From the
To elucidate the mechanisms that regulate the expression of
nuclear genes during biogenesis of mammalian mitochondria, the
expression pattern of the
Complex physiological responses require the simultaneous and
coordinated regulation of multiple genes. This is most evident in the
case of the regulation of biogenesis of mitochondria, where molecular
components of the organelle are encoded in two physically separated
genomes. Although it is assumed that nuclear genome activity controls
the program of organelle biogenesis, other events in the cytoplasm, on
the mitochondrial membranes and in the organelle interior, are likely
to play prominent roles in biogenesis of mitochondria (Attardi and
Schatz, 1988; Clayton, 1991; Nagley, 1991; Wallace, 1992).
The
regulation of the expression of nuclear encoded proteins involved in
mitochondrial energy metabolism has been described mostly at the
transcriptional level (Scarpulla et al., 1986; Williams et
al., 1987; Izquierdo et al., 1990; Torroni et
al., 1990; Izquierdo and Cuezva, 1993b). Specific
cis-acting elements and transcription factors for these genes
have been characterized (Li et al., 1990; Suzuki et
al., 1991; Chau et al., 1992; Chung et al.,
1992; Virbasius et al., 1993; Haraguchi et al., 1994;
Vander Zee et al., 1994; Gopalakrishnan and Scarpulla, 1994).
Recently, it has been suggested that some of these
trans-acting proteins may be directly involved in the
coordinated transcriptional expression of both genomes (Suzuki et
al., 1991; Virbasius and Scarpulla, 1994; Haraguchi et
al., 1994). However, the changes in the expression pattern of a
given protein during development could be the result of a combination
of regulatory mechanisms operating at multiple levels of gene
expression, i.e. gene transcription, mRNA stability,
translational efficiency, protein stability, etc. In this regard, it is
possible that other mechanisms operating at the post-transcriptional
level could share the control of biogenesis of mitochondria at
different stages of development. In fact, recent findings have provided
evidence that mitochondrial biogenesis in mammals is regulated at the
post-transcriptional level (Attardi et al., 1990; Polosa and
Attardi, 1991; Izquierdo and Cuezva, 1993a; Luis et al.,
1993).
Biogenesis of mitochondria during liver development results
both in an increase in the number of mitochondria per cell,
organelle proliferation (Rohr et al., 1971; Hommes,
1975), and in an increase in the functional capabilities of
mitochondria, organelle differentiation (Pollack and Sutton,
1980; Valcarce et al., 1988; Cuezva et al., 1990,
1993). During the last years, we have characterized mechanisms that
regulate the onset of mitochondrial function and differentiation in
liver (Valcarce et al., 1988, 1990; Izquierdo et al.,
1990; Valcarce and Cuezva, 1991; Izquierdo and Cuezva, 1993b; Luis
et al., 1993) and in other rat tissues (Luis and Cuezva, 1989;
Cuezva et al., 1990; Izquierdo and Cuezva, 1993a). Recently,
we have found that the increase in
In this work, we have addressed
the study of the mechanisms that control biogenesis of mitochondria
during both differentiation and proliferation of the organelle. The
study of the mechanisms controlling differentiation is important
because it has been found that
The F
Run-on experiments were
carried out according to Marzluff and Huang (1984) with the
modifications reported by Izquierdo and Cuezva (1993b). Nuclei
(2-3
To determine
whether rat
It has been described
that actinomycin D is also effective in inhibiting yeast mitochondrial
transcription in vitro (Barat-Gueride et al., 1987)
and in cultured liver cells (Chrzanowska-Lightowlers et al.,
1994). The results shown in Fig. 6 A revealed a
significant decline in the 12 S rRNA hybridization signal after
actinomycin D administration to the neonates, thus suggesting that the
drug is also effective as an inhibitor of mammalian mitochondrial
transcription in vivo. Although inhibition of mitochondrial
transcription could be directly or indirectly mediated, the decline in
12 S rRNA hybridization signals allowed a rough estimation of the
half-life of a transcript encoded in the mitochondrial genome. The
results obtained also revealed that 12 S rRNA has a higher stability in
neonatal than in adult liver (Fig. 6, A and C).
It should be mentioned that 12 S rRNA half-life could be subestimated
due to accumulation of this transcript in liver during the first 12
postnatal hours (Fig. 6 A).
The
Recent findings
indicate that the
The
opposite developmental profile between steady-state
The
degradation process of short-lived mammalian mRNAs, those encoded by
the early response gene family (Herschman, 1991), is dependent on
protein synthesis (Greenberg et al., 1986; Greenberg and
Belasco, 1993). When cellular protein synthesis is inhibited, the
stability of these mRNAs increases. Contrary to this situation and as a
further interesting contrast between different stages of development,
inhibition of protein synthesis in neonatal liver promotes a rapid and
profound reduction in steady-state
It is interesting to note that
the relative changes in the amount of
On the other hand, the parallelism
observed during liver development between steady-state levels of the
nuclear and mitochondrial encoded mRNAs (Fig. 4) further
reinforces the hypothesis, reported recently within different mammalian
cellular types (Izquierdo and Cuezva, 1993a), of the existence of a
conserved mechanism for coordination of the expression of the two
genomes. The parallel changes observed in RNA decay rates during
development for transcripts encoded in both genomes (Fig. 6) seem
to add additional support to this hypothesis.
