Departments of Biology and Kinesiology and Health Sciences, York University, Toronto, Ontario, Canada M3J 1P3
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ABSTRACT |
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The mitochondrial phenotype within cardiac muscle cells is dramatically altered by thyroid hormone. We report here that this can be accounted for, in part, by modifications in the rate of mitochondrial protein import. The import of matrix-localized precursor proteins malate dehydrogenase (MDH) and ornithine carbamoyltransferase was augmented, whereas the insertion of the outer membrane protein Bcl-2 was unaffected by thyroid hormone treatment. Coincident with increases in the import of these matrix-localized precursors were thyroid hormone-induced elevations in the outer membrane receptor Tom20 and the matrix heat-shock protein mthsp70. The phospholipid cardiolipin was not involved in mediating the thyroid hormone-induced increase in import, as judged from adriamycin inhibition studies. When the import reaction was supplemented with rat heart cytosol, we found that 1) MDH import was stimulated, but Bcl-2 import was inhibited and 2) thyroid hormone did not influence the effect of the cytosol on import rates. Thus distinct requirements exist for the mitochondrial import of precursor proteins, destined for different organellar compartments. Although import of these matrix-localized proteins was augmented by thyroid hormone treatment, the proteolysis of matrix proteins was unaffected as indicated by the degradation of cytob2(167)RIC-dihydrofolate reductase, a chimeric protein missorted to the matrix. Thus our data indicate that at least some thyroid hormone-induced modifications of the mitochondrial phenotype occur due to the compartment-specific upregulation of precursor protein import rates, likely mediated via changes in the expression of protein import machinery components.
cardiolipin; mitochondrial biogenesis; mitochondrial protein degradation; molecular chaperones; 20-kDa translocase of the outer mitochondrial membrane
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INTRODUCTION |
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MITOCHONDRIA CONTAIN hundreds of different proteins, but only a small fraction of those are encoded by mitochondrial DNA and are made inside the organelle. The remaining proteins are derived from nuclear DNA, synthesized in the cytosol, and then imported. This process requires the sequential participation of components located in the cytosol, the outer and inner mitochondrial membranes, and within the matrix (cf. Refs. 17 and 19 for reviews). Initially, precursor proteins, which are synthesized with specific targeting presequences, must be unfolded before being translocated from the cytosol into the mitochondria. This is facilitated by members of the 70-kDa heat-shock protein family (hsp70s), as well as the cytosolic chaperone termed the mitochondrial import stimulation factor (MSF; see Ref. 14). MSF directs precursors to receptor proteins composing the translocase of the outer mitochondrial membrane (Tom). Initially this interaction involves the heterodimer Tom37/70. The precursor is then transferred to Tom20/22 before its insertion in the outer membrane at a general import pore (12).
The mitochondrial inner membrane also possesses specific, independent transport components for the import of precursor proteins, referred to as the translocase of the inner mitochondrial membrane (Tim). Correct orientation of the precursor protein with the Tim subunits is likely provided by electrostatic interactions with the inner membrane phospholipid cardiolipin (CL), which has been shown to specifically interact with presequences (9, 11). Translocation of the remainder of the presequence, and all of the mature protein, requires interaction with the peripheral membrane protein Tim44, which is associated with the mitochondrial chaperone mthsp70 (20). This dimer functions to pull the precursor protein across the Tim channel in an ATP-dependent fashion (8). Because precursors can only be imported into mitochondria in an unfolded conformation, they must be refolded once in the matrix. This also depends on the assistance of chaperones, including hsp60, cpn10, and mthsp70 (18).
The maintenance of mitochondrial function and phenotype depends on the existence of a system for the synthesis and import of mitochondrial proteins, as well as a proteolytic system for the degradation of abnormal proteins (10). In the matrix, a serine protease termed PIM1 has been characterized (27). PIM1 promotes the degradation of misfolded proteins, and its activity is dependent on ATP hydrolysis, as well as the chaperone function of mthsp70 (10, 22, 26). Neither the protein import system nor the intramitochondrial proteolytic pathway has been investigated in cardiac mitochondria during conditions of altered mitochondrial biogenesis. To study this, we employed thyroid hormone 3,5,3'-triiodo-L-thyronine (T3), which produces striking alterations in mitochondrial number, composition, and function (21). In cardiac muscle, T3 treatment causes an increase in the mitochondrial volume fraction and in the area of mitochondrial cristae per unit of mitochondrial volume (13). In addition, hyperthyroidism results in increases in the content of several of the cytochromes (15) and in an approximately twofold increase in CL content (16). The purpose of this study was to investigate the possibility that the T3-related modifications in the cardiac mitochondrial phenotype could be attributable, in part, to alterations in either the protein import or protein degradation pathways.
