©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Expression of Subunit c Correlates with and Thus May Limit the Biosynthesis of the Mitochondrial FF-ATPase in Brown Adipose Tissue (*)

(Received for publication, November 14, 1994 )

Josef Houstek (1) (2)(§) Ulf Andersson (2) Petr Tvrdík (2)(¶) Jan Nedergaard (2) Barbara Cannon (2)

From the  (1)Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague, Czech Republic and the (2)Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, S-106 91, Stockholm, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A low content of mitochondrial ATPase in brown adipose tissue (BAT) has previously been found to contrast with high levels of the transcripts of the beta-subunit of the F(1) part of the ATPase and of the transcripts of the mitochondrial encoded subunits (Houstek, J., Tvrdík, P., Pavelka, S., and Baudysová, M.(1991) FEBSLett. 294, 191-194). To delineate which subunit limits the synthesis of the ATPase complex, we have studied the expression of the nuclear genes encoding subunits alpha, beta, and of the catalytic F(1) part and the b, c, d, and OSCP subunits of the F(0) part of the ATPase.

In comparison with other tissues of mice, high levels of transcripts of alpha-F(1), beta-F(1), -F(1), b-F(0), d-F(0), and OSCP were found in BAT. The only genes expressed at a low level in BAT were those of the c-F(0) subunit. The levels of c-F(0) transcripts were 4-70-fold lower in BAT than in other tissues. An analogous expression pattern of the ATPase genes was found in BAT of adult rat and hamster. In BAT of newborn lamb, which, in contrast to other mammals, has a high content of mitochondrial ATPase, correspondingly high levels of c-F(0) mRNA were found. Expression of the c-F(0) genes also correlated well with the ontogenic development of BAT in the hamster, being high during the first postnatal week when mitochondria are nonthermogenic and contain a relatively high amount of ATPase, but low on subsequent days when ATPase content decreases, as the thermogenic function develops.

It is suggested that expression of the c-F(0) genes and subsequent synthesis of the hydrophobic subunit c of the membrane-intrinsic F(0) part of the enzyme may control the biosynthesis of the ATPase complex in BAT. An analogous regulatory role of the c-F(0) subunit could be postulated in other tissues.


INTRODUCTION

The regulation of the biosynthesis and assembly of multisubunit mitochondrial enzyme complexes is still poorly understood, especially when a concerted modulation of the expression of genes encoded by both nuclear and mitochondrial genetic compartments is to be expected(1) . Of particular interest is the biogenesis of the ATPase, the central enzyme in oxidative phosphorylation, responsible for the production of most of the ATP in mammalian organisms.

The mammalian ATPase consists of 16 different polypeptides(2) , 6 of which comprise the globular catalytic F(1) part (subunits alpha, beta, , , and and the loosely attached ATPase inhibitor protein IF(1)) and 10 of which comprise the H-translocating, membrane-spanning F(0) part (subunits a, b, c, d, e, f, g, F6, OSCP, and A6L). The two parts of the enzyme are linked together by a stalk to which subunits , , , OSCP, F6, b, and d contribute(2) . Two subunits of the F(0) part (a and A6L) are products of mitochondrial genes(3) ; all the other subunits of the ATPase are nuclearly encoded. Tissue-specific gene expression has only been demonstrated for two subunits, alpha-F(1)(4) and c-F(0)(5, 6, 7) , of the complex. Eight subunits (alpha, beta, , and of F(1) and a, b, c, and OSCP of F(0)) appear to be absolutely essential for enzyme function as their homologous equivalents are found in all organisms(8) .

Transcriptional regulation of the mitochondrial proteins encoded in the nucleus is the predominant type of control of mitochondrial biogenesis (9, 10, 11) . Different cellular energy demands, resulting from different tissue requirements(12, 13) , adaptive changes(14) , or regulatory effects of hormonal and other factors(15) , may be met via the regulation of the expression of certain nuclear genes for mitochondrial proteins, and several regulatory sequence elements common to these genes have been identified, e.g.NRF-1, NRF-2, the so-called ``enhancer,'' or Mt1-Mt5(9, 15, 16, 17, 18, 19, 20) . However, not all nuclear genes encoding subunits of the oxidative phosphorylation complexes have these regulatory elements(11) ; in fact, only one or two of the subunits of each enzyme complex, including the ATPase, appear to be under the same transcriptional control(9, 11, 15, 16, 17, 18, 19, 20, 21, 22) . This implies that a differentiated expression circuitry exists for individual genes and that some genes may have a key role in the regulation of the biosynthesis of oxidative phosphorylation complexes.

