mRNA Levels for the Fermentative Alcohol Dehydrogenase of Saccharomyces cereuisiae Decrease upon Growth on a Nonfermentable Carbon Source*

The classical, fermentative alcohol dehydrogenase from Saccharomyces cerevisiae, which previously was thought to be constitutive, has been shown to be re-pressed by growth on nonfermentative carbon sources. “he rate of alcohol dehydrogenase I protein synthesis declined 6-fold within 3 to 4 h after yeast were trans- ferred from medium containing glucose to medium containing ethanol, and it declined 10-fold after glucose became depleted from the medium during diauxic growth. The decreased rate of alcohol dehydrogenase I protein synthesis was shown not to be the result of an increased rate of degradation of the alcohol dehydrogenase I protein. The decline in alcohol dehydrogenase I protein synthesis was correlated with a 6- to 10-fold decrease in the amount of functional alcohol dehydrogenase I mRNA within 3 to 4 h after transfer from glucose- containing medium to medium containing ethanol. A similar decrease in alcohol dehydrogenase I functional mRNA occurred when cells were depleted of glucose by diauxic growth. Total alcohol dehydrogenase I mRNA, as detected by hybridization to the cloned ADCl gene, was found in the same relative abundance as the amount of translatable alcohol dehydrogenase I mRNA during the different growth conditions. These results suggest that the alcohol dehydrogenase I protein is transcriptionally regulated.

The classical, fermentative alcohol dehydrogenase from Saccharomyces cerevisiae, which previously was thought to be constitutive, has been shown to be repressed by growth on nonfermentative carbon sources.
"he rate of alcohol dehydrogenase I protein synthesis declined 6-fold within 3 to 4 h after yeast were transferred from medium containing glucose to medium containing ethanol, and it declined 10-fold after glucose became depleted from the medium during diauxic growth. The decreased rate of alcohol dehydrogenase I protein synthesis was shown not to be the result of an increased rate of degradation of the alcohol dehydrogenase I protein.
The decline in alcohol dehydrogenase I protein synthesis was correlated with a 6-to 10-fold decrease in the amount of functional alcohol dehydrogenase I mRNA within 3 to 4 h after transfer from glucosecontaining medium to medium containing ethanol. A similar decrease in alcohol dehydrogenase I functional mRNA occurred when cells were depleted of glucose by diauxic growth. Total alcohol dehydrogenase I mRNA, as detected by hybridization to the cloned ADCl gene, was found in the same relative abundance as the amount of translatable alcohol dehydrogenase I mRNA during the different growth conditions. These results suggest that the alcohol dehydrogenase I protein is transcriptionally regulated.
When yeast are grown on a fermentable carbon source such as glucose, the fermentative, classical alcohol dehydrogenase (ADH 1') catalyzes the regeneration of NAD' from NADH with the concomitant production of ethanol from acetaldehyde. During such growth ADH I is present in a large amount in the cell as are many of the other principle fermentative enzymes in yeast (1, 2). However, when the fermentable carbon source is depleted, the yeast cells derepress the synthesis of a variety of other enzymes in order to utilize the previously excreted ethanol as an energy and carbon source via oxidative respiration and gluconeogenesis. Instead of using ADH I for the reverse reaction in the utilization of ethanol, another ADH isozyme, the glucose-repressible ADH (ADH 11) is synthesized (3). This latter enzyme seems kinetically better suited for the reverse reaction than ADH I (4).
Previous work on the control of yeast ADHs assumed on the basis of ADH I enzyme activity that it was synthesized constitutively (3, 5), i.e. synthesized in fixed amounts irrespective of the carbon source (10). However, there is no obvious need for yeast to maintain the synthesis of two ADHs during growth on ethanol. It seemed reasonable to investigate whether ADH I was indeed synthesized at comparable levels during growth on fermentable and nonfermentable carbon sources. In addition, it also seemed important to study the expression of ADH I since its promoter is being used to obtain the synthesis of foreign proteins in yeast (25-27). The results presented in this paper indicate that the amount of ADH 1 mRNA and the rate of ADH 1 protein synthesis decrease upon growth in ethanol-containing medium and during growth into stationary phase. The inverse regulation of the ADH isozymes and its relationship to the other fermentative enzymes in yeast is discussed.

