Regulation of Nuclear Genes Encoding Mitochondrial Proteins in ...

JOURNAL OF BACTERIOLOGY, Dec. 1995, p. 6836–6843 0021-9193/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 177, No. 23

Regulation of Nuclear Genes Encoding Mitochondrial Proteins in Saccharomyces cerevisiae TIMOTHY A. BROWN,1 CARLOS EVANGELISTA,2

AND

BERNARD L. TRUMPOWER1*

Dartmouth Medical School, Department of Biochemistry, Hanover, New Hampshire 03755,1 and State University of New York at Albany, Department of Biological Sciences, Albany, New York 122222 Received 15 May 1995/Accepted 25 September 1995

Selection for mutants which release glucose repression of the CYB2 gene was used to identify genes which regulate repression of mitochondrial biogenesis. We have identified two of these as the previously described GRR1/CAT80 and ROX3 genes. Mutations in these genes not only release glucose repression of CYB2 but also generally release respiration of the mutants from glucose repression. In addition, both mutants are partially defective in CYB2 expression when grown on nonfermentable carbon sources, indicating a positive regulatory role as well. ROX3 was cloned by complementation of a glucose-inducible flocculating phenotype of an amber mutant and has been mapped as a new leftmost marker on chromosome 2. The ROX3 mutant has only a modest defect in glucose repression of GAL1 but is substantially compromised in galactose induction of GAL1 expression. This mutant also has increased SUC2 expression on nonrepressing carbon sources. We have also characterized the regulation of CYB2 in strains carrying null mutations in two other glucose repression genes, HXK2 and SSN6, and show that HXK2 is a negative regulator of CYB2, whereas SSN6 appears to be a positive effector of CYB2 expression. To better understand carbon regulation of mitochondrial biogenesis, we have characterized the glucose regulation of CYB2, which encodes cytochrome b2, a soluble L-(1)-lactate– cytochrome c oxidoreductase located in the mitochondrial intermembrane space. CYB2 is an attractive model for identifying and characterizing mutants in glucose regulation of mitochondrial biogenesis, since it is highly expressed under nonrepressing conditions and, in contrast to most nucleus-encoded genes for mitochondrial proteins, glucose repression of CYB2 is stringent (23). Expression of CYB2 is heme dependent and is mediated through two positively acting cis elements and one negatively acting cis element (30). The two activating sequences lie 200 to 446 bp upstream of the transcriptional initiation site and synergistically activate CYB2. A negatively acting element lies downstream of these and is capable of repressing the activation from UAS1-B2. This upstream regulatory sequence region contains a 9-bp palindrome that is similar to the CAR1 repressor binding site, where the BUF1/2/3 repressor complex binds (31–33). We have isolated mutants that escape glucose repression of CYB2 and identified two of them as new alleles of GRR1/ CAT80 and ROX3. We have characterized the regulation of CYB2 by these two genes and also by HXK2 and SSN6, two genes previously implicated in regulation of glucose-repressed metabolic pathways.

When Saccharomyces cerevisiae strains are grown on glucose, they down regulate the biogenesis of mitochondria such that the volume occupied by mitochondria is decreased from 12 to 14% of the total cellular volume under derepressing conditions to 3 to 4% under repressing conditions. The glucose-repressed mitochondria also have poorly differentiated inner membrane cristae (51). Although this effect of glucose on yeast mitochondria was discovered over 40 years ago (14), surprisingly little is known about the mechanism of glucose repression of mitochondrial biogenesis. Much of what we know about glucose repression is from studies of the SUC and GAL genes, which are involved in the regulation of sucrose and galactose metabolism, respectively (27). Some genes which regulate repression of sucrose and galactose catabolic enzymes also regulate at least some mitochondrial enzymes. For example, mutations in HXK2, which encodes one of the hexokinases, release repression of mitochondrial cytochrome c reductase and cytochrome c oxidase activities (38). Mutations in CYC8 (SSN6) and CYC9 (TUP1) both cause overexpression of iso-2-cytochrome c (46), and mutations in GRR1 and CAT4 allow iso-1-cytochrome c to escape glucose repression (17, 48). However, the extent to which these genes may be general regulators of mitochondrial biogenesis remains unknown. Boker-Schmitt and coworkers isolated two constitutively highly respiring mutants, CCR-91 and CCR-96, in which respiration, mitochondrial cytochromes, and mitochondrial DNA synthesis were released from glucose repression (2). Significantly, the activities of maltase and malate synthase, two nonmitochondrial glucose-regulated enzymes, were not changed in the CCR-91 mutant, indicating that glucose repression of mitochondrial biogenesis is at least partially independent of repression of enzymes involved in disaccharide catabolism and the glyoxylate cycle.

