From the
In Saccharomyces cerevisiae cells with dysfunctional
mitochondria, such as in petites, the CIT2 gene encoding the
peroxisomal glyoxylate cycle enzyme, citrate synthase 2 (CS2), is
transcriptionally activated by as much as 30-fold, a phenomenon we call retrograde regulation. Two genes, RTG1 and RTG2, are required for both basal and elevated expression of CIT2 (Liao, X., and Butow, R. A.(1993) Cell 72,
61-71). Different blocks in the tricarboxylic acid cycle also
elicit an increase in CIT2 expression, but not to the extent
observed in petites. We have examined whether other genes of the
glyoxylate cycle exhibit retrograde regulation and the role of RTG1 and RTG2 in their expression. Of the glyoxylate cycle
genes tested, CIT2 is the only one that shows retrograde
regulation, suggesting that CS2 may be an important control point for
metabolic cross-feeding from the glyoxylate cycle to mitochondria.
Surprisingly, RTG1 and RTG2 are required for
efficient growth of cells on medium containing oleic acid, a condition
which induces peroxisome biogenesis; these genes are also required
together for oleic acid induction of three peroxisomal protein genes
tested, POX1 and CTA1 involved It has recently become clear that alterations in the functional
state of mitochondria and chloroplasts can result in changes in nuclear
gene
expression(1, 2, 3, 4, 5, 6, 7, 8, 9) .
We have called this pathway of interorganelle communication retrograde regulation and suggested that it is a mechanism for
the cell to adjust to changes in mitochondrial or chloroplast
activities(10) . One example in yeast that supports the idea
that retrograde regulation is an adaptive process to changes in
mitochondrial function is the dramatic elevation in expression of the CIT2 gene in cells with altered mitochondrial
activity(5) . CIT2 is a nuclear gene encoding the
glyoxylate cycle isoform of citrate synthase (CS2)(11) ; that
enzyme, together with other enzymes of the glyoxylate cycle, is located
in peroxisomes of Saccharomyces
cerevisiae(12, 13) . CIT2 expression is
increased 5-10 fold in cells in which the tricarboxylic acid
cycle is blocked because of a disruption of CIT1, the gene
encoding the mitochondrial isoform of citrate synthase,
CS1(14) . This increase in CIT2 expression compensates
for the loss of CS1 activity through metabolic cross-feeding between
the glyoxylate and tricarboxylic acid cycles(5) . The greatest
increase in CIT2 expression we have observed is in
respiratory-deficient The
increased abundance of CIT2 mRNA in cells with dysfunctional
mitochondria is due to transcriptional activation and is mediated in cis through an upstream activation site (UAS The
products of both RTG1 and RTG2 are required not only
for the elevated expression of CIT2 in cells with
dysfunctional mitochondria but also for its basal expression in
otherwise wild-type cells. Although neither RTG1 nor RTG2 are essential genes, cells with mutant alleles of either are
glutamate-aspartate auxotrophs and are unable to grow on acetate as a
sole carbon source. These growth requirements are characteristic of
cells with blocks in both the tricarboxylic acid and
glyoxylate cycles, suggesting that the RTG genes play a role
in the regulation of both(11) . As yet, we have not found any
significant lesions in enzymes of either cycle, other than the expected
absence of CS2 activity and a reduction ( These findings raise a number of questions
regarding retrograde regulation and other possible functions of the RTG genes. For example, do tricarboxylic acid cycle blocks,
other than a loss of CS1 activity, result in elevated CIT2 expression? Are other genes encoding glyoxylate cycle enzymes
subject to retrograde regulation? And what role, if any, do the RTG genes play in expression of other peroxisomal proteins? This
latter issue can be conveniently studied in yeast by growing cells in
medium containing oleic acid. Under these conditions, there is a large
induction of peroxisomal enzymes of the Here we show that a
tricarboxylic acid cycle block caused by a disruption of MDH1 (encoding the tricarboxylic acid cycle isoform of malate
dehydrogenase) elicits an elevation of CIT2 expression similar
to that observed in cells with a disruption of CIT1. We show
further that CIT2 is the only gene encoding a glyoxylate cycle
enzyme among three others tested that is subject to significant
retrograde regulation when its expression is compared between
isochromosomal
For quantitative Northern analysis, 2- to
8-fold serial dilutions of RNA samples were loaded on to the gel.