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METHODS |
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Animals and physiological measurements. Male Sprague-Dawley rats were injected intraperitoneally with either T3 (40 µg/100 g body wt) or vehicle (0.9% NaCl-propylene glycol; 40:60 vol/vol) for five consecutive days. Subsequently, the animals were anesthetized with pentobarbital sodium [40 mg/kg body weight (BW)]. To confirm that the hyperthyroid state was achieved, measurements of BW, heart rate (HR), mean arterial pressure (MAP), and the extent of left ventricular (LV) hypertrophy were routinely performed as done previously (4).
Mitochondrial isolation. Mitochondria
were isolated from the cardiac ventricular tissue as described
previously (4). The mitochondrial pellet was suspended in 350 µl of
10 mM HEPES (pH 7.4), 0.25 M sucrose, 10 mM sodium succinate, 2.5 mM
K2HPO4,
0.21 mM ADP, and 1 mM dithiothreitol. An aliquot of the mitochondrial suspension was stored at 20°C for subsequent enzymatic
analysis. Protein content was determined according to Bradford (1).
In vitro transcription and translation. Full-length cDNAs encoding malate dehydrogenase (rat MDH, from Dr. A. Strauss, Washington University, St. Louis, MO), ornithine carbamoyltransferase (rat OCT, from Dr. G. C. Shore, McGill University, Montreal, Canada), the hybrid protein containing the NH2-terminal 167 residues of cytochrome b2 fused to cytosolic mouse dihydrofolate reductase [yeast cytob2(167)RIC-dihydrofolate reductase (DHFR), from Dr. W. Neupert, University of Munich, Munich, Germany], as well as the outer membrane protein Bcl-2 (human Bcl-2 from Dr. G. C. Shore) were transcribed as described previously (4). Linearized plasmid DNA was incubated for 90 min with the appropriate RNA polymerase, ribonucleotide triphosphate substrates, and the cap analog m7G(5')ppp(5')G. RNA transcripts were subsequently isolated by phenol extraction followed by ethanol precipitation, and final concentrations were adjusted to 2.8 µg/µl on the basis of the absorbance at 260 nm. Precursor proteins were synthesized in rabbit reticulocyte lysate (Promega) in the presence of [35S]methionine (4).
Import of precursor proteins. Import reactions were performed as previously described (4, 23, 24) by incubating 25 µg of cardiac mitochondria with 10 µl of translation products at 30°C. After import with Bcl-2, mitochondria were washed in 0.1 M Na2CO3 (pH 11.0) and then incubated for 30 min on ice. All reaction mixtures were centrifuged (18,000 g for 15 min) to separate the mitochondria and supernatant fractions. The mitochondrial fraction was then resolved by 8% SDS-PAGE, and the gels were analyzed by electronic autoradiography (Instantimager; Packard, Meriden, CT). The extent of import was measured as the percentage of mature protein relative to the total protein added during the import reaction. In import assays containing adriamycin, the mitochondria were preincubated with various concentrations (90-360 µM) for 10 min at 30°C before the import reaction. For import reactions containing the rat heart cytosolic fraction, the translation products were preincubated with 25 µg of cytosolic protein for 10 min at 30°C before the import reaction.
Degradation of newly imported proteins. Import was terminated by adding 14.3 µM valinomycin and chilling on ice. Nonimported cytob2(167)RIC-DHFR precursor proteins were digested with proteinase K (130 µg/ml) for 20 min at 0°C. Digestion was stopped by the addition of phenylmethylsulfonyl fluoride (1 mM). To allow the degradation of newly imported proteins, samples were incubated at 30°C (26), and aliquots were withdrawn at various time points as indicated. Mitochondria were reisolated by centrifugation (18,000 g for 15 min), and the proteins were resolved by SDS-PAGE and autoradiography.
Preparation of cytosolic fraction. Cardiac muscle tissue was homogenized with 5 vol of buffer containing 10 mM HEPES, 220 mM mannitol, 70 mM sucrose, and 1 mM EGTA. The homogenate was centrifuged at 900 g for 10 min. The supernatant was centrifuged further at 5,000 g for 10 min and then at 100,000 g for 60 min. The resultant supernatant was then concentrated (Amicon ultrafiltration cell, YA10 membrane) to a final volume of <1 ml and was used as the cytosolic fraction in import experiments (23).
