Mutations Releasing Mitochondrial Biogenesis from Glucose

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1 JOURNAL OF BACTERIOLOGY, July 1982, p /82/ $02.00/0 Vol. 151, No. 1 Mutations Releasing Mitochondrial Biogenesis from Glucose Repression in Saccharomyces cerevisiae ELFRIEDE BOKER-SCHMI1T, SILVIA FRANCISCI,t AND RUDOLF J. SCHWEYEN* Institut fur Genetik und Mikrobiologie der Universitat Munchen, D-8000 Munich 19, West Germany Received 14 September 1981/Accepted 23 March 1982 Mutants which exhibit a constitutive glucose-insensitive expression of respiratory activity were selected by use of a triphenyltetrazolium staining technique. These mutants lack carbon catabolite repression, as was demonstrated by measuring cytochromes, the activity of succinate cytochrome c reduction, total cellular respiration, mitochondrial protein, and DNA synthesis. High growth rates of mutant cells in glucose medium and normal fermentative CO2 production exclude the possibility that this carbon catabolite insensitivity of mitochondrial functions is merely due to a decreased utilization of glucose. Accordingly, the activities of the two cytoplasmic enzymes measured, maltase and malate synthase, were glucose repressible to the same extent in the mutants as in the wild type. The mutations are dominant and showed nuclear inheritance. The results are discussed in terms of carbon catabolite-regulated expression of genes involved in the biogenesis of mitochondria. Carbon catabolite repression (17), a regulatory system that adapts the cellular enzymatic machinery to an economically advantageous use of carbon sources, is a well-documented phenomenon in the yeast Saccharomyces cerevisiae Ġlucose, the most convenient fermentable carbon source in this organism, represses the synthesis of functional components of several pathways that are not required for glycolytic metabolism of hexoses (e.g., Krebs cycle, respiratory chain and oxidative phosphorylation, glyoxylate shunt, and gluconeogenesis); it also represses the synthesis of enzymes that metabolize disaccharides (10, 20, 21). This phenomenon, as far as it concerns mitochondrial functions, involves the repression of synthesis of proteins made by two autonomous systems, the nucleocytoplasmic system and the mitochondrial system (23). Coordinate regulation of both systems is suggested by a variety of observations in connection with carbon catabolite repression-derepression and aerobic adaption of unaerobically grown cells. Especially instructive are the effects that temporary or permanent absence of mitochondrial protein synthesis have on the expression of nuclearly encoded organelle proteins and the effects that mutations in a variety of nuclear genes have on the expression of mitochondrially encoded proteins (27). Among the latter are two mutants which had been selected to have a constitutively glucoset Present address: Instituto di Fisologia Generale, Facolta di Science M.F.N. Universita di Roma, Rome, Italy. 303 derepressed phenotype (6, 18). Both show this constitutivity for the cytoplasmic and mitochondrial spectra of glucose-sensitive functions. In the present communication we describe mutants with a more specific release from carbon catabolite repression: the synthesis of components that contribute to oxidative energy conservation in mitochondria appears to be constitutively derepressed, whereas repression of the synthesis of cytoplasmic enzymes is still active. This study provides evidence for a separate control site for genes involved in mitochondrial biogenesis. MATERILS AND METHODS Strains. Mutant CCR-91 was derived from S. cerevisiae wild-type strain SM11-6C a ura- (capr ana') (26). Mutant CCR-96 was induced in ccr+ (normal carbon catabolite repression) spores (EM31-2A) of the third backcross of CCR-91 x SM11-6C ccr+. Strain (eryr olis part) (31) was used as the IL993-5C a ilvglucose-sensitive wild type for the first cross of this series. Media. YD medium (1% yeast extract, 2% agar) was supplemented with glucose (3, 5, or 7.5%), glycerol (3%) (YG medium), or galactose (3%) as the carbon source. Antibiotics were added to YG medium by the method of Schweyen and Kaudewitz (25). Minimal medium contained 0.7% Difco yeast nitrogen base (without amino acids), 3% glycerol or 3% glucose, and 2% agar. Sporulation was obtained on acetate agar plates (0.4% sodium acetate, 2% agar). Triphenyltetrazolium chloride ('TTC) overlay agar (19) consisted of 1.5% agar in M phosphate buffer (ph 7.0) and 0.1% TTC. Cell growth. Growth was followed by measuring the