Several experimental
systems have been described in which mRNAs are being stored
``masked'' until required to fulfill a predetermined
morphogenetic or differentiation program of the organism (Wickens,
1990; Jackson and Standart, 1990). The activation of these mRNAs is
usually mediated by specific covalent modification and/or
trans-activation of the stored mRNAs in response to given
stimuli (Bachvarova, 1992; Bouvet and Wolffe, 1994). This situation
mimics the differentiation process of mitochondria in rat liver. The
It is obvious that future investigations aimed at identifying
putative cis- and trans-acting factors of
In conclusion, our findings
indicate that the expression of nuclear encoded genes required for
mitochondrial biogenesis during liver development is exerted at various
levels. We hypothesize that mitochondrial biogenesis in mammals is
regulated primarily by the variable transcriptional expression of
certain genes that control key cellular functions (the catalytic
subunit of the ATP synthase complex is one example). To encompass or
finely tune the cellular outfit of mitochondria with energy demands,
other mechanisms exert a complex regulation of these genes at various
post-transcriptional levels during certain stages of development. These
findings are of utmost importance for understanding the mitochondrial
pathophysiology of mammals during adaptation to extrauterine life
(Valcarce et al., 1994).
We thank Prof. J. Satrstegui for critical reading of
the manuscript. Drs. A. M. Luis and J. F. Santarén are
acknowledged for help in measuring rates of protein synthesis and
running two-dimensional gels, respectively. We are grateful to M.
Chamorro and C. San Martn and to D. Jelenic for expert technical and
secretarial assistance, respectively.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-subunit of the ATP synthase gene has
been characterized in rat liver between day 20 in utero and 12
weeks postnatal. The parallelism existing between transcriptional
activity of the gene and the amount of
-F
-ATPase
protein in liver indicates that proliferation of mitochondria is
controlled at the transcriptional level. On the other hand, an
increased stability (4-5-fold) of
-F
-ATPase mRNA
during early neonatal life as well as a rapid postnatal activation of
translation rates affecting mitochondrial proteins appear to control
mitochondrial differentiation. Immunoelectron microscopy of the
F
-ATPase complex during liver development revealed that the
rapid postnatal increase in the in vivo rate of
F
-ATPase synthesis was mostly used for functional
differentiation of pre-existing organelles (Valcarce, C., Navarrete, R.
M., Encabo, P., Loeches, E., Satrstegui, J., and Cuezva, J. M. (1988)
J. Biol Chem. 263, 7767-7775). The findings support that
-F
-ATPase mRNA decay is developmentally regulated in
liver, indicating that gene expression is also controlled at this level
during physiological transitions that affect biogenesis of
mitochondria.
-F
-ATPase protein
experienced in liver during the first postnatal hour is due to the
activation of translation initiation as well as to a rapid increase in
the translational efficiency of the template as determined in vitro (Luis et al., 1993).
-F
-ATPase mRNA is
accumulated in fetal liver (Luis et al., 1993). As in previous
studies from our group (Izquierdo et al., 1990; Luis et
al., 1993; Izquierdo and Cuezva, 1993a), the nuclear encoded
-catalytic subunit of the mitochondrial ATP synthase has been used
as a reporter protein in mitochondrial biogenesis. Since previous
documentation of the differentiation process of mitochondria has always
been inferred from data provided in the isolated organelle (Valcarce
et al., 1988; Izquierdo et al., 1990), it is
necessary to ascertain, by immunoelectron microscopy techniques, the
occurrence of this process in situ. An additional important
question raised in this study is whether mitochondrial proliferation
and differentiation share the same molecular mechanisms of expression.
The findings reported reveal that whereas proliferation of the
organelle is controlled at the transcriptional level, differentiation
of mitochondria is controlled at the post-transcriptional level, both
by stabilization and translational activation of the transcript.
Animals
Timed pregnant albino Wistar rats
weighing 200 g were fed standard laboratory chow and water ad
libitum. The fetuses were delivered by rapid hysterectomy from
cervical dislocated pregnant rats on days 20 and 22 of gestation,
respectively. The newborns were maintained without feeding as described
previously (Valcarce et al., 1988) and killed by decapitation
at 1 and 6 h postnatal. Animals for other time points were taken at 5
days postnatal and 12 weeks (adult) of development.
In Vivo Determination of the Relative Rates of Synthesis
of the F
[ -ATPase
Complex
S]Methionine incorporation
into liver proteins of newborn rats were measured taking into
consideration the methods used for determining rates of protein
synthesis (Schimke, 1975). To determine the relative rate of synthesis
of the F
-ATPase complex, neonates were intraperitoneally
injected with 250 µCi of
L-[
S]methionine (1000 Ci/mmol) either
at the time of birth (0 h) or 1 h later (1 h old) (Izquierdo et
al., 1990). 10 min after receiving the tracer, newborns were
killed by decapitation, their livers (
0.3 g) were homogenized, and
mitochondria were isolated. Washed mitochondrial pellets were
solubilized in 1 ml of buffer containing 20 mM potassium
phosphate, 1% Triton X-100, 0.9% NaCl, pH 7.0; frozen and thawed three
times; and centrifuged at 40,000 rpm for 30 min at 4 °C. To measure
the rate of [
S]methionine incorporated into
cytosolic liver proteins, 2-µl aliquots of liver homogenates were
precipitated with trichloroacetate (Izquierdo et al., 1990).
-ATPase complex was immunoprecipitated from
solubilized mitochondrial proteins using rabbit anti-rat liver
mitochondrial F
-ATPase serum (Valcarce et al.,
1988) as described previously (Izquierdo et al., 1990). The
immunoprecipitates were washed as follows: (i) 2% Triton X-100 +
300 mM NaCl; (ii) 0.5 M NaCl + 0.1% Triton X-100
+ 10 mM Tris-HCl, pH 8; and (iii) 0.05% Triton X-100
+ 10 mM Tris-HCl, pH 8. The washed precipitates were
dissociated in loading buffer, resolved by SDS-12.5% polyacrylamide gel
electrophoresis, and further processed for fluorography as described
previously (Izquierdo et al., 1990; Luis et al.,
1993).
