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* The Wenner-Gren Institute and
Department of Biochemistry and Biophysics, The Arrhenius Laboratories, Stockholm University, Stockholm, Sweden; and
Department of Bioenergetics, Institute of Physiology and Centre for Applied Genomics, Prague, Czech Republic
3Correspondence: The Wenner-Gren Institute, The Arrhenius Laboratories, Stockholm University, SE-106 91 Stockholm, Sweden. E-mail: barbara.cannon{at}wgi.su.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: biogenesis • assembly • UCP1 promoter • transgenic mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In mammals, the mitochondrial ATP synthase contains 16 different nuclear- or mitochondrial-encoded subunits that are separated into two oligomers. The catalytic F1 oligomer extends into the mitochondrial matrix and is composed of five subunits that form the "headpiece" (
and β subunits) and central stalk (
, 
, and 
subunits) (1
, 2)
. The Fo oligomer is membrane-bound and composed of 10 different subunit types in mammals (a, b, c, d, e, f, g, (F6), A6L, OSCP), in which OSCP, F6, b, and d subunits form a peripheral stalk (3
4
5)
. Assembly of this multisubunit complex requires several steps that are well described for bacteria and yeast systems (6)
. However, in animal cells, the specific steps in the process of mitochondrial ATP synthase assembly have still not been fully characterized.
Brown adipose tissue (BAT) (7)
, the thermogenic organ of mammals, contains extremely high amounts of the mitochondrial respiratory chain enzymes but remarkably low amounts of the F1Fo-ATPase (8
, 9)
. Surprisingly, in contrast to this low protein content, at the mRNA level the majority of the F1Fo-ATPase subunits are present in abundant amounts (10)
. The only subunit with remarkably low levels of mRNA in this tissue is the c-Fo subunit (11)
.
In mammals, the c-Fo subunit is encoded in the nuclear genome by three different genes: ATP5G1, ATP5G2, and the recently described ATP5G3 (herein referred as isoforms P1, P2, and P3), which are translated into the same mature c subunit protein with different mitochondrial import presequences (12
, 13)
. Of these three isoforms, only the P1 isoform gene expression is actively regulated in response to various physiological stimuli (such as ontogenic development and cold acclimation), whereas the other isoforms are thought to maintain the basal levels of the c-Fo subunit (12
, 14
, 15)
.
To examine in vivo the significance of the level of P1 expression for the biogenesis of mitochondrial ATP synthase, we have here generated a transgenic mouse that overexpresses the c-Fo subunit P1 isoform specifically in BAT. We conclude that the levels of the c-Fo subunit P1 isoform are crucial for defining the final amounts of the F1Fo-ATPase in brown adipose tissue and suggest that this may be a generally critical factor in regulation of the assembly of F1Fo-ATPase and in determining the tissue-specific content of the enzyme in higher eukaryotes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was used to provide a BAT-specific overexpression of the P1 isoform of the c subunit of the F1Fo-ATPase. The EcoRI-HindIII fragment (
8.3 kb) of the UCP1 minigene was cloned into the vector pSV-SPORT1 (Life Technologies/Invitrogen, Carlsbad, CA, USA) (Fig. 1
). A unique XhoI endonuclease restriction site was introduced by site-directed mutagenesis into exon 1 of the minigene, and P1-c subunit cDNA was amplified from a mouse heart lambda-gt11 cDNA expression library (Clontech, Mountain View, CA, USA) using primers with XhoI sites added at the 5' ends (5'-gcgctcgagttgaaaaatgcagacca-3' and 5'-cggctcgagactggcatggagtcaaagc-3'). The cDNA fragment of the P1-c subunit included nucleotides 49 to 517, where the coding sequence started at nucleotide 57 and terminated at nucleotide 467.
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Generation and genotyping of transgenic mice
To eliminate the SV40 promoter sequence in the UCP1p1-pSV SPORT1 construct, it was digested with BsaWI restriction endonuclease (NEB, Ipswich, MA, USA), which has recognition sites only in the pSV-SPORT1 vector and not in the UCP1p1 insert (Fig. 1)
. The 9.1 kb BsaWI-digested fragment of the UCP1p1 construct was gel-purified (QIAEX II Gel Extraction Kit, Qiagen, Hilden, Germany) and injected into fertilized B6CBAF1/Crl oocytes. The pronuclear injections were performed at the Karolinska Center for Transgene Technologies (Stockholm, Sweden).