A particularly rewarding object for the study of mitochondrial biogenesis has been found in brown adipose tissue (BAT), (^1)a specialized mammalian thermogenic organ that utilizes a high oxidative capacity to produce heat instead of ATP. The molecular basis for this reaction is a H short-circuiting of the inner mitochondrial membrane due to the presence of a tissue-specific proton channel, the uncoupling protein (for review, see (23) and (24) ). In most species, BAT mitochondria contain only very low amounts of ATPase, and the ATPase/respiratory chain stoichiometry is 10-fold lower than in other tissues(25, 26, 27, 28) . BAT can thus be used as an unique natural model of selective suppression of the biosynthesis of the mammalian ATPase complex. In other respects, the ATPase complex is structurally and functionally normal, and both the F(1) and F(0) components are equally reduced in BAT(25, 26, 27, 28, 29) . However, analysis of the mRNA levels of both nuclearly and mitochondrially encoded ATPase subunits (beta-F(1) and a-F(0) (ATPase 6)) in BAT unexpectedly showed very high rates of expression of these ATPase genes (28, 30) . This clearly indicated that the catalytic beta-F(1) subunit is not transcriptionally suppressed in BAT and that also mitochondrially encoded F(0) subunits (a and A6L) are overexpressed. The key regulatory step in depressed biosynthesis of ATPase in BAT remains, however, unexplained.

In this study, experiments were undertaken to investigate whether control of ATPase biosynthesis could exist at the level of expression of other nuclear genes. Analysis of the transcripts for most of the essential subunits of the ATPase(8) , the alpha, beta, and subunits of the catalytic F(1) part and the b, c, d, and OSCP subunits of the F(0) part, was performed in BAT and in other tissues of several mammalian species as well as in different developmental states and in species with an elevated content of ATPase in BAT. The results unequivocally demonstrate selective transcriptional control of the genes encoding the c-F(0) subunit of the ATPase, which thus apparently has a key role in the regulation of the biosynthesis of ATPase in BAT.


EXPERIMENTAL PROCEDURES

Animals

Adult mice (NMRI strain; Eklunds, Stockholm, Sweden), rats (Sprague-Dawley; Alab, Stockholm), and Syrian golden hamsters (Mesocricetus auratus; Wenner-Gren Institute, Stockholm) as well as hamster pups were maintained at 22 ± 2 °C on a 12-h light/12-h dark cycle with standard diets and water adlibitum. Tissues from a 3-day-old newborn lamb were obtained from a local farm.

Mitochondria

Mitochondria from interscapular BAT, liver, and heart were isolated from 5% homogenates in 0.25 M sucrose, 10 mM Tris-HCl, pH 7.2, 1 mM EDTA, 2 µg/ml aprotinin according to standard procedures(31) . In the case of the lamb, a perirenal pad of BAT was used(32) . Protein concentration was determined by the method of Bradford(46) .

Northern Blot Analysis

Total RNA was isolated from up to 100-mg aliquots of each tissue, either fresh or frozen in liquid nitrogen, by a guanidinium/phenol/chloroform method using RNazol B (Biotecx Laboratories). 10-µg aliquots of total RNA were separated on a 1.25% formaldehyde-agarose gel and blotted onto Hybond-N membranes (Amersham Corp.). Prehybridization and hybridization were performed as described(33) . Membranes were washed in 0.1 times SSC and 0.2% SDS two times for 60 min at 52 °C. Membranes were stripped in 0.1% SDS for 15 min at 100 °C and rehybridized with no apparent loss of hybridization ability. The membranes were analyzed with Image Quant version 3.3 on a Molecular Dynamics PhosphorImager.

