ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

167, 627-637

(1975)

A Technique for the Isolation of Yeast Alcohol Dehydrogenase Mutants With Altered Substrate Specificity CHRISTOPHER Department

of Biology,

WILLS University

AND JULIA

of California,

PHELPS

San Diego,

California

92037

Received October 4, 1974 Electrophoretically distinguishable mutants of yeast alcohol dehydrogenase with specifically altered kinetics have been obtained using a modification of the ally1 alcohol selection technique of Megnet. The modification depends on the fact that mutant cells producing no detectable ADH or only the ADH sequestered inside the mitochondria cannot become petite. Ally1 alcohol is oxidized by yeast ADH to the poisonous compound acrolein, so petite cells possessing a cytoplasmic ADH will die unless the structural gene is altered to permit the enzyme to distinguish between ethanol and ally1 alcohol. The independent nuclear control of yeast mitochondrial ADH, suspected by other workers, is demonstrated. The fact that strains carrying this ADH cannot become petite provides direct genetic evidence that mitocbondrial and cytoplasmic NAD pools are separate in Saccharomyces cerevkiae.

A central problem in evolutionary biology, as pointed out by Lewontin (1) in his recent book (1974) is to determine what if any relationship exists between the variety of structures of allozymes and other isoalleles and their function. For example, what proportion of isoalleles exhibiting electrophoretic mobility changes is in fact selectively distinguishable? A definitive answer to this question, as Lewontin points out, remains elusive because of our inability to measure single-locus selection coefficients with any accuracy in nature, much less measure a sufficiently large sample of them. Model systems in the laboratory have the advantage that they can be manipulated so that selection can be measured very precisely. Perhaps their greatest disadvantage is that results obtained in the laboratory cannot as yet be generalized to nature. It is our hope that information obtained from model systems can, if sufficiently general physicochemical principles emerge, be so applied. Requirements for a satisfactory model system are: (1) an organism which can be grown under defined conditions and is

easily manipulable genetically; (2) a gene product which can be easily purified and preferably crystallized so that structural and functional studies are facilitated; (3) a selective scheme or schemes which bring pressure to bear on this gene and (hopefully) no other. In our system the first two requirements are satisfied and the third almost so. The organism employed is Saccharomyces cerevisiae and the enzyme is alcohol dehydrogenase (ADH, alcohol: NAD oxidoreductase, E.C. 1.1.1.1.). Alcohol dehydrogenase of S. cerevisiae is, unlike that of Schizosaccharomyces pombe (2) found in multiple forms. The two cytoplasmic enzymes have been determined to be under differential control. One, the ADH-4 of Lutstorf and Megnet (3), which is in all probability the same as the ADH-I of Ebisuzaki and GuzmanBaron (4), is constitutive under aerobic or anaerobic conditions but can be partially suppressed at very high concentrations of glucose (10%) or after a period of aerobic growth. It has, according to Lutstorf and Megnet, an apparent Michaelis constant of 9 mrvr ethanol at pH 8.8. The second, the ADH-2 of Lutstorf and Megnet (3) or 627

Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

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WILLS AND PHELPS

ADH-II of Ebisuzaki and Guzman-Baron (4), is completely repressed under anaerobic conditions or high glucose, but can be induced under aerobic growth with moderate amounts of glucose. When ethanol or glycerol is substituted as a carbon source, ADH-II becomes the predominant enzyme. Its apparent Michaelis constant is 1.2 mM ethanol, measured under the same conditions as ADH-I. The substrate specificities and kinetics of these two enzymes are so different as to strongly suggest that they are coded by different structural genes. Production of ADH-II can be repressed by addition of cycloheximide (3). In addition to these enzymes, a third NAD-dependent enzyme or enzymes is normally present. It has been variously reported as a diffuse, slowly migrating band (3) or five electrophoretically separable bands (5) which form the classic pattern seen when all combinations of fastand slow-migrating subunits of a tetramer come together. This enzyme is associated with the mitochondrial fraction (6, 7), and is of considerable theoretical interest because, although it is somewhat repressed on high glucose (S), it is present even under anaerobic conditions (see below). Because the enzyme is sequestered in the mitochondria, it can act directly on ethanol which diffuses into the mitochondria, making more of the energy in this molecule directly available for oxidative phosphorylation through the production of NADH within the mitochondrion (9). All these enzymes may be structurally related to each other, since they appear to form hybrids. It was our intention, in beginning work on this fairly well-characterized system, to investigate the possibility of evolving yeast ADHs to different substrate specificities by using selective agents. Before this could be done, the physiology and genetics of the system had to be investigated. It is still unclear just how many structural and regulatory genes are involved in ADH production. Further, normal cells cannot, as will be detailed below, be selected for altered ADH; they will mutate instead to strains lacking ADH activity. The purpose of this