Total RNA isolation and hybridization. Frozen LV muscle powders were processed for RNA isolation by using 50-mg samples, as previously described (3). Northern blots of total RNA (10 µg/lane) were prepared on Hybond-N membrane (Amersham, Mississauga, Canada). The cDNA probes encoding MDH and the large subunit of the MSF (MSF-L; from Drs. M. Sakaguchi, T. Omura, and K. Mihara, Kyushu University, Fukuoka, Japan) were radiolabeled by random priming (New England Biolabs, Beverly, MA). Membranes were prehybridized for 2 h, followed by overnight hybridization with the [32P]dCTP-labeled cDNA probe as done previously (3). Radioactive signal intensities were quantified by electronic autoradiography (Instantimager; Packard).
Immunoblot analyses. Mitochondrial or cytosolic samples were separated by SDS-PAGE and then electrotransferred (Enprotech, Hyde Park, MA) for 90 min on nitrocellulose membranes (Hybond-C; Amersham). The blots were probed with antibodies directed against hsp60 (StressGen Biotechnologies, Victoria, Canada), mthsp70 (grp75; StressGen Biotechnologies), MSF-L (from Drs. M. Sakaguchi, T. Omura, and K. Mihara, Kyushu University), Tom20 (from Dr. M. Mori, Kumamoto University, Kumamoto, Japan), or PIM1 (from Dr. M. Maurizi, National Institutes of Health, Bethesda, MD). The blots were quantified by monochromatic scanning (ScanMaker IIg; Microtek, Redondo Beach, CA) or laser densitometry (SL-504-XL; Biomed Instruments, Fullerton, CA).
Enzyme activities. Activities of individual enzymes were assessed spectrophotometrically (DU-64; Beckman) at 30°C. Cytochrome-c oxidase activity was measured as the rate of oxidation of reduced cytochrome c in solubilized isolated heart mitochondria (4). Mitochondrial MDH activity was determined on the basis of the rate of NADH oxidation at 340 nm by using oxaloacetic acid as a substrate (4).
Statistics. Independent t-tests (P < 0.05) were used for analysis of changes in BW, HR, MAP, LV-to-BW ratio, enzyme activities, and protein and mRNA levels. A two-way analysis of variance was used to evaluate the import and degradation data. All data are represented as means ± SE.
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RESULTS |
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T3 injection alters cardiac physiology, structure, and mitochondrial composition. The administration of T3 resulted in significant increases in HR (31% higher) and MAP (32% higher; Table 1). The LV-to-BW ratio, used as an index of cardiac hypertrophy, was elevated by 39.0 ± 2.0% in T3- compared with vehicle-injected animals. The activities (in U/mg mitochondrial protein) of the inner membrane enzyme cytochrome-c oxidase and the matrix enzyme MDH were significantly higher in cardiac mitochondria isolated from T3- vs. vehicle-injected animals (24 and 34%, respectively), indicating that mitochondrial composition was affected by T3 treatment.
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Mitochondrial degradation of imported cytob2(167)RIC-DHFR. Because the mitochondrial phenotype depends on the regulation of synthesis, import, and the degradation of proteins, we investigated the effects of T3 on the rate of degradation immediately after import. The protein cytob2(167)RIC-DHFR, which is missorted to the matrix as a result of specific point mutations in the presequence (26), was used for this evaluation, since preliminary data indicated that the half-time (t1/2) of MDH degradation was longer than that which could be reliably measured in isolated mitochondria. After import, cytob2(167)RIC-DHFR was degraded with t1/2 values of 103 and 100 min in mitochondria isolated from vehicle- and T3-injected animals, respectively. Thus T3 did not accelerate or reduce the degradation rate of this matrix-localized protein (Fig. 6). In concert with this result, T3 had no detectable effect on the expression of the protease PIM1 protein in cardiac mitochondria (immunoblot not shown). Quantification of several blots (n = 11) indicated a T3-to-vehicle PIM1 protein ratio of 1.21 ± 0.26 (P > 0.05).
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DISCUSSION |
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T3 can cause profound alterations in mitochondrial function, due in part to modifications in organelle protein and lipid composition (21). This is particularly evident in cardiac muscle cells (15, 16). Because most proteins located within mitochondria are nuclear encoded and imported into the organelle, we wanted to determine whether the effect of T3 on mitochondrial composition could be attributed, in part, to changes in protein import. Furthermore, because mitochondrial proteins are subject to intraorganelle proteolysis, protein levels could also be altered by modifications in the rate of degradation. Thus our main purpose was to examine the relative roles of import and degradation, and factors that modify these processes, on the mitochondrial phenotype as it is modified by T3.
