Molec. gen. Genet. 152, 193-200 (1977) © by Springer-Verlag 1977

Pyruvate Kinase Mutants of Saccharomyces cerevisiae: Biochemical and Genetic Characterisation P.K. M a i t r a and Zita L o b o Tara Institute of Fundamental Research, Bombay 400005, India

Summary. M u t a n t s o f Saccharomyces cerevisiae lacking p y r u v a t e kinase (EC 2.7.1.40) are described. These have less than 0.5% o f the pyruvate kinase activity of the wild type. All the other glycolytic enymes are present in n o r m a l a m o u n t s in these mutants. The m u t a t i o n is recessive and segregates in diploids as a single gene. Five alleles examined fail to complem e n t one another. Tetrad analysis and mitotic rec o m b i n a t i o n data place the m u t a t i o n on the left a r m o f c h r o m o s o m e I distal to cys 1. The majority of single-step s p o n t a n e o u s revertants on glucose regain the enzyme activity fully and this activity appears, by a n u m b e r of criteria, to be due to the same enzyme present in the wild type. Some of these revertants become nuclear petites. The mutants do neither grow on n o r ferment sugars but do grow on ethyl alcohol or pyruvate. Glucose addition to cultures growing on alcohol arrests growth until glucose is exhausted. The steady state rate of glucose utilization is slower than in the wild type. This is associated with the a c c u m u l a t i o n o f as m u c h as 5 gmoles P-enolpyruvate per g wet weight o f cells and p r o p o r t i o n a l a m o u n t s of 2-P-glyceric and 3-P glyceric acids. The m u t a t i o n is believed to involve some regulatory element in the synthesis o f pyruvate kinase.

Introduction Pyruvate kinase (EC 2.7.1.40) plays a crucial role in the glycolytic b r e a k d o w n o f sugars. F o r facultative organisms in particular, this enzyme is responsible for their ability to grow in absence o f oxygen generating A T P and also the electron acceptor either as pyruvate or acetaldehyde that confers on glycolysis its red-ox a u t o n o m y . It is expected that a functional block o f this enzyme would be deleterious to systems which sustain themselves anaerobically. A partial de-

ficiency o f pyruvate kinase in the h u m a n erythrocytes has been studied by Rose and W a r m s (1966). Recently mutants lacking this enzyme have been reported in Aspergillus nidulans ( P a y t o n and Roberts, 1976) and in Saccharomyces cerevisiae (Lain and M a r m u r , 1975 ; Clifton et al., 1977). All these mutants fail to grow on sugars either singly or in c o m b i n a t i o n with an otherwise permissible gluconeogenic c a r b o n source. The locus has been also m a p p e d in Aspergillus. We have been looking for sometime for mutants lacking the sugar carrier in the yeast S. cerevisiae. In course of this we have isolated a n u m b e r o f pyrurate kinase m u t a n t s with m u c h the same phenotype as reported by these authors. We report here a n u m b e r o f experiments characterising these mutants biochemically a n d genetically. Some o f these results have been presented at a recent meeting (Sinha et al., 1976).

Materials and Methods Strains. A wild type haploid stock of S. cerevisiae of mating type a was used as the parent strain. This was obtained from Dr. S.N. Kakar of Haryana Agricultural University at Hissar. The strain is prototrophic, carries the killer character (Sherman, Fink and Lawrence, 1974) and has a high respiratory activity. Other strains for crosses and genetic analysis were obtained from the Yeast Genetic Stock Center at Berkeley. N-Methyl-N1-nitro-N-nitrosoguanidine was used for isolating the pyruvate kinase mutants as described earlier (Maitra, 1970). Mutagenised cells were allowed to grow aerobically in YEP media containing peptone and yeast extract (10 g and 3 g per litre respectively) supplemented with 150 mM ethyl alcohol. Master plates were made in solid media with alcohol and replica plates in alcohol and 50 mM glucose separately. Alcohol-positive and glucose-negative colonies were picked, purified on alcohol medium and assayed for glycolytic enzymes in toluene lysates (Maitra and Lobo, 1971). Amongst nearly 30,000 colonies so screened, about 30 were glucosenegative of which 5 had lost greater than 99% of the wild type pyruvate kinase activity. To obtain spontaneous single-step revertants, plates containing the glucose media were heavily seeded with the mutant culture