Autoradiograms were quantified against the levels of actin mRNA, whose
abundance does not vary with growth conditions or among the various
strain derivatives(4) , with a Molecular Dynamics
PhosphorImager using ImageQuant software.
Figure 1:
Blocks in the tricarboxylic acid cycle
result in elevated CIT2 expression. Wild-type yeast strains
PSY142 (
The finding that the abundance of CIT2 mRNA is
greater in petites (which lack an electron transport chain and
oxidative phosphorylation apparatus) than in
Figure 2:
Retrograde regulation (
Figure 3:
Respiratory-competent
wild-type
Figure 4:
RTG1 and RTG2 are
required for full induction of expression of POX1 (acetyl-CoA
oxidase), CTA1 (catalase A), and PMP27 encoding a
peroxisomal membrane protein. Wild-type COP171U7
Figure 5:
Oleic acid induction of expression of
genes encoding glyoxylate cycle enzymes in wild-type and rtg mutant cells. The same total cellular RNA isolated from the
strains described in Fig. 4grown in the presence and absence of
0.2% oleic acid were used to determine the relative abundance of mRNAs
encoded by CIT2, MDH3, MLS1, and ILC1 in wild-type
As we have shown
previously, both RTG1 and RTG2 are required for basal
level and retrograde-induced expression of CIT2 in cells grown
on rich raffinose medium(6) . The requirement of RTG1 for CIT2 expression is also evident in the oleate
induction experiment of Fig. 5, where we could not detect any CIT2 mRNA in oleate-grown Surprisingly, in The present work extends our analysis of the retrograde
regulation of nuclear gene expression in response to the functional
state of mitochondria in S. cerevisiae. One of the goals of
the current study was to determine the specificity of the retrograde
response; that is, whether other lesions in the tricarboxylic acid
cycle besides the absence of CS1 activity would also elicit an increase
in CIT2 expression and whether other genes encoding glyoxylate
cycle enzymes might be subject to retrograde control. Regarding the
former, we have shown that Like many other
organisms with a glyoxylate cycle, yeast is able to utilize acetate as
a sole carbon source, relying on the glyoxylate cycle to provide two
2-carbon units for net gluconeogenesis(33) . Acetyl-CoA
generated from A comparison of mRNA abundance in
Although The
oleic acid-induced expression of a number of genes encoding peroxisomal
proteins in yeast has been shown to require a 5`-flanking cis-acting sequence called an ORE (oleate response element)
that is a target site for trans-acting transcription
factors(41, 42) . Although the RTG2 product
does not resemble a DNA-binding protein or known transcription factors,
the RTG1 product has been shown to be a basic helix-loop-helix
transcription factor that binds to the UAS Lewin et al.(12) and
McCammon et al.(13) localized glyoxylate cycle
enzymes in peroxisomes of yeast cells grown on medium containing oleic
acid. The present study represents, to our knowledge, the first
quantitative analysis of the effects of oleic acid on expression of
genes encoding glyoxylate cycle enzymes in yeast. While the oleic
acid-induced levels of expression are comparable to the induction of
expression of the P-C-P genes studied here, the results with
the rtg mutants are somewhat different. First, despite the
large induction of CIT2 mRNA in wild-type
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-oxidation of
long chain fatty acids and PMP27, which encodes the most
abundant protein of peroxisomal membranes. These data indicate that, in
addition to their role in retrograde regulation of CIT2, the RTG genes are important for expression of genes encoding
peroxisomal proteins and are thus key components in a novel, three-way
path of communication between mitochondria, the nucleus, and
peroxisomes.
petites, which lack
mitochondrial DNA. In those cells, the CIT2 mRNA abundance is
as much as 30-fold greater than in their isochromosomal
respiratory-competent (
) counterparts.