Tail biopsies and genomic DNA isolation of the founder mice were performed as described (17)
. Founders were screened by PCR for the presence of specific fragments amplified from the transgene insert (specific primers used were 5'-gccaggaacccatctctca-3' and 5'-ggtgatggtccctaggacacct-3'). UCP1p1 c subunit transgenic animals were maintained by crossing the heterozygous transgenic mice to wild-type C57 Bl/6 mice. Confirmed transgenic mice (Tg/0) were generally used for the experiments presented. For certain experiments, homozygous transgenic mice (Tg/Tg) were produced by intercrossing heterozygous mice and identified by quantitative analysis of transgene content in their genome. For these experiments, F1 generation offsprings of transgene homozygous mating pairs were used. All animals were maintained at 24°C with access to food and water ad libitum and kept at 12 h/12 h light/dark cycle.
RNA isolation and RT-PCR
Animals in experiments were kept at ambient temperatures (4°C or 30°C as indicated) for the times specified and were sacrificed with carbon dioxide. Dissected tissues were placed in excess amounts of RNAlater (Qiagen). Total RNA was extracted using Ultraspec (Biotecx, Houston, TX, USA) and RNeasy Kit (Qiagen) and treated with RNase-free DNase I (Qiagen), according to the instructions of the manufacturer. RNA integrity was assessed by agarose-formaldehyde gel electrophoresis. A total of 400 ng RNA and random decamers (Ambion, Foster City, CA, USA) was used to synthesize first-strand cDNA by M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). For all samples, RT-negative controls were made. For relative quantitative RT-PCRs, the linear range of each primer-target amplification was determined and a corresponding number of cycles was used for the PCR. Specific primers used for relative quantitative RT-PCR were: c-Fo subunit transgene-specific P1 isoform (ATP5G1): 5'-ccgggcaatctgggcttaacgggtcc-3' and 5'-cggctcgagactggcatggagtcaaagc-3'; c-Fo subunit total P1 isoform (ATP5G1): 5'-gcgctcgagttgaaaaatgcagacca-3'and 5'-cggctcgagactggcatggagtcaaagc-3'; c-Fo subunit P2 isoform (ATP5G2): 5'-tcacatggacgaagaggatgatgag-3' and 5'-ctctgccctgggagcagc-3'; c-Fo subunit P3 isoform (ATP5G3): 5'-gatgttcgcctgcgccaagctc-3' and 5'-cacacaccattctgggccccattaa-3'; total P1P3: 5'-attggctatgccaggaaccc-3' and 5'-tcacatggcgaagaggatgag-3'; β-F1 ATPase subunit (ATP5B): 5'-gcagggagagcagactggttttggagg-3' and 5'-gattctgcccaaggtctcaggaccaac-3'; b-Fo subunit (ATP5F1): 5'-gctgtcccgctcttcgcagacaatg-3' and 5'-cctccaaggccagggcaatgttattc-3'; COX1: 5'-gcttactcagccattttacctatgtt-3' and 5'-atacgagcagtacggctgtaataag-3'; citrate synthase: 5'-cttcggtcccttcccgccaggtcc-3' and 5'-agtcctcatagatgagctcccagtac-3'; UCP1: 5'-gccactgttgtcttcagggctgag-3' and 5'-ggtgatggtccctaggacacct-3'. The specificity of amplified PCR fragments for P1-P3 isoforms was confirmed by sequencing. The RT-PCR data were normalized using QuantumRNATM Universal 18S Internal Standard kit (Ambion).
Mitochondrial preparation
UCP1p1 transgenic and wild-type mice were single-caged and acclimated at 18°C for 1 wk, then transferred to 4°C and kept at this temperature for several weeks. The ability to acclimate to cold appeared unchanged in transgenic mice compared to wild-type mice: they maintained the same body temperature, they survived for at least a month in the cold, and they had an identical response to norepinephrine test injections (analyzed as in ref. 18
). Brown-fat mitochondria were prepared principally as described (19)
. The mitochondrial suspensions were kept on ice and used for no longer than 4 h.
