From the Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347
Received for publication, October 9, 2000, and in revised form, November 20, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Eucaryotic cells contain at least two general
classes of oxygen-regulated nuclear genes: aerobic genes
and hypoxic genes. Hypoxic genes are induced
upon exposure to anoxia while aerobic genes are
down-regulated. Recently, it has been reported that induction of some
hypoxic nuclear genes in mammals and yeast requires mitochondrial respiration and that cytochrome-c oxidase
functions as an oxygen sensor during this process. In this study, we
have examined the role of the mitochondrion and
cytochrome-c oxidase in the expression of yeast
aerobic nuclear COX genes. We have found that
the down-regulation of these genes in anoxic cells is reflected in
reduced levels of their subunit polypeptides and that
cytochrome-c oxidase subunits I, II, III, Vb, VI, VII, and VIIa are present in promitochondria from anoxic cells. By using nuclear
cox mutants and mitochondrial rho0
and mit Both procaryotes and eucaryotes respond to intermittent and
prolonged exposure to hypoxia (1). This response is crucial to survival
in most organisms and can range from an immediate change in energy
metabolism to the activation of gene expression pathways that help the
organism cope with a low oxygen environment (1-3). Currently, the
effect of oxygen concentration on gene expression is being studied
intensively in a number of different organisms (c.f. Ref.
4). Studies with each of these experimental systems have made it clear
that some genes (hypoxic genes) are up-regulated by exposure
to anoxic or hypoxic conditions while other genes (aerobic
genes) are down-regulated under these conditions. So far, most of the
studies on the effects of hypoxia on gene expression have focused on
the induction of hypoxic genes and have not addressed the
down-regulation of aerobic genes. These studies on the
induction of hypoxic genes have revealed that exposure to
hypoxia can initiate a complex series of events, which begin with some
sort of oxygen sensor and end with a signaling pathway that
up-regulates hypoxic genes (1, 5-10).
Currently, the best understood oxygen sensors and signaling pathways
are those in procaryotes (c.f. Ref. 1). Although progress has been made in understanding the transcriptional machinery involved in oxygen-regulated gene expression of eucaryotic genes, the underlying mechanisms of oxygen sensing and the signaling pathways that connect oxygen sensors to the transcriptional machinery in eucaryotes are
poorly understood. However, there is growing evidence that the
mitochondrial respiratory chain and cytochrome-c oxidase
function in oxygen sensing and in the induction of some
hypoxic genes in both yeast and mammalian cells (8, 9).
Early evidence for the involvement of cytochrome-c oxidase
in oxygen sensing in mammalian cells has come from spectral studies
(11-13) that examined the influence of azide, cyanide, and carbon
monoxide on chemosensory discharge, primarily in carotid body cells
(14). More recently, it has been found that part of the hypoxic
induction of some mammalian genes is sensitive to cyanide and that the
mitochondrial respiratory chain is required for the hypoxic induction
of a number of genes which are under the control of the HIF-1
transcription factor (10, 15).
Evidence for the involvement of the mitochondrial respiratory chain in
the induction of some hypoxic genes has also been reported for the yeast Saccharomyces cerevisiae (16). Carbon monoxide affects the induction of a subset of hypoxic genes in
this organism. It completely inhibits the induction of OLE1
(the gene for The above observations have increased interest in assessing how
cytochrome-c oxidase functions in oxygen-regulated gene
expression (c.f. Refs. 9 and 10). At present, little is
known about the oxygen sensing function of this enzyme. However, it is
clear that oxygen concentration has a profound affect on both its
biosynthesis and catalytic functions (c.f. Ref. 17 and 18).
This oligomeric protein of the inner mitochondrial membrane contains
subunit polypeptides encoded by both nuclear and mitochondrial genomes.
The three largest subunits (I, II, and III) are encoded by
mitochondrial genes (COX1, COX2, and COX3); they
form the catalytic core of the enzyme (17, 19). Oxygen affects the
expression of two of these genes post-transcriptionally. The other
polypeptide subunits are encoded by nuclear genes; some of them
modulate catalysis, whereas others function in the assembly or
stability of the holoenzyme. Active preparations of yeast
cytochrome-c oxidase contain at least six subunit
polypeptides (IV, Va or Vb, VI, VII, VIIa, and VIII) encoded by nuclear
COX genes (COX4, COX5a or COX5b,
COX6, COX7, COX9, and COX8, respectively) (20).
COX5a and COX5b encode interchangeable isoforms,
Va and Vb, of subunit V (21). The other nuclear-encoded subunits are
encoded by single-copy genes. Oxygen affects the levels of expression
of all of these nuclear genes at the level of transcription. All of
these genes except COX5b are aerobic genes;
COX5b is a hypoxic gene. The inverse regulation
of COX5a and COX5b by oxygen is especially
interesting because the isoforms encoded by these genes have
differential effects on holocytochrome-c oxidase activity.
By altering an internal step in electron transport between heme a and
the binuclear reaction center the "hypoxic" isoform, Vb, enhances
the catalytic constant (TN) of the enzyme 3-4-fold (22, 23). Hence,
these isoforms allow cells to assemble functionally different types of
holoenzyme in response to oxygen concentration.
We are using S. cerevisiae as a model system to further
explore the role of the mitochondrion and cytochrome-c
oxidase in adaptation to hypoxia. In this study, we focus on the role
of anoxia and the mitochondrion on the expression of aerobic
COX genes. Our findings provide support for the existence of a
signaling pathway, which acts independently of respiration, from the
mitochondrial genome to the nucleus.
Strains--
The S. cerevisiae strains used in this
study are described in Table I. Two
respiration-competent parental strains were used: D273-10B and JM43.
Strain JM43 was constructed by crossing D273-10B with strain AB35-13D
(MATa leu2-2, 112, ura3-52, his4-590, ade2), as described
(24) and then extensively backcrossed with D273-10B. These two strains
are isogenic except for the auxotrophic markers that were introduced
into JM43. Strain JM43 Growth Conditions--
Yeast cells were grown in either YPGal
(1% Bacto-yeast extract, 2% Bacto-peptone, 1% galactose) or a
semisynthetic galactose medium containing Tween 80, ergosterol, and
silicon antifoam (SSG-TEA; per liter: 10 g of galactose, 3 g
of Bacto-yeast extract, 1 g of KH2PO4,
0.8 g of (NH4)2SO4, 0.7 g
of MgSO4·7H2O, 0.5 g of NaCl, 0.4 g
of CaCl2, 5 µg of FeCl3, 0.1% Tween 80 (v/v), 20 µg of ergosterol, and 350 ppm Dow Corning FG-10 silicon
antifoam). To obtain a more uniform dispersion, the Tween 80, ergosterol, and silicon antifoam were sonicated in solution prior to
autoclaving. Amino acids and nucleotides were added, as appropriate, at
a concentration of 40 µg/liter. Cells were grown at 28-30 °C
either in YPGal in Delong flasks on a shaker (200 rpm) or in
semi-synthetic galactose medium in a New Brunswick Bioflo IIc
fermentor, equipped with a gas-flow train that allows cells to be
cultured at any desired oxygen concentration and to be shifted between
oxygen concentrations (36). The fermentor was inoculated with
precultures that were grown aerobically on a shaker at 28-30 °C to
mid-exponential phase. The temperature, pH, and sparge rate were
maintained as described (16). The dissolved oxygen concentration in the
fermentor was monitored continuously and the oxygen concentration
(µM) in the vessel was calculated from the measured
dissolved oxygen level and based upon the oxygen solubility in the
growth media at 28 °C, the ambient barometric pressure, and the
pressure with the vessel (cf. Ref. 36). Anoxic and
low-oxygen cultures (0-5 µM O2) were grown
in the dark to prevent photoinhibition of growth (37). Nominally
oxygen-free (anoxic) cultures were grown in O2-free
N2 containing 2.5% CO2. To prevent trace
O2 from entering the fermentor, the gas mixture was passed
through an Oxyclear O2 absorber (LabClear, Oakland, CA).