MATERIALS AND METHODS Strains and media. The yeast strains used in this study are described in Table 1. DFYPH10 is the progeny of a mating between DFY567 and YPH500. TBYDSSN6-1C was segregated from a cross between MCY1802 and YPH500. Yeast strains were transformed by a modified lithium acetate procedure (12). YP medium is 1% yeast extract and 2% peptone, supplemented with carbon sources. SD medium is minimal medium containing 0.7% yeast nitrogen base with ammonium sulfate, 2% dextrose, and the amino acids needed to satisfy auxotrophies. Dropout medium is the same formulation, except that all nutrients were included except for an amino acid or pyrimidine, depending on the prototrophic selection (44). Dextrose, galactose, and raffinose were sterilized by filtration. Glycerol and lactate were autoclaved to eliminate lactic anhydride. Plasmids. Large amounts of plasmid DNA were isolated by alkaline lysis and

* Corresponding author. Phone: (603) 650-1621. Fax: (603) 6501389. 6836

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TABLE 1. Yeast strains used in this study Strain

Genotype

Source or reference

YPH500 YPH499 BH1-10A BH3 W3031-A YM2953 YM2169 MCY1802 TByDSSN6-1C DFY567 DFYPH10

MATa ura3-52 his3-D200 leu2-D1 lys2-801 ade2-101 trp1-D63 MATa ura3-52 his3-D200 leu2-D1 lys2-801 ade2-101 trp1-D63 MATa ura3-52 his3-D200 leu2-D1 lys2-801 ade2-101 trp1-D63 rmr1-1 MATa ura3-52 his3-D200 leu2-D1 lys2-801 ade2-101 trp1-D63 rmr3-1 MATa ura3-1 his3-11, -15 leu2-3, -112 ade2-1 trp1-1 can1-100 MATa ura3-52 his3-D200 leu2-3, -112 lys2-801 ade2-101 grr1-1829::LEU2 MATa ura3-52 his3-D200 lys2-801 ade2-101 met2 gal80-538 LEU2::GAL1-lacZ MATa ura3-52 his4-539 lys2-801 ssn6D9 SUC2 MATa ura3-52 his3-D200 his4-539 leu2-D1 lys2-801 ade2-101 trp1-D63 ssn6-D9 MATa leu2-1 lys1-1 HXK2::LEU2 MATa his3-D200 leu2-D1 ade2-101 HXK2::LEU2

R. Sikorski (50) R. Sikorski (50) This work This work R. Rothstein M. Johnston (17) M. Johnston (58) M. Carlson This work D. Fraenkel (56) This work

cesium chloride gradient centrifugation (36). Smaller amounts of plasmid DNA were isolated by a phenol-free method (61). Plasmids were propagated in Escherichia coli DH5a (24). The HIS3 reading frame was amplified by PCR with the GeneAmp system (Perkin-Elmer) with genomic DNA from strain W303-1A as the template. Oligonucleotide primers were synthesized with a Biosearch Cyclone DNA synthesizer. The 59 and 39 primers had the respective sequences 59-CCGGATCCATG ACAGAGCAGAAAGCCC-39 and 59-CCCTGCAGCCTGATGCGGTATTTT CT-39. The amplified 685-bp fragment was digested with BamHI and PstI and subcloned into the CEN6 LEU2-containing vector pRS315 (50), resulting in the plasmid pTBH. The 59 regulatory region of the CYB2 gene is within 446 bases upstream of the initial codon (30). This region was amplified by PCR with the 59 and 39 oligonucleotides 59-CCCGAATTCACGGATACATCGGAAGGATC-39 and 59-CCC GGATCCATTGACTACTTTTGTTTGCT-39, respectively. The resulting amplicons were digested with EcoRI and BamHI, whose sites were engineered into the ends of the primer sequences. This fragment was ligated into pRS313 (50) and then subcloned into EcoRI-BamHI-treated YEp358, YIp358 (41), or pTBH. The resulting plasmids are pTBCYB2.358, YIpCYB2.7, and pTBCYB2/HC. YCp50 (43), YEp13 (5), and pBM1679 (17) have been described previously. YIpCYB2.7 was linearized at the NcoI site within the URA3 gene for directed integration into the ura3-52 loci of YPH500, BH1-10A, BH3-1, DFYPH10, and TBYDSSN6-1C. Transformants were selected for uracil prototrophy, and proper integration of the plasmid was confirmed by b-galactosidase assays and Southern analysis. Mutant selection. YPH500 and YPH499 were transformed with both pTBCY B2/HC and pTBCYB2.358. YPH500 was pregrown to mid-log phase in SD medium containing 2% raffinose–2% galactose and spread onto 10% dextrose plates containing 10 mM 3-aminotriazole at a density of 10,000 cells per plate. The plated cells were subjected to UV mutagenesis at a level sufficient to obtain 50% lethality as determined from corresponding YPD-grown cells. Putative mutants, identified by their ability to escape glucose repression of HIS3 expression from the plasmid pTBCYB2/HC and thus to grow without histidine, were isolated and assayed for CYB2-lacZ expression from the plasmid pTBCYB2.358. Putative mutants that also released repression of this second reporter were backcrossed to YPH499 containing the plasmids pTBCYB2/HC and pTBCY B2.358, and the phenotypes of the diploids were examined to establish the dominance or recessiveness of the mutations. Meiotic progenies were dissected, and segregation of the repression release was observed. Complementation analysis yielded at least four groups among 12 original isolates. b-Galactosidase and invertase assays. Cells were grown in 10 ml of dropout medium on a rotating wheel at 308C until late log phase (1 3 108 to 2 3 108 cells per ml) and then were disrupted in 60 mM Na2HPO4 z 7H2O–40 mM NaH2PO4 z H2O–10 mM KCl–1 mM MgSO4 z 7H2O–50 mM b-mercaptoethanol (pH 7) with 0.5-mm-diameter glass beads (Biospec Products) with 1 mM phenylmethylsulfonyl fluoride (22). Hydrolysis of o-nitrophenyl-b-D-galactopyranoside was measured at 420 nm, and b-galactosidase units were calculated as (optical density at 420 nm 3 1,000)/(ml of enzyme 3 min of incubation 3 mg/ml of protein). Protein was measured with the Bio-Rad dye-binding reagent (3). The values reported are means of four independent transformants unless otherwise stated. Standard error was typically 10 to 15% of the mean value. Invertase assays were modified from the glucose oxidase and peroxidase odianisidine procedure (21). Cells from 10-ml cultures were harvested in log phase, washed with an equal volume of water, resuspended in 0.5 ml of 50 mM K2HPO4–2 mM EDTA–2 mM b-mercaptoethanol (pH 7), and lysed at 48C by vortexing with glass beads. The values reported are averages of at least two enzyme dilutions which yielded absorbancies within the linear range of a standard curve generated from duplicate samples. Invertase activity is expressed in U/100 mg of protein. One unit is the amount of enzyme that yields 1 mM glucose per min at 308C at pH 4.9. Respiration rates. Respiration rates were measured polarographically with a Clark oxygen electrode and a Gilson K-ICC-H oxymeter. Cells were grown in YP

