©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
RTG Genes in Yeast That Function in Communication between Mitochondria and the Nucleus Are Also Required for Expression of Genes Encoding Peroxisomal Proteins (*)

Anna Chelstowska Ronald A. Butow (§)

From the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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.


INTRODUCTION

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 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.

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) 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.

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 (50%) in CS1 activity, which could account in any simple way for the rtg mutant phenotypes(16) .

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 -oxidation pathway accompanied by a general increase in the number of peroxisomes within the cell(17, 18, 19) .

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 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.


EXPERIMENTAL PROCEDURES

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).

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.

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.


RESULTS

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.


Figure 1: Blocks in the tricarboxylic acid cycle result in elevated CIT2 expression. Wild-type yeast strains PSY142 ( 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.



The finding that the abundance of CIT2 mRNA is greater in petites (which lack an electron transport chain and oxidative phosphorylation apparatus) than in 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.


Figure 2: 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 (rtg1rtg2) 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 rtg1rtg2 double mutant is slightly more impaired than the rtg1 mutant strain.


Figure 3: 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 H0, and resuspended in 1/4 volume of H0. 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) .

Respiratory-competent wild-type cells from strain COP161U7 and the rtg1, rtg2, and rtg1rtg2 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 rtg1rtg2 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.


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 and three derivatives, rtg1, rtg2, and the double disruption, rtg1rtg2, 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.


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 rtg1, rtg2, and rtg1rtg2 mutant cells.



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 rtg1 cells or the rtg1rtg2 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.

Surprisingly, in 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 rtg1rtg2 double mutant, indicating that under this growth condition RTG1 is epistatic to RTG2 for expression of these genes.


DISCUSSION

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 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.

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 -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.

A comparison of mRNA abundance in 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.

Although 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 rtg1rtg2 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) .

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 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 rtg1rtg2 double mutant strains suggests that the RTG genes may be required at some relatively early step in the oleic acid induction of peroxisome proliferation.

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 cells grown on medium containing oleic acid, no CIT2 mRNA was detected in the rtg1 or rtg1rtg2 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.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant 22525 and Grant I-0642 from The Robert A. Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 214-648-2053; Fax: 214-648-8856; butow{at}swmed.edu

Y. Jia, J. Etheredge, B. Rothermel, and R. A. Butow, unpublished results.

The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s).

H. Tabak, personal communication.


ACKNOWLEDGEMENTS

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.


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