paper is to present preliminary information on the genetics of the ADHs and the physiological properties of mutant strains that allow the enzymes to be altered by selection. EXPERIMENTALPROCEDURE Materials. Wild-type strains of S. cereuisiae X2180-1A (mating type a) and X2180-1B (mating type a) were derived from a diploid originally isolated by M. Resnick. This diploid is homozygous at all loci except the mating type locus, so that haploids derived from it are genetically very similar. Petites were isolated from these strains either from spontaneously occurring events or by treatment with ethidium bromide (10). A petite strain III. 1.7, with no detectable mitochondrial DNA, and its parent strain A664a/18A were kindly provided by Dr. J. Marmur. For biochemical studies, cells were grown in liquid at 30°C in a New Brunswick G25 air-bath shaker. In most cases, rotary shaking was sufficient, but for some mutant strains (particularly those that lacked detectable ADH activity) forced aeration was necessary. Aeration was provided from the laboratory air supply through two presterilized glass wool-filled traps. Anaerobic growth conditions were obtained by growing the cells in a New Brunswick 350-ml capacity Model C30 fermentor through which nitrogen was bubbled continuously. Oxygen was monitored with a galvanic oxygen electrode, sensitive to one part in 1000, throughout the anaerobic growth period. No detectable trace of oxygen was found throughout the experiments. For selection of mutants, a variety of solid selective media were employed. The basic complete medium was YEPD (1% yeast extract, 2% Bactopeptone, 2% dextrose, 2% agar, all w/v, all from Difco). The basic minimal medium consisted of 0.75% Yeast Nitrogen Base w/o amino acids, 2% dextrose and 2% agar, all w/v, all from Difco. Modifications of these basic formulas will be noted throughout the paper. Methods. Cells were disrupted by three different methods. The first employed an MSK liquid CO$cooled ball mill, in which 10 g wet weight of prewashed cells were placed in a tempered glass bottle along with 50 g of 0.5-mm diameter glass beads, prewashed in acid, and 10 ml of 0.1 phosphate buffer, pH 7.0, shaken at full speed for 30 seconds, decanted, and spun at 0°C at 13,060 rpm in the 534 head of a Sorvall RC2B centrifuge for 20 min. The supernatant was then treated with 15 ml/100 ml of 1.5% (w/v) protamine sulfate in the same buffer and again spun at 13,000 rpm for 20 min. The clear supematant could be frozen for several months at -76°C without noticeable loss of ADH activity. The second method, which gave results very simi-

