(Received for publication, November 16, 1995; and in revised form, January 8, 1996)
From the
Mitochondrial protein synthesis is acutely depressed during
anoxia-induced quiescence in embryos of Artemia franciscana.
Oxygen deprivation is accompanied in vivo by a dramatic drop
in extramitochondrial pH, and both of these alterations strongly
inhibit protein synthesis in isolated mitochondria. Here we show that
the oxygen dependence is not explained simply by blockage of the
electron transport chain or by the increased redox state. Whereas
oxygen deprivation substantially depressed protein synthesis within 5
min and resulted in a 77% reduction after 1 h, aerobic incubations with
saturating concentrations of cyanide or antimycin A had little effect
during the first 20 min and only a modest effect after 1 h (36 and 20%
reductions, respectively). Yet the mitochondrial NAD(P)H pools were
fully reduced after 2-3 min with all three treatments. This
cyanide- and antimycin-insensitive but hypoxia-sensitive pattern of
protein synthesis depression suggests the presence of a molecular
oxygen sensor within the mitochondrion. Second, we show for the first
time that acidification of extramitochondrial pH exerts inhibition on
protein synthesis specifically through changes in matrix pH. Matrix pH
was 8.2 during protein synthesis assays performed at the
extramitochondrial pH optimum of 7.5. When this proton gradient was
abolished with nigericin, the extramitochondrial pH optimum for protein
synthesis displayed an alkaline shift of 0.7 pH unit. These data
suggest the presence of proton-sensitive translational components
within the mitochondrion.
Exposure of brine shrimp embryos (Artemia franciscana)
to anoxia results in the largest acidification of intracellular pH ever
reported for eukaryotic cells, with pH falling from 7.7 to
6.8
in the first 20 min (1, 2) and to
6.3 upon longer
exposure(1) . This pH transient is thought to play a dominant
role in the induction of a reversible quiescent state under
anoxia(3) , which can last in excess of 2 years(4) .
The acidification of intracellular pH has been strongly implicated in
the arrest of both catabolic ((5, 6, 7) ;
reviewed in (8) ) and anabolic processes in the
cytoplasm(9, 10, 11, 12) .
Similarly, protein synthesis in mitochondria from these embryos is also
reduced markedly (up to 90%) in response to anoxia and acidic
pH(13) . In the present study, we examined mechanisms that
mediate the inhibitory effects of both oxygen deprivation and
extramitochondrial pH acidification on mitochondrial protein synthesis.
In A. franciscana mitochondria, the depression of protein synthesis in response to either oxygen deprivation (13) and/or pH acidification (14) appears to be global in that no qualitative differences are detectable in the array of translation products synthesized in response to these factors(13) . Furthermore, this depression is likely exerted post-transcriptionally; levels of selected mitochondrial mRNA (e.g. COX1) do not decrease during short term (6 h) anoxia in vivo(15) , yet mitochondrial protein synthesis in vitro is substantially depressed after 5 min of anoxia(13) . The depression of mitochondrial protein synthesis in yeast observed after longer term (hours) exposure to anoxia is also thought to be exerted, in part, post-transcriptionally (reviewed in (16) and (17) ). However, the precise mechanisms that mediate the acute effects of either oxygen deprivation or extramitochondrial pH acidification on translation within the mitochondrion are not known.
In isolated mitochondria from A. franciscana embryos, matrix ATP:ADP and GTP:GDP ratios decline with increasing time under anoxia at constant pH(13) . However, the addition of ATP (1 mM) at the onset of anoxia stabilizes the ATP:ADP ratio at aerobic values but does not rescue protein synthesis(13) . Thus, the anoxia-induced decrease in the matrix ATP:ADP ratio does not cause the inhibition of protein synthesis. Because we were unsuccessful in stabilizing the GTP:GDP ratio under anoxia(13) , the reduction in this ratio could contribute to the depression of protein synthesis during anoxia by altering rates of translational initiation and/or elongation. However, the fact that protein synthesis is rapidly depressed before purine nucleotide levels substantially change suggests that other factors are involved. For example, anoxia-induced changes in redox potential (see discussions in (18) and (19) ) are known to alter both transcriptional and translational rates in some organisms. Here, we have differentiated between the effects of oxygen limitation per se and redox alterations by comparing rates of mitochondrial protein synthesis under anoxia with aerobic rates in the presence of electron transport inhibitors (cyanide or antimycin A). Our data support the conclusion that redox changes caused by blockage of the electron transport chain do not explain the majority of inhibition in protein synthesis for isolated mitochondria under anoxia. Rather the data suggest the presence of a sensor for molecular oxygen (or oxygen byproducts) within the mitochondrion.