Western blot
Aliquots of freshly isolated mitochondrial suspensions were stored at –80°C after supplementation with protease inhibitor cocktail (Complete Mini®, Roche, Basel, Switzerland). Protein concentration was determined using the Lowry method. Mitochondrial protein samples were separated on 16.5% acrylamide-Tricine-SDS gels according to (20)
to allow good resolution of subunit c. Primary antibodies for c subunit (21)
, β subunit (Molecular Probes/Invitrogen, Carlsbad, CA, USA), subunit (Molecular Probes/Invitrogen) or COX1 (Molecular Probes/Invitrogen) were used. Membranes were incubated in ECL reagents (Amersham Biosciences, Piscataway, NJ, USA), chemiluminescence signal was detected with a CCD camera (Fuji, Tokyo, Japan) and quantitated using Image Gauge v. 3.45 (FujiFilm, Tokyo, Japan) software.
Blue-Native gel electrophoresis
Blue-Native gel electrophoresis was run essentially as described in (22)
. Briefly, 0.4 mg of pelleted mitochondria isolated from cold-acclimated UCP1p1 transgenic and wild-type mice was resupended in 25 µl of 0.75 M -aminocaproic acid (ACA), 75 mM BisTris, pH 7.0 and 40% glycerol, and 25 µl of 3% dodecyl-β-maltoside in 0.75 M ACA and 75 mM BisTris, pH 7.0 were added to the suspension. Samples were centrifuged at 5°C at 12 000 rpm for 30 min, 30 µl of supernatant was supplemented with 3 µl of sample buffer (ACA, BisTris, glycerol, and Coomassie G-250) and loaded on the gel (5–13.5% acrylamide gradient).
Measurements of F1-ATPase activity in isolated mitochondria
F1-ATPase activity in isolated brown-fat mitochondria of cold-acclimated transgenic and wild-type mice was determined spectrophotometrically by coupling the reaction to pyruvate kinase and lactate dehydrogenase and monitoring NADH oxidation at 340 nm. Frozen-thawed mitochondrial samples (100 µg) were added to a total of 1 ml of 350 mM NADH solution in 50 mM HEPES/KOH, 5 mM MgSO4, pH 8.0, containing 2.5 mM phosphoenolpyruvate (Sigma-Aldrich, St. Louis, MO, USA), 2 µM rotenone, 7 U pyruvate kinase (Sigma-Aldrich), 15 U lactate dehydrogenase (Fluka, Seelze, Germany), 0.05% dodecyl-β-maltoside; reaction mixture temperature was kept at 30°C. The enzymatic reaction was initiated by adding 2.5 mM ATP, and the decrease in NADH absorbance at 340 nm was monitored for 4 min. ATPase activity was then inhibited by addition of oligomycin (2 µg) to the reaction. The F1-ATPase-specific activity rate was estimated by subtraction of the oligomycin-insensitive rate.
Oxygen consumption
Oxygen consumption rates were monitored exactly as described in (23)
, with 5 mM pyruvate as substrate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and thus placed under the control of the UCP1 promoter (Fig. 1)
. Founder animals obtained were analyzed by PCR for the presence of the transgenic insert in the genomic DNA, and three founders (named k457.2, k459.1, and k459.7) were positively identified (Fig. 2
). The k459.1 founder that gave a very weak signal by PCR analysis failed to produce any transgenic offspring, but the other two founders, k457.2 and k459.7, produced transgenic offspring that were used for further experiments. Offsprings of the k459.7 founder line were also used to establish a homozygous line of transgenic animals.
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Analysis of P1 c subunit mRNA levels in UCP1p1 transgenic mice
We analyzed the expression of both the transgene-specific and endogenous P1 c subunit genes using RT-PCR. To activate the UCP1-promoter-regulated expression of the transgene in BAT, transgenic and control mice (F1 heterozygous littermates of the k459.7 and k457.2 founders) were transferred to 4°C for 24 h, a time known to be sufficient for activation of UCP1 gene expression (24)
. After 24 h cold exposure, tissues were dissected out and isolated total RNA was used for synthesis of cDNA.