SELECTION lar to the first, involved collecting the cells by centrifugation, washing once with 0.05 M phosphate buffer, pH 7.0, and removing the buffer by filtration in a Buchner funnel. An equal amount (w/w) of 80-100 mesh alumina, which had been washed repeatedly in distilled water to remove fines, was added to the cake in a prechilled mortar surrounded by crushed ice. The cells were ground vigorously for about 20 min, or until the mixture of cells and alumina became liquid in consistency. This method had the advantage that very large or very small amounts of cells could be ground. After grinding, the cells were extracted for 5 min with stirring in 4x the original cell amount (v/w) of buffer (the buffer varied depending on the experiment), then centrifugation and treatment with protamine sulfate was carried out as in the first method. No detectable difference was seen in the specific activities of the crude extracts prepared by these two methods. The third method was used to separate the cytoplasmic and mitochondrial fractions. The method of Leon and Mahler (11) was used without modification. It involves treatment of cells in sorbital medium buffered at pH 5.8 with glusulase (Endo Laboratories) to make spheroplasts, disruption of the spheroplasts by brief (15-20 s) agitation at low speed in a Waring Blendor and differential centrifugation in the appropriate high OP medium to separate the motochondria. The brownish-red .mitochondrial pellet was obtained in a yield comparable to that obtained by the above authors (l-2% of total cell protein). Both pellet and cytoplasmic supernatant could be saved without diminution of activity at -76°C. Electrophoresis of the cell extracts was performed by the horizontal starch method of Smithies (12). The gel buffer was Poulik, pH 8.6 (9.21 g Tris, 1.05 g citrate per liter) and the box buffer was borate, pH 8.1 (18.55 g boric acid, 2.4 g NaOH per liter). Electrophoresis was carried out for times varying from 2 ‘/z to 4 h at 250 V, resulting in an average current flow of about 60 mA. Visualization of the enzyme activity was accomplished by developing the gel in 100 ml of 0.1 M phosphate buffer, pH 7.0, in which was dissolved 25 mg NAD+, 25 mg nitro blue tetrazolium and 4 mg phenazine methosulfate (all from Sigma). To this solution, before it was poured on the gel, was added 1 ml 95% ethanol. It was found that the commercially available NAD+ usually contains a small amount of ethanol, so that faint ADH bands would appear even without the additio.n of substrate. Normally this did not pose a problem, but when NAD+-dependent enzymes other than ADH were being investigated this difficulty could he circumvented by dissolving the NAD in distilled water, freezing and lyophilizing overnight. Normally, satisfactory visualization of bands could be obtained from paper wicks dipped directly in the

629

OF YEAST ADH

crude extract. When there was very little activity present, however, the gel was poured around acrylamide slot formers and the slots so produced were filled with extract. This was particularly useful in visualizing the ADH obtained from gradient fractions. Glycerol in the medium was determined by the method of Burton (13), protein by the method of Lowry (14), ethanol by enzymatic reduction of NAD using commercial yeast ADH (Sigma). RESULTS

The Normal Yeast ADH Pattern

In the wild-type strains which we employed, the pattern of bands seen varied greatly depending on the extraction procedure. Cells grown with moderate aeration in liquid YEPD and ground in the MSK homogenizer or with alumina showed a great variety of bands, some of which correspond to those observed by other workers. Figure 1A shows an aluminaground strain grown for 48 hours on YEPD. Note the two dark rapidly-migrating bands with a faint band between them. The slowest of these is the constitutive ADH-I; the fastest is the inducible ADH-II, which in this culture has been induced to a greater extent than the constitutive ADH-I. Near the origin the set of five mitochondrial ADH bands can be seen clearly. There is also an intermediate set of bands, the number and intensity of which vary greatly depending on the physiological conditions of the cells and the conditions of disruption. Figure 1B shows a grande strain grown for 48 h under nitrogen. ADH-II is no longer present, nor is the faint hybrid band between ADH-I and ADH-II. All five mitochondrial bands are still present, however. The intermediate set of bands has now changed markedly in configuration; now only the slowest of these bands are present. Figure lC, in which the bands are displaced towards the origin because the gel was run for a shorter time, shows the pattern obtained from a diploid spontaneous petite, grown for 48 h on YEPD. As would be expected, since oxidative phosphorylation is now disrupted, the inducible AD-II is no longer present. In this particular petite, though not in all, only the

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+

0 A +

B

C

second shows the cytoplasmic fraction, consisting primarily of ADH I. (ADH-II was not present because satisfactory spheroplasts can only be obtained from logphase cells.) The third shows a mixture of the cytoplasmic and mitochondrial fractions. Note the absence of intermediate bands. Dr. Stephen D. Young in our laboratory (unpublished results) has succeeded in producing the intermediate bands by grinding together a strain containing only the mitochondrial bands with one containing only the cytoplasmic bands. This suggests that (a) the intermediate bands are indeed an artifact of extraction, (b) the cytoplasmic and mitochondrial enzymes may be sufficiently closely related to form hybrids although they apparently do not do so in the cell or (c) the “hybrid” bands are breakdown products which still retain activity. Molecular