Cells were harvested at mid-exponential phase. During harvest cells
were quick-chilled to RNA Isolation and Hybridization--
Total RNA was isolated from
washed cells as described previously (38). RNA samples were denatured
and separated on 1.8% agarose gels containing
MOPS1/formaldehyde buffer (20 mM MOPS, 40 mM sodium acetate, 8 mM
EDTA, and 220 mM formaldehyde (cf. Ref. 39). The
RNA was transferred to Nytran Plus charged nylon membranes (Schleicher
& Schuell) or GeneScreen (PerkinElmer Life Sciences) and hybridized as
described previously (40). Approximately 30 µg of RNA was loaded per
lane; loading was adjusted to give equal signals for hybridization to the ACT1 gene. DNA probes were prepared by random-primer
labeling of double-stranded DNA fragments using
[ Cell Fractionation--
Whole cell extracts were prepared by
shaking with glass beads (44). For preparation of mitochondrial and
cytosolic fractions, cells were grown to mid-exponential phase,
harvested, and spheroplasted as described (23), except that
cycloheximide was added to a concentration of 25 µg/ml to all buffers
used prior to the production of spheroplasts from anoxic cells. All
steps after spheroplasting were performed at 4 °C. Spheroplasts were
harvested by centrifugation (5 min at 3,000 × g),
washed gently in post-spheroplast buffer (1.5 M sorbitol, 1 mM Na2EDTA, 0.1% bovine serum albumin, pH
7.0), and sedimented at 3,000 × g for 5 min. Washed
spheroplasts were resuspended in lysis buffer (0.6 M
mannitol, 2 mM Na2EDTA, 0.1% bovine serum
albumin, pH 7.4), lysed in a Sorvall Omnimixer at low speed for 3 s and at full speed for 45 s, and then centrifuged for 5 min at
1,900 × g to pellet unbroken cells, nuclei, and
debris. The resulting supernatant was decanted and centrifuged for 10 min at 12,100 × g to pellet mitochondria. The
mitochondrial pellet was washed by resuspension in mitochondrial lysis
buffer minus bovine serum albumin (pH 7.0), homogenized with a
glass-Teflon homogenizer, and centrifuged at 1,651 × g
for 5 min. The resulting supernatant was decanted and centrifuged at
23,500 × g for 10 min to pellet the mitochondria. The
post-mitochondrial supernatant, collected after the 12,100 × g centrifugation, was used as the cytosol.
Western Immunoblotting--
Whole cell extracts were subjected
to SDS-polyacrylamide gel electrophoresis in gels containing 16%
acrylamide, 10% glycerol, and 4.8 M urea (20). Western
immunoblotting was done as described (45), except that proteins were
blotted onto Immobilon PVDF (Millipore) or Hybond ECL (Amersham
Pharmacia Biotech) nitrocellulose membranes, using the manufacturer's
instructions. Anti-cytochrome-c oxidase subunit-specific
sera were raised in rabbits to high performance liquid chromatography
purified yeast cytochrome-c oxidase subunits (20).
Immunoreactivity was detected either by 125I-protein A
followed by autoradiography, or horseradish peroxidase-linked secondary
antibodies followed by chemiluminescence using a chemiluminescence detection kit, as indicated in the figure legends. Immunoblots were
quantitated digitally using Kodak 1-D software.
Measurement of Mitochondrial Cytochromes and Respiratory Chain
Activities--
Absorption spectra of cytochromes were measured in
whole cells by low temperature difference spectroscopy at room
temperature, with an Aminco DW-2000 double beam dual wavelength
spectrophotometer, as follows. Cells were grown in YPGal to
midexponential phase, harvested by centrifugation, and suspended at
0.3 g wet weight per ml of 40 mM KPO4 (pH
7.4). A 1-ml sample was reduced with 5 mg of dithionite at room
temperature, then ethylene glycol was added to 30% and the sample was
frozen in liquid nitrogen. After freezing, the sample was allowed to
de-vitrify at room temperature for 2 min and was again frozen. The
absorption spectrum was scanned between 390 and 700 nm, using 575 nm as
the reference wavelength with the following settings: dual beam,
wavelength acquisition, slit width = 0.8 nm, scan rate = 2 nm/s. A second 1-ml sample was oxidized with 25 µl of 30%
H2O2 at room temperature for 2 min, adjusted to
30% ethylene glycol, and frozen in liquid nitrogen. After freezing, it
was allowed to de-vitrify, was refrozen, and its absorption spectra was
scanned as above. The difference spectrum was obtained by subtracting
the oxidized from the reduced spectrum. Whole cell respiration was
measured using a Yellow Springs Instruments oxygen electrode (21).
NADH-cytochrome c reductase activities and
cytochrome-c oxidase activities were determined
spectrophotometrically as described (44, 46).
Miscellaneous--
All gases (except air) were Matheson
certified standards and were obtained from U.S. Welding (Denver, CO).
Cell density was followed by measuring turbidity with a Summerson Klett
Meter fitted with a number 54 green filter.
Down-regulation of Aerobic Nuclear COX Genes and Their Protein
Products in Anoxic Cells--
Previously, we have found that mRNA
levels from COX4, COX5a, COX6,
COX7, COX8, and COX9 are all reduced
in hypoxic and anoxic cells and that the level of expression of these
genes is determined by oxygen concentration per se (40).
Each gene is down-regulated at reduced oxygen concentrations, with the
largest decrease in expression of these genes occurring at oxygen
concentrations below 1 µM O2. The effects of
oxygen concentration on the expression of these genes is conveniently
assayed by Northern blot hybridization using gels on which the
transcript load for ACT1, a gene whose expression is
unaffected by oxygen concentration (47), is held constant. Comparison
of transcript levels in normoxic and anoxic cells is shown in Fig.
1A. Transcript levels for
these genes in anoxic cells have been estimated previously at 17 (for
COX4), 23 (for COX5a), 31 (for COX6),
26 (for COX7), 25 (for COX8), and 39% (for
COX9) (40). It is surprising that these genes are
transcribed at all in anoxic cells, given that they encode subunits of
an enzyme, cytochrome-c oxidase, that uses oxygen as a
substrate, and given that anoxic cells lack cytochrome-c
oxidase activity and cytochromes aa3 (48).