DNA Isolation and Southern Blots
Rat genomic DNA
was isolated and digested with three restriction endonucleases
( EcoRI, BamHI, and PstI). DNA fragments were
resolved on 0.8% agarose gels and transferred to nylon membranes
(GeneScreen, DuPont NEN). An EcoRI- EcoRI
fragment (a-cDNA) and an EcoRI- HindIII fragment
(b-cDNA) from rat liver
-F
-ATPase cDNA (Garboczi
et al., 1988) were used as probes for hybridization
experiments. Probes were labeled with [
P]dCTP by
nick translation. A synthetic 41-mer oligonucleotide (Garboczi et
al., 1988; Izquierdo et al., 1990) contained in the
sequence of the EcoRI- EcoRI cDNA fragment was also
used as a probe after labeling with polynucleotide kinase (data not
shown). After hybridization, membranes were washed once using the
following protocol: (i) 6
SSC at room temperature for 10 min,
(ii) 2
SSC containing 0.1% SDS at 65 °C for 10 min, and
(iii) 0.1
SSC containing 0.1% SDS at 65 °C for 10 min.
Membranes were further exposed to x-ray films at
70 °C with
intensifying screens.
Northern Blot Analysis
Total liver RNA was
extracted from freeze-clamped rat livers and separated by
electrophoresis on formaldehyde-1.4% agarose gels (Izquierdo et
al., 1990; Luis et al., 1993). Denatured RNAs were either
stained with ethidium bromide or vacuum-blotted onto GeneScreenmembranes for hybridization procedures. Membranes were incubated
with [
P]dCTP-labeled cDNA probes. The rat liver
DNA probes used in this study were
-F
-ATPase (Garboczi
et al., 1988) and mitochondrial encoded subunits 6 and 8 of
the ATP synthase complex and subunit III of the cytochrome c oxidase and 12 S rRNA (Gadaleta et al., 1990), generously
provided by Drs. P. L. Pedersen (The Johns Hopkins University,
Baltimore) and P. Cantatore (University of Bari, Bari, Italy),
respectively. Rat liver
-microglobulin and human
-actin cDNAs were also used. Hybridization conditions have been
previously reported (Izquierdo et al., 1990). After
hybridization, membranes were washed according to the protocol
described above for Southern blots. For stripping labeled DNA probes,
membranes were incubated in sterile water at 90-100 °C for 20
min. Membranes were exposed to x-ray films. Autoradiograms falling
within the linear range of the films were analyzed by laser
densitometric scanning.
In Vivo RNA Half-life Determination
Animals used
for the determination of RNA half-lives were intraperitoneally injected
with 100 mg of actinomycin D (Sigma)/100 g of body weight. In adult
rats, a second dose of transcriptional inhibitor was administered 1 h
after the first dose. In neonatal rats, additional doses of actinomycin
D were administered 3 and 6 h after the first dose. To inhibit protein
synthesis, animals received an intraperitoneal injection of 10 mg of
cycloheximide (Sigma)/100 g of body weight. A second dose of
cycloheximide was administered 1 h later. The animals were killed at
various times post-injection, and the livers were processed as
described above.
Isolation of Nuclei and Run-on Transcription
Assays
Nuclei were isolated from 1 g of liver as described
previously (Izquierdo and Cuezva, 1993b). Briefly, the tissue was
minced and homogenized in 10 mM Tris-HCl, pH 8.0, containing
0.32 M sucrose, 3 mM CaCl, 2 mM
magnesium acetate, 0.1 mM EDTA, 1 mM dithiothreitol,
and 0.1 mM phenylmethylsulfonyl fluoride. Nuclei were prepared
by centrifugation through a 2 M sucrose cushion (Babiss et
al., 1986) with slight modifications (Kikuchi et al.,
1992). Isolated nuclei were resuspended in 25% glycerol, 50 mM
Tris-HCl, pH 8, 5 mM magnesium acetate, 0.1 mM
phenylmethylsulfonyl fluoride, 0.1 mM EDTA, and 5 mM
dithiothreitol. The nuclei were quickly frozen in liquid nitrogen and
stored at
70 °C for a few months.
10
) were used in run-on assays. The
transcription reaction was incubated at 25 °C for 60 min and
terminated by the addition of ribonuclease-free DNase I (23 units/ml;
Boehringer Mannheim) and 20 mM CaCl
. For specific
detection of radioactive nascent RNA transcript, 10-20
10
cpm of the isolated RNA was hybridized for 60 min at 60
°C and for 72 h at 42 °C with the following plasmid DNAs
immobilized on nylon filters: cytosolic phosphoenolpyruvate
carboxykinase (Yoo-Warren et al., 1981),
-F
-ATPase (Garboczi et al., 1988),
-actin (Gunning et al., 1983), and pUC18 (Yanisch-Perron
et al., 1985). Undigested plasmid DNAs (10 µg) were
denatured with 3 M NaOH at 65 °C for 60 min and
neutralized with 2 M NH
Ac, pH 7. Denatured DNAs
were applied to nylon membranes using a slot blot apparatus (Schleicher
& Schuell). After hybridization, membranes were washed according to
the following protocol: 1
SSC containing 0.1% SDS at 65 °C
for 15 min (two times) and 0.1
SSC containing 0.1% SDS at 65
°C for 15 min (two times). Membranes were exposed to x-ray films
and analyzed by densitometric scanning.