cDNA Probes

The cDNA probes were kindly provided by the following investigators. Rat beta-F(1) cDNA (34) was a 1.26-kilobase EcoRI fragment in pUC19 from Prof. P. L. Pedersen (The Johns Hopkins University, Baltimore) and the c-F(0) cDNA (6) was a human P1 full-length EcoRI fragment in pGEM-2 from Dr. P. Nagley (Monash University, Clayton, Australia). The following M13 clones of bovine cDNAs (4) were obtained from Dr. M. J. Runswick (Medical Research Council, Cambridge, United Kingdom): the alpha-F(1) cDNA was a 1.24-kilobase EcoRI/BamHI fragment in mp9, the -F(1) cDNA was a 0.97-kilobase BamHI fragment in mp8, the d-F(0) cDNA was a full-length EcoRI/BamHI fragment in mp9, and the b-F(0) cDNA was a 0.95-kilobase EcoRI/BamHI fragment in mp8. The OSCP probe was a 42-mer oligomer complementary to positions 160-201 of the rat OSCP cDNA. The cDNAs were labeled with the random primed DNA labeling kit (Boehringer Mannheim), and the OSCP probe was end-labeled with polynucleotide kinase (Amersham Corp.).

Western Blot Analysis

SDS electrophoresis was performed on 10% polyacrylamide slab gels(35) , and proteins were transferred by semidry electroblotting onto Hybond-C extra membranes (Amersham Corp.). For immunodetection in phosphate-buffered saline containing 0.05% Tween 20, rabbit antisera to bovine heart F(1)-ATPase, the bovine heart b-F(0) subunit(36) , the ovine c-F(0) subunit (obtained from Prof. M. Elleder, Faculty of Medicine, Charles University, Prague, Czech Republic), the rat heart cytochrome oxidase IV subunit, and hamster UCP (36) were used at dilutions of 1:1000-1:10,000. Immune complexes were visualized by a chemiluminescence method using a peroxidase-conjugated Fab fragment of goat anti-rabbit IgG (Bio-Rad) and an ECL kit (Amersham Corp.). Exposures made on Kodak X-Omat RP films were quantified on a Molecular Dynamics computing densitometer.


RESULTS

Expression of Nuclear Genes Encoding ATPase Subunits: Selectively Low Levels of c-F(0) Subunit mRNA in Thermogenically Active Brown Adipose Tissue

To investigate whether expression of a single subunit could be of regulatory importance for synthesis of the entire complex, the steady-state levels of transcripts for seven nuclearly encoded subunits of the mitochondrial ATPase complex were measured by Northern blotting in total RNA samples isolated from BAT of adult mice and compared with the expression in tissues with normal ATPase content. As shown in Fig. 1, in agreement with our previous studies(28) , the beta-F(1) transcripts were paradoxically high in BAT, as high as in heart, which has a 6-fold higher ATPase content than BAT, and much higher than in liver, which has a 3-fold higher ATPase content. Similarly high levels of the mRNAs for two other F(1)-ATPase subunits, alpha-F(1) and -F(1), were found in BAT (Fig. 1), indicating that they also had paradoxically high levels of expression when compared with the very low content of ATPase in BAT(28) . Analysis of the mRNA levels for the F(0)-ATPase subunits further revealed that also the levels of the b-F(0) and d-F(0) transcripts (Fig. 1) as well as the OSCP transcripts (data not shown) were high and thus corresponded well with the levels of F(1) subunits transcripts and to the high level of mitochondrially encoded F(0) subunits a and A6L (ATPase 6/8) transcripts(28) . However, the mRNA level of c-F(0) was markedly different. In contrast to the high expression of the genes for the other subunits, the expression of c-F(0) in BAT was exceptionally low (Fig. 1) and was thus the only subunit for which expression correlated with the low ATP synthase content.


Figure 1: Northern blot analysis of mitochondrial ATPase subunits mRNAs in mouse BAT, liver, and heart. 10-µg aliquots of total RNA from the indicated tissues were analyzed by means of reprobing the membrane with the cDNA probes described under ``Experimental Procedures.'' The results are representative of three to four similar experiments (see Table 1). The leftpanel shows the PhosphorImager scans, and the rightpanel the computation of these scans; the level of each subunit in heart was set to 1. The two mRNA species of alpha-F(1) were quantified together.