Weight of the Mitochondrial ADH

A number of estimates of the molecular weight of cytoplasmic ADH have been 0 made (15, 16) averaging about 150,000. To determine whether the mitochondrial enFIG. 1. Activity-stained ADH patterns. Crude ex- zyme is comparable in size to the cytoplastract was electrophoresed for two hours, as detailed in mic, a 15-35s glycerol density gradient Materials and Methods. (A) The pattern obtained layered with the mitochondrial fraction after alumina grinding of wild-type X2180. (B) The was run for 16 h in the SW50.1 swinging pattern obtained from the same strain after 48 h bucket head of a Beckman L3-50 centrifuge growth under nitrogen. (C) A diploid spontaneous at -2”C, using catalase and bovine serum petite; the heavily-staining band is actually ADH-I, albumin as markers according to the but in this preparation migration did not proceed as method of Martin and Ames (17). The far as in the other gels. (D, E, and F) The cytoplasmic, results are shown in Fig. 2. Similar results mitochondrial and pooled fractions, respectively, from disrupted spheroplasts. (G) The pattern ob(not shown) were obtained for the cytoplastained from the activity peak of a glycerol density mic enzyme. The mitochondrial and cytogradient of the mitochondrial enzyme. plasmic enzymes have similar molecular weights, within the limits of the resolution slowest-moving mitochondrial band is of the method. Figure 1G shows the electropresent in appreciable quantity. Once phoretic pattern obtained from the mitoagain, the intermediate band reflects the chondrial fraction with the greatest activposition of the cytoplasmic bands; in this ity on the gradient; all five bands are still case only the slowest of the intermediate present. The five-banded pattern seen with the bands is present. In Figs. lD, E, and F are shown the mitochondrial enzyme strongly suggests results when the cells are disrupted as that it is a tetramer made up of two slightly spheroplasts. The first shows the mitodissimilar types of subunits. It is already chondrial fraction, which contains predomknown from physicochemical studies that inantly the five slow-moving bands. The the cytoplasmic enzymes are tetramers

SELECTION

(18). Since cytoplasmic YADH is a halfsite enzyme, in which.under normal conditions only two of the four subunits are involved in catalysis (19), this may help to explain the formation of only a single hybrid band (20). Production

of ADH

Deficient

Mutants

By utilizing ally1 alcohol as a selective agent, Megnet (2) and Lutstorf and Megnet (3) were able to isolate mutants of Schizosaccharomyces porn be and S. cereuisiae partially deficient in ADH activity. We have been able to carry their method a step further and produce strains lacking any detectable ADH activity when grown under normal conditions. The technique is as follows: yeast ADH will normally oxidize ally1 alcohol (CH2= CHCH*OH) to acrylaldehyde (acrolein), which is poisonous to the cell. Its specific activity for ally1 alcohol is generally slightly higher than that for ethanol. Lutstorf and Megnet grew cells in glucose in the presence of very low concentrations (0.1 mM) ally1 alcohol, and picked up mutants

OF YEAST

631

ADH

deficient in ADH-I, although the inducible ADH-II remained unaltered. Our initial hope was to pick up mutants, using the same technique, of all the ADHs in the cell which would enable us to determine how many structural and regulatory genes were involved. Our wild-type strains were much more resistant to ally1 alcohol than were those of Megnet. Further, our strains grew quite well on 2% ethanol as a sole carbon source, provided that the minimal medium was supplemented with 0.1% Bacto-peptone. The following selective scheme was employed: 10’ cells/plate were plated out on medium containing 0.75% yeast nitrogen base without amino acids, 2% ethanol, and 0.1% Bacto-peptone, with the addition of 1 mM ally1 alcohol. Mutant colonies appearing after a week of incubation at 30°C were plated on the same medium containing 2 mM ally1 alcohol. The procedure was repeated through 5 and 10 mM ally1 alcohol. Continued selection on this medium was unsuccessful. When a large number of these mutant cells were plated on YEPD

BODlmin

-150-

Cafolase AOH BSA

0t s - 0.30

0.15 - 0.26

‘\

loo-

O.lO-

i i i

-024 -0.20

i

i

-0

I6

-0.12 50-

0.05- 0.06

Colalose

.