Tissue Processing for Electron Microscopy
Small
pieces of livers from 0-h, 1-h-old, and adult rats were fixed by
immersion in freshly prepared 4% paraformaldehyde (Merck, Darmstadt,
Germany) in 0.1 M Sörensen phosphate buffer, pH 7.2, for
2 h at 4 °C. Other tissue samples were fixed for 1 h with 1%
glutaraldehyde in phosphate-buffered saline (PBS;
(
)
0.01 M phosphate buffer, 0.15 M NaCl, pH
7.2) and 2% tannic acid. Tissue samples were rinsed in buffer, and the
free aldehyde groups were quenched by immersion in 50 mM
ammonium chloride in PBS for 60 min at room temperature (two changes of
30 min each), rinsed in PBS, and finally processed for embedding in
Lowicryl K4M (Chemische Wercke Lowi, Waldkraiburg, Germany) according
to the manufacturer's instructions. Gold interferential color
ultrathin sections were collected in Formvar/carbon-coated nickel grids
and stored until use.
Immunocytochemical Localization of the Mitochondrial
F
Grids containing thin
sections of fetal, neonatal, and adult rat liver were equilibrated for
5 min with PBS and then incubated in parallel with a 1:50-1:75
dilution in PBS of anti-rat liver mitochondrial F -ATPase Complex
-ATPase
serum (Valcarce et al., 1988). After brief rinses in PBS,
grids were incubated with protein A complexed to 15-nm gold particles
(BioCell Research Laboratories, Cardiff, United Kingdom), which were
diluted to A
= 0.4 in PBS containing 1%
bovine serum albumin, 0.01% Triton X-100, and 0.1% Tween 20. After 45
min of incubation with the protein A-gold complexes, grids were washed
twice in PBS and distilled water and air-dried. Counterstaining was
performed with 2% aqueous uranyl acetate (6 min) and 1% lead citrate
(45 s). Several series of standard controls for immunocytochemical
techniques were conducted in parallel to assess the specificity of the
immunoreactive signal. Quantitation of F
-ATPase protein
density in mitochondria (number of gold particles/square micrometer of
mitochondrial section) was performed according to Griffiths and
Hoppeler (1986).
Other Methods
Preparation of liver homogenates
(Izquierdo et al., 1990), isolation of liver mitochondria
(Valcarce et al., 1988), and one-dimensional (Luis et
al., 1993) and two-dimensional (Santarén et al.,
1993) Western blot analyses of the -subunit of the
F
-ATPase complex have been previously described in detail.
One Gene and One Protein for
The regulation of the
expression of the nuclear encoded genes involved in energy metabolism
in mammalian cells has been mostly described at two levels: (i)
differential expression of tissue-specific isoforms and (ii) variable
expression of functional single copy genes (for review, see Wallace
(1992)). The existence of more than one gene (Gay and Walker, 1985;
Breen, 1988; Walker et al., 1989; Pierce et al.,
1992) and the differential expression of isoforms, originated by
alternative splicing of the same transcript (Matsuda et al.,
1993a, 1993b; Endo et al., 1994), have been described for
other subunits of the mitochondrial ATP synthase.
-F
-ATPase
-F
-ATPase is encoded by a single gene or
whether there are additional genomic sequences related to the
-subunit, we analyzed rat genomic DNA by Southern blot
hybridization analysis with 5`- and 3`-fragments of the rat liver
-F
-ATPase cDNA (Fig. 1). The 5`-most cDNA
(a-cDNA) hybridized with single DNA fragments (
4.7-kb
EcoRI,
2.0-kb BamHI, and 3.5-kb PstI)
(Fig. 1 B, left panel). Also, an
additional faint band appeared in each track (Fig. 1 B,
left panel). The 3`-most cDNA (b-cDNA) hybridized
with 23-kb EcoRI, 2.3-kb BamHI, and two PstI
(0.7 and 1.8 kb) DNA fragments (Fig. 1 B, right panel). Likewise, with this probe, one or two additional
faint bands (25 kb in EcoRI, 5 and 3 kb in BamHI, and
2.2 and 4.5 kb in PstI) could be seen (Fig. 1 B,
right panel). These hybridization studies suggest
that rat
-F
-ATPase is encoded by a single copy gene.
The hybridization of the probes with a
-F
-ATPase
pseudogene (Ohta et al., 1988; Neckelmann et al.,
1989; Walker et al., 1989) could account for the appearance of
faint hybridization signals.
Figure 1:
Southern blot analysis
of rat genomic DNA. A, shown is a schematic representation of
DNA fragments derived from rat liver -F
-ATPase cDNA
with three restriction endonucleases. a- and b-cDNA represent the DNA fragments of
-F
-ATPase cDNA
used to hybridize digested genomic DNA. B, EcoRI-,
BamHI- and PstI-digested DNA fragments were resolved
on 0.8% agarose gels, transferred to nylon membranes, and hybridized
with
P-labeled a- and b-cDNA probes. Molecular size
markers are shown on the left in kilobases.
Two-dimensional Western blots of
mitochondrial proteins revealed that -F
-ATPase protein
had the same apparent molecular mass and isoelectric point (
49 kDa
and 5.3 pH units, respectively) at different stages of liver
development (data not shown) as previously reported for the adult liver
protein (Santarén et al., 1993), i.e. no
evidence for isoforms of the protein could be detected during
development. Altogether, the results herein reported indicate the
existence of a single copy
-F
-ATPase gene in rat
genome. This finding is in agreement with similar findings in humans
(Neckelmann et al., 1989) and in bovines (Breen et
al., 1988).
Expression of
The existence of one gene and one protein
product for -F
-ATPase during Liver
Development Reveals Two Different Processes of Mitochondrial
Biogenesis
-F
-ATPase in liver provided a good marker
to monitor mitochondrial biogenesis during development. Accumulation of
-F
-ATPase protein in liver showed almost a continuous
increase (Fig. 2 A). However, if the rate of accumulation
of
-F
-ATPase protein is taken into consideration, two
distinct periods of mitochondrial biogenesis can be distinguished
(Fig. 2 A). The first, defined by the highest rate,
occurs immediately after birth and results in a 2-fold increase in the
relative amount of
-F
-ATPase in just 1 h postnatal
(Fig. 2 A). The second, defined by a much lower rate,
results in a similar
2-fold increase between 6 h postnatal and the
adult stage (Fig. 2 A).