Insofar as the precise function of many of these nuclear-encoded
subunits in holocytochrome-c oxidase is not yet established
it is conceivable that they have functions in anoxic cells that are
unrelated to their function as subunits of holocytochrome-c
oxidase. Of course, this assumes that the transcripts for these
subunits are translated in anoxic cells and that the subunits
themselves are stable under anoxic conditions. To determine whether
this is the case we examined promitochondrial and cytosolic fractions
from anoxic cells, using cytochrome-c oxidase
subunit-specific antibodies. From Fig. 1B it is clear that
subunits VI, VII, and VIIa are present in promitochondria and that
subunits IV and VIII are essentially absent. As expected, the subunit V
isoform present in aerobic mitochondria is Va and the predominant
subunit V isoform present in promitochondria is Vb. To determine the
relative levels of these subunits in promitochondria we quantitated
immunoblots produced from two independent preparations of mitochondria
and promitochondria. Their average level in promitochondria is 0.2 (subunit IV), 9.5 (subunit Va), 52 (subunit VI), 23 (subunit VII), 51 (subunit VIIa), and 0% (subunit VIII) of their level in mitochondria.
These findings indicate that the lowered transcript levels from the
aerobic COX genes, in anoxic cells, are reflected in lowered
levels of the cytochrome-c oxidase subunits they encode. However, there is not a direct correspondence between the degree of
reduction in subunit polypeptide and transcript level, suggesting that
post-transcriptional events also affect subunit polypeptide levels. We
also found that the mitochondrially encoded subunits of
cytochrome-c oxidase are present in promitochondria (Fig.
2A), at average levels that
are 16 (for subunit I), 41 (for subunit II), and 20% (for subunit III)
of their level in mitochondria. Finally, we examined the cytosolic
fractions of normoxic and anoxic cells for the presence of
cytochrome-c oxidase subunits. From Fig. 2B it is
clear that these polypeptides are not readily detectable in the
cytosol. Together, these findings indicate that most
cytochrome-c oxidase subunits are present in anoxic cells
and that they are localized to promitochondria.
Mitochondrial Respiration Is Not Required for the
Down-regulation of Aerobic COX Genes during a Shift to Anoxia--
The
above results confirm that the aerobic nuclear COX genes are
down-regulated in response to reduced oxygen concentration. The
kinetics of this down-regulation for COX4 and
COX5a, after shifting cells from normoxic to anoxic
conditions, is seen in Fig. 3A,
lanes 2-6. It is obvious that transcript levels for each gene
decline rapidly and that both reach their anoxic levels by 2 h
after the shift (compare Fig. 3A, lanes 6 and 7).
Thus, transcript levels from these genes change quickly in response to
a shift from normoxia to anoxia. The down-regulation of these aerobic genes during a shift from normoxia to anoxia is interesting in light of
the recent finding that the mitochondrial respiratory chain is required
for the induction of some hypoxic nuclear genes in yeast and mammalian
cells (15, 16) during this sort of shift. Indeed, it raises the
question of whether the respiratory chain contributes to the
down-regulation of these aerobic genes during a shift from
normoxia to anoxia. To address this we examined the down-regulation of
these same two genes in the rho0 strain,
JM43 Mitochondrial Involvement in the Expression of Aerobic Nuclear COX
Genes--
To further explore a possible involvement of the
mitochondrion in the expression of the aerobic nuclear
COX genes we next compared the levels of the mRNAs
encoded by COX4, COX5a, COX6, COX8, and COX9 in
strains JM43 and JM43
Rhoo cells differ from rho+ cells both
genotypically and phenotypically. They differ genotypically in lacking
a mitochondrial genome and they differ phenotypically in lacking
mitochondrial respiration. To determine whether it is the absence of
respiration that leads to the down-regulation of the nuclear
COX genes we first analyzed the levels of their mRNAs in
two nuclear mutants, JM43GD6 and JM43GD9 (Fig.
5). These strains are isogenic with JM43
except that they carry null mutations in the nuclear
COX genes, COX6 and COX9,
respectively. Moreover, they are rho+ and carry a fully
functional mitochondrial genome, as judged by their ability to
complement a rho0 strain. Both JM43GD6 and JM43GD9 are
respiration-deficient (Table II). From Fig. 5 and Table
IV it is clear that levels of transcripts from COX4, COX5a, COX6, COX8, and COX9 in either
JM43GD6 or JM43GD9 are comparable to their levels in their
respiratory-competent parent strain, JM43, and higher than levels in
JM43
In a second experiment, we analyzed the levels of mRNA from the
aerobic nuclear COX genes in mutants that carry missense
mutations in two mitochondrial COX genes (cox1
and cox2) and in the cytochrome b gene,
cytb. These mutants are isochromosomal with the
respiratory-proficient strain, D273-10B, and like the rhoo
strain, D273-10B The Mitochondrial Genome Is Essential for Optimal Expression of
Aerobic Genes under Anoxic Conditions--
Because the three subunit
polypeptides (Atp6, Atp8, and Atp9) that make up the proton membrane
channel sector of the yeast ATP synthase are encoded by mitochondrial
genes (50) rho0 cells lack the ability to perform oxidative
phosphorylation. It is difficult to determine whether oxidative
phosphorylation is responsible for the down-regulation of the nuclear
COX genes observed in rhoo cells by using ATP
synthase mutants because yeast strains that carry mutations which
affect the function or assembly of the ATP synthase have highly
unstable mitochondrial genomes (51). In addition, uncouplers, which can
be used to inhibit oxidative phosphorylation in isolated mitochondria,
are problematic for intact yeast cells because they affect its plasma
membrane H+-ATPase (52). Fortunately, it is possible to
assess whether the lack of oxidative phosphorylation leads to a
down-regulation of nuclear COX genes directly in
rhoo cells by comparing their level of expression in anoxic
rho+ and rhoo cells. Promitochondria from
anoxic rho+ cells lack mitochondrial respiration and
oxidative phosphorylation but contain a mitochondrial genome, while
promitochondria from rho0 cells lack respiration, oxidative
phosphorylation, and a mitochondrial genome. If a defect in oxidative
phosphorylation is responsible for the down-regulation of nuclear
COX genes in rho0 cells one would expect the
level of expression of these genes to be the same in anoxic
rho0 and rho+ cells. Conversely, if it is the
lack of a mitochondrial genome per se that is responsible
then the level of expression of the nuclear COX genes should
be higher in anoxic rho+ cells than in anoxic
rho0 cells. To test which of these possibilities is
correct, strains JM43 and JM43
These findings imply that the down-regulation of nuclear COX
genes in rhoo cells is independent of both respiration and
oxidative phosphorylation, and that the mitochondrial genome is
essential for optimal expression of these genes even in anoxic cells.
This suggests that it is the absence of one or more mitochondrial
genes, or their ability to be expressed, that leads to the
down-regulation of aerobic COX genes in
rhoo cells. Although anoxia and the lack of a mitochondrial
genome both result in the down-regulation of aerobic nuclear
COX genes these results also indicate that these two
effectors of nuclear COX gene expression work independently
of one another.
The results of this study provide interesting new insight
concerning the regulation of aerobic COX genes in yeast.