) in
the 5` flanking region of the gene(5, 6) . We
previously reported that transcriptional regulation of CIT2 requires at least two trans acting factors encoded by the
single copy nuclear genes, RTG1 and RTG2(6) . RTG1 encodes a basic helix-loop-helix transcription factor
that binds to the CIT2 UAS
, while RTG2 encodes a protein of unknown function that probably acts
indirectly in the transactivation of CIT2 expression. A recent
report shows that the deduced sequence of the RTG2-encoded
protein has an ATP binding domain with similarity to bacterial enzymes
that hydrolyze the transcriptional regulators, ppGpp and
pppGpp(15) . We have recently identified a third gene (RTG3) required for CIT2 expression
(
)whose properties will be reported elsewhere.
50%) in CS1 activity,
which could account in any simple way for the rtg mutant
phenotypes(16) .
-oxidation pathway
accompanied by a general increase in the number of peroxisomes within
the cell(17, 18, 19) .
and
cells.
Finally, we show that RTG1 and RTG2 are required for
full induction of expression of genes encoding other peroxisomal
proteins, including an abundant protein associated with peroxisomal
membranes whose synthesis follows peroxisome biogenesis (20, 21) when cells are grown in medium containing
oleic acid. Thus, the RTG genes function not only in
communication between mitochondria and the nucleus, but are likely to
be involved in the oleic acid induction of peroxisome biogenesis as
well.
Yeast Strains
Yeast strains used in this study were COP161U7 (MATa, ade1 lys1 ura3 )
and ethidium bromide-induced (see below)
isolates.
Derivatives of
and
isolates
were made containing URA3 insertion mutations in RTG1 (
rtg1), RTG2 (
rtg2), or in
both chromosomal loci (
rtg1
rtg2) as described by
Liao and Butow(6) . Strain PSY142 (MAT
, leu2
lys2, ura3
) and its cit1 disruption derivative (cit1::LEU2) was kindly provided by
Dr. Paul Srere. To construct a disruption of MDH1 in PSY142,
30-mer PCR
(
)primers were made containing 21 bp
homologous to MDH1 sequence from position -144 to
-124 at the 5` end of a gene and from position +1187 to
+1207 at the 3` end of the gene(22) ; the remaining 9 bp
of each primer contained restriction site sequences for BamHI
(5`) and SalI (3`). The PCR product was cloned into the BamHI-SalI sites of Bluescript KS plasmid. A 735-bp HindIII-EcoRI internal fragment of MDH1 was
replaced by a 1170-bp HindIII restriction fragment containing
the URA3 gene after filling protruding 5` ends with Klenow
enzyme. The mdh1::URA3 fragment was excised from the above
plasmid and used to transform
PSY142 by a
one-step transformation procedure(23) . The disruption allele
was confirmed by Southern blot analysis. Various
derivatives of these strains were generated by growing cells to
saturation in YPD medium (1% yeast extract, 1% Bacto-peptone, and 2%
glucose) supplemented with 25 µg/ml ethidium bromide. The cells
were diluted 1:50 and grown once more in the same medium to stationary
phase. Individual colonies were checked by 4`,6`-diamino-2-phenylindole
staining for the loss of mitochondrial DNA.
Growth Conditions
Yeast strains were grown at 30 °C on YPR medium (1% yeast
extract, 1% Bacto-peptone, and 2% raffinose) additionally supplemented
with adenine (50 mg/liter). Solid medium contained 2% agar. For oleic
acid induction experiments, oleic acid (>99% purity, Aldrich) was
added to the medium at a final concentration of 0.2% (w/v) while
shaking vigorously to obtain an even emulsion. For most analyses, cells
were harvested by centrifugation at midlogarithmic phase of growth,
usually at an A between 0.9 and 1.1.