Figure 2:
Developmental changes in the relative
amount of -F
-ATPase protein in rat liver and in
isolated organelles. A, liver homogenates from fetal (days 20
and 22 of gestation ( lanes 1 and 2,
respectively)), neonatal (1, 6, and 5 days old ( lanes
3-5, respectively)), and adult ( lane 6)
rats. Left panel, Coomassie Blue-stained gel of 50
µg of liver proteins fractionated by SDS-polyacrylamide gel
electrophoresis. Molecular mass markers (200, 97, 69, 46, 30, 21, and
14 kDa) are shown on the left. Middle panel, representative
blot obtained from a parallel gel transferred to polyvinylidene
difluoride membranes and probed with affinity-purified
anti-
-F
-ATPase antibodies. Right panel,
quantitation of immunoreactive
-F
-ATPase protein
performed by laser densitometric scanning of the resulting bands. Data
are expressed in arbitrary units ( a.u.)/gram of liver and are
the means ± S.E. p < 0.05 (*), p < 0.025
(**), and p < 0.01 (***) when compared with the previous
value of the time course by Student's t test.
B, postnatal increase in
-F
-ATPase protein in
isolated liver mitochondria. 10 µg of day 22 fetal (0 h) ( lane 1), 1-h-old neonatal ( lane 2), and
adult ( lane 3) liver mitochondrial proteins were
resolved by SDS-polyacrylamide gel electrophoresis. The left panel shows a Coomassie Blue-stained gel ( upper) and a parallel
Western blot ( lower) processed with affinity-purified
anti-
-F
-ATPase antibodies. Note the rapid postnatal
increase ( right panel) in the steady-state amounts of
-F
-ATPase protein in isolated organelles. The values
are expressed as a percentage of
-F
-ATPase protein
detected in adult mitochondria and are the means ± S.E. *, p < 0.0025 when compared with the 0-h stage by Student's
t test. C, synthesis in vivo of the
mitochondrial F
-ATPase complex in fetal ( open bars) and neonatal ( hatched bars) rat
liver. F
-ATPase was immunoprecipitated from solubilized
mitochondrial proteins. Relative rates of F
-ATPase
synthesis are expressed as the ratio of the counts/minute in the
F
-ATPase complex to the counts/minute in cytosolic
proteins. The results are the means ± S.E. for three to four
experiments. p < 0.01 (*) and p < 0.0005 (**)
when compared with the 0-h stage by Student's t test.
Visualization of the two
different processes affecting organelle biogenesis during development
was illustrated when the steady-state amounts of
-F
-ATPase protein were expressed relative to the
amount of isolated mitochondrial proteins (Fig. 2 B). It
is shown that the relative
-F
-ATPase amount increases
2-fold between the fetal (0 h) and neonatal (1 h old) stages of
development. Interestingly, the relative content of this protein in
isolated mitochondria from adult liver is the same as that detected in
1-h-old samples (Fig. 2 B). These findings suggest that
the increase in liver
-F
-ATPase between 1 h postnatal
and the adult stage (Fig. 2 A) is due to an increase in
the number of differentiated mitochondria in liver. On the other hand,
the major period of organelle differentiation appears to occur during
the first hour after birth because the surge in liver
-F
-ATPase protein (Fig. 2 A) is
accounted for by the increase of the protein in isolated organelles
(Fig. 2 B).
A Selective Increase in the Relative Rate of Synthesis of
Mitochondrial Proteins Triggers Differentiation of the
Organelle
To analyze the mechanisms responsible for the rapid
postnatal increase in the amount of -F
-ATPase protein
in the liver, the in vivo rates of F
-ATPase
synthesis were determined in fetal (0 h) and neonatal (1 h old) rats
(Fig. 2 C). The rates of incorporation of
L-[
S]methionine into liver cytosolic
proteins increased 5-fold during the first postnatal hour
(Fig. 2 C). In contrast, the rates of incorporation of
L-[
S]methionine into the
immunoprecipitated F
-ATPase complex
(Fig. 2 C; see also Fig. 3in the work of Izquierdo
et al. (1990)) showed a 12-fold increase during the same time
period. The relative rates of synthesis of the F
-ATPase
complex were calculated in order to correct the possible differences in
(i) the dose and site of administration of the tracer, (ii) the rates
of uptake of L-[
S]methionine, and (iii)
the liver methionine pool size. The relative rate of synthesis was
expressed as the ratio of tracer incorporation into
F
-ATPase to that into liver cytosolic proteins. The
relative rate of F
-ATPase synthesis showed a 2-fold
stimulation during the first postnatal hour (Fig. 2 C).
These results illustrate the activation of translation initiation that
occurs in liver immediately after birth (Luis et al., 1993).
Even more important, they illustrate in vivo that the
synthesis of mitochondrial proteins is preferential over other
non-mitochondrial cellular proteins (Luis et al., 1993),
suggesting that new synthesis of the mitochondrial F
-ATPase
complex (Fig. 2 C) is used for differentiation of
pre-existing fetal mitochondria.
Figure 3:
Immunoelectron microscopy of the
F-ATPase complex evidences liver mitochondrial
differentiation. Shown is F
-ATPase immunogold labeling in
day 22 fetal ( a), 1-h-old neonatal ( b), and adult
( c) rat liver ultrathin sections from paraformaldehyde-fixed
samples. Gold particles decorate the inner mitochondrial membrane. In
d, quantitation of the gold particle density/square micrometer
of mitochondrial section evidences a major presence of the
F
-ATPase complex in neonatal ( 1h) and adult
( Ad) rat liver. *, p < 0.01 when compared with the
0-h stage by Student's t test.