194

P.K. Maitra and Z. Lobo: Pyruvate Kinase Mutants of Yeast

and incubated at 30 ° C. Occasional colonies showed up against a background of very faint smear. These were purified and maintained on glucose plates. The reversion frequency for all the mutants was of the order of 10-8. Table 1 shows a list of strains used.

Assay of Enzymes and Substrates. Low levels of pyruvate kinase were measured by estimating pyruvate produced from 1 m M Penolpyruvate in a reaction mixture containing cell extracts or toluene lysates, 1 m M ADP, 1 m M fructose 1, 6-diP, 10 m M MgC1 z and 50 m M triethanolamine hydrochloride neutralised to pH 7.3 with KOH. Reactions were stopped at specified intervals by adding perchloric acid to a final concentration of 0.7 N. Pyruvate was measured fluorometrically on the neutralised supernatants using

Table 1. Strains used Strain

Immediate parent

Markers

Manner in which obtained

HSC

a wild type

Dr. S.N. Kakar

X2928-3D-1C

adel gall leul his2 ura3 trpl metl4 adel a ~ lys7 trp3 met2 arg4

Yeast Genetic Stock Center

X3405-1A

cysl-5

NH4-11C

Yeast Genetic Stock Center Dr. S. Fogel

PK1 ; PK2; PK4; PK5

HSC

a pyruvate kinase mutant

Mutagenised

PK6

711 (Maitra, 1970)

a

Mutagenised

PK2R1R

PK2R1

Spontaneous reversion on alcohol

Table 2. Enzyme activities of mutants lacking pyruvate kinase, their revertants and the wild type parent. Enzyme assays and respiratory rates refer to stationary phase cultures Strain

Grown on

Enzyme units/mg protein"

Rate of respiration mpmole 02/ m i n x E650

HK

PFK

PK

Wild type Alcohol Glucose

1.70 3.01

0.09 0.18

2.14 3.06

38.6 21.6

PK2

3.66

0.11

0.01

25.3

Alcohol

PK2R1

Glucose

2.32

0.19

4.46

0.4

PK2R1R

Alcohol Glucose

2.32 2.31

0.09 0.12

2.72 3.64

9.8 6.5

PK2R4

Glucose

3,8

0.17

5.80

27.2

PK2R6

Glucose

1.4

0.24

0.085

13.6

PK5

Alcohol

1.7

0.11

0.006

55.0

PK5R3

Glucose

2.6

0.37

6.05

34.4

HK, hexokinase; PFK, fructose-6-P kinase; PK, pyruvate kinase

a

D P N H and lactic dehydrogenase. Appropriate blanks were included that lacked the enzyme solution or ADP. The assay was first order with respect to the enzyme activity. Standard fluorometric procedures were employed in determining high levels of pyruvate kinase, other glycolytic enzymes and intermediates (Maitra and Lobo, 1971). A unit of enzyme activity referred to 1 gmole substrate converted per minute at 23 ° C. For the assay of 1,3-diP-glycerate in cell suspensions, the reaction was stopped by adding 2 ml cold acetone to 1 ml of the suspension, the mixture shaken vigorously, chilled and centrifuged. The supernatant was kept in a shallow tray placed over ice and air blown over it till acetone could be smelt. An aliquot of the aqueous residue was used for estimating 1,3-diP-glycerate and 3-P-glycerate using glyceraldehyde 3-P dehydrogenase and P-glycerate kinase (Rose and Warms, 1970). The assay of the diphospho glycerate was completed within 8 minutes since addition of acetone to the yeast suspension. Acetone could extract only 20% of the 3-Pglycerate levels found in 0.7 N perchloric acid extracts. Since 1:3diP-glycerate could hardly be measured in acid extracts it was routinely assayed in acetone extracts prepared as above and the observed values multiplied by 5. For both p-enolpyruvate and 3-P-glycerate the corrected levels agreed to within 75% of the respective values observed in perchloric acid extracts, c~-Glycero-P and glycerol were estimated spectrophotometrically or fluorometrically in a glycine-hydrazine buffer using c¢-glycero-P dehydrogenase and glycerol kinase (Wieland, 1963). Ethyl alcohol was similarly assayed using alcohol dehydrogenase.

