(Received for publication, November 14, 1994 )
From the
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 -subunit of the F
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
,
, and
of the catalytic F
part and the b, c, d, and OSCP
subunits of the F
part of the ATPase.
In comparison with
other tissues of mice, high levels of transcripts of -F
,
-F
,
-F
, b-F
, d-F
, and OSCP were found in BAT. The only
genes expressed at a low level in BAT were those of the c-F
subunit. The levels of c-F
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
mRNA were found. Expression of
the c-F
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 genes and
subsequent synthesis of the hydrophobic subunit c of the
membrane-intrinsic F
part of the enzyme may control the
biosynthesis of the ATPase complex in BAT. An analogous regulatory role
of the c-F
subunit could be postulated in other tissues.
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 part (subunits
,
,
,
, and
and the loosely attached ATPase
inhibitor protein IF
) and 10 of which comprise the
H
-translocating, membrane-spanning F
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
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,
-F
(4) and
c-F
(5, 6, 7) , of the complex.
Eight subunits (
,
,
, and
of F
and a,
b, c, and OSCP of F
) 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), ()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
and F
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 (
-F
and
a-F
(ATPase 6)) in BAT unexpectedly showed very high rates
of expression of these ATPase genes (28, 30) . This
clearly indicated that the catalytic
-F
subunit is not
transcriptionally suppressed in BAT and that also mitochondrially
encoded F
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
,
, and
subunits of the catalytic F
part
and the b, c, d, and OSCP subunits of the F
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
subunit of the ATPase, which thus apparently has a key
role in the regulation of the biosynthesis of ATPase in BAT.
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 -F
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 -F
/c-F
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
-F
/c-F
ratios were observed in BAT; similarly high values were
calculated for the ratio between the mRNA of other ATPase subunits
measured and c-F
(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
, relative ratios for other comparisons always
came out close to the expected ratio of 1. This is illustrated in Fig. 2for the
-F
/
-F
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
transcript.
Figure 2:
Relative expression of -F
,
-F
, and c-F
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
-F
and c-F
mRNA and between
-F
and
-F
mRNA were
calculated. One representative experiment is shown. WAT, white
adipose tissue.
Quantitative evaluation of the
level of the c-F 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
-F
transcript was even higher in BAT than in
the heart, the ratio between the two transcripts (
-F
/c-F
) 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 genes in BAT. This implies that the control of
the expression of the c-F
genes may have a key
regulatory role in the biosynthesis of the mitochondrial ATPase in BAT.
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 ( +
)-F
subunits, the b-F
subunit, and the c-F
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
-ATPase (1:10,000), the b-F
subunit (1:5000),
and the c-F
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 -F
(Fig. 4) and other ATPase gene transcripts (data not
shown). Most notably, also the steady-state level of c-F
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
transcripts relative to those of
-F
were the same in all the investigated lamb tissues. The high
level of expression of the c-F
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 -F
and c-F
mRNAs in
heart, liver, and BAT of newborn lamb. The experimental procedures were
as described in the legend to Fig. 1.
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-ATPase and the c-F
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 ( +
)-F
antigens,
the level of the
-F
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
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
in BAT, but not with the expression of the
other ATPase genes. For comparison, it is seen that constantly high
levels of
-F
and c-F
transcripts were present in heart, where the ATPase is stably high
throughout this period (Fig. 6).
Figure 6:
-F
and c-F
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
-F
and c-F
transcripts as described in the legend to Fig. 1.
The specific correlation
between the expression of the c-F 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
mRNA levels correlates well temporally and quantitatively with
both the c-F
antigen amount and the
(
+
)-F
antigen amount, but there is no
correlation between the
-F
mRNA and the
-F
antigen. The same lack of correlation was found for
all the other transcripts investigated in this study (
-F
,
-F
, b-F
, d-F
, and OSCP;
data not shown). Thus, the c-F
transcript levels
may determine the resulting level of ATPase.
Figure 7:
Changes in the content of F-
and F
-ATPase subunits and their mRNA levels in hamster BAT
during development. The values for the content of F
protein
((
+
)-F
antigens) and c-F
protein were obtained from the immunoblotting experiment in Fig. 5; the values for
-F
and c-F
mRNAs are from Fig. 6. All data are
expressed as a percentage of the values at postnatal day
4/5.
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 and F
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
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
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
part of the enzyme complex: c-F
. 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.
Of the two genes that encode the c-F 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
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
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 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
mRNAs and not of mRNAs of other ATPase
subunits. Nevertheless, even then, c-F
would be the only
subunit of the ATPase produced at low levels.
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 -F
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) .
In yeast mitochondria, the F part has been shown to
assemble temporally in the order of subunits c-F
(ATPase
9), ATPase 8, and a-F
(ATPase 6), indicating that the c
subunit is the one that starts the assembly and is absolutely required
for the F
assembly(43) . The regulatory role of
c-F
, as proposed from our data, supports the view that also
in mammalian mitochondria, it is c-F
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 expression
and ATPase content indicates that the expression of the c-F
genes plays a pivotal role in the control of
ATPase biosynthesis. Only the expression of the c-F
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
mRNA, the
c-F
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
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
could exist in other
tissues.