First, they demonstrate that both oxygen and the mitochondrial genome exert a positive effect on the expression of these genes but that mitochondrial respiration per se has no effect. Second, they
show that a mitochondrial genome is required for optimal expression of
these genes under both normoxic and anoxic conditions. Third, they show
that the down-regulation brought about by reduced oxygen concentration
and the down-regulation brought about by the absence of a mitochondrial
genome are independent of one another, ruling out the possibility that
the down-regulation of these genes in hypoxic or anoxic cells is
mediated by the mitochondrion. Together, these findings imply that at
least one mitochondrial gene is involved in a signaling pathway to the
nucleus, that this pathway is operative under normoxic and anoxic
conditions, and that this pathway does not involve respiration.
Down-regulation of Aerobic Genes at Reduced Oxygen
Concentrations--
The finding that transcript levels from the
aerobic nuclear COX genes decline rapidly when
cells are shifted from normoxic to anoxic conditions has been observed
previously (40). As pointed out in this earlier study the decline in
transcript levels from these genes is too rapid to be attributable to
cessation of new transcript synthesis and simple dilution of
transcripts after the shift (40), and probably results from degradation
of these transcripts upon exposure to anoxia. It is interesting to
note, however, that transcripts from these genes do not disappear
completely after a shift, and that low levels exist even in anoxic
cells (Fig. 1). The proteins produced from most of these transcripts are present in the promitochondria of anoxic cells, indicating that
they are translated and targeted to the mitochondrial compartment even
under anoxic conditions. All three mitochondrially encoded subunits of
cytochrome-c oxidase are also present in the promitochondria of anoxic cells, confirming the results of a study done several years
ago (53). The finding that several of the subunit polypeptides of
cytochrome-c oxidase, including the two subunits (I and II) that make up its catalytic core, are expressed under conditions where
its substrate (i.e. oxygen) is unavailable is intriguing. One is led to wonder if they coassemble with one another into a protein
complex and what their function, if any, might be in the
promitochondria of anoxic cells.
Mitochondrial Effects on Expression of Aerobic Nuclear COX
Genes--
The results presented here, together with those of previous
studies with yeast and mammals, demonstrate that the mitochondrion can
affect the expression of nuclear genes in two fundamentally different
ways. In the first, mitochondrial respiratory function is essential for
the induction of some hypoxic genes as cells are shifted
from normoxia to hypoxia or anoxia (15, 16). In the second, the
mitochondrial genome, acting independently of its respiratory function,
is essential for optimal expression of aerobic nuclear genes under both
normoxic and anoxic conditions.
Recent studies with mammalian cells in culture have suggested that the
mitochondrial respiratory chain participates in the induction of
hypoxic genes via the production of reactive oxygen species
(15, 54, 55). It is not yet clear if reactive oxygen species are also
involved in mitochondrial control of nuclear gene expression in yeast.
So far, the only mitochondrially initiated pathway known to affect
nuclear gene expression in this organism is retrograde
regulation (49, 56, 57). This pathway is used, by yeast
cells, to sense the energy state of their mitochondria. It functions to
up-regulate some aerobic genes in response to the lack of
mitochondrial respiration. Components of this pathway include: two
subunits (Rtg1p and Rtg3p) of a heterodimeric transcription factor,
Rtg2p, a cytoplasmic protein that contains an hsp-like ATP-binding site
(58), and the Tup1-Cyc8 protein complex, which interacts with the
Rtg1-Rtg3 heterodimer and which can either activate or repress
transcription (59). It is unlikely that this pathway is involved in the
expression of nuclear COX genes because cells carrying
null alleles of RTG1 and RTG2 are
capable of respiration-dependent growth (57) and because
nuclear COX genes lack the R-box binding site for the
Rtg1-Rtg3 heterodimer.
We refer to the type of mitochondrial-nuclear cross-talk uncovered by
the studies presented here as intergenomic signaling. It is
distinguishable from retrograde regulation in three ways. First, mitochondrial respiration is important for retrograde
regulation but not for intergenomic signaling. Second
those genes that are subject to retrograde regulation are
up-regulated in the absence of mitochondrial respiration while those
genes that are subject to intergenomic signaling are
down-regulated in the absence of a mitochondrial genome. Third,
intergenomic signaling affects expression of respiratory
protein genes while retrograde regulation probably does not.
The finding that the transcription of aerobic nuclear
COX genes is reduced in cells that lack a mitochondrial
genome but not in cells that lack respiration suggests that it is the
absence of one or more mitochondrial genes, or their ability to be
expressed, that is involved. This is surprising because all known
mitochondrial gene products (proteins and RNA) are assumed to
participate either in the function or biogenesis of the mitochondrial
respiratory chain. This raises the possibility that an as yet
unidentified mitochondrial gene is involved. Until recently, our
understanding of the yeast mitochondrial genome sequence was based of a
conglomerate sequence derived from several polymorphic strains (60,
61). This sequence was incomplete and contained multiple errors (62). Recently, the entire mitochondrial genome sequence was determined for a
single yeast strain, FY1679 (62). The overall organization of the
mitochondrial genome of FY1679 is similar to the "short mitochondrial
genomes" of the two respiration-proficient strains, D273-10B and
JM43, used here except for two deletions and some differences in the
flanking regions of some protein-coding genes. Surprisingly, the
sequence of the FY1679 mitochondrial genome revealed seven new small
open reading frames. The function of these putative protein-coding
genes is not yet known so it is possible that one or more of them is
involved in intergenomic signaling. Alternatively, it is
possible that a mitochondrial gene product involved in respiration is
multifunctional and participates in both respiration and
intergenomic signaling. Precedent for the multifunctionality
of mitochondrial proteins comes from the finding that cytochrome
c can function both as an electron carrier in the
mitochondrial respiratory chain and in the activation of cytosolic
caspases during apoptosis (63), and from the finding that Atp6, which
is a mitochondrial gene product that functions as a subunit of ATP
synthase, is important for the stability of the mitochondrial genome
(51). Finally, it is possible that the physical presence of the
mitochondrial genome, or a complex of which it is a part, is required.
