, lane 1 vs. lane 4 and lane 7 vs. lane 10). The SS mitochondria obtained from 14-day-stimulated muscle illustrated a further increase in MDH import to 1.7 ± 0.09-fold (n = 6) above that of the SS mitochondria obtained from control muscle. However, no significant differences in MDH import were observed in IMF mitochondria isolated from stimulated muscle compared with IMF mitochondria from control muscle at 14 days.


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Fig. 2.   MDH protein import into SS and IMF mitochondria isolated from 7- and 14-day chronically stimulated and contralateral control muscles. A: autoradiogram of MDH protein import in presence of buffer (-), 35 µg of cytosolic fraction isolated from control muscles (C), or 35 µg of cytosolic fraction isolated from chronically stimulated muscles (S). Upper 35-kDa band represents precursor MDH protein bound to mitochondrial outer surface; lower 33-kDa band represents mature processed MDH protein within mitochondria. Imported MDH was quantified using electronic autoradiography. B: graphic representation of quantification of several experiments (n = 6-7) as shown in A, illustrating quantity of MDH import, expressed as percentage of MDH import into control mitochondria as a function of days of stimulation. Con, contralateral control mitochondrial protein import. * P <=  0.05 compared with appropriate control import values.  Significant difference in import between mitochondria isolated from 7- and 14-day-stimulated muscles (P <=  0.05).

Bcl-2 import into isolated mitochondria. To assess the potential adaptive responses of the import system that directs precursor proteins to a compartment distinct from the matrix space, we investigated the import of the outer membrane protein Bcl-2. A rapid increase in Bcl-2 insertion into the outer membrane was observed by 1 min, and this was further increased by 5 min of incubation in IMF mitochondria (Table 4). However, no significant differences were observed in the import of Bcl-2 in IMF mitochondria isolated from stimulated and control muscles.

                              
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Table 4.   Bcl-2 import into IMF mitochondria from control and 7-day-stimulated skeletal muscle

Influence of cytosolic fraction on protein import. To investigate the influence of the cytosol on protein import, we isolated a cytosolic fraction from stimulated and control muscles and measured the effect on the rate of protein import. The addition of up to 35 µg of cytosolic protein to the import mixture resulted in an approximately twofold increase in the ability of mitochondria isolated from control muscle to import protein (Fig. 3A; P <=  0.05, n = 48). No significant differences were observed in the import rate when the cytosolic fraction was derived from the control or the stimulated muscle (Fig. 3). Import into SS mitochondria isolated from stimulated muscles was similarly enhanced in the presence of 35 µg of control or stimulated muscle cytosolic fraction (Fig. 3B). In contrast, import in IMF mitochondria isolated from stimulated muscle was blunted in response to the addition of cytosol relative to the other mitochondrial subfractions. To determine whether the sensitivity of the mitochondria isolated from the chronically stimulated and control muscles differed in response to the addition of cytosolic fraction, lower amounts of cytosolic protein (1 and 5 µg) were used in the import reaction (Fig. 3). The addition of 1 µg of cytosolic protein led to very little increase in MDH import into SS and IMF mitochondria. A modest effect of 5 µg of cytosolic fraction on MDH import was apparent in both mitochondrial subfractions, but particularly in SS mitochondria isolated from control, nonstimulated muscle. Whether this result illustrates a differential sensitivity of the import process to added cytosol compared with SS mitochondria isolated from stimulated muscle requires further study.


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Fig. 3.   Influence of cytosolic fraction on MDH import into isolated SS and IMF mitochondria. A: protein import measured in SS and IMF mitochondria isolated from control nonstimulated muscles after pretreatment with 0, 1, 5, and 35 µg of cytosolic fraction isolated from control (C) or chronically stimulated (S) muscles (n = 6-7 experiments). B: MDH import measured in SS and IMF mitochondria isolated from 7-day chronically stimulated muscles after pretreatments outlined in A.