RNA Isolation and Analysis
Total yeast RNA was isolated from 50 ml of culture as
described by Schmitt et al.(24) . RNA was aliquoted
and stored at -80 °C. For Northern blot analysis of mRNA
abundance, 2.5-20 µg of RNA was separated on a 1.3% agarose
gel containing 6.4% formaldehyde by electrophoresis for 12-14 h
at room temperature. The fractionated RNAs were transferred to a Nytran
membrane (Schleicher & Schuell) by capillary blotting in 20
SSC (0.15 M NaCl, 0.15 M sodium citrate) and
cross-linked to the membrane. Hybridizations were performed using
Rapid-hyb buffer (Amersham) according to the manufacturer's
instructions. Prehybridization was carried out for 1 h at 65 °C.
Radiolabeled probes were added and hybridized for 2-3 h at 65
°C.
P-Labeled DNA probes for hybridization were
prepared by the random priming method using the Random Primed DNA
Labeling Kit (Boehringer Mannheim) or by PCR labeling using a PCR
Radioactive Labeling System (Life Technologies, Inc.).
[
-
P]dATP (3000 Ci/mmol, Amersham) was used
for generating radioactive probes. Blots were washed twice in 2
SSC and once in 0.4
SSC at 65 °C, briefly air-dried, and
exposed in PhosphorImager cassettes (Molecular Dynamics) or on
Hyperfilm-MP (Amersham).
Probes
Random Priming
Radioactive probes for the
analysis of mRNA abundance of the various genes analyzed in this study
generated by random priming (25) include the following
restriction endonuclease fragments: ACT1 (actin), 0.6-kb ClaI internal fragment from plasmid pGEM-actin(26) ; CIT2 (CS2), 1.1-kb SpeI-NcoI internal
fragment from plasmid pGCIT2-1083(5) ; CTA1 (catalase
A), 2.8-kb EcoRI fragment containing the coding and both 5`-
and 3`-untranslated regions (27) ; PMP27 (peroxisomal
membrane protein), 0.8-kb XbaI-BamHI fragment from
plasmid PMP27-pUC19 (20) containing 0.6 kb of coding sequence
and 0.2 kb of 3`-untranslated region of PMP27.Probes Generated by PCR
MDH1 (malate
dehydrogenase 1), coding sequence with flanking regions amplified from
position -144 to +1207(22) ; MDH3 (malate
dehydrogenase 3), internal fragment amplified from position +91 to
+981(28) ; ICL1 (isocitrate lyase), internal
fragment amplified from position +529 to +1512(29) ; MLS1 (malate synthase 1), internal fragment amplified from
position +533 to +1644(30) ; POX1 (acyl-CoA
oxidase), internal fragment amplified from position +828 to
+1764(31) . For these reactions, 1 µg of yeast genomic
DNA, isolated as described in (32) , was used as a template in
each PCR reaction. Primers used in all PCR reactions were synthetic
21-mers. PCR products were obtained using a DNA Thermal Cycler
(Perkin-Elmer Cetus). Thirty cycles with the following profile were
performed: 30 s at 94 °C, 2 min at 50 °C, and 2 min at 72
°C. PCR products were subsequently separated in 0.8% agarose and
gel-purified using a Geneclean Kit (Bio 101, Inc.). The purified PCR
products were used next as a template in labeling reactions by PCR or
random priming as noted above.