The identification of which mitochondrial gene(s) is (are) involved in
intergenomic signaling should help in understanding how the
mitochondrial genome sends "signals" to the nucleus. It will
require an exhaustive analysis of mit mutants, we have found that neither
respiration nor cytochrome-c oxidase is required for the
down-regulation of these genes in cells exposed to anoxia but that a
mitochondrial genome is required for their full expression under both
normoxic and anoxic conditions. This requirement for a mitochondrial
genome is unrelated to the presence or absence of a functional
holocytochrome-c oxidase. We have also found that the
down-regulation of these genes in cells exposed to anoxia and the
down-regulation that results from the absence of a mitochondrial genome
are independent of one another. These findings indicate that the
mitochondrial genome, acting independently of respiration and oxidative
phosphorylation, affects the expression of the aerobic nuclear
COX genes and suggest the existence of a signaling pathway
from the mitochondrial genome to the nucleus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-9 fatty acid desaturase) and CYC7 (the
gene for iso-2 cytochrome c) and partially inhibits the
induction of COX5b (the gene for cytochrome-c
oxidase subunit Vb) but has no effect on the induction of seven other
hypoxic genes (HEM13, HMG1, HMG2, ERG11, CPR1(NCP1), ANB1, and AAC3). This finding revealed two classes of
yeast hypoxic genes: carbon monoxide-sensitive and carbon
monoxide-insensitive. By using mutants deficient in the two major yeast
carbon monoxide-binding hemeproteins (cytochrome-c oxidase
and flavohemoglobin) it was found that cytochrome-c oxidase
but not flavohemoglobin is required for the induction of the carbon
monoxide-sensitive genes. These studies also revealed that
OLE1 and CYC7 are not induced in rho0
strains (which lack a mitochondrial genome and respiration), in strains
that are respiration deficient but retain a mitochondrial genome, and
in the presence of respiratory inhibitors. Together, these findings
indicate that the mitochondrial respiratory chain is involved in the
expression of some hypoxic yeast genes and that
cytochrome-c oxidase is a likely oxygen sensor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0 and D273-10B
o
were derived from their respective parent strains by treatment with
acriflavine, for 18 h, and then selecting for rhoo
colonies, as described (25). The absence of DNA was initially assessed
by crossing to JM8, a rhoo tester strain and then verified
by cesium chloride centrifugation in the presence of
4,6-diamidino-2-phenylindole (26). Strain JM43GD9 was produced
as described previously (27). Strain JM43GD6 was constructed as
follows. The COX6 gene in JM43 was disrupted by use of the
plasmid pVISt3 (28), which is derived from pUC19. The URA3
gene was inserted into the HindIII site that is within the
COX6 coding region on pVISt3 and the resulting plasmid was used to transform JM43 by the lithium acetate procedure (29). Confirmation that the COX6 gene was disrupted was achieved
by Southern blot analysis, Northern blot analysis, and Western
immunoblotting of mitochondrial proteins. Strains aM10-150-4D, M9-3,
DS-80, and aM17-162-4D were derived from strain D273-10B; they were
kindly provided by Dr. Alexander Tzagoloff. The mitochondrial DNA in aM10-150-4D carries a 7.5-kilobase deletion that spans the region from
47.5 to 60 map units and that completely removes the cox1 genetic locus (30, 31). Strain aM17-162-4D carries a
mit
mutation in COB2 region of the the
cytb gene (32). Strain DS-80 is a rho
strain
that has a mitochondrial genome consisting of a tandemly repeated
segment of mtDNA that is 2.6 kilobase pairs in length. This
2.6-kilobase pair repeat spans the paromomycin-resistant locus and
encodes the 15-s rRNA gene. Strain M9-3 carries a
mit
mutation in the cox3 gene (33).
Strain VC36 carries a cox2 promoter mutation which prevents
transcription of cox2 (34). Genetic complementation tests
were routinely performed to be sure that the neither the nuclear
cox mutants nor the mit
mutants
used in this study were rhoo. The nuclear cox
mutants, JM43GD6 and JM43GD9, were checked for their ability to
complement the respiratory deficiency in JM6 and the
mit
mutants, aM10-150-4D, VC36, and
aM-17-162-4D, were checked for their ability to complement the
respiratory deficiency in M9-3. Plasmids were propagated in
Escherichia coli HB101 or DH5
and purified by alkaline
hydrolysis (35).
Yeast strains used in this study
4 °C, washed twice with ice cold diethylpyrocarbonate-treated distilled water, and either processed for
RNA immediately or frozen in liquid N2. Cycloheximide was added to anoxic cultures at a final concentration of 25 µg/ml prior
to harvesting.
-32P]dCTP or (DuPont NEN; cf. Ref. 35)
"All-In-One" random prime mixture (Sigma). Either plasmid-based
probes or PCR fragments were used. The plasmid based probes were as
follows: the probe for COX4 was an 800-bp XbaI
fragment, derived from pUC-4SH (41); the probe for COX5a was
a 500-bp PstI fragment from plasmid
p5ap5002; the probe for
COX6 was a 500-bp StuI/BglII fragment
from pVISt3 (28); the probe for COX8 was a 370-bp
XbaI-StyI fragment from pUC-8XS (42); the probe
for COX9 was a 240-bp ClaI-NheI
fragment derived from pVIIaS3 (27); and the probe for ACT1
was a 520-bp StyI fragment from pRB155 (43). A PCR fragment
of each gene was obtained by amplifying genomic DNA. The sequence of
the PCR primer pairs and their coordinates are as follows: the
COX4 fragment corresponded to bp 2 to 461 of the gene and
was amplified with the primer pair
5'-GGATTCTGAGGTTGCTGCTTTGGTT-3' and 5'-ACCGGCCAAATCGATTCTCA-3'; the
COX5a fragment corresponded to bp 10 to 454 of the gene and was amplified with the primer pair 5'-AACACTTTTACTAGAGCTGGTGGACT-3' and
5'-ATTGGACCTGAGAATAACCACCCC-3'; the COX6 fragment
corresponded to bp 8 to 444 of the gene and was amplified with the
primer pair 5'-CAAGGGCCATATTCAGAAATCCAGT-3' and
5'-AGAAGAGCTTGGAAATAGCTCTCC-3'; the COX8 fragment
corresponded to bp 1 to 231 of the gene and was amplified with the
primer pair 5'-ATGTTGTGCCAACAGATGAT-3' and 5'-AGCACCTGACTTTTTCAATT-3';
the COX9 fragment corresponded to bp 11 to 165 and was
amplified with the primer pair 5'-CTCCAATTACTGGTACGATCA-3' and
5'-TTTCCTCTCAGCTAGCTCTG-3'; and the ACT1 fragment
corresponded to bp 3 to 546 of the gene and was amplified by the primer
pair 5'- GGATTCTGAGGTTGCTGCTTTGGTT-3' and 5'-ACCGGCCAAATCGATTCTCA-3'. After amplification, PCR fragments were gel-purified on a 1%
TAE-agarose gel (35). Stringency washes were performed as described
previously (21). Signal intensity was measured with either an AMBIS
Radioanalytical Imaging System or a Molecular Dynamics Storm 860 PhosphorImager. To quantify the transcripts, the relative signal
strength was normalized to the level of ACT1 mRNA. To
determine whether levels of actin mRNA are affected by
mitochondrial genotype, we prepared total nucleic acids from JM43 and
JM43
o cells grown to mid-exponential phase in YPGal,
chemically quantitated the amounts of RNA and DNA, and probed a
Northern blot of the RNA with the 32P-labeled
ACT1 probe. When normalized to cellular DNA the amount of
actin mRNA did not vary by more than 5%, indicating that the expression of ACT1 is unaffected by mitochondrial genotype.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (50K):
[in a new window]
Fig. 1.
Effects of anoxia on nuclear COX
gene transcript and protein levels. Panel A,
Northern blot. Total RNA was isolated from JM43 cells grown either
under normoxic (air) or anoxic conditions (O2-free
N2 containing 2.5% CO2) and hybridized with
probes to COX4, COX5a, COX6, COX7, COX8, COX9, and
ACT1, as described under "Experimental Procedures." The
ACT1 trancript was used as an internal control for loading.