Expression and function of MSF. To determine the possibility that the MSF was involved in the import process, expression of this cytosolic chaperone was evaluated. The tissue concentration of the mRNA encoding MSF-L was not significantly different from the control muscle after 7 or 14 days of stimulation (Fig. 1A, Table 2; n = 6-7). However, quantities of the MSF-L protein were significantly increased by 1.6 ± 0.1- and 1.4 ± 0.2-fold in the cytosolic fractions after 7 and 14 days of chronic stimulation, respectively, compared with the contralateral control muscles (Fig. 1B, Table 3; n = 6).

To confirm the role of MSF in the stimulation of MDH protein import, antibody inhibition studies were performed (1). Isolated cytosolic fractions were preincubated with antibodies specific for the two subunits of MSF before incubation in the import reaction. A large reduction (~50%) in the quantity of MDH import was observed when the MSF-L or MSF-S1 antibody was used (Fig. 4, lanes 4 and 5). When the cytosolic fraction was preincubated with both antibodies together, MDH import was not further reduced. Therefore, the remaining stimulatory influence on import is due to the presence of factors other than MSF within the cytosol. The observed response was specific, since cytosolic fractions that were preincubated with anti-cytochrome c antibody or preimmune serum were still capable of enhancing the rate of import (Fig. 4, lanes 6 and 7).


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Fig. 4.   Autoradiogram illustrating MDH protein import into isolated skeletal muscle mitochondria under influence of cytosolic factors. TL, 5 µl of rabbit reticulocyte lysate containing radiolabeled precursor MDH protein, which was not incubated with mitochondria. All subsequent lanes represent protein import into 25 µg of isolated mitochondria in presence of 12 µl of rabbit reticulocyte lysate containing radiolabeled MDH. Additions to import reaction were as follows: 35 µg of cytosolic fraction (lane 1), equivalent amount of buffer by volume (lane 2), 35 µg of BSA (lane 3), 35 µg of cytosolic fraction preincubated with 1 mg/ml of anti-MSF-L (lane 4), 35 µg of cytosolic fraction preincubated with 1 mg/ml of anti-MSF-S1 (lane 5), 35 µg of cytosolic fraction preincubated with 1 mg/ml of anti-cytochrome c (lane 6), and 35 µg of cytosolic fraction preincubated with 1 mg/ml of preimmune serum (lane 7).

Components involved in the protein import pathway. Because our data indicated that a change in the mitochondrial import machinery might be implicated in the enhanced import rates resulting from chronic contractile activity, the expression of the outer membrane import receptor Tom20 and the intramitochondrial chaperone mtHSP70 was evaluated. The mRNA encoding Tom20 was not significantly elevated in 7- or 14-day-stimulated muscles compared with the control muscles (Fig. 1A, Table 2). Despite this lack of change at the mRNA level, significant two- to threefold increases were observed in the protein content of Tom20 in the SS and IMF mitochondria after 7 and 14 days of contractile activity (Fig. 1B, Table 3).

Quantities of the mRNA encoding the intramitochondrial chaperone mtHSP70 were increased by 1.4 ± 0.1-fold (P <=  0.05, n = 6) after 7 days of contractile activity (Fig. 1A). This effect was no longer evident after 14 days (Table 2; n = 6). The mtHSP70 content evaluated in isolated mitochondria obtained from chronically stimulated muscle was significantly increased after 7 and 14 days (Fig. 1B, Table 3). It was 1.4 ± 0.1- and 3.0 ± 0.5-fold greater in SS and IMF mitochondria, respectively, than in control mitochondria after 7 days of stimulation (n = 6). This was further increased to 2.2-fold in SS mitochondria and 8- to 7-fold in IMF mitochondria by 14 days of stimulation (n = 6).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The aim of this study was to investigate potential adaptations in the protein import system during mitochondrial biogenesis. For this purpose we chose the treatment of chronic contractile activity in skeletal muscle. This experimental model has proven to be highly effective in eliciting mitochondrial biogenesis (for reviews see Refs. 6 and 8). Our previous work using the contraction conditions utilized in the present study illustrated a 1.6-fold increase in tissue cytochrome c oxidase enzyme activity after 7 days of stimulation, indicative of an increase in mitochondrial biogenesis (30). We also demonstrate in the present study that the extractable protein content of the isolated SS and IMF mitochondrial subfractions was significantly elevated after 7 and 14 days of stimulation.