- 0.04 - BSA

ADH -

FRACTION

NO.

FIG. 2. Determination of molecular weight of mitochondrial yeast ADH on a 1535% glycerol density gradient using catalase and bovine serum albumin as markers. The calculated molecular weight of the ADH at maximum activity was 140,084, approximately the same as published figures for the cytoplasmic ADH. Note the heavy shoulder on the peak, however; this was repeatable and may indicate some binding of the mitochondrial ADH to membrane elements or other molecules.

632

WILLS AND PHELPS

with 2% (v/v) ally1 alcohol, however, mutant colonies appeared after a week at a frequency of about 10ml. These mutants could grow, though with difficulty, at concentrations of up to 10% ally1 alcohol. Ten independent mutants of this type were derived from each mating type. All possible crosses were made, and none exhibited complementation; all the diploids were capable of growing on 2% ally1 alcohol. Extracts of these mutants, grown in liquid YEPD and tested on gels or in the spectrophotometer by the method of Vallee and Hoch (21), showed no detectable ADH activity. These strains would no longer grow on ethanol as a sole carbon source. When the cells were grown on YEPD, washed in 0.05 M phosphate buffer, pH 7.0, and transferred for 24 h to liquid medium containing 2% ethanol as a sole carbon source, they did however show a slight amount of ADH activity detectable in the spectrophotometer though not in gels. This activity corresponded to 0.13 unitsfmg protein, compared to 2.4 units/mg for normal extracts of wild-type cells (one unit reduces 1 micromole of NAD per minute at 25°C under the assay conditions). Because the activity could not be visualized on a gel, it was impossible to determine which of the ADH’s was responsible. That more than one mutational step had to occur in order to produce these ADHnull strains is apparent from two facts: the selection had to proceed stepwise through a series of increasing concentrations of ally1 alcohol, and it has not proved possible, despite repeated attempts using spontaneous mutation, nitrosoguanidine or EMS treatment, to go in a single step from wild-type to ADH-null. When the ADH-null strains XW520-9A (a) or XW520-9B (a) were backcrossed to wild-type, from a total of 18 tetrads only one null mutant was observed. This indicates a large number of mutant genes (5-7). Out of this total of 72 spores three showed a high rate of mutation to null. One of these was in the same tetrad as the single null mutant, and happened to be of the opposite mating type. When nonresistant

cells of this strain were crossed with the null, the null character segregated out 2:2 in 5 tetrads. On investigation it was found that the nonnull spores in these tetrads carried only the five mitochondrial bands. The null spores showed, as before, no detectable ADH activity. We can therefore say with some certainty that the uppearante of the mitochondrial enzymes is controlled by a single nuclear gene, which is independent of those controlling the cytoplasmic enzymes. Since electrophoretic mobility mutants of the mitochondrial bands have not yet been obtained, it is not possible to tell whether this mutation is regulatory or structural, though we suspect it may be the former. In order to be structural it would have to affect both subunits of the mitochondrial ADH. This might in turn imply that one mitochondrial ADH subunit is produced by posttranscriptional modification of the other. Physiology of the Mutant Strains The mutant strain of Lutstorf and Megnet, A63, had ADH-II still present, though it did not appear until glucose was depleted. As a consequence, the operation of Neuberg’s second form of fermentation would be expected to result in high concentrations of glycerol before glucose depletion. This form of fermentation, which does not result in a net gain of ATP, results when the NAD-dependent cytoplasmic glycerol phosphate dehydrogenase reduces dihydroxyacetone phosphate to glycerol3-phosphate, which is in turn converted to glycerol. As glucose diminishes and ADHII is turned on, surplus NADH is converted to NAD+ with the concomitant production of ethanol, and the glycerol concentration will drop. This is in fact what is seen (their Fig. 7). When our ADH-null strains were grown with forced aeration and tested for glycerol production, a continued rise of glycerol in the medium was seen past the 24-h period, as would be expected since the ADH-II could no longer be turned on (Fig. 3). When a strain carrying the mitochondrial ADHs only, XW521-lA, was tested under identical conditions, it showed an early rise in glycerol production followed