medium, washed with 20 mM morpholineethanesulfonic acid (MES) adjusted to pH 6 with ethanolamine, and suspended to 50% (wt/vol) in MES buffer. The respiratory capacity for each strain was determined with 10 mg of cells in aerated MES buffer at 258C. Oxidative phosphorylation, which otherwise might limit respiration, was uncoupled with 6 mM carbonyl cyanide m-chlorophenylhydrazone (Sigma Chemical Co.). Although the cells contained endogenous substrate, 100 mM ethanol was added to ensure that excess oxidizable substrate was available throughout the measurement. Cloning of RMR1. A genomic library from the strain DC-5 in the yeast vector YEp13, a gift from Mike Douglas, was used to transform the rmr1 mutant strain BH1-10A. Approximately 26,500 independent transformants were pooled and resuspended in water. A leucine dropout 10% dextrose culture was inoculated with an aliquot of the cell suspension. After 3 days, floating cells were separated from the aggregated cells at the bottom of the vessel and inoculated into fresh selective medium. This selection was repeated after 4 days. Nonflocculating cells in the suspended phase of the latter culture were spread onto leucine dropout plates. Total DNA was isolated from each of 48 nonflocculating isolates (26) and transformed into E. coli to amplify plasmid DNA, which was mapped with restriction endonucleases. Cloning of the rox3-404 (RMR1) mutant allele. The rox3-404 mutant allele was recovered by gap repair with the plasmid YEpROX3-X, a multicopy plasmid containing the TRP1 marker and a ROX3 HindIII fragment in which a XhoI site had been placed at the last codon (45). The 700-bp BglII-XhoI fragment, comprising the entire ROX3 coding region, was deleted. The gapped plasmid was transformed into BH1-10A, with selection for the TRP1 marker. DNA was prepared from the Trp1 transformants (44) and used to transform E. coli DH5a to ampicillin resistance, recovering those plasmids in which the gap had been repaired by gene conversion from the mutant ROX3 locus. Physical mapping of ROX3/RMR1. Agarose plugs containing chromosomes from S. cerevisiae YPH80 were purchased from New England Biolabs and separated on a 1% agarose gel with a CHEF II apparatus (Bio-Rad). Electrophoresis was performed in 0.53 Tris-borate-EDTA with 200-V 70-min pulses for 15 h followed by 120-min pulses for 10 h. Chromosomes were transferred to BioTrans nylon membrane after two 15-min washes in 0.25 M HCl, followed by two more 15-min washes in 0.5 M NaOH–1.5 M NaCl before neutralization. Blots were hybridized in Quikhybe (Stratagene) and probed with the 2.7-kbp HindIIIcomplementing fragment of RMR1. The same probe was hybridized to a set of blots containing grids of ordered lambda clones containing 98% of the yeast genome (a gift from Linda Riles, Washington University, St. Louis, Mo.) under the hybridization conditions of Riles and Olson (42b). DNA sequencing. DNA sequencing of PCR-amplified DNA fragments and RMR1 fragments was done by the double-strand template method (57), with substitution of the plasmid preparation method of Zhou et al. (61), with the Sequenase kit from U.S. Biochemicals. Both dideoxynucleoside triphosphates and 7-deaza-dGTP nucleotide analogs were used. Nucleic acid and protein databases were searched by the FASTA-FASTP algorithms with the Genetics Computer Group sequence analysis software on a Digital VAX117-80 computer at the Dartmouth College Computing Center.