2 304 BOKER-SCHMITT, FRANCISCI, AND SCHWEYEN J. BACTERIOL. turbidity of liquid medium at 578 nm in an Eppendorf photometer. Mutagenesis and isolation of CCR- mutants. (i) Treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Late-log-phase cells of strains SM11-6C and EM31-2A were suspended at 108/ml in YD medium. N-Methyl- N'-nitro-N-nitrosoguanidine was added to a final concentration of 100,ug/ml, and the cells were incubated for 60 min at 30 C. Cells were washed, suspended at 2 x 106/ml in YD medium containing 7.5% glucose, and grown to the early stationary phase. (ii) Screening of CCR- mutants. Cells from the cultures treated with N-methyl-N'-nitro-N-nitrosoguanidine were spread onto YD plates containing 7.5% glucose to give rise to about 100 colonies per plate. The colonies were then tested by overlaying 20 ml of TTC agar (at 40 C) on the plates. Red and white colonies were scored 1 h after overlay. Differential staining of colonies is based on TTC reacting with the respiratory chain via cytochrome oxidase and reducing it to formazan (28). Formazan accumulates as an insoluble, red pigment in the cells. Actively respiring cells produce red colonies on high-glucose agar plates, whereas cultures incapable of respiration produce white or pink colonies (22). Red colonies were isolated, suspended in YG medium, and grown to the early stationary phase. For further tests, cells from these cultures were spread onto YD plates (7.5% glucose) and onto YG plates. Plates bearing 50 to 100 colonies were overlaid with TTC agar. Colonies which showed the same red color after growth on glycerol and high-glucose media were finally isolated as mutants that were constitutively derepressed in mitochondrial functions (CCR-). They were found with a frequency of about Crosses and tetrad analysis. Mutant CCR-91 derived from S. cerevisiae wild-type strain SM11-6C was crossed with wild-type strain 1L993-5C, and mutant CCR-96 derived from strain EM31-2A was crossed with strain SM11-6C. Diploids were sporulated on acetate agar. Asci were digested with glucuronidase (Boehringer Mannheim Corp., Mannheim, West Germany) in water for 20 min and were dissected with a micromanipulator (E. Leitz, Inc., Wetzlar, West Germany). Spores were grown to colonies on YD plates and tested by replica plating on different media and by overlaying with TTC agar. Respiration and fermentation. Oxygen uptake was determined polarographically with an Eschweiler oxygen electrode in a 2-ml cell with a thermoregulated (23 C) water jacket and magnetic stirrer and was related to turbidity. CO2 production and 02 uptake were determined in a standard Warburg respirometer at 30 C in YG medium supplemented with 4% glucose (ph 5). Spectra. For spectral analysis, wild-type and mutant cells were grown in YD medium (7.5% glucose) and in YG medium (3% glycerol) to the log phase (1 x 107 to 2 x 107 cells per ml). The cultures were centrifuged, and the pellets were suspended in 50% glycerol to about 109 cells per ml and frozen in liquid nitrogen in a homemade aluminium cuvette of 3-mm path length. The spectra were recorded on a Shimadzu UV 210A spectrophotometer equipped with a halogen lamp (B. Lang, unpublished data). Mitochondrial protein synthesis. Wild-type and mutant CCR-91 cells were grown in minimal medium with glycerol (3%) at 23 C to the log phase and were supplemented with glucose (7.5%) or galactose (3%). At various times, three samples of cells (107/ml) were removed from each of the glucose-, glycerol-, and galactose-grown cultures and incubated with [3H]leucine (0.7,uCi/ml, specific activity, 60 Ci/nmol; Amersham Buchler Corp., Braunschweig, West Germany) for 45 min (i) without antibiotics, (ii) in the presence of cycloheximide (100 p.g/ml), or (iii) in the presence of cycloheximide and erythromycin (4 mg/ml) (5). The synthesis was stopped by the addition of trichloroacetic acid to a final concentration of 7%. Samples were heated at 90 