Our results document that mitochondria derived from the cardiac muscle of T3-treated animals exhibit an ~40-60% higher import rate of matrix proteins compared with mitochondria from vehicle-treated animals. This observation indicates that mitochondrial protein import is an adaptable process that could be partially responsible for the phenotypic differences exhibited in mitochondria in response to thyroid hormone. Consistent with this is the relatively close parallelism between the T3-induced increases in MDH enzyme activity and MDH precursor protein import. Nonetheless, it should be noted that similar augmentations in the import rates of matrix-targeted proteins were also observed in a recent study that utilized the experimental model of chronic contractile activity in skeletal muscle to elicit mitochondrial biogenesis (23). These observations indicate that mitochondrial protein import can adapt to multiple stimuli that induce mitochondrial biogenesis.
In distinct contrast to the increases observed in matrix protein import
rates, the rate of import of the mitochondrial outer membrane protein
Bcl-2 was not affected by T3. This
apparent discrepancy may be explained by the divergent import
requirements, and pathways, of matrix and outer membrane proteins.
Although both matrix-targeted and outer membrane proteins interact with
components of the Tom machinery, insertion of the outer membrane
proteins is not influenced by the Tim complex, by components found
within the matrix, or by the electrochemical membrane potential .
Thus to identify some of the mechanisms responsible for the enhanced
import rate induced by T3, we
measured the expression of three important components of the import
machinery. We reasoned that, since Tom20 is involved at the
intersection of the import pathways of both outer membrane and matrix
proteins, its expression may be related to any change in import rate
observed. The 1.7-fold increase in Tom20 levels with
T3 treatment closely paralleled
the increase in import of the matrix-destined precursor proteins.
However, this increased expression of Tom20 did not influence the
import of Bcl-2, which remained unaffected by
T3 treatment. Thus either Tom20 is
used differentially in the import of outer membrane proteins, or its level is not rate limiting for the insertion of outer membrane proteins. This may be because, before its induction by
T3, it was already present at a
sufficiently high concentration to support the import of outer membrane proteins.
It has been suggested that mthsp70 may exert a regulatory role in the import process (5). The results presented here demonstrate that the quantity of mthsp70 is increased by thyroid hormone and that this increase is also matched by an increased rate of import of matrix-destined proteins. This suggests that mthsp70 may be important in mediating the enhanced import rate induced by T3. A coordinated increase in the expression of hsp60, a protein that cooperates with other chaperones to refold many matrix proteins (18), was also observed. It should be noted that we have previously observed some discordance between the expression of mthsp70 and hsp60 and rates of precursor protein import (4). In a study of the aging process, we found an accelerated import rate in cardiac mitochondria obtained from senescent (28 mo) rats compared with that observed in young (4 mo) and old (22 mo) animals. However, the expression of mthsp70 and hsp60 was elevated in mitochondria from both the old and senescent animals. This suggests, at least during aging, that mthsp70 is sufficiently high and does not act in a singular fashion to regulate the import rate of matrix proteins in cardiac mitochondria.
In addition to the protein components of the import machinery, which may determine the import rate, the phospholipid composition of the inner membrane may also play a role. Precursor proteins appear to interact not only with the different receptor proteins but also with membrane lipids, with a preference for negatively charged phospholipids (7). Of special interest is CL, which has been previously implicated in the import process (4, 6, 9, 11, 24). CL involvement has been demonstrated, in part, through the use of the drug adriamycin, which appears to bind with high specificity to CL (2). Because it is known that T3 treatment results in up to a twofold increase in the CL content in cardiac mitochondria (16), we hypothesized that this difference in CL content would play a role in modifying the rate of protein import. Our data confirm CL involvement in protein translocation by demonstrating a dose-dependent inhibition of import in response to the addition of increasing concentrations of adriamycin. Surprisingly, no significant difference in import sensitivity to adriamycin was observed between these two groups. Thus we conclude that T3-inducible increases in import rate are not achieved via variations in CL content.