Quantitative Immunoelectron Microscopy Evidences
Mitochondrial Differentiation after Birth
Immunoelectron
microscopy of the mitochondrial F-ATPase complex in rat
liver ultrathin sections showed that gold particles decorated the inner
mitochondrial membrane (Fig. 3). Quantitation of
F
-ATPase gold particle density/square micrometer of
mitochondrial section in fetal, neonatal, and adult liver revealed a
significant increase at the 1-h time point (Fig. 3). The
percentage of the increase in F
-ATPase immunolabeling at 1
h ranged from
50 to
250%, depending upon whether tissue
samples were fixed in paraformaldehyde (Fig. 3) or glutaraldehyde
(data not shown), respectively. Interestingly, the mitochondrial gold
particle density in 1-h-old and adult hepatocytes was the same
(Fig. 3), in agreement with the results of Western blotting
experiments (Fig. 2 B). The differences observed in the
increase in the mitochondrial F
-ATPase complex between
fetal and neonatal samples estimated by electron microscopy of in
situ organelles (Fig. 3) and Western blotting of the
isolated organelles (Fig. 2 B) may be explained by the
differences in the conservation of antigenic determinants between the
two fixation procedures of the electron microscopic technique. In fact,
when liver samples were fixed in glutaraldehyde, the results obtained
by both techniques were comparable (data not shown).
Proliferation of Liver Mitochondria Occurs when
Steady-state mRNA Levels Show a Sharp Decline
Fig. 4
summarizes the developmental changes in liver steady-state
levels of various mitochondrial (nuclear (-F
-ATPase)
and mitochondrial (cytochrome c oxidase subunit III +
ATPase 6 and 8 and 12 S rRNA) encoded) and non-mitochondrial
(
-microglobulin and
-actin) transcripts.
Interestingly, the developmental profile for the two mRNAs of the ATP
synthase complex was similar although they are encoded in different
genomes. They steadily accumulated in liver, reaching maximum levels at
6 h postnatal. It should be noted that when differentiation of the
organelle occurred (during the first postnatal hour), there were no
changes in steady-state levels for these two transcripts, as reported
earlier (Luis et al., 1993) (Fig. 4). Surprisingly, at
the time when most of the
-F
-ATPase protein
accumulated in liver (between the 5-day neonatal and adult stages; see
Fig. 2A), steady-state transcript levels were at a
minimum (Fig. 4). The specific developmental profile for these
two mitochondrial mRNAs was further supported by the expression pattern
of the other three transcripts (Fig. 4). While mitochondrial 12 S
rRNA showed no changes during development, steady-state mRNA levels of
-microglobulin and
-actin showed the opposite
developmental profile (Fig. 4).
Figure 4:
Developmental changes in the relative
amount of -F
-ATPase mRNA in rat liver. 30 µg of
total RNA extracted from fetal livers at days 20 and 22 of gestation
( 20d-F and 22d-F, respectively), from neonatal livers
at 1 h, 6 h, and 5 days postnatal ( 1h-N, 6h-N, and
5d-N, respectively), and from adult livers were analyzed by
Northern blot hybridization procedures with the indicated probes. A
parallel ethidium bromide-stained gel shows 28 S and 18 S rRNAs. The
relative abundance of the RNAs (arbitrary units
( a.u.)/microgram of RNA) was determined by laser densitometric
scanning of the autoradiograms. The values shown are the mean of two
different total liver RNA preparations. COIII, cytochrome
c oxidase subunit III;
-microg.,
-microglobulin.
Nuclear Transcription Rates of the
Nuclear run-on assays in isolated liver nuclei
from different stages of development revealed that transcription rates
of the -F
-ATPase Gene during Mitochondrial
Biogenesis
-F
-ATPase gene (Fig. 5) did not parallel
the sharp reduction in steady-state
-F
-ATPase mRNA
levels (Fig. 4). In fact,
-F
-ATPase
transcription rates in adult nuclei (Fig. 5) were found to be at
least 3-fold higher than at any other stage of liver development ( p < 0.025 and p < 0.005 when compared with the 5-day
neonatal and day 20 fetal stages, respectively). The transcription
rates of the
-F
-ATPase gene during development closely
resembled those of another housekeeping gene,
-actin
(Fig. 5). Comparison of the relative
-F
-ATPase/
-actin transcription rate during
development (data not shown) revealed that
-F
-ATPase
gene transcription increased (
3-5-fold) toward the adult
stage. Determination of transcription rates of the inducible
phosphoenolpyruvate carboxykinase gene further supported the results
obtained from nuclear run-on assays (Fig. 5). Transcription rates
of the phosphoenolpyruvate carboxykinase gene, in agreement with
previous reports (Garcia Ruiz et al., 1978; Lyonnet et
al., 1988), were switched on immediately after birth to allow
development of gluconeogenesis and the supply of glucose to neonatal
tissues (Patel et al., 1982; Mayor and Cuezva, 1985).
Figure 5:
Developmental changes in transcription
rates of the -F
-ATPase gene. Liver nuclei were
isolated from fetal (days 20 and 22), neonatal (1 h, 6 h, and 5 days
old), and adult rat liver. Nuclear run-on assays were carried out for
determining transcription rates of the phosphoenolpyruvate
carboxykinase,
-F
-ATPase, and
-actin genes.
A, representative autoradiograms hybridized with radioactive
nascent RNA transcripts at the indicated time periods during liver
development. Note the rapid postnatal activation of phosphoenolpyruvate
carboxykinase ( PEPCK) gene transcription. B,
quantitation of transcription rates (arbitrary units
( a.u.)/10
cpm) of the genes after densitometric
scanning of the resulting hybridization signals. The results shown are
the means ± S.E. for three to four independent
experiments.