RESULTS Mutant selection. To identify mutants in which expression of CYB2 is not repressed by glucose, we fused the HIS3 gene to the upstream regulatory region of the CYB2 gene and used this CYB2:HIS3 gene fusion on a CEN-containing yeast plasmid, pTBCYB2/HC, to cover the histidine auxotrophy of a his3D200 yeast strain. Under glucose repressing conditions, expression of HIS3 from the CYB2 promoter is repressed and the yeast should not grow without histidine. Mutants that escape from

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this CYB2-mediated repression of HIS3 should grow without histidine. In preliminary experiments, we found that repression of the CYB2 promoter was incomplete, and there was enough expression of the HIS3 gene under repressing conditions to allow growth without histidine. We thus supplemented the histidine dropout medium with 10 mM 3-amino-1,2,4-triazole, a competitive inhibitor of the HIS3 gene product, imidazoleglycerophosphate dehydratase, in order to titrate the low level of HIS3 expression and inhibit growth. A similar strategy has been used to find negative regulators of GAL1 (18). After mutagenesis of approximately 7 3 105 cells with UV light, 32 putative mutants capable of growth on dextrose were isolated and assayed for b-galactosidase expression from the pTBCYB2.358 plasmid to confirm the release of repression. Twelve of the 32 isolates had elevated levels of b-galactosidase expression. Although the remaining 20 were not further characterized, we assume these included up-regulating mutations in the HIS3 reporter plasmid and mutants defective in aminotriazole uptake. Expression of the b-galactosidase reporter in the 12 mutants was elevated from 10- to 80-fold under repressing conditions, and none of the mutants was completely unresponsive to glucose repression. All of the mutants were recessive. We named these mutated genes RMR, for release of mitochondrial repression. We were interested in identifying additional phenotypes in these mutants that would facilitate genetic characterization and subsequent cloning of the responsible genes by complementation. Two of the rmr mutants had additional phenotypes that cosegregated with the release of repression. The mutation in RMR1 segregated with a severely flocculating phenotype, and that in RMR3 segregated with an elongated cellular morphology. RMR3 is allelic with GRR1/CAT80. The morphologic and invertase expression data in Fig. 1 indicate that the rmr3 mutation in strain BH3 is an allele of the previously described GRR1/CAT80 (1, 13, 17). The elongated sausage shape was an early indication of the allelism of RMR3 with GRR1. This led us to assay for abnormal invertase regulation, which has been described for mutations in GRR1 (17, 53). The right side of Fig. 1 depicts the invertase expression. Normal morphology and invertase expression of YPH500 contrast with the elongated morphology and the reversed invertase regulation in both mutant strains BH3 (rmr3-1) and YM2953 (grr1-1829). Both of the aberrant phenotypes in BH3 are complemented back to normal by the GRR1-containing plasmid pBM1679 (a gift from Mark Johnston). In addition, when BH3 and YM2953 were mated, the resulting diploid maintained the recessive mutant phenotypes. These results indicate that RMR3 and GRR1 are allelic. Interestingly, both the cell morphology and reversed invertase regulation phenotypes in BH3 are more severe than those caused by grr1-1829. Flocculation is induced by glucose in BH1-10A. Insensitivity to glucose repression and severe flocculation cosegregated with the nonreverting mutation in rmr1. In 8% dextrose, BH110A formed a single oval mass of cells in the bottom of the culture tube. Single suspended cells apart from this mass were extremely rare. Growth in 2% dextrose permitted some cells to be released from this mass in late growth phases. However, no flocculation occurred during growth in galactose, raffinose, or ethanol-glycerol. Addition of 15 mM EDTA to chelate divalent cations eliminated the flocculation, which is, therefore, probably calcium dependent. BH1-10A [rho0] cells isolated after treatment with ethidium bromide maintained the flocculating phenotype, indicating that the mitochondrial genome is not necessary for this type of flocculation as in other nuclear backgrounds (16, 25). BH1-10A grown anaerobically in YP 8%

J. BACTERIOL.

FIG. 1. The mutation in RMR3 is not complemented by a grr1-1829 mutant but is restored by a plasmid carrying GRR1. The left panels show differential interference micrographs of the morphologies of YPH500 and the mutant strains. The right panels show invertase activities of the same strains when grown under repressing (8% dextrose) or derepressing conditions (2% raffinose). YPH500 is the normal control, BH3 is the rmr3-1 mutant, BH3 [pBM1679] maintains a plasmid with GRR1, YM2953 has the grr1-1829 mutant, and the bottom panel shows the rmr3-1/grr1-1829 diploid.