The interpretation of these types of data may be complicated by problems resulting from technical differences between the analyses of the levels of different transcripts (e.g. due to different labeling intensities) as well as from possible biological differences in translation efficiency between the different transcripts. To avoid such problems, we present here the data for the different transcript levels, adjusted to those observed in the ATPase-richest tissue investigated: the heart. Thus, the level of each transcript in the heart was set to 1 (Fig. 1); this also means that the ratio between the levels of all transcripts in the heart is 1. When the beta-F(1)/c-F(0) ratio was calculated in this way for most tissues in the mouse, the expected ratios close to 1 were obtained (Fig. 2). However, very high values for the beta-F(1)/c-F(0) ratios were observed in BAT; similarly high values were calculated for the ratio between the mRNA of other ATPase subunits measured and c-F(0) (data not shown). An analogous pattern was found in the rat (Fig. 2). In contrast to this pattern observed when any transcript level was related to that of c-F(0), relative ratios for other comparisons always came out close to the expected ratio of 1. This is illustrated in Fig. 2for the beta-F(1)/alpha-F(1) ratio, but was found for all other transcript ratios, supporting the validity of the ratio method to identify the unique behavior of the c-F(0) transcript.


Figure 2: Relative expression of alpha-F(1), beta-F(1), and c-F(0) genes in different tissues of mouse and rat. Northern blot analyses of seven different tissues from mouse and three different tissues from rat were performed and evaluated as described in the legend to Fig. 1. The expression of each subunit in heart was set to 1. The relative ratios between beta-F(1) and c-F(0) mRNA and between beta-F(1) and alpha-F(1) mRNA were calculated. One representative experiment is shown. WAT, white adipose tissue.



Quantitative evaluation of the level of the c-F(0) transcript indicated that the gene in BAT was expressed about 70-fold lower than in the heart, about 14-fold lower than in the liver, and about 4-fold lower than in white adipose tissue (Table 1). As the level of the beta-F(1) transcript was even higher in BAT than in the heart, the ratio between the two transcripts (beta-F(1)/c-F(0)) was >100-fold higher in BAT than in the heart.

It can be concluded that there is a selective decrease in the expression of the c-F(0) genes in BAT. This implies that the control of the expression of the c-F(0) genes may have a key regulatory role in the biosynthesis of the mitochondrial ATPase in BAT.

High c-F(0) Expression in ATPase-rich Brown Adipose Tissue of Lamb

If a general regulatory role in the control of the biosynthesis of ATPase should be ascribed to the c-F(0) subunit, it would be predicted that the expression of this subunit should be higher in BAT in species having a higher ATPase content. It has earlier been observed that lamb is such a species. Lamb BAT produces heat by the same UCP-based mechanism of uncoupling of oxidative phosphorylation as BAT in rodents(32, 37) , but, unlike in rodents, the ATPase is not reduced in lamb BAT: mitochondria from BAT of newborn lamb have a high activity of oligomycin-sensitive ATPase and high amounts of F(1)-ATPase(32) . We have now also investigated by Western blotting the content of the ATPase subunits in different tissues of lamb and compared the pattern with that in mouse. As expected, in the mouse, the content of each investigated subunit of the ATPase was much lower in BAT mitochondria than in liver mitochondria (Fig. 3). It may especially be noted that the level of subunit c was about 5-7-fold lower in BAT than in liver, in fair agreement with the 7-19-fold lower content of the c-F(0) transcript in BAT than in liver (ranges from different experiments, cf. also Table 1).


Figure 3: Western blot analysis of the ATPase content in BAT and liver of mouse and newborn lamb. Isolated mitochondria (3-µg protein aliquots) from mouse and lamb liver and BAT were analyzed for the content of (alpha + beta)-F(1) subunits, the b-F(0) subunit, and the c-F(0) subunit by probing the appropriate parts of the membrane (molecular mass regions of 90-45, 30-20, and 14-5 kDa, respectively) with specific rabbit antibodies to F(1)-ATPase (1:10,000), the b-F(0) subunit (1:5000), and the c-F(0) subunit (1:1000) as detailed under ``Experimental Procedures.''