SELECTION

by a drop to near control levels (Fig. 4). Ethanol was produced by this strain, indicating that the mitochondrial ADHs were functional. Because the second form of fermentation produces no net gain in ATP, ADH-null cells which cannot rely on oxidative phosphorylation should not be able to survive as petites. As can be seen from Fig. 5, treatment of null mutants with ethidium bromide, which normally induces cytoplasmic petites with almost 100% effectiveness within one or two cell divisions, results in a killing curve instead. Repeated attempts to produce ADHnull mutants from petite cells, and to isolate spontaneous petites from ADH-null cells, have failed.

ok---%-

20

/ ; 30 TIMElhrrl

I 40

,

, 50

FIG. 3. Production of glycerol over time in wildtype and ADH-null strains grown in a rotary shaker with moderate aeration. XW520-9A was a consistently more rapjd grower than XW520-9B; this probably explains the more rapid rise in glycerol concentration.

---.___ *------.---....._._.__ ._._.... iLLLLL”““““‘r”r*’ IO 20 30 40 50

0

TIME (hrsl

FIG. 4. Glycerol production in a strain carrying only mitochondrial ADH compared with the wildtype. After an initial burst in each case, glycerol fell to a low and constant level. Slightly more glycerol was produced in the mutant than in the wild type.

OF YEAST ADH

633

Figure 5 also shows that treatment of the strain XW521-lA, which has the mitochondrial ADH, results in a killing curve as well. Selection for ADHs With Altered Electrophoretic Mobility

The fact that petite strains cannot become null provides a technique for selecting an alcohol dehydrogenase with an altered substrate specificity. At the same time, it has proved possible to determine whether the two cytoplasmic enzymes, ADH-I and ADH-II, are coded by different structural genes. While the grande strain A664a/18A from which Marmur’s group derived III-l-7 (the cytoplasmic petite lacking any trace of mitochondrial DNA), has an ADH-I indistinguishable electrophoretically from our wild-type strains, its ADH-II migrates slightly more slowly. This mobility difference segregates 2:2 in tetrads. The Marmur petite strain 111-l-7, upon electrophoresis, exhibits only ADH-I and the mitochondrial bands. Large numbers of these petite cells (approximately lO’/plate) were spread on plates consisting of YEPD, 2% ethanol (conditons under which the ADHs were fully derepressed), and levels of ally1 alcohol that were increased stepwise throughout the selection procedure. Spontaneous mutants picked up at each level of ally1 alcohol concentration were cloned and then plated at the next highest concentration. Four separate lines were produced that could grow well when selected consecutively at concentrations of 25, 50, 60, 100, and 120 mM ally1 alcohol. One clone was taken from each line grown on 120 mM plates. Upon electrophresis, three of the four strains were found to be apparently unchanged, though the activity of the enzyme in crude extracts was greatly reduced. The fourth showed a pronounced shift of ADH-I towards the origin. Upon backcrossing to X2180-lA, this mutant ADH-I was found to segregate independently of the slowly-migrating ADH-II. All the spores of this tetrad were, of course, grande, so that ADH-II could be detected. It seems likely, therefore, as was inferred from non-genetic data by Lutstorf

634

WILLS

AND

PHELPS

300 c

. -

X2180-IA 0 - PETITE COLONIES

0 TIME (MINUTES)

1

I

I

20

40

60

--------_ --_--__-_

120

180

TIMEfMINUTESI

FIG. 5. (A) Normal induction of petites in wild-type cells by treatment with 10 pg/ml ethidium bromide. (B) Killing curves resulting from treatment of ADH-null and strains with mitochondrial ADH with 10 pg/ml ethidium bromide. The few cells that survived this treatment were found to be grande cytoplasmic mutants resistant to ethidium bromide treatment.