dextrose also flocculated, indicating that oxygen or heme regulatory effects do not influence this phenotype. RMR1 is allelic with ROX3, a regulator of CYC7. Nonflocculating library transformants of the mutant BH1-10A were isolated after enrichment in liquid media. Plasmids from 48 complemented isolates were restriction mapped. Forty-six of these contained a plasmid with the same 5.7-kb insert, and 1 contained an overlapping larger insert with a size of 9 kb. These were called pTBRMR1.2 and pTBRMR1.4, respectively (Fig. 2). Both of these plasmids were transformed back into BH110A to verify their ability to rescue the mutant phenotypes. Further confirmation was obtained by allowing plasmid loss to occur, which in every case resulted in a reappearance of the original mutant phenotype. Restriction fragments of the insert from pTBRMR1.2 were subcloned into the vector YCp50 and transformed back into the mutant BH1-10A. A 1.3-kb BamHI-HindIII fragment was the smallest fragment capable of complementing the flocculating phenotype of BH1-10A (Fig. 2). The sequencing of this and an adjacent 1.4-kb HindIII-BamHI fragment revealed the identity of this gene as ROX3 through a homology search (45). Interestingly, the smallest complementing fragment lacks the first 17 codons of the reading frame. This result confirms that of Rosenblum-Vos and coworkers (45), who found that this 59

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TABLE 2. Effects of the rox3/rmr1 mutation on GAL1 and invertase expressiona GAL1-lacZ expression (U/100 mg of protein) with:

Invertase activity (U/100 mg of protein) with:

Genotype

Normal rmr1-1 (rox3-404) gal80-538 rmr1-1 (rox3-404) gal80-538

8% dextrose

2% galactose

3% glycerol

10% dextrose

2% galactose 1 2% raffinose

6 88 124 176

10,727 3,842 12,602 8,170

9 17 8,451 10,131

3 7

717 1,748

a The GAL1-lacZ expression values are the means of eight meiotic segregants from a cross between YM2169 and BH1-10A. Invertase values are the means of six segregants from a cross between MCY1802 and BH1-10A.

FIG. 2. Restriction mapping and localization of RMR1 by determination of the smallest complementing fragment. Two plasmids with overlapping inserts were isolated by their ability to complement the flocculating phenotype of BH110A and were mapped by digestion with HindIII (H), XbaI (X) BamHI (B), SphI (S), and ClaI (C). Fragments of pTBRMR1.2, identified by the bars below the restriction map, were subcloned into YCp50 and transformed into BH1-10A. Subclones which complemented the flocculating and repressing phenotypes are scored as positive. AmpR, ampicillin resistance; 2 m, 2 mm plasmid.

truncation bounded by the BamHI site was also capable of complementing normal expression of the CYC7 gene. Sequencing of the ROX3 (RMR1) mutant allele from BH110A genomic DNA revealed a G-to-A transition at position 404, resulting in an amber termination at codon 135 in a reading frame of 220 codons. This mutant allele will subsequently be referred to as rox3-404. ROX3/RMR1 is located near the telomere on the left arm of chromosome 2. An autoradiograph of a yeast chromosome blot probed with a 2.7-kb HindIII fragment that encompasses ROX3/RMR1 indicated the fragment hybridizes to chromosome 2 (results not shown), and hybridization of the same fragment with an ordered set of lambda clones containing most of the S. cerevisiae genome identified ROX3/RMR1 as a new leftmost marker on chromosome 2 (coordinates of the lambda clones were interpreted by Linda Riles). DNA sequencing has localized ROX3 approximately 12 kb distal to TEL1 (42a). Effects of ROX3/RMR1 mutation on GAL1 and SUC2 expression. Since ROX3/RMR1 has not been described as a regulator of glucose repression, we were interested if our mutation affected the carbon regulation of other genes in addition to CYB2. Two model genes frequently used to study carbon source effects are the GAL1 and SUC2 genes (27). For the GAL1 analysis, we isolated meiotic segregants containing the integrated reporter construct GAL1-lacZ (59). The effect of the rox3/rmr1 mutation on GAL1-lacZ expression was assayed

in a normal background and in a gal80-538 context. The GAL80-encoded protein is a negative regulator of the GAL4 transcriptional activating protein (28, 35). In 8% dextrose, there is a small release of repression in the rox3-404 mutants with and without the gal80-538 mutation (Table 2). Interestingly, gal80 and rox3/rmr1 effects are additive, indicating independent mechanisms of repression. In 2% galactose, the rox3-404 mutation has a striking negative effect on GAL1 expression that is only partially relieved by the gal80538 mutation. In glycerol, a nonfermentable carbon source, there is little effect. These results indicate that ROX3/RMR1 has both negative and positive regulatory functions. It is a weak negative regulator of GAL1 under glucose repressing conditions and acts independently of the GAL80-encoded protein. Under galactose-inducing conditions, ROX3/RMR1 is needed for full expression of GAL1 and therefore may have an activating function on GAL1 which is responsive to galactose, but not glycerol, and which is also independent of the GAL80-encoded protein. In contrast, glucose repression of invertase, encoded by the SUC2 gene, is normal in the rox3/rmr1 mutant (Table 2). Derepressed expression of invertase in cells grown on raffinose plus galactose, however, is about 2.5 times the normal level of expression. This result indicates that either ROX3/RMR1 is normally involved in down regulating SUC2 in raffinose plus galactose or this mutation enhances access to the transcriptional machinery of the SUC2 gene. Effects of rox3/rmr1 and grr1/rmr3 mutations on cell respiration. The BH1-10A and BH3 mutants were isolated by selection for insensitivity of CYB2 to glucose repression. To test whether this release of repression has a pleiotropic effect on the biogenesis of mitochondria, we measured the respiratory activities of the mutants after growth under repressing or nonrepressing conditions (Fig. 3) and examined the growth characteristics of the mutants in nonrepressing carbon sources raffinose and glycerol (Fig. 4). When grown under repressing conditions, BH1-10A (rox3/ rmr1) has about twice the respiratory activity as the parent, YPH500 (Fig. 3). There is an even more dramatic release of repression of mitochondrial function in BH3 (grr1/rmr3), in which respiratory activity after growth on dextrose is four- to fivefold greater than that of YPH500. Clearly, in both mutants, the rate-limiting steps in respiration are at least partially released from glucose repression. The rox3-404 mutation also results in a slightly higher respi-