To examine tissue specificity of the transgene P1 expression, we used primers that were complementary to UCP1 5'UTR and P1 c subunit 3'UTR, which allowed us to generate a transgene-specific PCR fragment. As seen in Fig. 3
A, a specific PCR fragment corresponding to the UCP1p1 insert was found only in BAT of UCP1p1 transgenic mice (Tg/0) and not of wild-type control mice (wt). The absence of the PCR fragment in heart confirms the tissue-specific expression of the UCP1p1 insert.
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The transgene UCP1p1 expression in mice derived from the founder k457.2 was also analyzed by the same PCR method and found to be very low (results not shown). Therefore, for further experiments, offsprings of the founder k459.7 were used.
Relative mRNA levels of total P1-c subunit were analyzed by RT-PCR with P1 subunit-specific primers and with 18S-specific primers, used as an internal control (Fig. 3B
). The substantial difference in amounts of P1 subunit c mRNA between BAT and heart tissue in the wild-type animals was in good agreement with previously published results (11)
. In BAT, the amount of the total P1 subunit c was significantly increased in the UCP1p1 transgenic animals (Tg/0) compared to the wild-type controls (wt) (Fig. 3C
).
The mRNA levels of the other isoform of subunit c, P2, were also checked by relative RT-PCR. As expected, no significant difference in P2 mRNA amounts was found between transgenic and wild-type mice (Fig. 3D
). Similarly, we observed no difference in the levels of the P3 isoform (not shown). Total c subunit mRNA levels (transgene P1 plus endogenous P1 plus endogenous P2 and P3 isoforms) were determined with primers within the coding region (which is identical in all isoforms) and was found to be augmented in transgenic animals, up to 60% as compared to wild-type (data not shown).
We also examined the mRNA levels of other ATP synthase subunits that belong to either F1 (β subunit) or Fo oligomer (b subunit). The transcription of the β subunit is high in BAT; the mRNA levels in BAT are as high as those in heart (11)
. Our results (Fig. 3D
) were in agreement with these observations and also showed that the β subunit gene in transgenic and control animals was expressed at the same level. Similar results were shown for the transcription of the b-Fo subunit (Fig. 3D
). Transcript levels of one of the subunits of Complex IV, cytochrome c oxidase subunit 1 (COX1), were also measured by RT-PCR, as well as citrate synthase and UCP1 mRNA levels (Fig. 3D
). All of them were found to be expressed at the same level in BAT of transgenic animals (Tg/0) compared to control (wt) mice (UCP1 gene expression is absent in heart). Thus, in the brown adipose tissue of UCP1p1 transgenic animals, only the P1-c subunit mRNA levels were augmented, whereas the levels of other transcripts of mitochondrial proteins were unchanged.
Analysis of protein content of F1Fo-ATPase and whole-complex assembly in UCP1p1 transgenic mice
Mitochondria were isolated from cold-acclimated (at least 3 wk at 4°C) UCP1p1 transgenic and control mice, and several mitochondrial proteins were analyzed by Western blot (Fig. 4
). In UCP1p1 transgenic animals, total subunit c protein amount (Fig. 4A
) was increased by 50% compared to wild-type mice (Fig. 4B
), principally in proportion to the increase in total subunit c mRNA.
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However, strikingly, the increase in c subunit protein amount was accompanied by a clearly significant increase in β subunit protein content (Fig. 4C, D
), despite the unaltered levels of β subunit mRNA (Fig. 3D
). A similar increase in protein levels was found for the subunit (Fig. 4E, F
).
To analyze the ATP synthase complex (Complex V) levels in brown-fat mitochondria of transgenic and wild-type mice, we used Blue-Native gel electrophoresis, which allowed us to resolve the individual mitochondrial respiratory complexes in their native state (Fig. 4G
). In mitochondria isolated from UCP1p1 transgenic mice (Tg/0), the level of Complex V was notably increased compared to wild-type mitochondria (wt), whereas the levels of complexes I, III, and IV were not changed in transgenic mice compared with control mice (Fig. 4G
). The increase in the whole ATP synthase complex in transgenic mice clearly shows that the increase in protein levels of a single ATP synthase subunit, the c subunit, was sufficient to lead to the increase in the assembly of the whole complex.