In addition to this increase in cellular mitochondrial content, our data also indicate that chronic contractile activity alters mitochondrial composition. For example, the matrix enzyme MDH was increased by 1.6- and 1.9-fold in the SS and IMF subfractions derived from stimulated muscle, respectively. These alterations are due to contractile activity-induced modifications in the expression, synthesis, and posttranslational processing of MDH. Although a number of previous studies utilizing chronic contractile activity to study mitochondrial biogenesis have focused on changes in gene expression (9, 17, 19, 32, 33), none has assessed the potential changes occurring in the targeting and import of proteins into mitochondria. Here we show for the first time that skeletal muscle mitochondria respond to chronic contractile activity, in part, via modifications in the import pathway. In addition, although the pattern of increase differed between the IMF and SS mitochondria, the adaptive changes were evident in both subfractions. These adaptations include increases in the absolute rate of precursor protein import into the organelle as well as increases in the expression of key components involved in the import process.

MDH is a matrix protein that is nuclear encoded and synthesized in the cytosol as a larger, precursor protein (pMDH) possessing a cleavable presequence. This presequence interacts with cytosolic chaperones (e.g., MSF), which direct MDH to the outer mitochondrial membrane import receptors Tom70-Tom37 and then to the 20-kDa translocase protein referred to as Tom20. This protein, along with Tom22, forms a receptor subcomplex that functions to accept precursor proteins at the surface of the mitochondrion (23). It appears that all precursor proteins that are internalized within the mitochondrion, as well as many destined for the outer membrane, interact with Tom20 (18, 23). Tom20 then forms part of the translocation pore (12) for transfer of the precursor to the functionally independent inner membrane import machinery. Translocation intermediates bound to the inner membrane translocase (Tim) proteins are capable of interactions with intramitochondrial chaperonins such as mtHSP70. mtHSP70 cooperates with Tim44 to function as the main components of an "import motor" designed to pull the precursor into the matrix and prevent retrograde transport (25, 26). Thus it is evident that Tom20 and mtHSP70 fulfill critical roles in protein import. Our data illustrate that the protein expression of both of these components is increased in response to contractile activity. In addition, our previous work (22) showed that chronic contractile activity also results in large increases in the expression of the intramitochondrial chaperonins heat shock protein 60 (HSP60) and cpn10. Thus, although the rate-limiting component in the import process is not established, our data are consistent with the interpretation that the increase in import rates observed for MDH is attributable in part to increases in the expression of members of the import machinery. The increases in import cannot be attributed to variations in the precursor protein supply or to differences in cytosolic chaperone levels, since equal quantities of precursor protein were present in the import reaction, and muscle cytosolic components were not present (Fig. 2). In addition, the increases in MDH protein import coincide reasonably well with the greater MDH content in each mitochondrial subfraction, suggesting that rates of intramitochondrial MDH degradation remain unaltered as a result of contractile activity. However, this remains to be determined using direct measurements.

Chronic contractile activity does not appear to have the same influence on the import of proteins located in the outer membrane of the mitochondrion, because the import of the outer membrane protein Bcl-2 was not affected by this treatment (Table 4). Because the import of outer membrane proteins also relies on interactions with the Tom receptor complex (18), this suggests that the upregulation of Tom20 is not sufficient to mediate an increase in import rates, at least for proteins destined for the outer membrane. This also implies that the mechanisms involved in the import of matrix and outer membrane proteins are sufficiently divergent that the same treatment (i.e., contractile activity) does not modify the rate of import within a similar time frame (7-14 days). The difference may be related to the involvement of the inner membrane phospholipid cardiolipin, which is known to be important in the recognition and binding of the presequence (15) and is involved in the import of matrix proteins (28). Because chronic contractile activity leads to a rapid increase in cardiolipin content within the muscle (29) and because cardiolipin is localized almost exclusively in the inner membrane, its presence may be a factor determining the different import responses of proteins destined for the inner and outer membranes.