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FIG. 3. Effects of rox3/rmr1 and grr1/rmr3 mutations on respiration of yeast cells grown on repressing or nonrepressing carbon sources. Solid bars, YPH500; hatched bars, BH1-10A; shaded bars, BH3-1.

ratory rate when the yeast cells are grown in ethanol-glycerol (Fig. 3), supporting the idea that rox3-404 is a general negative regulator that responds to signals other than those in glucose repression. Spectral analysis of this mutant indicates that cytochromes cc1, b562, and aa3 are increased 30 to 50% relative to those in YPH500 after growth in ethanol-glycerol (results not shown), which correlates with the differences in respiratory rates and growth in glycerol. In contrast, BH3 (grr1/rmr3) respires more slowly than YPH500 or BH1-10A when grown on ethanol-glycerol (Fig. 3) and likewise grows more slowly on the nonrepressing carbon sources (Fig. 4). This defective growth and respiration in nonfermentable carbon sources may be specific for the BH3 (grr1/

FIG. 4. Effects of rox3/rmr1 and grr1/rmr3 mutations on growth of yeast cells on nonrepressing carbon sources. Cultures were pregrown in YP–2% dextrose, inoculated at 5 3 105 cells per ml into YP medium containing 2% raffinose or 3% glycerol, and grown at 308C.

FIG. 5. Effects of rox3/rmr1, grr1/rmr3, ssn6, and hxk2 mutations on expression of CYB2. A CYB2:lacZ fusion, integrated into the URA3 locus, was used to measure CYB2 expression in the various mutant backgrounds. YPH500, the wild-type strain, BH1-10A (rox3-404), BH3-1 (grr1/rmr3), TBDSSN6-1C (ssn6D9), and DFYPH10 (HXK2::LEU2) were pregrown in 2% dextrose–uracil dropout medium and inoculated into YP medium containing the indicated carbon sources. Cells were harvested in late log phase and assayed for b-galactosidase.

rmr3) mutation, however, since a disruption of the GRR1 locus has no severe growth defect on glycerol (17). It is possible that the abbreviated form of Grr1p confers mitochondrion specific defects. CYB2-lacZ expression in rox3/rmr1, grr1/rmr3, ssn6, and hxk2 mutants. To further identify and characterize genes that play a role in the carbon regulation of CYB2, we integrated a CYB2-lacZ fusion at the ura3-52 locus of several strains carrying mutations in rox3/rmr1, grr1/rmr3, ssn6, and hxk2. The effects of carbon source on expression of CYB2 in several mutant backgrounds are shown in Fig. 5. The rox3-404 mutation releases glucose repression of CYB2 about twofold, relative to YPH500. This release of repression is much lower than that from the 2mm plasmid reporter in pTBCYB2.358 (data not shown). More striking is the effect of the BH1-10A/rox3-404 mutation on the derepression or activation of the CYB2 promoter, in which the level of expression is only about 30% of that in YPH500 when grown on ethanolglycerol or lactate-glycerol (Fig. 5). This again implicates ROX3 as both a positive and negative effector of gene expression. The mutant grr1/rmr3 allele in BH3 releases glucose repression of CYB2 about eightfold compared with YPH500 (Fig. 5). As in BH1-10A, this strain is also defective in derepression or activation of the CYB2 promoter. Maximal expression in this mutant was only about half of that seen in the parental strain, YPH500. Our mutant search failed to identify any isolates containing either the tup1 or ssn6 alleles. These two genes encode proteins that belong to a large complex that has a role in transcriptional repression (29, 59). Although neither of these proteins has DNA binding specificity, their pleiotropic repressing function might gain specificity by recruiting other promoter binding proteins such as those encoded by the MIG1 and MATa2 genes (29). The TUP1/SSN6 complex is involved in glucose repression of the GAL and SUC genes. However, the participation of this complex in glucose repression of mitochondrial components is not as clear. The effect of the ssn6D9 mutation on carbon source control of CYB2 expression is also shown in Fig. 5. Glucose-repressed