To further substantiate the dependence of the increase in ATP synthase amount on the increase in c subunit protein, we created homozygous transgenic UCP1p1 mice (Tg/Tg) and checked the P1 transgene-specific mRNA levels (Fig. 5
A) and P1 total mRNA levels (Fig. 5B
). These mRNA levels were further increased compared with UCP1p1 heterozygous transgenic mice (Tg/0). In the homozygous UCP1p1 mice, the protein content of c subunit, as well as subunit, was also markedly augmented, compared with wild-type (wt; Fig. 5C
). In Fig. 5D, we
demonstrate that it was possible to quantitatively titrate the protein amount of (at least) the constituents of the catalytic F1 oligomer, exemplified here by β subunit, as a result of altering the mRNA level of only one of the constituents of the Fo oligomer.
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Thus, analysis of the protein content of ATP synthase subunits indicates that if provided with additional amounts of c subunit protein, the total ATP synthase content is proportionally augmented in brown-fat mitochondria. It indicates that during the final steps of ATP synthase assembly, it is the limited availability of the c subunit protein that defines the final (low) amount of the enzyme in brown-fat mitochondria.
Functional activity of the ATP synthase in brown-fat mitochondria of UCP1p1 transgenic mice
To demonstrate that the increased protein content of the ATP synthase subunits subsequently results in an increased content of the fully functional enzymatic complex, we used several approaches to measure ATP synthase activity in isolated brown-fat mitochondria of UCP1p1 transgenic and wild-type mice.
Direct F1-ATPase activity was examined in brown-fat mitochondria of the transgenic (Tg/0) and the control (wt) mice (Fig. 6
). The F1-ATPase activity was measured spectrophotometrically in an enzyme assay system that is coupled to NADH oxidation (Fig. 6A
). Significantly increased activity of oligomycin-sensitive F1-ATPase in transgenic mice (Fig. 6B
) confirmed our hypothesis that the increase in one subunit (Fo-c subunit) led to an increase in the amount of the whole enzyme, which was also functionally active.
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Another approach, oxygen consumption measurement, was used to monitor ATP synthase activity in isolated brown-fat mitochondria. In Fig. 7
A and B, mitochondrial respiration was stimulated by addition of substrate (pyruvate). In brown-fat mitochondria, the addition of substrate alone leads to a large increase in respiration. This is caused by the activity of uncoupling protein 1 (UCP1) [for review see (7)
]. The mitochondria may be recoupled by the addition of GDP, which totally blocks the activity of UCP1. In wild-type (Fig. 7A
) and transgenic (Fig. 7B
) mice, the response to GDP was similar, as expected. In wild-type mice, subsequent induction of respiration on addition of ADP (Fig. 7A
) showed the characteristic extremely low functional activity of the ATP synthase in BAT mitochondria, in agreement with previous reports (9)
. However, in the UCP1p1 transgenic mice the ADP-mediated stimulation of ATP synthase activity (Fig. 7B
) was more evident compared to wild-type (although it was still low compared to both UCP1 activity and total respiratory capacity, as observed after addition of the artificial uncoupler FCCP).
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To allow for better quantification of ATP synthase activity in the mitochondria of the UCP1p1 transgenic mice, we used an alternative experimental design. Here UCP1 activity was inhibited by GDP prior to addition of substrate; more mitochondria could thus be added (Fig. 7C, D
), and the ADP-induced and oligomycin-inhibited respiration (that corresponds to ATP synthase activity) became much more evident. Quantitatively, the oligomycin-sensitive respiration in brown-fat mitochondria was significantly greater in the UCP1p1 transgenic mice compared to wild-type controls (Fig. 7E
), thus demonstrating an increased content of functional ATP synthase in the UCP1p1 transgenic mitochondria. Thus, the increase solely in subunit c mRNA levels was sufficient to induce a proportional increase in fully functional assembled ATP synthase.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Strikingly, however, the majority of the ATP synthase subunit genes are expressed coordinately with other OXPHOS enzymes, and the mRNA levels for most subunits are thus very high compared to that in most other tissues (10
, 11)
. The remarkable issue is thus the means by which the very low levels of ATP synthase are achieved in brown adipose tissue. We earlier observed low mRNA levels of only one of the ATP synthase subunits, the c-Fo subunit P1 isoform. This finding led us to suggest that selective modulation of the c-Fo subunit protein level might be crucial for defining the final low levels of the ATP synthase in brown adipose tissue (11
, 15)
. However, no evidence for this hypothesis has been available until now.