RESULTS
ADH I Protein Synthesis-The rate of ADH I protein synthesis during growth on a nonfermentable carbon source was quantitated by a radioimmune assay. Yeast were grown on glucose-containing medium overnight and transferred to medium containing ethanol. At various times after transfer, the yeast cells were pulse-labeled for 15 min with [:%]methionine, and the ADH protein was isolated by immunoprecipitation with antibody specific to ADH (1). The total radioactive proteins and the radioactive immunoprecipitates were subsequently fractionated by SDS-polyacrylamide gel electrophoresis and identified by fluorography. Densitometric analysis of these fluorograms was carried out in order to calculate the percentage of total radioactively labeled protein which was ADH (6). Because the strains used in these experiments contained both ADH I and ADH I1 activity, ADH I protein had to be differentiated from that of its related and antigeni- 1165 cally similar isozyme ADH II-F. 3 In the first two series of experiments, ADH I and ADH 11-F were distinguished after limited proteolysis of the immunoprecipitates with Staphylococcus aureus protease (6,9). This method was used previously to quantitate ADH 11-F protein synthesis (6). Fig. 1 displays a typical fluorogram of the total proteins synthesized after transfer of yeast to medium containing ethanol, and Fig.  2 displays the proteolytic cleavage patterns of the immunoprecipitated ADH.
As can be seen by the distribution of radioactive polypeptides in Fig. 1, significant changes in the synthesis of some polypeptides occurred when yeast were transferred to medium containing ethanol, although the total rate of [:'%]methionine incorporation remained relatively unchanged. The data in Fig.   2 indicate that ADH I protein synthesis declined in the first 2 h of growth on a nonfermentable carbon source. Synthesis of ADH I1 was first detected approximately 1 h after release from glucose repression. This result is in sutstantial agreement with previous results on ADH I1 regulation, which was analyzed in strains deficient in ADH I enzyme activity (6). were taken and ADH I protein synthesis was quantitated as described above. In addition, a fraction of the original yeast culture was grown continuously on glucose in the presence of ["S]methionine in order to show that incorporation of [""Slmethionine into protein would have proceeded unimpeded throughout this interval. Fig. 4 (see Miniprint) displays the change in total ["S]methionine incorporation and ADH I synthesis during the experiment.
In cells continuously incubated with ["S]methionine, radioactivity accumulated linearly with respect to time into total protein ( Fig. 4a, Miniprint) and into ADH I (Fig. 4b, Miniprint). In cells labeled with [""Slmethionine and then grown on either glucose-or ethanol-containing media in the presence of excess nonradioactive methionine, total incorporation of radioactivity into proteins did not change after removal of the radioactive label (Fig. 4 a ) . This control experiment indicates that the chase with unlabeled methionine had stopped the incorporation of exogenous radioactivity into yeast proteins. slight increase in radioactive ADH I protein was observed. These results strongly suggest that the decline in ADH I protein synthesis during growth on a nonfermentable carbon source was not a result of a major change in the rate of ADH I protein degradation upon switching cells to medium containing ethanol, nor was it a result of a high turnover rate for the ADH I protein.
Amount of Translatable ADH Z mRNA-To determine whether the decline in ADH I protein synthesis resulted from a change in the amount of ADH I mRNA, the amount of translatable ADH I mRNA was measured. RNA was extracted from yeast a t various times after placing them in medium containing ethanol. The RNA was translated in a wheat germ cell-free system in the presence of [:%]methionine after which the radioactive ADH was immunoprecipitated from a portion of the sample. The immunoprecipitate and a sample of the total radioactive proteins were analyzed by polyacrylamide gel electrophoresis and fluorography. The percentage of total protein synthesis which was ADH I was calculated as described in the preceding sections after analyzing the immunoprecipitated ADH by limited proteolysis. Fig. 3b (see Miniprint) depicts the change in the percentage of ADH I and ADH I1 synthesis in vitro (equivalent to the amount of translatable ADH I and ADH I1 mRNA) as a function of time after transfer of the yeast to ethanol-containing medium. The values represent the average of three separate experiments.
As seen in Fig. 36 (see Miniprint), the percentage of functional ADH I mRNA declined from approximately 1.1 to 0.20% within 3 to 4 h after switching the cells to ethanolcontaining medium. This decline was coincident with the change in ADH I protein synthesis as seen in Fig. 3a. After 3 h, the amount of translatable ADH I mRNA leveled off. The amount of translatable ADH I1 mRNA and the timing of its appearance were similar to that previously found in cells deficient in ADH I activity, i.e. ADH I1 mRNA appeared after about 1 h (6).