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expression in this mutant is the same as that in YPH500, suggesting that SSN6 is not involved in repression of CYB2. However, this mutant is severely impaired in its ability to derepress or induce expression from the CYB2 promoter. Expression in ethanol-glycerol is only 10% and that in lactateglycerol is only 23% of that in YPH500. Finally, the HXK2 gene, encoding one of the isozymes of hexokinase, has repeatedly been shown to be a general regulator of glucose repression. However, not all glucose-repressed genes are affected by HXK2 null mutants, including PET494 and PRB1 (37, 40). Our results (Fig. 5) indicate that CYB2 is not a member of this HXK2-independent set of glucose-regulated genes. The release of glucose repression in this mutant is about five times that of the wild-type strain. Derepressed expression in ethanol-glycerol is the same as that in YPH500, although in lactate-glycerol medium, the level of induction of CYB2 is lower than normal. DISCUSSION The CYB2 gene is a useful paradigm for glucose regulation of mitochondrial biogenesis. We have taken advantage of the unusually tight regulation of the glucose repression of CYB2 to identify a set of mutants that are released from mitochondrial repression (RMR genes). Here, we have characterized two of these mutants and have identified their mutations within the previously described genes GRR1 and ROX3. In addition, we have characterized the regulation of CYB2 in SSN6 and HXK2 mutants. The GRR1 locus, also named CAT80 (13) and COT2 (10), is a global regulator of glucose repression that, when mutated, releases the repression of invertase, maltase, galactokinase, and cytochrome c oxidase activities (1). The predicted protein structure derived from GRR1 yields few clues toward a function other than the presence of 12 tandem repeats that are implicated in protein-protein interactions (17). Among the many phenotypes of grr1 mutants are an elongated cell morphology and a reversed carbon regulation of invertase (SUC2) expression in which the gene is glucose induced and raffinose repressed (17, 53). We have described here a similar effect of grr1/rmr3 on the respiratory capacity of the mutant BH3, which is greater when the cells are grown in glucose than when they are grown in ethanol and glycerol (Fig. 3). This is a reversal of the normal response. However, we do not see reversed regulation of the CYB2 gene. CYB2 expression on glucose is about nine times higher than normal, and expression on nonfermentable carbons is half that of normal. The GRR1 locus is needed for repression by glucose and for maximal derepression of CYB2 expression. GRR1 is thought to encode a cytoplasmic protein that is involved in sensing or signaling nutrient availability and subsequently affecting gene expression (17, 42, 53, 54). Mutations in GRR1 cause a variety of nutrient-related defects, including the transport systems for hexose, divalent cations, aromatic amino acids, and possibly leucine. Our CYB2 regulation data extend this model to include a role for GRR1 in mitochondrial biogenesis which is not limited to a defect in glucose transport. We have shown that a grr1/rmr1 mutation causes slow growth in both raffinose and glycerol. More importantly, the mutant is defective in respiration when grown on nonfermentable carbon sources. The lowered level of expression of the CYB2 gene suggests that this general defect has a transcriptional basis. If the GRR1 locus encodes a sensor or signaling protein, we propose that it is involved in sensing carbon sources other than glucose.

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Grr1p appears to be a multifunctional protein. A suppressor of the repression defects of grr1 was isolated in the gene RGT1 (15, 54). This suppressor covers the glucose repression and divalent cation transport defects, but it does not suppress the abnormal morphology, which separates multiple independent defects associated with the GRR1 locus. The abnormal morphology and slow growth also indicate a defect in cell cycle regulation. In addition, growth defects in a GRR1 null mutant can be suppressed by multiple copies of the 59 noncoding region of the HXT4 gene, which encodes a GRR1-affected putative glucose transporter. However, the abnormal SUC2 regulation is not suppressed (42). This is further evidence that the role of GRR1 in carbon regulation of gene expression is not limited to glucose transport defects. The ROX3 gene was identified and cloned through mutations that cause increased expression of a CYC7/galK fusion vector (45). The CYC7 gene encodes iso-2-cytochrome c, a minor isozyme of cytochrome c. ROX3 is an essential gene, since the null mutation is lethal under any growth conditions. Mutations in ROX3 have effects on the level of aerobic CYC7 expression, in which a slight increase in the level of steady-state message is seen under both repressing and derepressing conditions. The altered carbon regulation of CYB2 in the rox3-404 mutant is analogous to that of CYC7. CYB2 mRNA is undetectable under anaerobic conditions (23), and as far as we know, it shares only the carbon regulation of ROX3 with that of the CYC7 gene. CYB2 and CYC7 might be expected to have parallel regulation, since their gene products are functionally related. Cytochrome b2 oxidizes lactate to pyruvate, transferring electrons directly to cytochrome c. It is unclear whether cytochrome b2 preferentially transfers electrons to the CYC7- or CYC1-encoded isozymes of cytochrome c (6). However, the parallel regulation of CYB2 and CYC7 through the ROX3 protein is not shared with CYC1, since ROX3 mutations have little effect on the levels of CYC1 mRNA (45). Under anaerobic and glucose repressing conditions, the level of CYC7 expression is disproportionately greater than the level of CYC1 expression (55). Therefore, the ROX3-expressed protein may have a role in the differential regulation of the two isozymes of cytochrome c. The coordinately carbon-regulated CYC7 and CYB2 genes share at least two cis elements within their promoter regions. Both contain a HAP1 binding region that is responsible for part of the transcriptional activation of each of the genes (20, 55). In addition, CYC7, CYB2, and GAL1/10 share a consensus 9-bp palindromic sequence shown to be a BUF1/2/3 repressor binding site present within a variety of genes (33). The upstream repressing sequence of the CYB2 gene which contains the BUF1/2/3 site is also essential for maximum expression of CYB2 (30), which correlates well with our ROX3 mutant data. The heterotrimeric BUF1/2/3 complex is essential and is also implicated in transcriptional activation and replication (4, 31, 32). Interestingly, the BUF1/2/3 binding site is located at the GAL UAS-g, where both positive and negative effects could be applied. The release of the rate-limiting step(s) in respiration in the rox3-404 cells is further evidence of the participation of this gene in mitochondrial biogenesis. The cytochrome content in this mutant correlated with the increase in the respiratory capacity and is also consistent with the increased growth ability in nonfermentable carbon sources. These results indicate that the rox3-404 cells are releasing repression through a general regulatory mechanism. The severe flocculation caused by the ROX3/RMR1 mutation places this gene in a category with other flocculation loci (39). Of these flocculating mutants, rmr1, rgr1 (47), urr1 (18),