Genetic Analysis. Mating was done in YEP-alcohol plates and diploids isolated by prototrophic selection on minimal-glucose plates. Sporulation was induced in standard sporulation plates (Sherman et al., 1974) devoid of glucose without going through a presporulation medium. Spores were digested with glusulase or snail gut juice and were analysed by tetrad dissection using a sliding micromanipulator from Carl Zeiss with an Ergaval microscope. The dissection slab was made of YEP medium solidified with 2% agar. For occasional random spore analysis the enzyme-treated spore suspension Was freed from diploid cells by repeated extraction with water of a mineral oil phase in which spores stayed preferentially (Emeis and Gutz, 1958). Spores were allowed to germinate in YEP-alcohol plates. All other methods were as described by Sherman et al. (1974). The segregation of pyruvate kinase phenotype in spore arrays was followed by measuring the enzyme activity in toluene lysates. A loopful of cells (107-10 s cells) from a streak on YEP-alcohol plates was suspended with a platinum wire in 0.5 ml of 50 m M potassium phosphate buffer containing 2 mM 2-mercapto-ethanol and 2 mM EDTA at pH 7.4 to which 50 ~tl of toluene was added. The suspension was shaken at 37 ° C on a reciprocating shaker for 30 minutes and this was used (1 to 100 gl) directly for enzyme assay. The toluene tysate was kept at 23 ° C and not in an ice bath as pyruvate kinase is cold-labile.

Results 1. Glycolytic E n z y m e Activities in Pyruvate Kinase M u t a n t s The mutants we all t h e g l y c o l y t i c kinase. In Table of these enzymes revertants. Their

describe here were found to have enzyme activities except pyruvate 2 we show the levels of a number for two of these mutants and their respiratory rates are also shown;

P.K. Maitra and Z. Lobo: Pyruvate Kinase Mutants of Yeast these refer to rates of oxygen consumption using 100 m M ethanol determined polarographically as described earlier (Maitra, 197l). These results show that the mutants have less than 1% of the wild type levels of pyruvate kinase activity, with little change in hexokinase or fructose 6-P kinase activity. It was found earlier that growth on sugars increases the level of glycolytic enzymes in yeast (Maitra and Lobo, 1971). Since the pyruvate kinase mutants do not grow on glucose, we have recorded the activity of alcoholgrown cultures. However, growth on maltose does not repair the reduced activity of these mutants. This is therefore not a special feature of particular condition of growth as addition of glucose to a suspension of fresh alcohol-grown cells either at 23 ° C or at 37 ° C fails to induce any detectable activity of pyruvate kinase in these mutants. Mixing experiments rule out the possibility that the lack of enzyme activity is due to an inhibitor or proteolytic activity in these extracts. Furthermore, all the revertants collected on glucose, except PK2R6, regain pyruvate kinase activity in amounts slightly greater than the wild type levels. Revertant PK2R1 deserves a special mention; unlike the parent PK2 it has lost the ability to grow on alcohol or lactate presumably as a result of its inability to respire. The incidence of this respiration-deficient character has been found in a fraction of glucose-revertants of the majority of pyruvate kinase mutants. It is unlikely that this was due to a cytoplasmic petite mutation as shown by the ability of PK2R1 to revert on oxidative substrates with an attendant regain of respiratory activity. The simultaneity of the two events, getting back the pyruvate kinase activity and the loss of respiratory capacity in spontaneous reversion, implicates a single genetic event in this transition.