These results were confirmed using a more direct assay for in vitro-synthesized ADH I and ADH I1 protein by separating by size the ADH I and ADH 11-S proteins. On SDS-containing polyacrylamide gels, the subunit polypeptides of ADH 11-S migrate with a lower apparent molecular weight than the subunits of ADH I. The separation of ADH 11-S and ADH I polypeptides on an SDS-polyacrylamide gel is shown in ered to be a derivative of ADH I since it was only present when ADH I was present. In the original photographs, this ADH I band was present at the fist two time points described below.
The amount of functional ADH I and ADH 11-S mRNA present during growth on glucose-containing medium and during growth on ethanol-containing medium was determined in an experiment similar to the one described above. Total RNA was extracted at various times after transfer of the cells at time zero from medium containing ethanol to that containing glucose. The reciprocal experiment was also performed: RNA was extracted at various times after transfer of cells at time zero from glucose-containing medium to that containing ethanol. Equal microgram quantities of RNA were translated in vitro in the presence of ["'S]methionine. The radioactive ADH polypeptides were immunoprecipitated and identified by fluorography after separation on SDS-polyacrylamide gels.
A fluorogram showing the change in the relative levels of ADH I and ADH 11-S mRNA as a function of time is presented in Fig. 5, and this change is depicted quantitatively in Fig. 6 (see Miniprint). In Fig. 5, a polypeptide of unknown origin can be seen to have precipitated along with ADH. This species was not observed in the immunoprecipitates in the previous experiments, suggesting that it was not an ADH degradation product produced in the cell-free translation system. Transfer from ethanol growth medium to medium containing glucose resulted in about a 3-to 5-fold increase in ADH I mRNA level with a corresponding 4-fold decrease for the amount of ADH I1 mRNA (Fig. 6a, Miniprint). In the reciprocal experiment, transfer from medium containing glucose to medium containing ethanol (Figs. 5 and 66), ADH I mRNA declined 10-fold within 3 to 4 h. At the same time, ADH I1 mRNA became derepressed. These results are similar to those obtained above using limited proteolysis to distinguish the two enzymes, although the degree of ADH I mRNA decline appears to differ in the two experiments. However, this difference is probably attributable to the fact that the data obtained by separating the ADH isozymes by size were not normalized to the total amount of translatable mRNA but to the amount of RNA present as measured by its absorbance at 260 nm. These combined results indicate that the decrease in ADH I protein synthesis upon growth on a nonfermentable carbon source resulted from a decrease in the amount of translatable ADH I mRNA.
ADH Z Regulation during Growth into Stationary Phase-ADH I protein synthesis and mRNA levels were also analyzed in cells inoculated into medium containing glucose and grown continuously into stationary phase. These experiments were undertaken in order to analyze ADH regulation in conditions in which the glucose became depleted naturally from the medium. Cells from a stationary phase culture of strain 358-21 were inoculated into medium containing 1% glucose and allowed to grow for 26 h. Between 14 and 16 h after inoculation, the glucose was depleted from the medium and cell division ceased (data not shown). A second period of cellular division, albeit at a much slower rate than the initial logarithmic growth, commenced at about 20 h (22). The rate of ADH protein synthesis was measured as described above. Fig. 7a (see Miniprint) depicts the rate of ADH I and ADH I1 protein synthesis as function of time during the course of the experiment. The lack of ADH protein synthesis until 4 h reflected the fact that little total protein synthesis occurred until that time. This resulted from the use of an inoculum that was taken from stationary phase cells that were essentially dormant. ADH I was synthesized during the growth on glucose medium, but its rate of synthesis declined about 5-fold between 12 and 14 h after the start of the experiment. This

Regulation of Yeast
Alcohol Dehydrogenase I decline in ADH I protein synthesis was coincident with the cessation of cellular division and the depletion of glucose from the medium (22). Little or no ADH I1 protein synthesis occurred when glucose was present in the medium, but ADH I1 synthesis increased after the depletion of glucose from the medium, which is in agreement with previous results on the study of ADH 11 regulation (see above) (6). The amount of functional ADH I mRNA was quantitated as described above by extracting RNA at times throughout the growth into stationary phase, translating the RNA in the cell-free system, and identifying the amount of ADH I polypeptide synthesized by limited proteolysis and fluorography. Fig. 7b (see Miniprint) presents the percentage of total translatable mRNA which was respectively ADH I and ADH I1 during growth into stationary phase. The amount of functional ADH I mRNA was found to decline at the same time that ADH I protein synthesis was shown to decline in Fig. 7a.