and Megnet (8), that ADH-I and ADH-II are coded by separate structural loci. The existence of an ADH-I with such pronouncedly different overall charge permitted a first test of the hypothesis that the mitochondrial enzymes are also coded independently. XW530A, which carries a normal ADH-II and a mutant ADH-I, was electrophoresed in alternate slots with X2180-1A for four hours, in order to enhance any possible mobility differences between the bands of the mitochondrial enzymes of the two strains. None could be detected (Fig. 6). This preliminary evidence suggests that the cytoplasmic and mitochondrial enzymes do not share subunits in common, except when hybrids are formed. That the mutant enzyme was in fact conferring the resistance to ally1 alcohol was checked by crossing the strain carrying it to a grande wild-type. Tetrads from this cross were converted to cytoplasmic petites by treatment with ethidium bromide, and these petite tetrads replica-plated onto YEPDE plus 120 mM ally1 alcohol. Two out of the four meiotic products grew in all seven tetrads tested. When one tetrad was investigated by electrophoresis, it was

found that the mutant ADH-I was present in the two meiotic products that grew. This indicated that the majority of the ally1 alcohol resistance could be traced to the mutant enzyme, though it does not entirely rule out a role of other genes. The kinetics of this mutant have been investigated. The mutant and wild-type ADH-I were purified by affinity chromatography (Wills and Phelps, in preparation) and Michaelis-Menten kinetics for ally1 alcohol and ethanol determined at pH 8.8. The K, for ethanol remained about the same in both (9.55 mM and 8.89 mM for the wild-type and mutant respectively; the K, for ally1 .alcohol showed a drop in the mutant to 7.13 from 12.9 mM. There was a large shift in the ratio of the ally1 alcohol to ethanol V, that for the wild-type being 2.34 and that for the mutant being 1.09. DISCUSSION

An extension of the ally1 alcohol selective system of Megnet is proving to be a powerful tool in dissecting the genetics and physiology of yeast ADHs. Much further work is required to determine the number and nature of the mutants that together make up the ADH-null phenotype. Un-

SELECTION

OF YEAST

635

ADH

operating. In batch culture, null mutants can only be grown successfully under forced aeration. Excess glycerol-3-phosphate from the DHAP-G3P cycle is converted to glycerol. Null mutants therefore cannot survive as petites. Rather more difficult to understand is the fact that strain XW521-lA, which has the mitochondrial ADH but not the cytoplasmic ones, also cannot survive as a petite. An explanation consistent with the foregoing is as follows: when the strain carrying only the mitochondrial ADHs is by the grande, NAD + is produced DHAP-G3P shuttle. This is sufficient to drive glycolysis fairly normally, since the mitochondrial ADH is still available to produce ethanol as the glycolytic endproduct. As a result, there is not as much surplus G3P, and after an initial peak glycerol production drops off. When mitochondrial function is disrupted, DHAP builds up, no more cytoplasmic NAD+ is produced, and glycolysis comes to a halt in spite of the presence of the mitochondrial ADH. In a grande strain of this type,

doubtedly they are a combination of structural gene mutations, a mutation or mutations than enhance glucose repression of ADH-I and ADH-II, a mutation or mutations that prevent the oxygen induction of ADH-II, and-the only one that has been successfully isolated so far-a single-step mutation that in some fashion stops the production of all five mitochondrial ADHs. It is still unclear whether this last mutation is structural or regulatory. A physiological explanation of the behavior of the strains lacking all ADH activity and those lacking cytoplasmic ADH activity seems to be fairly straightforward, and is diagrammed in Fig. 7. With no cytoplasmic ‘4DH, all cytoplasmic NAD + must be produced by the DHAPG3P pathway. The G3P diffuses into the mitochondrion, passes on its electrons at the level of coenzyme Q, and is reconverted in the process by a flavoprotein enzyme to DHAP. In the null mutants, energy production must be absolutely dependent on the electron transport chain of the mitochondria being intact. Glycolysis is not +

A FIG. 6. Normal comparison, mitochondrial cytoplasmic

6

A

6

A

B

(B) and mutant (A) forms of ADH-I electrophoresed for four hours. For ease of three samples of each were placed on the same gel. Note that relative positions of the ADH hands have not shifted in the normal and mutants strains, though the ADH shows a marked cathodal shift.