C for 20 min, washed on glass fiber filters, dried, and counted in 3 ml of a scintillation cocktail containing 4 g of PPO (2,5-diphenyloxazole) and 0.1 g of dimethyl-popop (1,4-bis-[5-phenyloxazolyl]benzene) per 1 liter of toluene in an Isocap 300 (Searle- Nuclear-Chicago) liquid scintillation counter. The cycloheximide-resistant, erythromycin-sensitive incorporation is regarded as mitochondrial (24). Estimation of mitochondrial DNA content. Cultures of the wild-type and mutant CCR-91 cells were labeled with [14C]adenine (0.1 uci/ml) during growth overnight in minimal medium containing 3% glycerol and 0.3% Casamino Acids. Cells were harvested, washed with the above-mentioned medium, and suspended in minimal medium containing 7.5% glucose, 3% glycerol, or 3% galactose to about 1 x 107 to 5 x 107/ml. At various times, samples were incubated with [3H]adenine (10 pxciiml) for 45 min. Mitochondria were prepared after lysis of spheroplasts (14) and centrifuged on NaJ gradients. The gradients were fractionated and prepared for counting as described previously (15). Enzyme assays. Protein was measured by the method of Lowry et al. (16); determination of maltase activity was as described by Zimmermann and Eaton (32). Succinate:cytochrome c oxidoreductase was assayed by the method of Arrigoni and Singer (1), with cytochrome c as the electron acceptor; malate synthase was determined by the method of G. H. Dixon and H. L. Kornberg (Biochem. J. 3p:72, 1959). RESULTS Actively respiring yeast cells rapidly reduce TTC to 2,3,5-triphenylformazan, an insoluble red pigment. Vital staining by overlaying colonies with TTC agar is useful for discrimination between respiration-competent wild-type cells and respiration-deficient mutants; the former turn red, whereas the latter stay white (19, 22). We observed that colonies of carbon catabolite-repressed cells (grown on a high-glucose substrate) and derepressed cells (grown on a low-glucose or nonfermentable substrate) also show a distinct difference in staining in that repressed colonies are white or slightly pink, in contrast to the derepressed red colonies. From a series of wild-type strains studied, we chose strains SM11-6C and EM31-2A, which showed good differentiation in the TTC test, to isolate mutants which stain red irrespective of the carbon source and concentration on which they were grown. After mutagenic treatment, a series of mutant

3 VOL. 151, 1982 GLUCOSE REPRESSION IN YEAST MITOCHONDRIA 305 colonies was isolated which, according to this TTC staining, was regarded as insensitive to carbon catabolite repression (CCR-). Since the red color is assumed to be due to rapid reduction of TTC via the respiratory chain, we expected the CCR- mutants isolated to be constitutively derepressed in mitochondrial functions. Two of them, CCR-91 and CCR-96, were further characterized. Genetics. Mutant CCR-91 was crossed with the wild-type (ccr+) strain IL993-5C. When tetrads were analyzed by TTC staining, the majority were found to segregate as 2 CCR-:2 ccr+ and the remainder mainly as 3 CCR-:1 ccr+. After CCR- spores were backcrossed three times with parental strain SM11-6C ccr+, the fraction of tetrads segregating 2:2 was close to 100%. We thus conclude that CCR-91 contains a single nuclear mutation. Mutant CCR-96, induced in a spore (EM31-2A) of the third backcross, gave the same 2:2 segregation when crossed with SM11-6C ccr+. When diploids from CCR- x ccr+ crosses were stained by TTC, a colony phenotype close to that of CCR- x CCR- crosses was observed. This indicates that the mutations in CCR-91 and CCR-96 are dominant or semidominant. An allelism test was performed by analyzing tetrads of the cross CCR-91 x CCR-96. Among 78 tetrads studied, no ccr+ spore was detected. This points to location of the two mutations in the same gene or in two closely linked genes. The similarity of phenotypic expression (see below) favors the assumption of two allelic mutations. Physiology. Growth rates of mutant and wildtype cells on different carbon sources were compared (data not shown). In the nonfermentable substrate glycerol and in the fermentable substrate galactose, no difference in the growth rates of mutant and wild-type cells was observed. In the readily