To more closely approximate the cellular environment, as well as to investigate the role of cytosolic chaperones in the import process, protein import reactions were performed in the presence and absence of a muscle cytosolic fraction. Addition of cytosolic fraction to the import reaction resulted in significant increases in mitochondrial protein import. These results confirm the findings of others using different tissues (12, 13, 27). We have also confirmed that MSF, a component of the cytosol, is involved in stimulating the import of MDH, since preincubation of the cytosolic fraction with anti-MSF antibodies reduced the effect of the cytosol. However, this treatment of the cytosol did not completely eliminate its stimulatory effect, implying the existence of other cytosolic factors that enhance MDH import into mitochondria. Despite this enhancement of import rates in the presence of cytosol, we did not find a difference in the ability of the cytosolic fraction isolated from the control or the stimulated muscle to accelerate protein import. This was surprising, since we detected a significant contractile activity-induced increase in the protein content of MSF-L in the cytosolic fraction. It may be that the quantity of MSF present in the cytosolic fraction of control muscle is more than sufficient to support an accelerated import rate, even in mitochondria obtained from stimulated muscle. Alternatively, stimulated muscle may also contain a higher level of a factor that counteracts the function of MSF. Because the cytosolic fraction is obviously a complex mixture of proteins with positive and negative (E. E. Craig and D. A. Hood, unpublished observations) influences on the import process, further fractionation of the cytosol, variations in the import reaction conditions, and in vivo experiments are required to answer this question.

It is interesting to note the gene expression response of selected components of the protein import system during chronic contractile activity. The protein content of all components investigated (MSF, Tom20, and mtHSP70) increased significantly after 7 and 14 days of chronic contractile activity. These changes occurred despite various responses in the expression of steady-state mRNAs encoding these proteins. For example, the mRNAs encoding Tom20 and MSF-L appeared to be unaffected by 7 or 14 days of chronic contractile activity. Our previous work has also indicated that, despite large increases in the protein levels of HSP60 and the inducible cytosolic isoform of mtHSP70 after 10 days of contractile activity, mRNA levels were not different from those found in control muscle. This suggests that these mRNAs responded transiently to increases in contractile activity before the first measured time points in these studies. The transient nature of the mRNA response has previously been shown for HSP70 (19, 24) and other immediate early genes (17), and the observed behavior of the mRNAs encoding mtHSP70 and MDH supports this idea. These mRNAs were increased by 7 days of contractile activity but then returned to levels that were not different from those found in control muscle by 14 days. Thus the accumulation of mtHSP70 and MDH protein levels is likely a result of a combination of transient increases in mRNA levels by 7 days, accompanied by a subsequent decrease in the rate of protein degradation. The lack of detectable differences in the levels of Tom20 and MSF-L mRNA by 7 days indicates that the transient mRNA increase may have occurred before this time, but studies designed to define the adaptations at earlier time points (e.g., 1, 3, and 5 days) are necessary to confirm this.

In summary, we have shown for the first time that skeletal muscle responds to chronic contractile activity by modifying the rate of mitochondrial precursor protein import, mediated in part via modifications in the expression of important components of the import machinery. This work sets the stage for in vivo studies designed to evaluate the physiological role of the protein import pathway in regulating mitochondrial biogenesis.

    ACKNOWLEDGEMENTS

We are grateful to Dr. G. C. Shore (McGill University, Montreal, PQ, Canada) for providing the Bcl-2 vector, to Dr. A. Strauss (Washington University, St. Louis, MO) for supplying the MDH vector, to Dr. K. B. Freeman (McMaster University, Hamilton, ON, Canada) for supplying the MDH antibody, to Dr. N. Nomura (Kazusa DNA Research Institute) for the donation of the Tom20 cDNA, to Dr. M. Mori (Kumamoto University School of Medicine) for providing the Tom20 antibody, to Dr. R. Morimoto (Northwestern University) for providing the mtHSP70 cDNA, and to Drs. M. Sakaguchi, T. Omura, and K. Mihara (Kyushu University) for providing the MSF-L cDNA and antibody and the MSF-S1 antibody.