However, in lamb, the relationship was opposite, and the BAT mitochondria here contained 1.3-1.7-fold more ATPase subunits than those from liver (Fig. 3). Thus, the evidence from immunoblotting was in agreement with that expected from earlier studies with other methods (32, 37) .

As shown in Fig. 4, this high content of ATPase subunits in BAT of lamb was accompanied by high steady-state levels of the mRNAs for beta-F(1) (Fig. 4) and other ATPase gene transcripts (data not shown). Most notably, also the steady-state level of c-F(0) mRNA (Fig. 4) was high in BAT, almost as high as in lamb heart and much higher than in lamb liver. Thus, in contrast with the situation observed in mice and rats ( Fig. 1and Fig. 2and Table 1), the levels of c-F(0) transcripts relative to those of beta-F(1) were the same in all the investigated lamb tissues. The high level of expression of the c-F(0) gene(s) in lamb BAT thus, as predicted, corresponded well with the high content of mitochondrial ATPase in this species.


Figure 4: Northern blot analysis of beta-F(1) and c-F(0) mRNAs in heart, liver, and BAT of newborn lamb. The experimental procedures were as described in the legend to Fig. 1.



Developmental Regulation of c-F(0) Expression in Brown Adipose Tissue

It has been shown in several rodent species that perinatal recruitment of BAT thermogenic function, characterized by the onset of ucp gene expression, is preceded by a period when the mitochondria in BAT are nonthermogenic. These mitochondria are thus similar to mitochondria from other tissues in that they completely lack the UCP(38, 39) , but are equipped with active respiratory chain and ATPase. In newborn hamster, this nonthermogenic period lasts until the end of the first postnatal week when the nonthermogenic-thermogenic transition begins. In agreement with previous measurements of ATPase activity and F(1)-ATPase content(38) , we have here observed in BAT of neonatal (5-day) hamsters a high content of (alpha + beta)-F(1) antigens (Fig. 5). This was paralleled by a high content of the c-F(0) antigen. During postnatal development, a pronounced decrease in the content of (alpha + beta)-F(1) subunits as well as of the c-F(0) subunit was observed. This postnatal pattern contrasted with the appearance of the UCP antigen and with the pronounced increase in cytochrome oxidase IV antigen during this period (Fig. 5).


Figure 5: ATPase, UCP, and cytochrome oxidase content in BAT during postnatal development in the hamster. 18-µg protein aliquots of homogenate from BAT of 5-day, 15-day, and adult hamsters (60 days) were analyzed by Western blotting as described in the legend to Fig. 3. The appropriate parts of the membrane were probed with antibodies against F(1)-ATPase and the c-F(0) subunit (see legend to Fig. 3) as well as with antibodies against UCP (30-45-kDa region; 1:10,000) and the cytochrome oxidase IV subunit (COXIV; 20-14-kDa region; 1:1000).



Regarding the mRNA levels, a markedly different pattern was observed during postnatal development. In contrast to the decreasing amount of the (alpha + beta)-F(1) antigens, the level of the beta-F(1) mRNA (Fig. 6) as well as of other ATPase subunit transcripts (data not shown) continuously increased in BAT with age. However, the level of c-F(0) mRNA continuously decreased. Thus, the changes in the amount of ATPase subunits (Fig. 5) and the enzyme activity (38) correlated only with the expression of c-F(0) in BAT, but not with the expression of the other ATPase genes. For comparison, it is seen that constantly high levels of beta-F(1) and c-F(0) transcripts were present in heart, where the ATPase is stably high throughout this period (Fig. 6).


Figure 6: beta-F(1) and c-F(0) mRNA levels in BAT and heart during postnatal development in the hamster. Total RNAs from BAT and heart of hamster pups of the indicated ages and from adult hamsters (60 days) were analyzed for the beta-F(1) and c-F(0) transcripts as described in the legend to Fig. 1.