Blocks in the Tricarboxylic Acid Cycle Result in
Elevated Expression of CIT2
The response of CIT2 gene
expression to dysfunctional mitochondria is readily observed in a
comparison of the abundance of CIT2 mRNA in versus
cells. In a typical Northern
blot of total cellular RNA from
and
cells of strain PSY142, CIT2 mRNA is 25 times more
abundant in the
derivative compared with
cells (Fig. 1, lanes 1 and 2) when both strains are grown on rich raffinose medium. A
less severe alteration in mitochondrial function can also result in an
increase in CIT2 expression; for example, in
cells that have a disruption of the CIT1 gene. As shown
in Fig. 1, lanes 3 and 4, CIT2 mRNA
abundance is 10-fold greater in
cit1::URA3 cells than in the wild-type. One possibility to account for this
increase in CIT2 expression is that it is a specific response
to the absence of any mitochondrial CS1 activity, a plausible notion
considering the metabolic cross-feeding of citrate generated in the
glyoxylate cycle from peroxisomes to mitochondria(33) . To
examine this point, we have measured CIT2 mRNA levels in
cells with a different lesion in the
tricarboxylic acid cycle generated by a disruption of MDH1,
the gene encoding the mitochondrial isoform of malate
dehydrogenase(22) . As shown in Fig. 1, lanes 5 and 6,
cells with an MDH1 disruption also have elevated levels of CIT2 mRNA, which
in this experiment is 6.5-fold greater than the wild-type
control. This result rules out the possibility
that increased expression of CIT2 is the result of a defect in
the tricarboxylic acid cycle specifically involving the CIT1 gene.
and
) and PSY142
derivatives bearing a disruption of CIT1 (
cit1) or MDH1 (
mdh1) were
grown to midlog phase on YPR medium. Total RNA was isolated from each
strain and fractionated on a 1.3% agarose gel, blotted, and hybridized
with specific probes for CIT2 and actin as described under
``Experimental Procedures.'' Each pair of lanes represent a
2-fold load difference in RNA (20 and 10 µg). The signals for each
probe were quantified by PhosphorImager analysis. The relative CIT2 mRNA abundance shown below the blots represents the average of the
two loads expressed as the values normalized to the signal for actin
mRNA.
cells which lack tricarboxylic acid cycle activity suggests that
multiple lesions in mitochondrial function may account for the high
levels of CIT2 expression in petites. This view is consistent
with a previous observation that the abundance of CIT2 mRNA in
cells with a CIT1 disruption can be
further increased to the level observed in petites if the cells are
grown in the presence of the respiratory chain inhibitor, antimycin
A(5) . Taken together, these findings indicate that increases
in CIT2 expression can be induced, probably cumulatively, by a
variety of blocks in mitochondrial function.
Specificity of the Retrograde Response among Glyoxylate
Cycle Genes
Given the metabolic interplay between the glyoxylate
and tricarboxylic acid cycles(33) , a key question is whether
the expression of other glyoxylate cycle genes besides CIT2 respond to the functional state of mitochondria. To examine this,
we have compared the mRNA levels in and
derivatives of strain COP161U7 for three additional
glyoxylate cycle genes, ICL1 (isocitrate lyase), MLSI (malate synthase), and MDH3 (malate dehydrogenase 3).
Northern blots of total RNA isolated from these cells were hybridized
with probes specific for each of these genes as described under
``Experimental Procedures'' and in the legend to Fig. 2. The results of these experiments (Fig. 2) show
that of these genes, only CIT2 is subject to significant
retrograde regulation. In this experiment, the CIT2 mRNA
abundance is 10-fold greater in the
petite than in
cells, whereas the mRNA levels for MLSI and MDH3 are no greater than 2-fold. The probe used for ICL1, which is a 983-bp fragment derived by PCR from the
coding region of the gene (see ``Experimental Procedures''),
hybridizes about equally to two closely migrating transcripts, neither
of which shows a retrograde response. Thus, at this level of analysis,
clearly not all glyoxylate cycle genes are subject to retrograde
regulation.
versus
) of genes encoding enzymes of the
glyoxylate cycle is specific for CIT2. A
and a
derivative of strain COP161U7 were grown
to midlog phase in YPR medium. Total RNA was isolated from each strain
and analyzed by Northern blotting using probes specific for CIT2,
MDH3, MLS1, ICL1, and actin as described under ``Experimental
Procedures.'' The gel shows four RNA loads off 20, 10, 5, and 2.5
µg.