Matrix purine nucleotides do
not change in response to pH during aerobic incubations of A.
franciscana mitochondria(13) , and therefore alterations
in the mitochondrial energy status cannot account for the observed
depression of protein synthesis at low pH. In addition, alterations in
pH do not appear to inhibit the import of amino acids(13) . One
hypothesis explored in the present study is that protons have a direct
effect on components of the mitochondrial translational machinery
through changes in matrix pH. The pH sensitivity of translational
components in the cytoplasm of these embryos has previously been
documented(11) , and initial studies indicated that substantial
changes in matrix pH do occur in response to extramitochondrial
pH(13) . Here we have extended our measurements of matrix pH
and examined the pH sensitivity of protein synthesis in the presence of
nigericin, a H/K
exchanger that
abolishes mitochondrial
pH(20) . We predicted that if
matrix pH is directly affecting protein synthesis, then an alkaline
shift in the extramitochondrial pH optimum should occur with nigericin
that is similar in magnitude to the
pH in the absence of
nigericin. This prediction is supported by the data presented.
Respiration data were analyzed with
DatGraf software (Oroboros, Innsbruck, Austria). Oxygen concentration
in the chamber (c, nmol O
/ml) was
calculated from pO
measurements based on O
solubility in the respiration medium at 25 °C and ambient
barometric pressure. Oxygen flux (J
, pmol O
s
ml
) was
calculated as the time derivative of c
.
Corrections were made for 1) consumption of oxygen by the electrode and
back diffusion of oxygen into the chamber in the absence of
mitochondria, 2) the exponential time constant of the oxygen sensor,
and 3) transient changes in c
resulting from the
injection of solutions. K
values for KCN and
antimycin A were calculated with the WILMAN 4 kinetics package
(Michigan State University) according to the methods of
Wilkinson(21) .
Because 3-hydroxybutyrate and acetoacetate
were used to titrate mitochondrial redox state, it was important to
verify the presence of -hydroxybutyrate dehydrogenase activity in A. franciscana embryos. Dechorionated embryos were homogenized
in 3 volumes of medium consisting of 50 mM Tris-HCl (pH 8.0),
2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, and 1 µg/ml soybean trypsin inhibitor and centrifuged at
1000
g for 10 min. 30 µl of supernatant was
assayed at 340 nm in a 1-ml reaction mixture containing 150 mM Tris-HCl (pH 8.0), 1.9 mM
-NAD, and 27 mM 3-hydroxybutyrate.
-Hydroxybutyrate dehydrogenase activity
was 0.26 ± 0.01 units/g wet tissue mass (mean ± S.E., n = 6).
Figure 1: Comparison of the effects of antimycin A, KCN, and anoxia on protein synthesis in isolated mitochondria from A. franciscana embryos. Filled circles are aerobic controls, squares are assays containing 100 µM antimycin A, triangles are assays containing 500 µM KCN, and open circles are anoxic assays. The data are the means ± S.E. for at least three independent assays from multiple mitochondrial preparations at each time point.
Figure 2:
Change in matrix pH during transitions
between state 2 and state 3 respiration in isolated mitochondria from A. franciscana embryos. The pH of the medium was 7.4. 5 mM succinate was added at time 0, 10 mM KHPO
was added at 10 min, and 125
µM ADP was added at 15 min. The duration of state 3
respiration was determined in parallel assays of oxygen consumption and
is indicated on the figure.
Fig. 3shows the
steady-state values of matrix pH (A) and pH (B)
as a function of extramitochondrial pH values that span the relevant
physiological range (pH 6.3-7.9). Interestingly, both matrix pH
and
pH decreased with decreasing extramitochondrial pH. Others (26, 27, 28) have reported a modest increase
in
pH with decreasing extramitochondrial pH over a similar pH
range. At present, the explanation for this difference in A.
franciscana mitochondria is not clear. However, in the context of
the anoxic embryo in vivo, a key point supported by Fig. 3is that as cytoplasmic pH plummets, the matrix pH would
apparently experience an acidification of even larger magnitude.