The specific correlation between the expression of the c-F(0) gene(s) and the content of the mitochondrial ATPase in BAT is outlined in Fig. 7. It can be seen that the decrease in c-F(0) mRNA levels correlates well temporally and quantitatively with both the c-F(0) antigen amount and the (alpha+beta)-F(1) antigen amount, but there is no correlation between the beta-F(1) mRNA and the beta-F(1) antigen. The same lack of correlation was found for all the other transcripts investigated in this study (alpha-F(1), -F(1), b-F(0), d-F(0), and OSCP; data not shown). Thus, the c-F(0) transcript levels may determine the resulting level of ATPase.


Figure 7: Changes in the content of F(1)- and F(0)-ATPase subunits and their mRNA levels in hamster BAT during development. The values for the content of F(1) protein ((alpha + beta)-F(1) antigens) and c-F(0) protein were obtained from the immunoblotting experiment in Fig. 5; the values for beta-F(1) and c-F(0) mRNAs are from Fig. 6. All data are expressed as a percentage of the values at postnatal day 4/5.




DISCUSSION

BAT is one of the most dynamic mammalian tissues known with respect to mitochondrial biogenesis. In this study, three different physiological conditions have been employed that are characterized by profoundly different levels of the biosynthesis of mitochondrial ATPase in BAT. Steady-state levels of mRNAs for the most essential and representative ATPase subunits of both the F(0) and F(1) parts of the enzyme have been determined. Under all the conditions studied, the expression of genes for only one subunit of the ATPase, the c-F(0) subunit, was found to correlate well with the tissue content of ATPase and with its respective changes. Although it cannot be completely excluded that expression of one of the nonessential subunits may also be involved, it is reasonable to suggest that the expression of the c-F(0) gene(s) seems to be pivotal for ATPase biosynthesis and is selectively regulated. The amount of ATPase assembled in this tissue thus appears to be governed by a limiting availability of only one subunit of the F(0) part of the enzyme complex: c-F(0). As documented by high steady-state mRNA levels, the genes of the other ATPase subunits are always highly expressed in BAT with an intensity that correlates with the expression of the genes of the respiratory chain complexes rather than with the resulting level of ATPase.

Regulation of Gene Expression

At the level of gene expression, selective control of the biosynthesis of ATPase relative to the respiratory chain enzymes can be achieved only via nuclear genes. beta-F(1) and -F(1) genes do not seem to be appropriate for such control as they bear regulatory sequence elements allowing their coordinate transcription with the genes of the respiratory chain enzymes(9, 11, 17, 18, 19, 20) . In agreement with this, it seems to be the expression of the c-F(0) genes that is selectively controlled in BAT.

Of the two genes that encode the c-F(0) subunit(6, 7, 42) , P2 has been found to be expressed in all tissues tested, while P1 transcripts have been found only in heart and brain and at a low level in kidney(7) . The level of c-F(0) mRNA that have been measured here in BAT is the sum of the mRNA of both genes since the human cDNA used (HUM 1 in (6) ) covers the full length of the mature c-F(0) mRNA and would therefore hybridize well with both P1 and P2 transcripts. Accordingly, not only P1, but also P2 transcription must be suppressed in thermogenically active rodent BAT.

The low level of c-F(0) transcripts could result from fast degradation, but then there would have to operate under the steady-state conditions a highly selective degradation of c-F(0) mRNAs and not of mRNAs of other ATPase subunits. Nevertheless, even then, c-F(0) would be the only subunit of the ATPase produced at low levels.

Translational or Post-translational Regulation

High levels of mRNA are expected to lead to a high production of the corresponding proteins. The very high levels of ATPase mRNAs (except c-F(0)) encountered in BAT would therefore be expected to lead to an overproduction of these subunits, unless some compensatory mechanism exists. This would seem to be the case for the beta-F(1) subunit of the ATPase since the transcripts from BAT were shown to have a severalfold lower translational efficacy than transcripts from other tissues(30) , partly compensating for the excess of beta-F(1) mRNA. A similar post-transcriptional mechanism might be effective also with other overproduced transcripts of ATPase subunits, although the nature of such a mechanism is unknown. The possibility that all subunits except c-F(0) are synthesized in excess but subsequently degraded cannot be excluded.