fermentable substrate glucose, both mutants grew slightly faster than the wild type. This excludes the possibility that carbon catabolite insensitivity is simply due to a mutational defect in glucose uptake or in glycolysis. The content of cytochromes in glucoserepressed wild-type cells is highly reduced compared with that in derepressed cells. Cytochrome aa3 is most sensitive (4, 5, 13, 20). Figure 1 shows low-temperature spectra of whole cells, comparing mutants CCR-91 and CCR-96 grown in high-glucose medium with the respective wild-type strains grown in high-glucose or glycerol medium. In wild-type cells, the absorption band of cytochrome aa3 (600 nm) is strong in derepressed cells and almost lacking in repressed cells. The effect of glucose on other cytochromes is significant but less pronounced. In contrast, mutants CCR-91 and CCR-96 grown in high-glucose medium had cytochrome contents similar to those of wild-type cells and mutant cells grown under derepressing conditions. This supports the notion that the biogenesis of mitochondria in these mutants may be widely insensitive to carbon catabolite repression. Respiration and fermentation rates were determined by measuring the 02 uptake and CO2 production during carbon catabolite repression, i.e., after high glucose concentrations had been added to cultures of mutants and respective wild types grown under derepressing conditions. Figure 2 (A and B) shows the kinetics of repression as revealed from 02 consumption by whole cells. After a delay of 2 to 3 h, 02 consumption by wild-type cells dropped considerably, whereas it remained at an initial high rate in the mutants. This is in accordance with the high cytochrome content and TTC staining properties of mutant cells grown in glucose medium. Diploids from CCR- x ccr+ crosses showed respiration rates similar to those of the haploid CCRmutants (data not shown); this result further documents the dominance of the mutations in CCR-91 and CCR-%. Upon addition of glucose to glycerol-grown cells, CO2 production increased at virtually the same rates and to similar maximal levels in CCR- mutants and the respective wild types (Fig. 2C). This rapid increase in fermentative CO2 production, which is commonly observed in aerobically fermenting yeasts (7, 11, 29), reveals that glucose uptake and fermentation are virtually unaffected by the two CCR- mutations studied. This agrees well with the observed high growth rates of the mutants in glucose medium. So far, insensitivity to carbon catabolite repression in CCR-91 and CCR-96 has been shown for mitochondrial parameters. To test whether there is an effect on cytoplasmic functions, the activities of two enzymes were determined; one of them, maltase, may be regarded as a representative of the enzymes that metabolize disaccharides, and the other, malate synthase, may be regarded as a representative of the glyoxylate cycle. In wild-type cells neither of them is inactivated by glucose, but the synthesis of both is sensitive to repression (10, 21, 30). This sensitivity was not affected by the mutation in CCR-91 (Table 1). The mutant showed the same extent of repression by glucose as did the wild type. We conclude from this result that the mutation in CCR-91 causes a release from carbon catabolite repression in mitochondria, and possibly in some other functional systems, but not in disaccharide metabolism or in the glyoxylate cycle. Succinate:cytochrome c oxidoreductase, which was determined in a parallel assay (Table 1), was

4 306 BOKER-SCHMITIT, FRANCISCI, AND SCHWEYEN J. BACTERIOL..0.0 ICJ~~~~~~~~~~~. [~~~~a] I Iii C~~~~~~~~~~~~~~~wvlnt Inm IIh wavelength (nm) II wqvelength (nm) FIG. 1. Low-temperature absorption spectra of whole cells of wild types and mutants. The wild types and mutants were grown in YD medium (7.5% glucose) and in YG medium (3% glycerol). The cultures were centrifuged, the pellets were suspended in 50%o glycerol and frozen in liquid nitrogen, and absolute spectra were recorded as described in the text. The fine vertical lines mark, from left to right, the absorption of cytochromes c (547 am), cl (554 nm), and b (558 am). (A) Low-temperature absorption spectra of wild-type SM11-6C grown in glucose medium (a) and in glycerol medium (b) and of mutant CCR-91 grown in glucose medium (c). (B) Lowtemperature absorption spectra of wild-type EM31-2A grown in glucose medium (a) and in glycerol medium (b) and of mutant CCR-96 grown in glucose medium (c). insensitive in CCR-91, as was expected from the observation of glucose-insensitive respiration. Similar results were obtained with mutant CCR- 96 (data not shown). Synthesis of macromolecules in mitochondria. Overall mitochondrial protein synthesis is sensitive to carbon catabolite repression (12, 29). The kinetics of this repression in strain SM11-6C is shown in Fig. 3. Whole cells grown in glycerol and treated as described above had a rate of cycloheximide-resistant, erythromycin-sensitive incorporation which equalled 6% of the rate of incorporation in the absence of any inhibitor. This represents the contribution of the mitochondrial protein-synthesizing machinery. Upon addition of glucose, a rapid decrease of this rate, approaching a value of less than 1%, was observed. The reaction of this parameter was very fast and preceded that of the repression of 02 consumption (see Fig. 2A and B). It is logical to assume that in growing cells a reduction in mitochondrial protein synthesis (with some delay) results in a partial depletion of components of the respiratory chain (some of which are made on mitochondrial ribosomes) and thus in a reduction of 02 consumption. In contrast to the wild type, mutant CCR-91 showed no repression of the overall mitochondrial protein synthesis. This is in agreement with the observations on cytochromes, respiration, and succinate:cyto-

5 VOL. 151, 1982 GLUCOSE REPRESSION IN YEAST MITOCHONDRIA 307 x c b A 10I 81 6I O~o o 0) C I1. B 10! 8[ 6 4 o 0o o o 0 0 AS~A~ 4) 2 2 [ a# r- 0 x -E 1- I~ ~ a A a h chrome c oxidoreductase. The rapid response of the mitochondrial protein synthesis in the wild type and the absence of this response in CCR-91 indicate that carbon catabolite repression acts at the level of gene expression. The amount of mitochondrial DNA (mtdna) also varies with the degree of glucose repression (4). Figure 4 shows the kinetics of repression of mtdna synthesis upon addition of glucose to glycerol-grown cells. In wild-type strain SM11-6C mtdna synthesis was reduced from about 17% of the total cellular DNA to about 6%. The kinetics of this repression are slow and its extent is small; it cannot account for the more rapid and pronounced repression of protein synthesis. The synthesis of mtdna in mutant CCR-91 grown in glucose and glycerol media was similar to that in the derepressed wild type. DISCUSSION The aim of this study was to isolate mutants which are insensitive to carbon catabolite repression in mitochondrial functions (CCR-). The screening for such mutants by use of the h FIG. 2. Rates of oxygen uptake and fermentative CO2 production of cells of wild types and CCRmutants in YG medium. In A and B, glucose (final concentration, 7.5%) was added to growing derepressed cells at zero time; the rate of oxygen uptake was determined at the times indicated and related to the optical density of the culture. (A) 0, wild-type SM11-6C in glycerol medium; *, wild-type SM11-6C in glucose medium; A, mutant CCR-91 in glycerol medium; A, mutant CCR-91 in glucose medium. (B) 0, wild-type EM31-2A in glycerol medium; *, wildtype EM31-2A in glucose medium; A, mutant CCR-96 in glycerol medium; A, mutant CCR-96 in glucose medium. (C) Glucose (final concentration, 4%) was added to growing derepressed celis. 0, wild-type EM31-2A; *, mutant CCR-96; A, wild-type SM11-6C; A, mutant CCR-91. TTC overlay staining of colonies was successful. After mutagenesis, some colonies were found which, in spite of high glucose concentrations, stained red like derepressed wild-type cells. The TABLE 1. Levels of glucose-sensitive enzymes in derepressed (3% glycerol) and repressed (7.5% glucose) wild-type and mutant cells of S. cerevisiaea Sp act (nmol/min per mg of Strain Growth protein) Malate substrate Maltase Mytasae SDH Wild-type glycerol SM11-6C glucose Mutant glycerol CCR-91 glucose a The activities of the cytoplasmic enzymes maltase and malate synthase and the mitochondrial enzyme succinate:cytochrome c oxidoreductase (SDH) were determined as described in the text.