Previous in vitro studies have confirmed the requirement for cytosolic protein factors in the efficient mitochondrial import of various precursor proteins (14). Investigations of skeletal muscle indicated that the levels of the MSF protein could be increased by chronic contractile activity and that MSF was actively involved in enhancing the import of MDH in the matrix (23). In contrast to our results in skeletal muscle, the data obtained in the present study do not suggest that MSF is inducible by T3, since no differences in mRNA transcript or protein levels were detected between T3- and vehicle-injected animals. We then examined the effect of supplementing the protein import reaction with a concentrated cytosolic fraction to determine whether other, previously unidentified, cytosolic factors may be upregulated by T3, which may modify the rate of protein import. Although the addition of cytosol clearly altered the rate of protein import, our results were not consistent with a T3-mediated induction of cytosolic chaperones, since no difference existed in the extent of stimulatory or inhibitory activity of the cytosolic fraction derived from T3- or vehicle-treated animals. Furthermore, the effect of cytosol varied depending on the precursor protein. When cytosol was added to MDH import reactions, a marked stimulation of MDH import was observed. In contrast, the addition of the cytosolic fraction to Bcl-2 import reactions did not stimulate precursor import but instead resulted in a significant inhibition. These results support the concept that distinct cytosolic requirements exist in the mitochondrial import of individual precursor proteins (9, 25). These differences may correspond to variable affinities of the different precursors for molecular chaperones (9) or may be due to variations in the tendency of precursors to fold, misfold, or aggregate (25).
In addition to protein import, we wanted to establish whether the T3-induced alterations in mitochondrial phenotype could be attributed to modified rates of intramitochondrial protein degradation. In the matrix, the ATP-dependent protease PIM1 has been shown to promote the degradation of misfolded proteins (26). Its activity is dependent on the chaperone function of the mthsp70 system (26). In view of the T3-mediated upregulation of mthsp70 in cardiac cells, we hypothesized that PIM1 would be induced in a coordinated fashion and that the overall degradation rate mediated by the PIM1-mthsp70 system would be increased. However, immunoblot analyses revealed no effect of T3 on PIM1 expression. In addition, the proteolysis of matrix-localized cytob2(167)RIC-DHFR remained unaffected by T3. Thus our data suggest that the increased expression of mthsp70 (by 1.6-fold) does not influence the rate of protein degradation of newly imported matrix proteins.
How does T3 modify the mitochondrial phenotype? The experiments reported here suggest that this is due, in part, to modifications in the rate of precursor protein import. Rates of protein degradation, as well as the influence of cytosolic chaperones, while likely important in maintaining steady-state concentrations of mitochondrial proteins, appear to have a lesser effect in mediating the T3-induced enhancement of mitochondrial biogenesis.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. G. C. Shore (McGill University, Montreal, Canada) for providing the OCT and Bcl-2 vectors, to Dr. A. Strauss (Washington University, St. Louis, MO) for supplying the MDH vector, to Drs. W. Neupert and H.-C. Schneider (University of Munich, Munich, Germany) for supplying the cytob2(167)RIC-DHFR vector, to Dr. N. Nomura (Kazusa DNA Research Institute, Chiba, Japan) for the donation of the Tom20 cDNA, to Dr. M. Mori (Kumamoto University School of Medicine, Kumamoto, Japan) for providing the Tom20 antibody, to Dr. M. Maurizi (National Institutes of Health, Bethesda, MD) for the Lon antibody, and to Drs. M. Sakaguchi, T. Omura, and K. Mihara (Kyushu University, Fukuoka, Japan) for providing the MSF-L cDNA and antibody.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests: D. Hood, Dept. of Biology, York Univ., Toronto, ON, Canada M3J 1P3.
Received 17 June 1998; accepted in final form 3 September 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bradford, M. M.
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
255-260,
1976[Medline].
2.
Cheneval, D.,
M. Müller,
R. Toni,
S. Ruetz,
and
E. Carafoli.
Adriamycin as a probe for the transversal distribution of cardiolipin in the inner mitochondrial membrane.
J. Biol. Chem.
260:
13003-13007,
1985
3.
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].
4.
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
5.
Dekker, P. J. T.,
F. Martin,
A. C. Maarse,
U. Bomer,
H. Muller,
B. Guiard,
M. Meijer,
J. Rassow,
and
N. Pfanner.
The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44.
EMBO J.
16:
5408-5419,
1997
6.
Eilers, M.,
T. Endo,
and
G. Schatz.
Adriamycin, a drug interacting with acidic phospholipids, blocks import of precursor proteins by isolated yeast mitochondria.
J. Biol. Chem.
264:
2945-2950,
1989
7.
Endo, T.,
and
G. Schatz.
Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding.
EMBO J.
7:
1153-1158,
1988[Abstract].
8.
Glick, B. S.
Can hsp70 proteins act as force-generating motors?