In other cases, post-transcriptional events do seem to be involved in the regulation of ATPase biosynthesis also, i.e. in the developing liver of the newborn rat(40, 41) . The postnatal switch of liver energy provision is made possible by a rapid development in liver bioenergetic functions due to a preferential postnatal increase in the rate of synthesis of mitochondrial proteins. This includes also the ATPase, and using beta-F(1) as a reporter gene, it was shown that these developmental changes in the liver resulted from both induction of gene expression and specific changes in the translational efficacies of the cytosolic nuclearly encoded mitochondrial mRNAs due to their prenatal accumulation and postnatal rapid mobilization onto cytosolic polyribosomes(41) . This type of biosynthetic control, however, would not discriminate between the ATPase and the other oxidative phosphorylation enzymes and could therefore not explain the asynchronous changes in their biosynthesis found in the early developmental stages of BAT of different rodent species (Fig. 5)(38, 39) .

ATPase Assembly

An important aspect of ATPase biosynthesis is the order in which the subunits and parts of the complex assemble. In yeast mit mutants that were unable to synthesize the F(0) part, an unimpaired biosynthesis of the F(1) subunits was found. This catalytically active F(1)-ATPase was even intramitochondrially located and membrane-attached, but of course incompetent with respect to ATP synthesis(43) . Similarly, in other yeast mutants lacking nuclear genes required for ATPase assembly, F(1) subunits are present as aggregated proteins(45) . Also, in ethidium bromide-inhibited mouse fibroblasts lacking mitochondrially encoded F(0) subunits (44) , no suppression of the synthesis of immunodetectable F(1) subunits was found. Thus, in both cases, nuclearly encoded F(1)-ATPase could be synthesized in the absence of the F(0) part. In contrast, under the physiological conditions as in the experiments reported here as well as in the preceding studies of ATPase in BAT, the results are indicative of a primary role of the F(0) part in the overall assembly of the mammalian ATPase complex in vivo. Despite the overexpression of the F(1) genes with respect to c-F(0), the catalytic F(1) and the membrane-spanning F(0) parts of the enzyme are always present in corresponding quantities in BAT ( Fig. 3and Fig. 5)(25, 26, 27, 29) .

In yeast mitochondria, the F(0) part has been shown to assemble temporally in the order of subunits c-F(0) (ATPase 9), ATPase 8, and a-F(0) (ATPase 6), indicating that the c subunit is the one that starts the assembly and is absolutely required for the F(0) assembly(43) . The regulatory role of c-F(0), as proposed from our data, supports the view that also in mammalian mitochondria, it is c-F(0) that begins the assembly of the membrane sector part of the ATPase and thus of the whole enzyme complex. Other factors may, of course, also be important for the assembly(45) .

In conclusion, the striking correlation presented here between c-F(0) expression and ATPase content indicates that the expression of the c-F(0) genes plays a pivotal role in the control of ATPase biosynthesis. Only the expression of the c-F(0) genes is selectively down-regulated in BAT. The other ATPase genes are apparently highly expressed in parallel with the oxidative enzymes. Due to the low amount of c-F(0) mRNA, the c-F(0) subunit could be synthesized in very small amounts, whereas it is possible that the other parts of the ATPase are produced at a higher rate, and further studies will be required to elucidate the fate of the respective excess transcripts and their protein products. As the c-F(0) subunit is probably the first and therefore the limiting part of the assembly of functional ATPase, the entire complex would be assembled in very low amounts in BAT. It would therefore seem that a very efficient control point for the regulation of ATPase biosynthesis has been utilized in BAT. It is not impossible that analogous regulatory roles of c-F(0) could exist in other tissues.


FOOTNOTES

*
This work was supported in part by Academy of Sciences of the Czech Republic Grant 511407 and by a grant from the Swedish Natural Science Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a guest researcher fellowship from the Swedish Natural Science Research Council. To whom correspondence should be addressed. Tel.: 422-475-2434 or 2433; Fax: 422-471-9517.

Recipient of a postdoctoral fellowship from the Swedish Natural Science Research Council.

(^1)
The abbreviations used are: BAT, brown adipose tissue; UCP, uncoupling protein.


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