6 308 BOKER-SCHMITT, FRANCISCI, AND SCHWEYEN 7-6 GO - oine u 1 > O 2.- E C I- F * in in In *-; > S 0v a$ = c.4- c Go >%4-4,0 *`1~ h FIG. 3. Rate of mitochondrial protein synthesis (as percentage of total cellular protein synthesis), determined as cycloheximide-resistant, erythromycin-sensitive incorporation of [3H]leucine in whole cells of wild-type SM11-6C and mutant CCR-91 grown in glycerol medium and treated as described in the text. At zero time, glucose (final concentration, 7.5%) was added to growing derepressed cells. 0, wild-type SM11-6C in glycerol medium; *, wild-type SM11-6C in glucose medium; A, mutant CCR-91 in glycerol medium; A, mutant CCR-91 in glucose medium. mutants built up a highly active respiratory chain under conditions which in wild-type cells cause a strong repression. In accordance with the proposed mechanism of reduction of TTC (28) and previous in vivo studies with respiration-deficient mutants (2, 19), we conclude that rapid TTC reduction in vivo mainly depends upon the flux of electrons in the respiratory chain. The two mutants described carry single nuclear mutations that determinie the CCR- phenotype; both mutations are dominant. (Some other mutants isolated by the same screening methods and having phenotypes similar to those of CCR- 91 and CCR-96 also showed nuclear inheritance.) Two classes of mutational alterations may be envisaged which could cause this carbon catabolite insensitivity of mitochondrial functions. (i) Glucose, the repressing substrate regularly used in the experiments described above, is taken up and fermented at a reduced rate only; therefore, a signal which in the wild type represses the synthesis of the respiratory chain and of enzymes of other pathways is lacking or weak. (ii) The mutations interfere with a component of a regulating circuit which controls (a) all functions known to be sensitive to carbon catabolite repression or (b) only one branch of a complex regulatory system. The results presented exclude a defect according to lypothesis i since mutant cells grew on glucose at least as rapidly as wild-type cells, and since fermentation of glucose by mutant cells, as revealed from the CO2 production rate, was indistinguishable from that by wild-type cells in both amount and time course. Hypothesis iib is in excellent agreement with the data presented: maltase and malate synthase, two cytoplasmic enzymes whose synthesis is highly repressible, exhibited the same sensitivity in mutants as in the wild type, whereas the synthesis of components of the respiratory chain became glucose resistant due to the mutations in CCR-91 and CCR-96. The fact that mitochondrial functions are released from carbon catabolite repression whereas at least some cytoplasmic functions still are repressed points to either two independent regulatory circuits or two branches of the same regulatory system with partially autonomous regulation. It is tempting to postulate that one branch controls the synthesis of cytoplasmic enzymes involved in utilization of sugars which are less convenient to the cell than glucose (e.g., maltose or sucrose) (33) and the synthesis of glyoxylate cycle and gluconeogenetic enzymes (20, 21), whereas the other branch regulates the synthesis of all mitochondrial components that contribute to oxidative energy conservation. Only the latter branch has changed from repressible in the wild type to constitutive in the mutants. Evidence for coreg < 15 C- 1 Z lo 0 = 0 u I. J. BACTERIOL..o,..,..o..A \N1, h FIG. 4. DNA synthesis (as percentage of total cellular DNA synthesis) determined by [3H]adenine incorporation in DNA. Separation of mtdna and nuclear DNA was achieved by NaJ gradient centrifugation, and cells were treated as described in the text. At zero time, glucose (final concentration, 7.5%) was added to growing derepressed cells of wild-type SM11-6C and mutant CCR-91. 0, wild-type SM11-6C in glycerol medium; 0, wild-type SM11-6C in glucose medium; A, mutant CCR-91 in glycerol medium; A, mutant CCR-91 in glucose medium.