Figure 3:
Matrix pH (A) and pH (B) as a function of extramitochondrial pH in isolated
mitochondria from A. franciscana embryos. The line of unity is
indicated in A. BCECF-loaded mitochondria were placed in
respiration medium, and matrix pH was measured under steady-state
conditions. The data are the means ± S.E. for three independent
determinations at each pH.
To
provide a baseline for subsequent experiments with nigericin (described
below), it was important to quantify precisely the pH for isolated
mitochondria under actual conditions of protein synthesis. The temporal
profile of change in matrix pH during a 1-h protein synthesis assay at
an extramitochondrial pH of 7.5 is shown in Fig. 4. All
components required for oxidative phosphorylation and protein synthesis
were supplied together at time 0. Matrix pH rapidly increased during
the first 5 min and thereafter remained relatively constant to the end
of the 1-h assay. Steady-state values for matrix pH and
pH during
protein synthesis assays at three different extramitochondrial pH
values are given in Table 4. The initial change (0-5 min)
in matrix pH during these assays was not related to the occurrence of
protein synthesis per se because similar changes in matrix pH
were observed in the presence of puromycin (Fig. 4, filled
symbols). Rather, the rapid alkalinization in matrix pH is likely
the result of oxidative phosphorylation, because this same initial
pattern was observed during a 10-min preincubation with succinate and
ADP prior to initiation of the protein synthesis assay (data not
shown).
Figure 4: Matrix pH in mitochondria from A. franciscana embryos during protein synthesis assays at pH 7.5. Open symbols are assays performed in the absence of puromycin, and closed symbols are assays performed with puromycin (0.4 mg/ml). The data are the means ± S.E. for three independent determinations at each time point.
Finally, it is appropriate to note that at an
extramitochondrial pH of 7.5 matrix pH did not significantly change
during a 3-h exposure of isolated mitochondria to oxygen deprivation (Table 5). We find this constancy of the pH gradient remarkable
in the absence of active proton translocation via the electron
transport chain. Yet by comparison, Andersson et al.(29) showed that the pH was maintained for at least
30 min under anoxia in rat liver mitochondria. Both observations
suggest that mechanisms exist to depress proton leakage across the
inner membrane during anoxia.
Figure 5: Shift in the pH optimum for protein synthesis in the presence and the absence of nigericin in isolated mitochondria from A. franciscana embryos. Open symbols are assays performed in the absence of nigericin, and filled symbols are assays performed with 100 µM nigericin. The data are the means ± S.E. for three independent determinations at each pH.
This study has revealed two previously undescribed features
of mitochondrial bioenergetics that concern the mechanisms by which
oxygen deprivation and acidic extramitochondrial pH acutely depress
organellar protein synthesis. First, the rapid reduction of protein
synthesis by 80% that occurs when oxygen is removed cannot be
explained by blockage of the electron transport chain per se or by the associated change in redox state. Rather, as developed
below, a case is made for direct signaling through the removal of
molecular oxygen and/or oxygen byproducts that is mediated by an oxygen
sensor. Such mechanisms for acutely down-regulating
mitochondrial protein synthesis under anoxia are distinct from other
identified controls of gene expression that are operative during longer
term (hours) anoxia in yeast (reviewed in Refs. 17, 30, and 31).
Second, our results indicate that the pronounced sensitivity of
mitochondrial protein synthesis to extramitochondrial pH is a direct
result of associated changes in the matrix pH, as opposed to
alterations in the
pH or the import of amino acids for example.
Sensitivity of cytoplasmic protein synthesis to pH has been previously
documented for A. franciscana embryos(11) , as well as
for other cell types(32, 33, 34) , yet a
direct proton effect on biosynthesis within the mitochondrion has never
been reported. This latter observation is of particular interest in the
context of cells that experience transients in cytoplasmic pH, because
it suggests that one signal can simultaneously serve to down-regulate
biosynthesis in two cellular compartments. Furthermore, the result
implicates the presence of proton-sensitive components within the
mitochondrial translational machinery.