636

WILLS AND PHELPS

*NAD+

G3-PFlavoprofein ,GP dehydrogenose

NADH 3 tH+

OHAPr GlyAdeyde 3-phosphate I

Net qoin of 2 ATPs through

A

b

ATP

I?- ATP

; !

Because null strains cannot become petite and petites cannot become null, we expect that these strains will provide a powerful method for selecting yeast alcohol dehydrogenases with altered specificity for a variety of substrates. This technique has the advantage that enzymes with altered kinetics have a high probability of being selected for, in contrast with the situation encountered by Hartley and co-workers (22) in which mutants of the organism (Klebiella aerogenes) could grow more rapidly by simply making more enzyme. ACKNOWLEDGMENTS

*Cytaplosmic

sources

of NAOt

We wish to acknowledge helpful discussions with Drs. Peter Carlson, Raymond Valentine, and Stephen D. Young, and the assistance of Ms. Susan McOmber and Messrs. Gary Bennett and Jeffrey Root. Dr. Julius Marmur kindly supplied a petite strain free of mitochondrial DNA. Supported by Grants GM14700 and GM19967 from the Public Health Service.

7. The glycolysis and oxidative phosphorylation pathways that are likely to be affected by the ADH mutants discussed in the paper. On the left are diagrammed the only cytoplasmic sources of NAD’ available to the cell. ADH-null mutants have no mitochondrial or cytoplasmic ADH activity (blocks symbolized by heavy lines). Glycolysis is therefore not operative in these cells, and the only energy source available is through the DHAP-G3P shuttle with the resultant production of ATP in the mitochondrion. Excess G3P is converted to glycerol. A petite null mutant cannot survive, since the electron transport chain of the mitochondria is disrupted and neither glycolysis nor oxidative phosphorylation function. A mutant blocked in only the cytoplasmic ADHs (block symbolized by a dotted line) is also unable to survive as a petite. Here, glycolysis is operative but is directly coupled to oxidative phosphorylation, since the only source of cytoplasmic NAD+ comes from the shuttle. The existence of the mitochondrial ADH allows ethanol to be produced as an end-product of glycolysis, but the NAD+ produced remains sequestered inside the mitochondrion. Because glycolysis is at least partially operative, glycerol does not build up markedly. When the electron transport chain is disrupted, however, so is the DHAP-G3P cycle. No more cytoplasmic NAD+ is produced, and the cell dies.

10.

glycolysis and oxidative phosphorlyation are coupled. If our interpretation is correct, these strains provide direct genetic evidence for the separation of the NAD pools in the mitochondria and cytoplasm of yeast.

11. LEON, S., AND MAHLER, H. R. (1968) Arch. Biohem. Biophys. 126, 305-319. 12. SMITHIES, 0. (1955) Biochem. J. 61, 629-641. 13. BURTON, R. M. (1957) in Methods of Emzymology, (Colowick, S. P., and Kaplan, N. O., eds.), Vol. III, pp. 246-249, Academic Press, New York.

FIG.

REFERENCES 1. LEWONTIN, R. C. (1974) The Genetic Basis of Evolutionary Change, Columbia University Press, New York. 2. MEGNET, R. (1967) Arch. Biochem. Biophys. 121, 194-201. 3. LUTSTORF, U., AND MEGNET, R. (1968) Arch. Bio&em. Biophys. 126,933-944. 4. EBISUZUKI, K., AND GUZMAN-BARON, E. S. (1957) Arch. 5.

6. 7. 8.

9.

Biochem.

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