Changing Patterns of
The lack of correlation
between steady-state mRNA levels (Fig. 4) and transcription rates
of the -F
-ATPase mRNA
Half-life during Development
-F
-ATPase gene (Fig. 5) prompted us to
estimate in vivo the half-life of the messenger during
development. This approach is possible if transcription rates are
blocked after administration of an inhibitor (Belasco and Brawerman,
1993). Administration of actinomycin D to rat neonates prevented the
postnatal accumulation of phosphoenolpyruvate carboxykinase mRNA in
their livers (Fig. 6 A). Similarly, administration of
actinomycin D to adult rats promoted the disappearance of this
short-lived mRNA (Fig. 6 A). These findings indicate that
mRNA synthesis is blocked after actinomycin D administration. Thus,
quantitation of steady-state amounts of the transcripts at various
times after administration of the inhibitor (Fig. 6 A)
and calculation of RNA decay assuming first-order rate kinetics
(Fig. 6 B) (Belasco and Brawerman, 1993) allowed the
estimation of
-F
-ATPase mRNA half-life.
Figure 6:In vivo determination of
-F
-ATPase mRNA half-life in neonatal and adult liver.
Transcription rates were inhibited by intraperitoneal administration of
actinomycin D to neonatal and adult rats (+). Untreated
age-matched neonates were processed to illustrate the inhibitory effect
of actinomycin D on transcription rates of the phosphoenolpyruvate
carboxykinase ( PEPCK) gene (
). A, total RNA
was prepared from livers at various times during neonatal development
and after administration of the inhibitor and analyzed by Northern blot
hybridization procedures with the probes indicated on the left.
Quantitation of mRNA content was carried out by laser densitometric
scanning of the resulting hybridization signals. B,
calculation of RNA half-lives was done assuming first-order kinetics.
The log of steady-state
-F
-ATPase mRNA levels
(arbitrary units ( a.u.)/microgram of total RNA) is represented
at various times after administration of actinomycin D to neonatal
( closed circles) and adult ( closed triangles) rats. The log of steady-state
-F
-ATPase mRNA levels in untreated neonates is also
shown ( open circles). B represents the experiment
shown in A. Note that the 6-h time point is not represented
because of artifactual RNA loading in the gel. C, shown are
the calculated RNA half-lives. The results shown are the means ±
S.E. for three to four independent experiments. p < 0.0025
(*) and p < 0.0005 (**) when compared with the neonatal
stage by Student's t test.
Interestingly, the disappearance of the hybridization signals for
the nuclear encoded -F
-ATPase transcript after
administration of the inhibitor was more pronounced in adult than in
neonatal liver (Fig. 6 A; please note differences in the
time scale). In fact, calculation of the apparent mRNA half-life
(Fig. 6 B) revealed that the neonatal
-F
-ATPase mRNA was 4-5 times more stable than
the adult transcript (Fig. 6 C).
A Role for Labile Protein(s) in Stabilizing
The
experiments described above open up the possibility that the stability
of the -F
-ATPase mRNA during Development?
-F
-ATPase transcript in fetal and early
neonatal liver depends on mechanisms such as translation rates of the
template itself (Greenberg et al., 1986; Greenberg and
Belasco, 1993) or on the presence of a specific trans-acting
factor(s) that could modulate its stability at that stage of
development. As a preliminary approach to address this question, an
inhibitor of cytosolic protein synthesis was administered to newborn
and adult rats, and the steady-state mRNA levels for
-F
-ATPase and ATPase 6 and 8 and for 12 S rRNA were
determined in liver at various times after cycloheximide administration
(Fig. 7).
Figure 7:
Inhibition of protein synthesis increases
-F
-ATPase mRNA decay in neonatal liver. 10 mg of
cycloheximide/100 g of body weight was intraperitoneally administered
to newborns at the time of birth. At 0, 30, 60, and 120 min ( lanes
1-4, respectively) after administration of the first dose of
inhibitor, livers were removed, and RNA was extracted. 30 µg of
total RNA were processed for Northern blotting and hybridized with the
probes indicated on the left. Note the sharp reduction in the
-F
-ATPase mRNA hybridization signal at 30 min after
cycloheximide administration, whereas subtle changes occur for the
other two transcripts, indicating a specific effect of the inhibitor on
-F
-ATPase mRNA levels. Two different experiments
( A and B) are shown.
12 S rRNA and the mitochondrial encoded ATPase 6 and
8 mRNA showed no major changes after administration of the inhibitor.
However, the nuclear encoded -F
-ATPase mRNA showed a
sharp decline in the neonatal liver 30 min after cycloheximide
administration (Fig. 7). By 1 h post-injection, the mRNA levels
again reached normal levels (Fig. 7). Interestingly,
administration of the cytosolic protein synthesis inhibitor to adult
rats did not promote such rapid (30 min) changes in the steady-state
levels for any of the three transcripts studied (data not shown). These
results, obtained in vivo, should be interpreted with caution
because of possible side effects of cycloheximide in other cellular
functions. However, they suggest that the stability of the transcript
during early stages of development is more dependent on ongoing protein
synthesis than at later stages (adult). On the other hand, since
determination of protein synthesis in liver (Valcarce et al.,
1988) after 2 h of cycloheximide administration to neonates indicates a
significant reduction in the amount of radioactivity incorporated into
protein (4026 ± 267 and 303 ± 49 dpm/mg of protein for
saline- and cycloheximide-injected age-matched neonates, respectively),
we suggest that the transient nature of the
-F
-ATPase
mRNA response (Fig. 7) may represent an adaptation of liver cells
to inhibition of protein synthesis.