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tup1 (46, 58), sin4/tsf3 (9), ssn6, and six other ssn genes (7) are all implicated in glucose repression. Interestingly, ROX3/ RMR1 is unique among these genes in that the rox3-404 mutant confers glucose-induced flocculation which is relieved by growth in other carbon sources. The flocculation is not influenced by mitochondrial DNA (16, 25) or inappropriate respiratory activity. In addition to the two RMR genes characterized here, we have described the CYB2 regulation in HXK2 and SSN6 null mutants. HXK2, encoding hexokinase II, has been identified several times as a key regulator of glucose repression (19, 38). In the HXK2 mutant, expression of CYB2 in high concentrations of glucose is below that of the derepressed level, but expression of CYB2 is still well above the normal level. These results extend the previous findings that HXK2 regulates glucose repression of GAL1/10, SUC2, and CYC1 (34). SSN6 is an epistatic suppressor of the SNF1 protein kinase (8). It was also identified as CYC8 in conferring glucose-insensitive expression of CYC7 (46). Mutants with mutations in SSN6 share some of the phenotypes of TUP1 mutants, including mating-type defects and flocculation. The products of these two genes exist in a protein complex inside the nucleus (49, 58) and are thought to be part of a general repressor complex (29). Since the products of SSN6 and TUP1 were both identified as releasing glucose repression of CYC7, we considered it likely that other mitochondrial proteins would be under similar control. However, the glucose repression of CYB2 is intact in ssnD9 mutants. This does not exclude the possibility that the TUP1-encoded protein may have a role in glucose repression of CYB2. When recruited to a heterologous promoter via fusion with lexA bacterial DNA binding domains, SSN6 represses transcription in a TUP1-dependent manner (29). However, TUP1 repressive effects are independent of SSN6 in a similar system (52), suggesting that only some of the repressive effects of TUP1 are mediated through SSN6. In addition, these two proteins have discreet roles in establishing chromatin structure in MATa2/MCM1 operator regions (11). The nonrepressed expression of CYB2 was defective in the ssn6D9 context. Because this is a null mutation, we conclude that SSN6 normally has a role in CYB2 activation or derepression. Recently, the TUP1/SSN6 complex was reported to have a positive effect on HAP1 activity (60). Our results are consistent with this finding, since a major activator of CYB2 is the HAP1 (CYP1) complex (20, 30). Mutants in glucose repression of CYB2 represent members of a new panel of mutations in the RMR genes. We have cloned RMR1, which had been previously identified as ROX3, a regulator of CYC7. However, this is the first indication that this gene is a regulator of glucose repression. The gene has pleiotropic regulatory effects on SUC2, GAL1, CYB2, and respiratory functions in addition to the previously described effects on CYC7 and ANB1. We have also characterized the mutational effects in RMR3 (GRR1) which cause defects in CYB2 expression and respiratory function. Several lines of evidence indicate that ROX3, GRR1, and SSN6 all have roles in both activation and repression of a variety of genes. Interestingly, none of the RMR mutants was completely insensitive to glucose repression of CYB2. It seems likely that glucose repression of CYB2 is therefore regulated by several independent mechanisms. Further characterization of the RMR mutants will be useful in extending our understanding of this global regulatory mechanism as it pertains to mitochondrial biogenesis in S. cerevisiae. ACKNOWLEDGMENTS This research was supported by National Institutes of Health Research grants GM20379 to B.L.T. and GM26061 to Richard Zitomer.

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