The above data set is consistent with the
presence of a molecular oxygen sensor within the mitochondrion that
serves to mediate the rapid depression of protein synthesis observed
under anoxia. Particularly relevant to this premise is the recent
characterization of a cyanide- and antimycin-insensitive but
hypoxia-sensitive form of cytochrome b that is apparently
resident in the plasma membrane of carotid body cells and HepG2 cells.
This cytochrome b is an integral component of
the NAD(P)H oxidase and serves as an oxygen sensor that initiates an
oxygen signaling cascade(38, 39) . In our studies with
isolated mitochondria, we gave special experimental attention to the
difference between the hypoxia sensitivity and the cyanide/antimycin
insensitivity of protein synthesis because it had previously been
reported that the erythropoietin pathway in oxygen-sensing cells could
be modulated by hypoxia but not by cyanide poisoning (40, 41) or by other respiratory chain
inhibitors(42) . The present work documents such an inhibitory
signature for mitochondrial protein synthesis and now points to an
oxygen-sensing mechanism located within this organelle.
It is
appropriate to note that oxygen-linked byproducts could in principle
explain the differential effect of anoxia and the inhibitors of the
electron transport chain as well. Reactive oxygen species (e.g. superoxide free radical, HO
) cannot be
produced in the absence of oxygen but are generated aerobically by
mitochondria(43, 44) . The presence of electron
transport inhibitors like antimycin A accentuate the leakage of single
electrons from electron transport chains, thereby giving rise to
univalent reductions of molecular oxygen and increased levels of
reactive oxygen species(45, 46) . Interestingly,
specific protein sensors for both superoxide free radicals (SoxR) and
H
O
(OxyR) have been described in bacteria (47, 48, 49, 50) . The sensing
mechanism of the SoxR protein involves the oxidation of an iron-sulfur
center by the superoxide anion(50, 51) ; such a
mechanism presumably would not be blocked by cyanide. The SoxRS regulon
is also activated by the free radical nitric oxide(52) , and
nitric oxide synthase has recently been localized within the
mitochondrion(53, 54) .
In the well studied case of
the yeast Saccharomyces cerevisiae, synthesis of
mitochondrial-encoded subunits of cytochrome c oxidase is
depressed in response to oxygen deprivation both in vitro(55, 56) and in vivo (reviewed in Refs.
16, 17, and 30). Translational regulation of mitochondrial-encoded
subunits of cytochrome c oxidase in response to oxygen
deprivation has been shown, in part, to be exerted by nuclear-encoded
translational activators that bind to specific mitochondrial mRNAs and
regulate the association with the small ribosomal subunit (reviewed in (16) ). As these activators become depleted within the
mitochondrion during anoxia, protein synthesis is
depressed(57) . However, for A. franciscana embryos
there is no direct evidence that nuclear-encoded proteins, whose
expression is dependent on oxygen, influence rates of mitochondrial
protein synthesis; protein synthesis rates under aerobic conditions are
identical for mitochondria isolated from aerobic embryos compared with
those of mitochondria isolated in the presence of cycloheximide from
embryos exposed to 6 h of anoxia. ()Rather, in A.
franciscana mitochondria the available data suggest that the
anoxia-induced depression of protein synthesis is globally mediated at
the post-transcriptional level(13, 15) .
Although the pH sensitivity of protein synthesis has been noted in mitochondria from other organisms (e.g. rat heart(59) , yeast(60) , and mouse adrenal gland(61) ), the pH effect on mitochondria from A. franciscana embryos is much more acute. The optimal pH range is narrower, and there is greater absolute difference between minimal and maximal rates over a similar range of pH. The data presented in this study indicate that there are proton-sensitive translational components within the mitochondrion. Thus, in conjunction with studies on the pH sensitivity of both catabolic (3, 5, 6, 7) and anabolic metabolism (9, 10, 11) in the cytoplasm of these embryos, our studies suggest that transitions in intracellular pH provide an intracellular signal integrating metabolic depression in both the mitochondrial and cytoplasmic compartments of A. franciscana embryos during transitions between active and anoxia-induced quiescent states.