2. Genetic Characlerisation The pyruvate kinase mutants were crossed to a wild type strain, X2928-3D-1C of mating type c~, and the resultant diploids were sporulated to give spore progenies carrying the pyruvate kinase mutation in association with different genetic markers in both mating types. Such experiments demonstrated that the pyruvate kinase deficiency segregated as a single gene giving in spore tetrads 2 spores which are glucose-positive and contain wild type levels of pyruvate kinase activity and 2 spores that arc glucose-negative and enzymenegative as well. These segregants were further backcrossed to reduce the mutagenised genetic background. Pyruvate kinase mutants from the third of such backcrosses were diploidised with wild type strains, either X2928-3D-1C or the other parental strain of a mating

195 Table 3. Activity of pyruvate kinase in haploids and diploids nlU/E650

Strain a PYK PYK PK2 + PK4 + PK5 + PK2 PK5

-

Parent haploid Parent haploid

372 326

Wild type diploid

300

Diploid

192

Diploid

13l

Diploid

184

Diploid

1.4

type to give diploids carrying the mutation in heterozygous state. Intercrosses between pyruvate kinase mutants themselves were also made as also between the two wild types. These experiments showed that pK + diploids are glucose-positive while the heteroPKallelic diploids are glucose-negative. The mutations therefore are recessive and non-complementing. Results in Table3 establish this point quantitatively. These experiments also suggest that the enzyme activ-

PK~]~- is roughly half

ity in heterozygous diploids \ P K - !

(PK+~ of that of the homozygous diploid \ p K + [ or that of the wild type haploid parent. We name the gene as pykl.

3. Mapping pykl Indication that pykl gene was linked to adel was found in random spore analysis of diploids hetero-

adel + or as + pykl adel pykl The recombinant fraction constituted from + + 34% (pykl-1, pykl-2, pykl-6) to 44% (pykl-5, pykl-4) amongst a population of approximately 150 random spores analysed from each diploid• Recovery of the two parental and the two recombinant classes was also within the expected range of fluctuations. To prove further that pykl is located on chromosome I, we have performed trisomic analysis (Mortimer and Hawthorne, 1975) of pykl-2 and pykl-5 employing a disome for this chromosome, the strain X3405-1A. The diploids were respectively zygous for these two loci, either as

adel + lys7 trp3mel2 arg4 + + adel + c~ adel pykl-2 + + + + leul metl4 a

196

P.K. Maitra and Z. Lobo: Pyruvate Kinase M u t a n t s of Yeast

and

a

adel + lys7 adel + adel pykl-5 +

trp3 met2 arg4 +

+

+

+ trpl

Sporulation of these diploids gave the following tetrad types based on glucose+: glucose- g r o w t h : - P K 2 gave 13 of 3+: 1-, 8 of 2+: 2- and 5 of 4+: 0 - tetrads; PK5 gave 6 of 3+: 1-, 1 of 2+: 2 - and no 4+: 0 tetrads. The glucose-negative spores thus became fewer in number when pyk mutants were crossed with a chromosome I disome, unlike in a cross of normal haploids as shown below. The segregation of arg4 was examined, it showed the expected 2 + :2- segregation. This result clearly indicates pykl to be located on chromosome I. Table 4 describes the result of several pyk crosses + analysed by tetrads. The relatively large number of N P D tetrads shows that pykl is only weakly linked to adel. This is also consistent with the second division segregation frequency of pykl calculated from the spore array relative to trpl. This ranged between 53 and 70%, for the various alleles. To decide whether pykl is located in the same chromosome arm of linkage group I, a mitotic crossover experiment was performed. The diploid used was

adel pykl. If pykl is distal to adel on the right arm, a

+ + single crossover between the centromere and adel would give glucose-negative red sectors due to simultaneous homozygosity of this pair of markers. The diploid was plated on YEP-alcohol plates with about