These results indicate that the decline of ADH 1 functional mRNA levels that was observed when cells were transferred to medium containing ethanol also occurred when cells were depleted of glucose by growth into stationary phase. ADH I1 mRNA began to accumulate at about 14 h, a time which was coincident with the time of increase in the rate of ADH I1 protein synthesis shown in Fig. 7a.
ADH Z mRNA Levels as Detected by Hybridization-The preceding analysis only detected functional ADH I mRNA. In order to quantitate ADH I mRNA irrespective of its ability to be translated in vitro, total ADH I RNA was detected by hybridization to the cloned ADCl (ADH I) gene (20). The interpretation of these analyses is not straightforward because the ADCl and ADR2 (ADH 11) genes are 90% homologous in nucleotide sequence (23,29), and no hybridization conditions were found which allowed the ADCI gene to hybridize solely to ADH 1 mRNA. Nonetheless, the quantitation of ADH I mRNA hybridization was made possible, as shown below, by the fact that each respective ADH gene hybridized about 6fold better to its homologous mRNA than to the heterologous mRNA.
In the first experiment, samples of total yeast RNA were analyzed which contained primarily o d y one or the other of the ADH mFtNAs as detected by the in vitro translation assay. The RNAs which were chosen were extracted from strain 358-21 12 and 26 h after growth on glucose-containing medium (see Fig. 76, Miniprint). These were the respective times when only ADH I mRNA was detected and when ADH I1 mRNA was in 20-fold excess over the amount of ADH I mRNA that was present. Total RNA was fractionated by electrophoresis on agarose gels after glyoxylation (24). The RNA was blotted from the gel into nitrocellulose (21) and hybridized to nick-translated fragments of either ADCI or ADR2. The resultant autoradiograms are depicted in the inset in Fig. 8 (see lanes a and b, Miniprint). Lanes a and b of the upper part of the inset show the hybridization of the ADCl gene to the yeast RNA samples which contained primarily ADH I or ADH 11, respectively, as determined by in vitro translation. Although equal amounts of ADH mRNA were present in each sample, as assayed by the in vitro translation assay, the ADCl gene hybridized about 6-fold better to the sample containing the ADH I mRNA than to that containing the ADH I1 mRNA as ascertained after densitometric analysis of the autoradiograms. When the ADR2 gene was used as the hybridization probe, it hybridized about &fold better to the sample containing ADH I1 mRNA (Zowerpart of inset, lane b ) than to that containing primarily ADH I mRNA (lower, lane a). Furthermore, the sizes of ADH RNA species in the two samples confirm the identity of the RNAs since S1 endonuclease assays indicate that ADH I mRNA is smaller (50 base pairs) than ADH I1 mRNA (23, 29). As seen in lane a of the inset in Fig. 8, the RNA (identified above as ADH I) which hybridized best to the ADCl gene is slightly smaller than the RNA in lane b of the inset (identified as ADH 11) which hybridized best to the ADRP gene. These results confirm that ADH I mRNA declines when yeast are grown into stationary phase, indicating that ADH I RNA is not present at a constant concentration irrespective of growth condition.
Total ADH RNA was also analyzed during growth on ethanol-containing medium. Total yeast RNA extracted from cells that were transferred to medium containing ethanol was separated by agarose gel electrophoresis, blotted to nitrocellulose, and hybridized to each of the ADH genes as described above. Fig. 8, upper inset (see Miniprint) presents an autoradiogram displaying the RNAs which hybridized to the ADCl gene as a function of time after transfer. As can be seen, the total amount of ADH mRNA that hybridized to the ADCl gene declined within the first 3 h of the experiment. After 2 h, the amount of total RNA hybridizing to the ADCl gene increased. An autoradiogram displaying the RNAs which hybridized to the ADR2 gene after the transfer to ethanolcontaining medium is presented in the lower section of the inset in Fig. 8. These results are presented quantitatively in Fig. 8. The amount of each ADH RNA present is normalized to the amount of rRNA in each sample. The amount of each ADH RNA was determined after assuming that each gene hybridized 6-fold better to the homologous RNA than to the heterologous RNA (see above) and by assuming based on S1 endonuclease assays (29) that no ADH I1 RNA was present a t the zero time point. As shown in Fig. 8, the amount of ADH I RNA declined 7-fold within the first 3 h after transfer to medium containing ethanol. Both the kinetics of ADH I RNA decrease and the kinetics of ADH I1 RNA increase were identical with those found for the mRNA levels as measured in the in vitro translation system (Fig. 3b, Miniprint).