Cells with Null Alleles of RTG1 and RTG2 Have a Reduced
Ability to Utilize Oleic Acid as a Sole Carbon Source
RTG1 and RTG2 are required for both basal expression of CIT2 in wild-type cells and for its elevated expression in
cells with dysfunctional mitochondria(6) . A surprising
collateral phenotype of cells with mutant alleles of RTG1 or RTG2 is that they are unable to utilize acetate as a sole
carbon source. That phenotype is usually associated with some block in
the tricarboxylic acid cycle and is not observed in cells which lack
only CS2 activity(6, 11) . These observations raised
the question of whether cells with mutant alleles of RTG1 and RTG2 could utilize the two carbon acetyl-CoA units generated
by -oxidation of fatty acids. To test this, we determined whether
COP161U7 cells with null alleles of RTG1 (
rtg1) or RTG2 (
rtg2) or both (
rtg1
rtg2) could grow on medium containing oleic
acid as the sole carbon source. Both wild-type
and the null mutant derivatives were serially diluted on YP
medium containing 0.2% oleic acid and 0.25% Tween 40. As a control, the
same dilutions were plated on YPD medium. Fig. 3shows that
while all strains grow comparably well on YPD, all of the rtg mutant strains show impaired growth on oleic acid medium compared
with the wild-type strain, COP161U7. A closer inspection of the plates
suggests that oleate growth for the
rtg2 and
rtg1
rtg2 double mutant is slightly more impaired
than the
rtg1 mutant strain.
rtg1 and
rtg2 strains show impaired growth on medium containing oleic acid as
the sole carbon source. Wild-type COP161U7 cells and the rtg mutants as indicated were grown to midlogarithmic phase in YPR
medium, harvested, washed with H
0, and resuspended in 1/4
volume of H
0. 10-fold dilutions were made, and 4 µl of
each were applied on the medium as shown in the figure. Plates were
incubated at 30 °C for 3 days.
RTG1 and RTG2 Are Required for Full Induction of
Peroxisomal Oleate-responsive Genes
The reduced ability of the rtg mutant cells to efficiently utilize oleic acid in the
growth medium could be due to an impairment in the induction of
peroxisomal enzymes of the -oxidation pathway. To investigate this
possibility, we have carried out quantitative Northern blot analysis to
measure the effects of null alleles of RTG1 and RTG2,
both singly and in combination, on the mRNA levels for three
peroxisomal proteins known to be highly induced in cells grown on oleic
acid(18, 19) . These are acetyl-CoA oxidase and
catalase A (encoded by the POX1 and CTA1 genes,
respectively), and a major peroxisomal membrane protein, Pmp27 (encoded
by the PMP27 gene), whose abundance in cells closely follows
peroxisomal proliferation and is required for peroxisome division and
segregation(20, 21) .
cells from strain COP161U7 and the
rtg1,
rtg2, and
rtg1
rtg2 mutant
derivatives were grown on YPR medium in the presence or absence of 0.2%
oleic acid. Under these conditions, all of the strains will grow, and
the presence of oleic acid in the medium should induce enzymes of the
-oxidation pathway. The levels of acetyl-CoA oxidase, catalase A,
and Pmp27p mRNAs were then determined by Northern blot analysis of
total RNA isolated from these cells using specific hybridization probes
for each (see ``Experimental Procedures''). A summary of the
quantification of these Northern blots for a representative experiment
is presented in Fig. 4. First, as expected, growth of wild-type
cells in the presence of oleic acid results in a large increase in the
abundance of the mRNAs for all three of these peroxisomal proteins.
Second, relative to wild-type cells, the mRNA levels are reduced to
60-70% in the
rtg1 mutant and 20-40% in the
rtg2 mutant. The effects of these mutations are roughly
additive, since in the
rtg1
rtg2 double disruption
strain, oleate induction is further reduced to only 10-15% of the
mRNA levels observed in the wild-type strain. These data are consistent
with the impaired growth of the rtg mutant strains on medium
containing oleic acid (Fig. 3). Taken together, these findings
indicate that the RTG genes, while required specifically for CIT2 expression, are also important for oleic acid induction
of other peroxisomal proteins.
and three
derivatives,
rtg1,
rtg2, and the double disruption,
rtg1
rtg2,
were grown to midlogarithmic phase on YPR medium with or without the
addition of 0.2% oleic acid. Total RNA was isolated from each, and
Northern blot analysis was performed using probes specific for each of
the genes indicated in the figure. RNA levels were quantified by
PhosphorImager analysis, and the relative mRNA abundance for each was
normalized to the level of actin mRNA.