Lane 1, blot of RNA from normoxic cells. Lane 2,
blot from anoxic cells. Panel B, Western blots of
mitochondria and promitochondria with subunit-specific antisera to
cytochrome-c oxidase subunits IV-VIII. Subunit-specific
antisera to subunits IV, VI, VII, VIIA, and VIII were made from high
performance liquid chromatography purified subunits. The antiserum to
subunit V was made to a synthetic peptide which duplicates the 20-amino
acid sequence common to both Va and Vb at their carboxyl terminus.
Proteins were separated on a 16% SDS-polyacrylamide gel containing
10% glycerol and 4.8 M urea and transferred to
nitrocellulose. Cytochrome-c oxidase subunits were detected
with a subunit-specific primary antibody followed by horseradish
peroxidase-linked secondary antibodies. Immunoreactivity was detected
by chemiluminescence using a chemiluminescense detection kit
(PerkinElmer Life Sciences). 10 µg of mitochondrial protein was
loaded in each lane. Lane 1, aerobic mitochondria.
Lane 2, anaerobic promitochondria.
View larger version (55K):
[in a new window]
Fig. 2.
Presence and location of mitochondrially
encoded cytochrome-c oxidase subunits in anoxic
cells. Panel A, Western blots of mitochondria and
promitochondria with subunit-specific antisera to
cytochrome-c oxidase subunits I, II, and III. Lane
1, mitochondria. Lane 2, promitochondria. Panel
B, Western blots of cytosolic fractions from normoxic and anoxic
cells with anti-holocytochrome-c oxidase antiserum.
Lane 1, purified cytochrome-c oxidase. Lane
2, cytosol from normoxic cells. Lane 3, cytosol from
anoxic cells. 10 µg of protein were loaded in each lane.
Cytochrome-c oxidase subunits were detected by
chemiluminescence as in Fig. 1.
o. This strain lacks a mitochondrial genome and is
respiration-deficient (Table II). From
Fig. 3B it is clear that transcript levels from both
COX4 and COX5a are reduced in
JM43
o. It is also clear that they decline rapidly after
the shift with decay kinetics that are similar, if not identical, to
those observed for JM43 (Fig. 3A). These results indicate
that neither mitochondrial respiration nor cytochrome-c
oxidase is required for the reduction in COX4 and
COX5a transcript levels as cells adapt to anoxic
conditions.
View larger version (39K):
[in a new window]
Fig. 3.
Down-regulation of COX4 and
COX5a expression in JM43 and
JM43 o cells after a shift from
normoxia to anoxia. Cells were grown to early to mid-log
phase in air. At zero time, the first sample was taken, and the sparge
gas was shifted from air to 2.5% CO2 in
O2-free nitrogen. Cells were harvested for RNA at different
times after the shift, and total RNA was isolated and hybridized.
Panel A, JM43. Top, Northern blot of total RNA
probed for mRNAs from ACT1, COX4, and COX5a. Lanes
1 and 7 are Northern blots from cells grown under
normoxic conditions (air) and anoxic conditions (2.5% CO2
in O2-free N2), respectively. Lanes
2-6 represent Northern blots of RNA from cells taken at different
times after a shift from normoxic to anoxic conditions. Lane
2, 0 min; lane 3, 1 h; lane 4, 2 h; lane 5, 3 h; lane 6, 4 h.
Bottom, kinetics of transcript decay for COX4 and
COX5a after a shift from normoxia to anoxia. Panel
B, JM43
o. Top, Northern blot of total
RNA probed for mRNAs from ACT1, COX4, and
COX5a. Lanes 1-5 represent Northern blots of RNA
from cells taken at different times after a shift from normoxic to
anoxic conditions. Lane 1, 0 min; lane 2, 1 h; lane 3, 2 h; lane 4, 3 h; lane
5, 4 h. Bottom, kinetics of transcript decay for
COX4 and COX5a after a shift from normoxia to
anoxia. The mRNAs from COX4 and COX5b were
quantitated on a PhosphorImager and normalized to ACT1
mRNA. The normalized level for each is reported relative to its
level in aerobic cells.
Levels of respiration in mutant strains
0 grown in air. From Northern blot
analysis of RNA from JM43 and JM43
o it is clear that the
steady state levels of mRNA from all five COX genes are
reduced in the rhoo strain (Fig.
4A). To quantitate the
down-regulation of these genes in rhoo strains, we used
Northern blots representing three separate cultures of JM43 and
JM43
o. In addition, we compared the level of expression
of these genes in three different cultures of D273-10B, a
respiration-competent strain, D273-10B
o, a
respiration-deficient rho0 derivative of D273-10B, and
DS-80, a rho
derivative of D273-10B. Northern blots were
quantitated by counting the radioactivity associated with each band,
and normalizing to mRNA from ACT1, a gene whose
expression is not affected by the presence or absence of a
mitochondrial genome (see "Experimental Procedures," and Ref. 49).
From Table III it is clear that the levels of the nuclear aerobic COX transcripts are
reduced by 2.5-3.5-fold in the both of the rhoo strains
and in the rho
strain, relative to their respective
parental strains. Further evidence for the down-regulation of
COX4, COX5a, COX6, COX8, and COX9 in a
rho0 mutant strain comes from comparing the levels of their
protein products in JM43 and JM43
o. The Western
immunoblot in Fig. 4B demonstrates that the levels of
subunits IV and VI are reduced while subunits Va, VIIa, and VIII are
absent in JM43
o. Subunits IV and VI are present in
JM43
o at 20 and 16%, respectively, of their level in
JM43.
View larger version (74K):
[in a new window]
Fig. 4.
Expression of nuclear COX
genes is reduced in rhoo cells. Panel
A, Northern blot. Poly(A+) RNA was isolated from
strains JM43 and JM43 o cells grown to mid-exponential
phase and hybridized. All transcript levels were normalized to mRNA
from ACT1, a gene whose transcription is unaffected by the
mitochondrial genome (see "Experimental Procedures"). The position
of each COX gene mRNA is indicated. Lane 1,
JM43; lane 2, JM43rhoo. Panel B,
Western immunoblot of cytochrome-c oxidase polypeptide
subunits in whole cell extracts prepared from JM43 and JM43
o cells. Cells were grown to mid-exponential phase,
broken by shaking with glass beads, and subjected to SDS-polyacrylamide
gel electrophoresis in gels containing 16% acrylamide, 10% glycerol,
and 4.8 M urea. Western immunoblotting was done as
described (45), except that proteins were blotted onto Immobilon PVDF
membranes (Millepore). Immunoreactive bands were identified using
125I-Protein A and autoradiography. 10 µg of protein were
loaded on each lane.
Down-regulation of aerobic nuclear COX genes in rho0 and
rho strains
o and DS80, and JM43 for JM43
o).
Values and standard deviations are given for three independent
determinations for each strain and their respective parents.
o. It should be noted that both JM43GD6 and JM43GD9
are null mutants in an essential nuclear-encoded subunit of
cytochrome-c oxidase. They are devoid of
cytochrome-c oxidase activity and spectrally detectable
cytochromes aa3 (Fig.
6) but retain normal levels of the other
respiratory chain complexes (Ref. 27, and data not shown). In short,
they lack an assembled active holocytochrome-c oxidase but
have an otherwise normal respiratory chain. The finding that these two
strains express the nuclear aerobic COX genes at the normal
levels found in their respiration-competent parent indicates that the
presence or absence of a functional holocytochrome-c oxidase
does not affect the expression of these genes.