DISCUSSION
Previous studies on the regulation of ADH I dealt with its activity as analyzed by starch gel electrophoresis (3, 5). From these studies, it was concluded that ADH I was synthesized constitutively. The results reported here have shown, however, that ADH I is not a constitutive enzyme: the amount of total and functional mRNA coding for ADH I and the rate of ADH I protein synthesis declined 6-to 10-fold after transfer to a nonfermentable carbon source or during growth into stationary phase. Yet, after this decrease, ADH I protein synthesis remained at 0.5-1.0% of total yeast protein synthesis.
Since ADH I was shown not to turn over rapidly, even this reduced rate of its synthesis during growth on a nonfermentable carbon source or during stationary phase would maintain a relatively high level of ADH I activity (i.e. the appearance of its being synthesized constitutively). Thus, the results reported here are not inconsistent with the previous studies. They do indicate, although, the limitation in applying the word "constitutive" to enzymes whose actual rates of protein synthesis have not been obtained.
The data presented in this paper suggest that the ADCl gene is transcriptionally regulated. Recent studies using an endonuclease S1 protection assay also show that total ADH I mRNA declines during growth on ethanol-containing medium (29). In addition, when the ADCl promoter is placed in front of the yeast cytochrome c structural gene, cytochrome c mRNA declines during growth on ethanol-containing medium: implying that the amount of transcription from the ____ -' A. Sledziewski, personal communication.

Regulation of Yeast
Alcohol Dehydrogenase I 1169 ADCl promoter decreases during growth on ethanol-containing medium. The recent finding that the mRNA levels for the fermentative enolase also decrease during growth on nonfermentable carbon sources (28) is suggestive that all the glycolytic enzymes may be regulated. Attempts by us to isolate regulatory mutants which control ADH I synthesis have proven unsuccessful. One mutant, gcrl, which affects several glycolytic enzymes has been described, but its effect on ADH I has not been investigated (11). However, it has been shown to depress the mRNA levels of several other glycolytic enzymes. 5 The results in this paper also indicate that the ADH I1 mRNA is not present during growth on glucose-containing medium, a result which has been c o n f i e d by an S1 endonuclease assay (29). It should be noticed that the amount of ADH I1 mRNA and its rate of protein synthesis is much greater during natural depletion of glucose from the medium (diauxic growth) than during growth on ethanol-containing medium. Whether ADH I and ADH I1 are coordinately regulated in an inverse fashion cannot be ascertained, although regulatory genes affecting ADH I1 mRNA expression appear to have no effect on ADH I expression (22).
The size of the ADH I and ADH I1 mRNAs as measured after agarose gel electrophoresis (1.2 kilobases) conforms to the size as determined by S1-mapping (29). A larger, less abundant RNA also hybridized to the ADCl gene. This RNA species is found during ethanol growth conditions and has been identified as an ADCI transcript commencing 1.2 kilobases in front of the ADCl gene (31). This larger transcript presumably is capable of being translated into an ADH I gene product, for an RNA twice the size of the normal ADH I mRNA has been found to make an ADH I protein in the wheat germ cell-free system. 6 While the gluconeogenic and fermentative reversible reactions in yeast are considered to be catalyzed by the same enzyme, the case of the ADH isozymes indicates that different enzymes for the same reaction may be expressed depending on the needs of the cell. This would occur in much the same way and for a similar purpose as for the classical lactate dehydrogenase isozymes in mammals. Kinetic characteristics of one enzyme would make it more effective for the gluconeogenic pathway whereas a different enzyme would be used for the reverse fermentative pathway. The kinetic parameters of the ADH isozymes have been found to be consistent with their expression (4). The existence in yeast of multiple genes and/or multiple proteins of glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphoglucose isomerase, enolase, and hexokinase suggests that this multiplicity serves this function (12)(13)(14)(15)(16)(17). Recent evidence that the enolase genes are differentially expressed depending on the available carbon source lends additional credence to this view (28). Similar findings have been presented for the two hexokinase isozymes (13,14). This type of general enzyme usage for reversible reactions would add another level of control over the gluconeogenic and fermentative pathways in yeast other than through the regulation of the irreversible M. J. Holland, personal communication. C. L. Denis, unpublished observations. reactions and the flux of substrates through the pathways. Determination of the kinetic parameters of these other isozyme pairs for both the forward and backward reactions which they catalyze would be instrumental in substantiating this pattern.