Oleic Acid Induction of Expression of Genes Encoding
Glyoxylate Cycle Enzymes
The same RNA samples from the strains
used in the previous experiment were analyzed by Northern blotting to
determine whether oleic acid also induces expression of the glyoxylate
cycle genes, CIT2, MDH3, MLS1, and ICL1. As shown in Fig. 5, the mRNA abundance of all four of these genes was
greatly increased in wild-type cells when grown in medium containing
oleic acid. These data are consistent with the experiments of McCammon et al.(13) , which indicated from cytochemical and
cell fractionation studies of oleate grown yeast cells, that the
enzymes of the glyoxylate cycle are peroxisomal.
rtg1,
rtg2, and
rtg1
rtg2 mutant cells.
rtg1 cells or the
rtg1
rtg2 strain, despite the fact that in wild-type
cells, CIT2 mRNA abundance is increased
30-fold when
those cells are grown in the presence of oleic acid. The mRNA levels
for MDH3, MLS1, and ICL1 in the oleate-grown
rtg1 mutant was decreased to 20-35% of the
wild-type level.
rtg2 cells grown in
the presence of oleic acid, CIT2 mRNA was not only detectable
but its abundance was about 40% of the wild-type level. Hence, the RTG2 requirement for basal CIT2 expression in cells
grown on rich raffinose medium (6) is evidently bypassed when
cells are grown on medium containing oleic acid. MDH3 expression in
rtg cells was reduced by only 25% of
wild-type, and, for MLS1 and ICL2, the mutation
resulted in an increase (2- and 2.5-fold, respectively) in the
abundance of the mRNAs for these genes, suggesting that RTG2 can also act as a repressor. The ability of oleic acid to increase
expression of MDH3, MLS1, and ICL1 is essentially
blocked in the
rtg1
rtg2 double mutant, indicating
that under this growth condition RTG1 is epistatic to RTG2 for expression of these genes.
cells containing a
null allele of the tricarboxylic acid cycle gene MDH1 also
have an elevated level of CIT2 mRNA comparable to that
observed in
cit1 cells. This
finding suggests that CIT2 expression is not likely to be
dependent specifically on mitochondrial CS1 activity. How CIT2 expression senses the functional status of the tricarboxylic acid
cycle is not clear. In
cells with a null allele
of either CIT1 or MDH1, the increase in CIT2 mRNA abundance relative to wild-type
cells
is not as great as is observed in
petites. Together
with our previous finding that CIT2 expression also increases
(about 2-fold) by simply growing wild-type
cells
on a fermentable carbon source in the presence of a respiratory chain
inhibitor(5) , we conclude that CIT2 retrograde
control is likely to be a complex phenomenon involving the cumulative
effects of different mitochondrial lesions.
-oxidation of fatty acids can be a source of these
2-carbon units, which can shuttle from peroxisomes into mitochondria
through the organelle-specific acylcarnitine transferases (34) . We suggested previously that the retrograde response of CIT2 expression is a means of increasing the flux of carbon,
and particularly citrate, from the glyoxylate cycle to mitochondria,
perhaps in response to conditions where tricarboxylic acid cycle
activity is limiting(5, 10) . The findings that cells
with mutant alleles of either RTG1 or RTG2 cannot
grow on acetate and are auxotrophic for glutamate/aspartate, which are
phenotypes characteristic of cells with blocks in both the
tricarboxylic acid and glyoxylate cycles(11) , is consistent
with the function of the RTG genes in effecting communication
between these pathways.