View larger version (52K):
[in a new window]
Fig. 5.
Northern blot analysis of nuclear COX
gene mRNAs in the nuclear cox mutants JM43GD6 and
JM43GD9. Poly(A+) RNA was prepared, electrophoresed,
blotted, and hybridized with gene specific probes for ACT1,
COX6, COX4, COX9, COX5a, and COX8. Northern blots
of RNA from JM43 (lane 1), JM43 o (lane
2), JM43GD6 (lane 3), and JM43GD9 (lane 4).
A short truncated COX6 transcript is observable in JM43GD6
(lane 3).
Relative transcript levels in respiration-deficient strains
View larger version (13K):
[in a new window]
Fig. 6.
Low temperature absorption spectra of
cytochromes in strains JM43, JM43GD6, and JM43GD9. Cells were
grown on liquid YPGal medium to mid-exponential phase and resuspended
at 0.3 g wet weight per ml in 40 mM KPO4
(pH 7.4). Cells were either oxidized (with
H2O2), or reduced (with dithionite), brought to
30% ethylene glycol, frozen in liquid nitrogen, de-vitrified, and the
spectra scanned. The spectra shown are difference spectra (reduced
minus oxidized) recorded on an Aminco DW-2000 double beam dual
wavelength spectrophotometer. The -absorbance bands for cytochromes
c, c1, b, and
aa3 are noted.
o, derived from D272-10B, they are
respiration deficient (Table II). Aside from carrying mit
mutations in cox1, cox2, or cytb these strains
carry a rho+ mitochondrial genome, as judged from their
ability to complement one another and produce respiration-proficient
diploid strains when mated. From Fig. 7
and Table IV it is clear that the levels of transcripts from
COX4, COX5a, COX6, COX8, and COX9 in the
aM17-162-4D, aM10-150-4D, and VC36 are equivalent to those levels
observed in the respiratory competent parent strain, D273-10B.
Together, these data with JM43GD6, JM43GD9, aM17-162-4D, aM10-150-4D,
and VC36 demonstrate that a deficiency in respiration per se
cannot account for the decrease in COX gene mRNA levels
observed in rhoo cells.
View larger version (58K):
[in a new window]
Fig. 7.
Northern blot analysis of nuclear
COX gene mRNAs in strains D273-10B,
D273-10B 0, aM10-150-4D, VC36, and
aM17-162-4D. Poly(A+) RNA was prepared,
electrophoresed, blotted, and hybridized with gene specific probes for
ACT1, COX6, COX4, COX9, COX5a, and
COX8. Northern blot of mRNA from D273-10B (lane
1); D273-10B
o (lane 2); aM10-150-4D
(lane 3); VC36 (lane 4); and aM17-162-4D
(lane 5).
0 were cultured in 2.5%
CO2 in oxygen-free N2 and mRNA levels from COX4, COX5a, COX6, COX8, and COX9 were measured. From Fig.
8 it is clear that the levels of
expression of these genes is reduced in JM43
o relative
to JM43. Their expression in anoxic JM43
o cells was
between 16 and 36% of their levels in anoxic JM43 cells.
View larger version (42K):
[in a new window]
Fig. 8.
Down-regulation of nuclear COX
genes in anoxic JM43 o
cells. Total RNA was isolated from anoxic cultures of JM43
and JM43
o cells and subjected to Northern blotting with
probes to ACT1, COX4, COX5a, COX6, COX8, and
COX9. The mRNAs from each COX gene were
quantitated on a PhosphorImager and normalized to ACT1
mRNA. Transcript levels relative to ACT1 mRNA are
reported relative to their levels in aerobic cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutants
in individual mitochondrial genes and rho
mutants that
retain different parts of the mitochondrial genome. The limited set of
mitochondrial mutants (e.g. am17-162-4D, aM10-150-4D, VC36, and
DS-80) used in this study indicate that the ctyb, cox1, cox2, and 15 S rRNA genes, by themselves, are not involved
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. John Trawick and Norbert Kraut for early contributions to this study and Dr. Alexander Tzagoloff for strains.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL63324 and GM30228.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a National Institutes of Health postdoctoral fellowship.
§ To whom correspondence should be addressed. Tel.: 303-493-3823; Fax: 303-492-8783; E-mail: Poyton@spot.Colorado.EDU.
Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M009180200
2 C. Dagsgaard, and R. O. Poyton, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid; PCR, polymerase chain reaction; bp, base pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Bunn, H. F.,
and Poyton, R. O.
(1996)
Physiol. Rev.
76,
839-885 |
2. |
Hochachka, P, W.,
Buck, L. T.,
Doll, C.,
and Land, S. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9493-9498 |
3. |
Hochachka, P. W.
(1998)
J. Exp. Biol.
201,
1243-1254 |
4. | Semenza, G. L. (2000) Adv. Exp. Med. Biol. 475, 303-310[Medline] [Order article via Infotrieve] |
5. | Acker, H. (1994) Ann. N. Y. Acad. Sci. 718, 3-10[Medline] [Order article via Infotrieve] |
6. | Huang, L. E., Ho, V., Arany, Z., Krainc, D., Galson, D., Tendler, D., Livingston, D. M., and Bunn, H. F. (1997) Kidney Int. 51, 548-552[Medline] [Order article via Infotrieve] |
7. |
Ratcliffe, P. J.,
Maxwell, P. H.,
and Pugh, C. W.
(1997)
Nephrol. Dial. Transplant.
12,
1842-1848 |
8. | Semenza, G. L. (1999) Cell 98, 281-284[Medline] [Order article via Infotrieve] |
9. | Poyton, R. O. (1999) Respir. Physiol. 115, 119-133[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Chandel, N. S.,
and Schumacker, P. T.
(2000)
J. Appl. Physiol.
88,
1880-1889 |
11. | Wilson, D. F., Mokashi, A., Chugh, D., Vinogradov, S., Osanai, S., and Lahiri, S. (1994) FEBS Lett. 151, 370-374[CrossRef] |
12. | Wilson, D. F., Mokashi, A., Lahiri, S., and Vinogradov, S. A. (2000) Adv. Exp. Med. Biol. 475, 259-264[Medline] [Order article via Infotrieve] |
13. | Lahiri, S., Buerk, D. G., Chugh, D., Osanai, S., and Mokashi, S. (1995) Brain Res. 684, 194-200[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Acker, H.,
and Xue, D.
(1995)
New Physiol. Sci.
10,
211-216 |
15. |
Chandel, N. S.,
Maltepe,
Goldwasser, E.,
Mathieu, C. E.,
Simon, M. C.,
and Schumacker, P. T.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11715-11720 |
16. |
Kwast, K. E.,
Burke, P. V.,
Staahl, B.,
and Poyton, R. O.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5446-5451 |
17. | Poyton, R. O., and McEwen, J. E. (1996) Annu. Rev. Biochem. 65, 563-607[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Burke, P. V.,
and Poyton, R. O.