7 VOL. 151, 1982 ulation of both branches comes from a recent report (18) describing a recessive mutant, carrying girl-i, which, like CCR-91 and CCR-96, exhibits glucose-insensitive respiration and, unlike the CCR- mutants, also exhibits constitutive expression of maltase and some other cytoplasmic enzymes. Since the glrl-l mutant appears to be normal in glucose uptake and fermentation, the authors conclude that the altered function is likely to be in a primary regulatory step (18). This step may link two coregulated branches of the carbon catabolite-sensitive system which we postulate. A common regulation of all glucose-sensitive functions in yeasts also has been postulated by Ciriacy (6) based upon the study of a dominant mutant (CCR-80) which was selected primarily for reduced repression of respiratory functions, but which turned out to be also reduced in repression of various cytoplasmic enzymes. Unlike CCR-91, CCR-96, and the glrl-l mutant, CCR-80 is strongly impaired in fermentation, suggesting a primary defect in hexose uptake, hexose metabolism, or both. Evidence for regulation of different branches of a common system by separate regulatory sites, as we propose for the expression of the mitochondrial functions, comes from studies by Zimmermann and co-workers (8, 9, 33, 34). They describe a series of mutations in three different genes which affect carbon catabolite repression-derepression of limited spectra of cytoplasmic enzymes only. Mitochondrial biogenesis involves the coordinate expression of information encoded by two genomes, the nuclear and the mitochondrial (23). Many experiments have been designed to illustrate the interdependency of the two genomes during carbon catabolite derepression and respiratory adaptation of unaerobic cells (27). The study of constitutively derepressed mutants like those described here provides further evidence for the coordinate expression and gives access to the study of its regulation. We have shown that CCR-91 and CCR-96, both of which contain dominant mutations in nuclear DNA, release mitochondrial gene expression from carbon catabolite repression. The finding that the respiratory chain is fully equipped implies that the expression of the many nuclear genes which contribute to its synthesis is also released from repression. This concommittant release and the dominant nature of the mutations suggest that a regulatory function that acts on the expression of both nuclear and mitochondrial genes has been altered. The level at which the expression of information for mitochondrial components is regulated remains unknown. Only in the case of the nuclear gene coding for cytochrome c (cycl) has it been demonstrated that glucose represses tran- GLUCOSE REPRESSION IN YEAST MITOCHONDRIA 309 scription (35). In mitochondria there are strong regulatory effects exerted by glucose, as is revealed by a highly reduced level of overall protein synthesis and reduced amounts of mtdna (4, 5). The kinetics of the repression of protein synthesis, which we showed in this study, are fast (70% reduction within 1 h of growth). This indicates an efficient regulation at the level of transcription and translation. The repression appears to affect all mitochondrially made proteins, with some minor quantitative differences only (12). More pronounced differences have been observed in the pattern of isoaccepting trnas in mitochondria. In glucose-repressed cells, one of two serine isoacceptors, which are coded for by different mitochondrial genes, could not be detected by a trna acylation test (3). Whether repression acts at the level of transcription, transcript processing, modification, or (in the case of polypeptides) protein synthesis is still an open question. The possibility that it occurs via a decrease in the quantity of mtdna, i.e., by a shortage in the template for transcription, is excluded by the data we presented: the total decrease in mtdna synthesis is low, and the kinetics of the decrease are low compared with the repression of protein synthesis. ACKNOWLEDGMENTS We thank W. Bandlow, M. Ciriacy, L. Frontali, and F. Zimmermann for helpful suggestions and discussions. S.F. was awarded a fellowship of the Instituto Pasteuer Fondazione Cenci Bolognetti, held in Munich in 1979 and This work was supported by the Deutsche Forschungsgemeinschaft. LITERATURE CITED 1. Arrigoni, O., and T. P. Singer Limitations of the phenazine methosulfate assay for succinic and related dehydrogenases. Nature (London) 193: Bachhofen, V., R. J. Schweyen, K. Wolf, and F. 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