and in
cells of the three
other glyoxylate cycle genes examined, MLS1, ICL1, and MDH3, shows that at this level of analysis, the
retrograde response of glyoxylate cycle genes is specific for CIT2. Together with our previous finding that increased CIT2 expression compensates for a reduction in tricarboxylic
acid cycle activity(5) , this observation suggests that CS2
activity could be a limiting step in the glyoxylate cycle. We are
cautious, however, in noting that it is not straightforward to pinpoint
a rate-limiting step in the in vivo operation of a metabolic
pathway(35) , particularly if the pathway has branch
points(36) . Thus, while the possibility is attractive that CS2
activity is an important control point in the metabolic interaction
between the glyoxylate and tricarboxylic acid cycles, that notion will
require further study.
rtg1 and
rtg2 strains are unable to use acetate directly as a sole carbon
source, it remained possible that they could utilize acetyl-CoA
generated by
-oxidation of long chain fatty acids in the growth
medium. We found, however, that the mutants were also compromised in
their ability to utilize oleic acid as a carbon source. The latter
defect could be due to an inability of the mutant cells to efficiently
utilize acetyl-CoA generated by
-oxidation of oleic acid or to
some other defect in the pathway of oleic acid metabolism. In an
attempt to clarify this issue, we examined the oleic acid induction of
mRNAs for three peroxisomal proteins: two of them, POX1 and CTA1 (encoding acetyl-CoA oxidase and catalase A,
respectively), are involved in
-oxidation; the third, PMP27, encodes the most abundant protein associated with
peroxisomal membranes in yeast and is required for oleic acid induction
of wild-type peroxisomal profiles(20, 21) . PMP27 expression is thus a convenient marker for peroxisome
proliferation. The results of these experiments show that both RTG1 and RTG2 are required roughly additively for full oleic
acid induction of expression of these peroxisomal protein genes
(abbreviated as P-C-P): in the
rtg1
rtg2 double mutant, oleic acid induction is inhibited by 85-90%.
Moreover, each of the P-C-P genes is affected similarly in the
single rtg mutant strains where the apparent requirement for RTG2 is greater than RTG1. Taken together, these data
point to a similarity of function between the RTG genes and PAS (peroxisome assembly) genes that are required for
peroxisome
biogenesis(37, 38, 39, 40) .
upstream of CIT2 and is essential for its expression(6) . One
possibility, therefore, is that RTG1 is also a specific
component of ORE complexes. However, the RTG1 product does not
appear to bind to the ORE upstream of the FOX3 gene(41) , which encodes the oleic acid-inducible
peroxisomal enzyme 3-oxoacyl-CoA thiolase and whose induction by oleic
acid also requires RTG1.
(
)We cannot
rule out the possibility, however, that the RTG1 product
interacts with other OREs. In any case, the similarity of inhibition of
oleic acid induction of the P-C-P gene in the
rtg1,
rtg2, and
rtg1
rtg2 double mutant strains suggests that the RTG genes may be
required at some relatively early step in the oleic acid induction of
peroxisome proliferation.
cells grown on medium containing oleic acid, no CIT2 mRNA was detected in the
rtg1 or
rtg1
rtg2 mutant strains. Similar results were
obtained with
rtg1 cells grown on
medium containing oleic acid (data not shown), underscoring the
absolute requirement of RTG1 for CIT2 expression(6) . Second, unlike the P-C-P genes,
oleic acid induction of the glyoxylate cycle genes shows a greater
requirement for RTG1 than for RTG2. In fact, the
absence of the RTG2 product results in an
2-fold
elevation in the mRNA abundance for MLS1 and ICL1 in
cells grown on oleic acid medium. These data show that the RTG2 product can act as both a positive and negative regulator of
oleate-induced peroxisomal gene expression. Finally, despite the
complexities of the various responses, it is apparent that the RTG genes play a key role in a threeway communication between
mitochondria, the nucleus, and peroxisomes.
We thank Dr. H. Tabak for communicating results to us
prior to publication. We also thank Drs. J. Goodman and M. Skoneczny
for gifts of plasmids. Finally, we thank our colleagues for a critical
reading of the manuscript and for helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.