(1998)
J. Exp. Biol.
201,
1163-1175 |
19. | Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-itoh, K., Nakashima, R., Yaono, R., and Yoshikawa, S. (1996) Science 272, 1136-1144[Abstract] |
20. | Poyton, R. O., Goehring, B., Droste, M., Sevarino, K. A., Allen, L. A., and Zhao, X-J. (1995) Methods Enzymol. 260, 97-116[Medline] [Order article via Infotrieve] |
21. | Trueblood, C. E., and Poyton, R. O. (1987) Mol. Cell. Biol. 7, 3520-3526[Medline] [Order article via Infotrieve] |
22. |
Waterland, R. A.,
Basu, A.,
Chance, B.,
and Poyton, R. O.
(1991)
J. Biol. Chem.
266,
4180-4186 |
23. |
Allen, L. A.,
Zhao, X. J.,
Caughey, W.,
and Poyton, R. O.
(1995)
J. Biol. Chem.
270,
110-118 |
24. | Cumsky, M. G., Ko, K., Trueblood, C. E., and Poyton, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2235-2239[Abstract] |
25. | Mattick, J. S., and Nagley, P. (1977) Mol. Gen. Genet. 152, 267-276[Medline] [Order article via Infotrieve] |
26. | Williamson, D. H., and Fennell, D. J. (1975) Methods Cell Biol. 12, 335-351[Medline] [Order article via Infotrieve] |
27. |
Wright, R. M.,
Dircks, L. K.,
and Poyton, R. O.
(1986)
J. Biol. Chem.
261,
17183-17191 |
28. | Wright, R. M., Rosenzwieg, B., and Poyton, R. O. (1989) Nucleic Acids Res. 17, 1103-1120[Abstract] |
29. | Ito, H., Fukada, Y., Murata, K., and Kimura, M. (1983) J. Bacteriol. 153, 163-168[Medline] [Order article via Infotrieve] |
30. | Morimoto, R., Lewin, A., and Rabinowitz, M. (1979) Mol. Gen. Genet. 170, 1-9[Medline] [Order article via Infotrieve] |
31. |
Bonitz, S. G.,
Coruzzi, G.,
Thalenfeld, B. E.,
Tzagoloff, A.,
and Macino, G.
(1980)
J. Biol. Chem.
255,
11922-11926 |
32. | Alexander, N. J., Vincent, R. D., Perlman, P. S., Miller, D. H., Hanson, D. K., and Mahler, H. R. (1979) J. Biol. Chem. 254, 2471-2479[Abstract] |
33. | Slonimski, P. P., and Tzagoloff, A. (1976) Eur J. Biochem. 61, 27-41[Abstract] |
34. |
Cameron, V. L.,
Fox, T. D.,
and Poyton, R. O.
(1989)
J. Biol. Chem.
264,
13391-13394 |
35. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , pp. 1-38, Cold Spring Harbor Laboratory, New York |
36. |
Burke, P. V.,
Kwast, K. E.,
Everts, F.,
and Poyton, R. O.
(1998)
Appl. Environ. Microbiol.
64,
1040-1044 |
37. | Sulkowski, E., Guerin, B., Defaye, J., and Slonimski, P. P. (1964) Nature 202, 36-39[Medline] [Order article via Infotrieve] |
38. | Elder, R. T., Loh, E. Y., and Davis, R. W. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2432-2436[Abstract] |
39. | Tsang, S. S., Yin, X., Guzzo-Arkuran, C., Jones, V. S., and Davison, A. J. (1993) BioTechniques 14, 380-381[Medline] [Order article via Infotrieve] |
40. |
Burke, P. V.,
Raitt, D.,
Allen, L. A.,
Kellogg, E. A.,
and Poyton, R. O.
(1997)
J. Biol. Chem.
272,
14705-14712 |
41. | Maarse, A. C., VanLoon, A. P. G. M., Riezman, H., Gregor, I., Schatz, G., and Grivell, L. A. (1984) EMBO J. 3, 2831-2837[Abstract] |
42. |
Patterson, T. E.,
and Poyton, R. O.
(1986)
J. Biol. Chem.
261,
17192-17197 |
43. | Ng, R., and Abelson, J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3912-3916[Abstract] |
44. |
McEwen, J. E.,
Ko, C.,
Kloeckener-Gruissem, B.,
and Poyton, R. O.
(1986)
J. Biol. Chem.
261,
11872-11879 |
45. | Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract] |
46. | Tzagoloff, A., Akai, A., and Needleman, R. J. (1975) Biol. Chem. 250, 8228-8235[Abstract] |
47. |
TerLinde, J. J. M.,
Liang, H.,
Davis, R. W.,
Steensma, H. Y.,
van Dijken, J. P.,
and Pronk, J. T.
(1999)
J. Bacteriol.
181,
7409-7413 |
48. | Rogers, P. J., and Stewart, P. R. (1973) J. Bacteriol 115, 88-97[Medline] [Order article via Infotrieve] |
49. | Liao, X., Small, W. C., Srere, P. A., and Butow, R. A. (1991) Mol. Cell. Biol. 11, 38-46[Medline] [Order article via Infotrieve] |
50. |
Petersen, P. L.,
and Amzel, L. M.
(1993)
J. Biol. Chem.
268,
9937-9940 |
51. |
Contamine, V.,
and Picard, M.
(2000)
Microbiol. Mol. Biol. Rev.
64,
281-315 |
52. | Serrano, R. (1980) Eur. J. Biochem. 105, 419-424[Abstract] |
53. | Woodrow, G., and Schatz, G. (1979) J. Biol. Chem. 254, 6088-6093[Abstract] |
54. |
Chandel, N. S.,
Budginger, G. R. S.,
and Schumacker, P. T.
(1996)
J. Biol. Chem.
271,
18672-18677 |
55. |
Duranteau, J.,
Chandel, N. C.,
Kulisz, A.,
Shao, Z.,
and Schumacker, P. T.
(1998)
J. Biol. Chem.
273,
11619-11624 |
56. | Butow, R. A. (1988) Philos Trans. R. Soc. Lond. B Biol. Sci. 319, 127-133[Medline] [Order article via Infotrieve] |
57. | Liao, X., and Butow, R. A. (1993) Cell 72, 61-71[Medline] [Order article via Infotrieve] |
58. |
Rothermal, B. A.,
Shyjan, A. W.,
Etheridge, J. L.,
and Butow, R. A.
(1995)
J. Biol. Chem.
270,
29476-29482 |
59. |
Conlan, R. S.,
Gounalaki, N.,
Hatzis, P.,
and Tzamarias, D.
(1999)
J. Biol. Chem.
274,
205-210 |
60. | de Zamaroczy, M., and Bernardi, G. (1986) Gene (Amst.) 41, 155-177 |
61. | Tzagoloff, A., and Myers, A. M. (1986) Annu. Rev. Biochem. 55, 249-285[CrossRef][Medline] [Order article via Infotrieve] |
62. | Foury, F., Roganti, T., Lecrenier, N., and Purnelle, B. (1998) FEBS Lett. 440, 325-331[CrossRef][Medline] [Order article via Infotrieve] |
63. | Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. (1996) Cell 86, 147-157[Medline] [Order article via Infotrieve] |
64. |
Li, M.,
Tzagoloff, A.,
Underbrink-Lyon, K.,
and Martin, N.
(1982)
J. Biol. Chem.
257,
5921-5928 |