We have now addressed this question by introducing an increased amount of the P1 isoform of the c-Fo subunit in brown adipose tissue. This was accomplished through a transgenic construct where the P1 isoform of the c-Fo subunit was expressed under the control of the brown adipose tissue-specific UCP1 promoter. This approach allowed for both tissue specificity and for enabling us to directly control subunit c expression, as the UCP1 promoter activity is under physiological control and expression can thus be selectively induced by exposing the animals to a decreased environmental temperature. Indeed, in the brown adipose tissue of the transgenic animals, we found that cold-stimulated expression of the P1-c subunit transgene resulted in a significant increase in total mRNA levels of the P1-c subunit (transgene and endogenous) (Fig. 3)
. The total c subunit (P1–P3) mRNA levels in the transgenic animals were thus augmented, and this led to a parallel increase in protein levels of the c-Fo subunit in brown-fat mitochondria (Fig. 4)
. Thus, the protein level of this subunit seems to be directly controlled by the corresponding mRNA level.
Remarkably, although the levels of mRNA coding for the other subunits of the F1Fo-ATPase were unchanged [followed here as the β and the b subunits mRNA level (Fig. 3D
)], elevated amounts of the F1-ATPase subunits were found at the protein level in transgenic animals [followed here as the and β subunits (Fig. 4C
–F)] apparently as a response to the increased availability of the c-Fo subunit (Figs. 3
, 4
A–E). Moreover, the whole Complex V assembly was increased in transgenic mice (Fig. 4G
), showing the crucial influence of the increase in c subunit protein levels on elevation of the total ATP synthase amount. This observation was substantiated by similar experiments in homozygous transgenic mice where a further increase in mRNA and protein content of c subunit resulted in further proportional increase in other subunits of the ATP synthase (Fig. 5)
. In transgenic animals, the subunits were not only present in increased amounts compared to wild-type, but they were also assembled into a functionally active enzyme, as was evidenced by increased ATPase activity (Fig. 6)
. Thus, there was no other limitation in the mitochondrial topography that prevented ATP synthase assembly. Most noticeable was that the increased ATP synthase protein complex could indeed carry out functional ATP synthesis, implying that the interaction between the F1 and the Fo oligomers was of the same qualitative efficiency as that found in the endogenously controlled amount of ATP synthase (Fig. 7)
.
The specific steps in mitochondrial ATP synthase assembly have still not been characterized in animal cells. We advocate that BAT is a valuable model that provides an insight into this process. The lack of coordinated expression of different ATP synthase subunits in brown adipose tissue, with most subunits being transcribed at a high level but the c-subunit only being present at a very low level (11
, 15)
, does not lead to accumulation of ATP synthase assembly intermediates (J. Houstek, U. Andersson, B. Cannon, unpublished results). This observation, together with our present findings, indicates that the assembly of the ATP synthase in brown adipose tissue goes through at least one critical step, when supposedly preassembled F1-complex "checks" for the availability of the subunit c ring/Fo-complex in the mitochondrial membrane. If these subcomplexes are not incorporated into functional enzyme due to the unavailability of a sufficient amount of subunit c, excessive subcomplexes are presumably destabilized and rapidly degraded by mitochondrial proteases. We cannot, however, exclude at this point that the c subunit expression levels do not directly affect the synthesis rate of other ATP synthase subunits.
Thus, we have demonstrated here in vivo the validity of a hypothesis that in brown adipose tissue, the amount of the ATP synthase and its assembly are regulated specifically by the mRNA and protein levels of a single subunit, the c-Fo subunit, which results in the final low levels of the ATP synthase. Such selective regulation of expression of the c-Fo subunit P1 isoform may provide the means of changing ATP synthase levels in response to physiological stimuli in a tissue-specific context and identifies regulation of the expression of the c-Fo subunit as a possible major determinant in controlling the bioenergetic balance of a given tissue.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: AstraZeneca R&D Södertälje, SE-151 85 Södertälje, Sweden
Received for publication March 22, 2007. Accepted for publication July 5, 2007.
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