Cell
80:
11-14,
1995[Medline].
9.
Hajek, P.,
and
D. M. Bedwell.
Characterization of the mitochondrial binding and import properties of purified yeast F1-ATPase subunit precursor.
J. Biol. Chem.
269:
7192-7200,
1994
10.
Langer, T.,
and
T. Neupert.
Regulated protein degradation in mitochondria.
Experientia
52:
1069-1076,
1996[Medline].
11.
Leenhouts, J. M.,
Z. Török,
V. Mandieua,
E. Goormaghtigh,
and
B. de Kruijff.
The N-terminal half of a mitochondrial presequence peptide inserts into cardiolipin-containing membranes.
FEBS Lett.
388:
34-38,
1996[Medline].
12.
Mayer, A.,
W. Neupert,
and
R. Lill.
Mitochondrial protein import: reversible binding of the presequence at the trans side of the outer membrane drives partial translocation and unfolding.
Cell
80:
127-137,
1995[Medline].
13.
McCallister, L. P.,
and
E. Page.
Effects of thyroxin on ultrastructure of rat myocardial cells: a stereological study.
J. Ultrastruct. Res.
42:
136-155,
1973[Medline].
14.
Mihara, K.,
and
T. Omura.
Cytosolic factors in mitochondrial protein import.
Experientia
52:
1063-1068,
1996[Medline].
15.
Nishiki, K.,
M. Erecinska,
D. F. Wilson,
and
S. Cooper.
Evaluation of oxidative phosphorylation in hearts from euthyroid, hypothyroid and hyperthyroid rats.
Am. J. Physiol.
235 (Cell Physiol. 4):
C212-C219,
1978[Abstract].
16.
Paradies, G.,
F. M. Ruggiero,
G. Petrosillo,
and
E. Quagliariello.
Enhanced cytochrome oxidase activity and modifications of lipids in heart mitochondria from hyperthyroid rats.
Biochim. Biophys. Acta
1225:
165-170,
1994[Medline].
17.
Pfanner, N.
Mitochondrial import: crossing the aqueous intermembrane space.
Curr. Biol.
8:
R262-R265,
1998[Medline].
18.
Ryan, M. T.,
D. J. Naylor,
P. B. Høj,
M. S. Clark,
and
N. P. Hoogenraad.
The role of molecular chaperones in mitochondrial protein import and folding.
Int. Rev. Cytol.
174:
127-193,
1997[Medline].
19.
Schatz, G.
The protein import system of mitochondria.
J. Biol. Chem.
271:
31763-31766,
1996
20.
Schneider, H.-C.,
J. Berthold,
M. F. Bauer,
K. Dietmeier,
B. Guiard,
M. Brunner,
and
W. Neupert.
Mitochondrial hsp70/MIM44 complex facilitates protein import.
Nature
371:
768-774,
1994[Medline].
21.
Soboll, S.
Thyroid hormone action on mitochondrial energy transfer.
Biochim. Biophys. Acta
1144:
1-16,
1993[Medline].
22.
Suzuki, C. K.,
M. Rep,
J. M. van Dijl,
K. Suda,
L. A. Grivell,
and
G. Schatz.
ATP-dependent proteases that also chaperone protein biogenesis.
Trends Biochem. Sci.
22:
118-123,
1997[Medline].
23.
Takahashi, M.,
A. Chesley,
D. Freyssenet,
and
D. A. Hood.
Contractile activity-induced adaptations in the mitochondrial protein import system.
Am. J. Physiol.
274 (Cell Physiol. 43):
C1380-C1387,
1998
24.
Takahashi, M.,
and
D. A. Hood.
Protein import into subsarcolemmal and intermyofibrillar skeletal muscle mitochondria.
J. Biol. Chem.
271:
27285-27291,
1996
25.
Terada, K.,
I. Ueda,
K. Ohtsuka,
T. Oda,
A. Ishiyama,
and
M. Mori.
The requirement of heat shock cognate 70 protein for mitochondrial import varies among precursor proteins and depends on precursor length.
Mol. Cell. Biol.
16:
6103-6109,
1996[Abstract].
26.
Wagner, I.,
H. Arlt,
L. van Dyck,
T. Langer,
and
W. Neupert.
Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.
EMBO J.
13:
5135-5145,
1994[Abstract].
27.
Wang, N.,
S. Gottesman,
M. C. Willingham,
M. M. Gottesman,
and
M. R. Maurizi.
A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease.
Proc. Natl. Acad. Sci. USA
90:
11247-11251,
1993[Abstract].