    FOOTNOTES

This work was supported by the Natural Sciences and Engineering Research Council of Canada.

Address for reprint requests: D. A. Hood, Dept. of Biology, York University, Toronto, ON, Canada M3J 1P3.

Received 31 October 1997; accepted in final form 27 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Alam, R., N. Hachiya, M. Sakaguchi, S.-I. Kawabata, S. Iwanaga, M. Kitajima, K. Mihara, and T. Omura. cDNA cloning and characterization of mitochondrial import stimulation factor (MSF) purified from rat liver cytosol. J. Biochem. (Tokyo) 116: 416-425, 1994[Abstract].

2.   Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 255-260, 1976[Medline].

3.   Cogswell, A. M., R. J. Stevens, and D. A. Hood. Properties of skeletal muscle mitochondria isolated from subsarcolemmal and intermyofibrillar regions. Am. J. Physiol. 264 (Cell Physiol. 33): C383-C389, 1993[Abstract/Free Full Text].

4.   Connor, M. K., M. Takahashi, and D. A. Hood. Tissue-specific stability of nuclear- and mitochondrially encoded mRNAs. Arch. Biochem. Biophys. 333: 103-108, 1996[Medline].

5.   Craig, E. E., and D. A. Hood. Influence of aging on protein import into cardiac mitochondria. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H2983-H2988, 1997[Abstract/Free Full Text].

6.   Essig, D. A. Contractile activity-induced mitochondrial biogenesis in skeletal muscle. Exerc. Sport Sci. Rev. 24: 289-319, 1996[Medline].

7.   Hallman, M., and P. Kankare. Cardiolipin and cytochrome aa3 in intact liver mitochondria of rats. Evidence of successive formation of inner membrane components. Biochem. Biophys. Res. Commun. 45: 1004-1010, 1971[Medline].

8.   Hood, D. A., A. Balaban, M. K. Connor, E. E. Craig, M. L. Nishio, M. Rezvani, and M. Takahashi. Mitochondrial biogenesis in striated muscle. Can. J. Appl. Physiol. 19: 12-48, 1994[Medline].

9.   Hood, D. A., R. Zak, and D. Pette. Chronic stimulation of rat skeletal muscle induces coordinate increases in mitochondrial and nuclear mRNAs of cytochrome c oxidase subunits. Eur. J. Biochem. 179: 275-280, 1989[Abstract].

10.   Hoppeler, H. Exercise-induced ultrastructural changes in skeletal muscle. Int. J. Sports Med. 7: 187-204, 1986[Medline].

11.   Hoppeler, H., P. Lüthi, H. Claassen, E. R. Weibel, and H. Howald. The ultrastructure of the normal human skeletal muscle. Pflügers Arch. 344: 217-232, 1973[Medline].

12.   Komiya, T., and K. Mihara. Protein import into mammalian mitochondria. J. Biol. Chem. 271: 22105-22110, 1996[Abstract/Free Full Text].

13.   Komiya, T., M. Sakaguchi, and K. Mihara. Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria. EMBO J. 15: 399-407, 1996[Abstract].

14.   Krieger, D. A., C. A. Tate, J. McMillin-Wood, and F. W. Booth. Populations of rat skeletal muscle mitochondria after exercise and immobilization. J. Appl. Physiol. 48: 23-28, 1980[Abstract/Free Full Text].

15.   Leenhouts, J. M., Z. Török, V. Chupin, and B. de Kruijff. A molecular model for the specific cardiolipin-presequence interactions. Biochem. Soc. Trans. 23: 968-971, 1995[Medline].