Table 4. Linkage ofpykl gene with adel

ariel + + pykl"

All diploids were of the configuration -

adel-pykl segregation

Allele

in tetrads

1 2 4 5 6

PD

NPD

T

18 32 45 30 10

3 4 1 9 2

20 50 64 58 13

Only 4-spored asci showing 2 + : 2 - segregation of both pykl and

adel were taken into account pyk I - 4

I

cys I

I

ode I

•

i

Fig. l, Location of pykl-4 on c h r o m o s o m e I of Saccharomyces cerevisiae (Mortimer and Hawthorne, 1975)

1000 cells per plate and the plates were subjected to ultraviolet irradiation at approximately 30% killing. 32 Red sectors were picked up after growth in darkness and examined for growth on glucose plates. All but one of the 32 clones remained glucose-positive. We conclude from this that pykl is not on the same arm of chromosome I as on which adel is located. Attempts were made to examine the linkage of pykl with cysl, a chromosome IL marker kindly sent by Dr. S. Fogel. These experiments were inconclusive as spores from the diploid carrying these markers germinated poorly. Figure 1 shows the location of pykl-4 on chromosome I.

4. Nature of the Mutation Unlike PK4 and PK5, the isolates PK1, PK2 and PK6 give a very faint smear of growth on YEP-glucose plates on prolonged incubation at 23°C or at 30 ° C; PK4 and PK5 which do not give any such background growth are presumably tighter mutations compared to the other three. At 37 ° C, however, no growth of any of the mutants could be seen on YgPglucose. One of the leaky mutants, PK2, gave on reversion in glucose media the revertant PK2R6 that could grow on glucose without having regained any more pyruvate kinase activity than the parent PK2. Although we do not know how exactly PK2R6 grows on glucose, it has been observed that such reversion is restricted only to the leaky alleles PK1, PK2 and PK6; the tighter mutations PK4 and PK5 do not seem to yield such pyruvate kinase-negative revertants. In order to examine the nature of the enzyme in such revertants, PK2R6 was grown on glucose thus increasing the basal pyruvate kinase activity and the enzyme was purified about 400-fold by a modification of the method outlined by Hunsley and Suelter (1969), the final enzyme having a specific activity of 10 units per mg protein. By a number of criteria such as the Km for ADP and for K ÷ ions, cold lability and thermostability, sedimentation in a sucrose density gradient and the pronounced stimulation of the reaction rate with fructose-1, 6-diP, the enzyme from the revertant PK2R6 resembled the enzyme from the wild type. The very low enzyme activity in the mutants precluded their examination by methods dependent on catalytic activity. However, an investigation of the enzymes from the revertants has been made and the results are summarised in Table 5. These data show that the enzymes from the revertants or from the mutant PK2 are no different from the wild type pyruvate kinase to the extent these two parameters might reveal. It is expected that if in any of the mu-

P.K. Maitra and Z. Lobo: Pyruvate Kinase Mutants of Yeast

tants the structure of pyruvate kinase were changed, the revertant enzyme might have shown an altered behaviour. As already mentioned, some of the reversion mutations confer on the revertants the character of nuclear petites in that they lose respiration and further spontaneous revertants selected for growth on alcohol get back the ability to respire. Revertants PK1R1, PK2R1, PK2R5 and PK4R1 belonged to this category. 5. Features o f Growth

The pyruvate kinase mutants fail to form colonies on YEP or minimal medium plates supplemented with Table 5. Properties of pyruvate kinase from wild type and glucosepositive revertants of pykl mutants. Centrifuged crude extracts prepared by a French pressure cell were heated at a protein concentation of approximately 1 mg per ml (made up with bovine serum albumin where necessary) for various lengths of time, chilled and the enzyme activity assayed immediately. The inactivation was first order in all the cases