-subunit of the ATP synthase plays a key role in
mitochondrial function. It is the catalytic subunit of the complex and
thus a bottleneck for oxidative phosphorylation. Here, we show the
result of mitochondrial differentiation in rat liver at the subcellular
level (Fig. 3). This finding illustrates in situ that
new synthesis of
-F
-ATPase protein (Fig. 2) is
mostly used for increasing the relative amount of this protein in
pre-existing organelles, confirming previous results obtained at the
molecular and functional levels with the isolated organelles (Valcarce
et al., 1988; Izquierdo et al., 1990). Furthermore,
the two processes that affect biogenesis of mitochondria during
development, proliferation and differentiation, differ in time and
intensity scales and do not share the same molecular mechanisms for
their expression, most likely reflecting the different physiological
states and energy demands of the liver during development.
Differentiation is controlled at the post-transcriptional level by
increasing the stability (Fig. 6) and translational efficiency
(Fig. 2; see Luis et al. (1993)) of mitochondrial
transcripts. Proliferation is controlled at the transcriptional level
(Fig. 5). In both differentiation and proliferation processes,
the participation of additional levels of regulation at the
post-translational level cannot be excluded, i.e. differential
regulation of protein stability during development.
-F
-ATPase gene is under complex
transcriptional regulation (Webster et al., 1990; Torroni
et al., 1990; Stepien et al., 1992). It is
interesting to note that the
-F
-ATPase gene (Ohta
et al., 1988; Neckelmann et al., 1989) contains a
putative nuclear respiratory factor-2-responsive element (Virbasius and
Scarpulla, 1991), in common with other genes involved in energy
metabolism. It also contains an enhancer element, in common with human
cytochrome c
and subunit E1
of the pyruvate
dehydrogenase complex (Tomura et al., 1990). Similarly, other
cis-acting elements have been found in common between the ATP
synthase
-subunit and other oxidative phosphorylation genes (Li
et al., 1990; Suzuki et al., 1991; Chau et
al., 1992). On the other hand, thyroid hormones have been shown to
effectively regulate the transcriptional expression of the
-F
-ATPase gene (Izquierdo et al., 1990;
Izquierdo and Cuezva, 1993b). Ubiquitous and tissue-specific nuclear
factors that interact with overlapping promoter elements (OXBOX/REBOX)
of the gene have been described (Li et al., 1990; Chung et
al., 1992; Haraguchi et al., 1994). Interestingly, these
factors interact differentially with cis-acting elements
depending on cellular conditions (Chung et al., 1992). Thus,
it seems reasonable to suggest that differential transcription of the
-F
-ATPase gene during liver development (Fig. 6)
could result, among other factors, from the differences in thyroid
hormone availability and functional maturation of the thyroid
transduction pathway that accompany mammalian development.
-F
-ATPase mRNA levels (Fig. 4) and transcription
rates of the gene (Fig. 5) indicated the existence of a mechanism
for controlling mRNA decay that is differentially expressed during
development of the mammalian liver. The post-transcriptional regulation
of
-F
-ATPase mRNA availability in fetal and neonatal
liver is further supported by the higher half-life of the transcript at
this stage of development (Fig. 6). Little, if anything, is known
about the existence of specific cis-acting elements,
endonucleolytic pathways, mechanisms of mRNA decay, and
trans-acting factors involved in determining the stability of
nuclear encoded transcripts for mitochondrial proteins.
-F
-ATPase mRNA
levels (less than half of the original values in <30 min;
Fig. 7
). This finding could argue against the idea that nuclear
templates encoding for mitochondrial proteins are degraded as a result
of their translational activation after birth (Fig. 2 C;
see Luis et al. (1993)). On the contrary, it may suggest that
-F
-ATPase mRNA stability is dependent on ongoing
protein synthesis, a mechanism that seems to be differentially
regulated during liver development.
-F
-ATPase/gram
of liver (Fig. 2 A) parallel the changes in the number of
mitochondria/hepatocyte (Rohr et al., 1971; Hommes, 1975) and
almost parallel the changes in the amount of mtDNA/cellular DNA during
rat liver development (Cantatore et al., 1986). On the other
hand, it appears that representation of the mitochondrial translational
machinery, when expressed relative to the cellular one (assessed by 12
S rRNA) (Fig. 4), is kept constant throughout development.
Therefore, we suggest that maintenance of the constancy of the level of
12 S rRNA results from the concerted operation of constitutive
transcription of mtDNA (Cantatore et al., 1986), which will
increase as development proceeds, and mechanisms of regulation
controlling mitochondrial rRNA decay, which are also expected to
increase during development. This is consistent with the observed
difference in 12 S rRNA half-life estimated at two stages of liver
development (Fig. 6).
-F
-ATPase mRNA accumulated in liver during fetal
stages of development changes its sedimentation behavior (Luis et
al., 1993) and translational efficiency immediately after birth,
as assessed both in vivo (Fig. 2 C) and in
vitro (Luis et al., 1993). At the present time, we have
no explicit finding that could explain such differences in the physical
(Luis et al., 1993) and functional (Fig. 2 C;
Luis et al., 1993) behavior of
-F
-ATPase
mRNA.
-F
-ATPase mRNA sorting, stabilization, and
translational activation will contribute to a better picture of the
mechanisms that control the post-transcriptional regulation of
mitochondrial biogenesis in mammals. In this regard, it is worth
mentioning that some mitochondrial proteins are bifunctional, i.e. they are catalysts of metabolic pathways, and they also present
mRNA binding activities (Preiss et al., 1993; Preiss and
Lightowlers, 1993; Klausner et al., 1993; Beinert and Kennedy,
1993). This situation opens up new trends for positive and negative
feedback autoregulation of the post-transcriptional expression of the
genes involved in mitochondrial biogenesis. We believe that the
developing rat liver provides an excellent experimental system in which
to address future studies at this level.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.