16.   Martin, T. Predictable adaptations by skeletal muscle mitochondria to different exercise training workloads. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 88: 273-276, 1987.

17.   Michel, J. B., G. A. Ordway, J. A. Richardson, and R. S. Williams. Biphasic induction of immediate early gene expression accompanies activity-dependent angiogenesis and myofiber remodeling of rabbit skeletal muscle. J. Clin. Invest. 94: 277-285, 1994[Medline].

18.   Millar, D. G., and G. C. Shore. Assembly of mitochondrial membrane proteins. In: Membrane Protein Assembly, edited by G. von Heijne. New York: Chapman and Hall, 1997, p. 151-172.

19.   Neufer, P. D., G. A. Ordway, G. A. Hand, J. M. Shelton, J. A. Richardson, I. J. Benjamin, and R. S. Williams. Continuous contractile activity induces fiber type specific expression of HSP70 in skeletal muscle. Am. J. Physiol. 271 (Cell Physiol. 40): C1828-C1837, 1996[Abstract/Free Full Text].

20.   Neupert, W. Protein import into mitochondria. Annu. Rev. Biochem. 66: 863-917, 1997[Medline].

21.   Ogata, T., and Y. Yamasaki. Scanning electron-microscopic studies on the three dimensional structure of mitochondria in the mammalian red, white, and intermediate muscle fibers. Cell Tissue Res. 241: 251-256, 1985[Medline].

22.   Ornatsky, O. I., M. K. Connor, and D. A. Hood. Expression of stress proteins and mitochondrial chaperones in chronically stimulated skeletal muscle. Biochem. J. 311: 119-123, 1995[Medline].

23.   Pfanner, N., and M. Meijer. Mitochondrial biogenesis: the Tom and Tim machine. Curr. Biol. 7: R100-R103, 1997[Medline].

24.   Puntschart, A., M. Vogt, H. R. Widmer, H. Hoppeler, and R. Billeter. Hsp70 expression in human skeletal muscle after exercise. Acta Physiol. Scand. 157: 411-417, 1996[Medline].

25.   Schatz, G. The protein import system of mitochondria. J. Biol. Chem. 271: 31763-31766, 1996[Free Full Text].

26.   Schneider, H.-C., B. Westermann, W. Neupert, and M. Brunner. The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import. EMBO J. 15: 5796-5803, 1996[Abstract].

27.   Sheffield, W. P., G. C. Shore, and S. K. Randall. Mitochondrial precursor protein. J. Biol. Chem. 265: 11069-11076, 1990[Abstract/Free Full Text].

28.   Takahashi, M., and D. A. Hood. Protein import into subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. J. Biol. Chem. 271: 27285-27291, 1996[Abstract/Free Full Text].

29.   Takahashi, M., and D. A. Hood. Chronic stimulation-induced changes in mitochondria and performance in rat skeletal muscle. J. Appl. Physiol. 74: 934-941, 1993[Abstract].

30.   Takahashi, M., D. T. M. McCurdy, D. A. Essig, and D. A. Hood. delta -Aminolaevulinate synthase expression in muscle after contractions and recovery. Biochem. J. 291: 219-223, 1993[Medline].

31.   Takahashi, M., A. Rana, and D. A. Hood. Portable electrical stimulator for use in small animals. J. Appl. Physiol. 74: 942-945, 1993[Abstract].

32.   Williams, R. S., S. Salmons, E. A. Newsholme, R. E. Kaufman, and J. Mellor. Regulation of nuclear and mitochondrial gene expression by contractile activity in skeletal muscle. J. Biol. Chem. 261: 376-380, 1986[Abstract/Free Full Text].

33.   Yan, Z., S. Salmons, Y. L. Dang, M. T. Hamilton, and F. W. Booth. Increased contractile activity decreases RNA-protein interaction in the 3'-UTR of cytochrome c mRNA. Am. J. Physiol. 271 (Cell Physiol. 40): C1157-C1166, 1996[Abstract/Free Full Text].


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