Wild type PK1R1 PK2 b PK2R1 PK2R2 PK2R3 PK2R4 PK2R5 PK2R6 c PK4RI PK5R1 PK5R2 " b

t1~2, 53 ° C

'Kin',

2.4, 4, 3.5 3.7 3.1 2.8 2.3 2.9 2.5 1.8 2.3, 5.2, 3.7 3.7, 3.0 3.2 3.8

0.05

FDP, mM a

6. Glucose Metabolism

The near-complete block at the pyruvate kinase step in these mutants raises a number of biochemical puzzles. In the absence of any bypass the following stoichiometry may prevail when respiration is blocked:

0.04 0.04 0.05

Glucose + 2 A T P + 2 N A D + + 2 P i ~ 2 P E P + 2 A T P + 2 N A D H + 2 H ÷.

Table 6. Growth rates of pyruvate kinase mutants, their revertants and the wild type in aerobic cultures. Yeast extract-peptone medium was supplemented with 150 mM alcohol or 50 mM sugars. Samples from shaken flasks at 32 ° were diluted and its extinction at 650 mg measured. Results refer to exponential growth

Wild type PK2 PK5 PK2R1 PK2RIR PK2R3 PK2R6 PK5R3

glucose, fructose and mannose; however, they grow on plates supplemented with alcohol, acetate and pyruvate. Maltose supports slow growth while the response is variable on galactose ; the leaky alleles PK1, PK2 and PK6 are scored as positive after five days while PK4 and PK5 show as a faint smear on longer incubation. This property is preserved after a number of backcrosses with wild type and is therefore not due to a randomly mutagenised background. Results in Table 6 describe the doubling time of some of these mutants and their revertants. Addition of glucose to cultures of these mutants growing on alcohol lead to a rapid arrest of growth. Removal of the added glucose by centrifugation or washing causes resumption of growth at the same rate as before sugar addition. It will be shown in the next section that cells accumulate large amounts of P-enolpyruvate, 2-P-glycerate and 3-P-glycerate under these conditions. Pyruvate kinase mutants of S. cerevisiae thus behave no differently from other known mutants in which accumulation of non-metabolisable phosphorylated intermediates lead to growth stasis (Yarmolinsky etal., 1959; Cozzarelli etal., 1965; B6ck and Neidhardt, 1966; Fraenkel, 1968; Bernheim and Dobrogosz, 1970; Maitra, 1971).

0.04 0.06

PEP 1 mM, A D P 1 raM, KC1 5 0 m M Purified 20-fold over crude extract levels Purified 400-fold

Strains

197

Doubling time (h) in medium containing Alcohol

Glucose

Maltose

Galactose

3.0 4.0 4.6 50.0 2.5 2.5 2.7 4.0

1.5 25.0 75.0 2.5 1.3 5.0 4.0 2.0

3.0 8.0 11.5 50.0 3.5 2.5 3.2 3.0

2.7 9.5 9.0 6.0 4.2 2.8 Not done 2.3

A number of difficulties, however, arises in such a formulation. Glucose utilisation should stop when the intracellular phosphate is depleted; the lack of an electron acceptor for N A D H should set a limit to the reaction unless the production of glycerol is stoichiometric with glucose. And finally the cell should end up in an ATP-deficient state by virtue of endogenous energy-utilising processes. We have examined the known products of glucose fermentation under anaerobic conditions simulated by completely inhibiting the respiration with 0.5 m M KCN. The results of such an experiment with a leaky mutant PK2 and tight mutant PK4 are shown in Figure 2. Data for the wild type are given for comparison. The basic features of these results, re-examined for a number of mutants after 3 or 4 backcrosses to the wild type, are essentially similar to those shown in Figure 2 for the mutagenised stocks.

198

P.K. Maitra and Z. Lobo : Pyruvate Kinase Mutants of Yeast 0

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