Isolation andCharacterization Saccharomyces Mutant ... · After 1 h of nystatin treatment, the culture wasplated onto solid S lac-tate mediumat a dilution sufficient to yield 100

JOURNAL OF BACTERIOLOGY, Apr. 1977, p. 232-241Copyright C 1977 American Society for Microbiology

Vol. 130, No. 1Printed in U.S.A.

Isolation and Characterization of a Saccharomyces cerevisiaeMutant Deficient in Pyruvate Kinase Activity

GEORGE F. SPRAGUE, JR.'Departments ofBiology and of Molecular Biophysics and Biochemistry, Yale University, New Haven,

Connecticut 06510

Received for publication 16 November 1976

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvatekinase activity has been isolated. The mutant strain is capable of growth whensupplied with lactate as the carbon source but not capable of growth whensupplied with dextrose or other fermentable sugars or glycerol as the carbonsource. Genetic analysis demonstrated that the phenotype of the pyruvatekinase-deficient strain was due to a single nuclear mutation, which was desig-nated pykl, and preliminary genetic mapping experiments located the pykllocus on chromosome , 30 centimorgans from the adel locus. Adenine nucleotidelevels in the mutant and parental strains were compared when the cells weresubjected to various growth and starvation conditions. When carbon supply andenergy production were dissociated by supplying the mutant strain with dex-trose, adenine nucleotide levels fell dramatically. This result suggests that theinitial reactions of glycolysis are not rate limiting, nor are they readily inhibitedby feedback controls. R

Glycolysis and the hexose monophosphateshunt are the major pathways of carbohydratemetabolism utilized by the yeast Saccharomy-ces cerevisiae (for review, see reference 28).However, only two mutant S. cerevisiae strainsdefective in carbohydrate metabolism havebeen reported: one deficient in phosphogluco-isomerase activity (20), and one deficient inphosphomannoisomerase activity (15). The iso-lation of additional mutants defective in carbo-hydrate metabolism should make it possible todetermine the catalytic capabilities of glycoly-sis and the hexose monophosphate shunt and todetermine the actual pathway used when cellsare provided with a particular carbon source.Mutants of Escherichia coli defective in carbo-hydrate metabolism have been used success-fully to examine the properties ofglycolysis andthe hexose monophosphate shunt in that orga-nism (10).In this paper, I describe the isolation and the

biochemical and genetic characterization of anS. cerevisiae mutant strain deficient in pyru-vate kinase activity. Microbial mutants defi-cient in pyruvate kinase activity had not beenreported until recently, when an Aspergillusnidulans mutant lacking this activity wasbriefly described (24). However, several heredi-tary hemolytic anemias in humans (5, 22) anddogs (29) that appear to be the result of quanti-

1 Present address: Institute of Molecular Biology, Uni-versity of Oregon, Eugene, OR 97403.

tative and qualitative differences in erythro-cyte pyruvate kinase activity have been re-ported.Pyruvate kinase catalyzes the terminal and

net energy-producing reaction of glycolysis.Furthermore, several lines of evidence suggestthat this enzyme functions as a control site inglycolysis. First, the pyruvate kinase reactionis essentially irreversible in vivo and is by-passed by pyruvate carboxylase and phospho-enolpyruvate (PEP) carboxykinase activitieswhen yeast cells are supplied with a gluconeo-genic carbon source such as lactate (Fig. 1) (12).Second, results obtained from the oscillatoryglycolysis system of the yeast Saccharomycescarlsbergensis suggest that pyruvate kinase isa key enzyme in regulating glycolytic flux (16).Finally, the kinetic properties of pyruvate ki-nase purified from both S. cerevisiae and S.carlsbergensis also suggest a regulatory role forthis enzyme (13, 17, 18). In particular, enzymeactivity is modulated by a number of positiveand negative effectors including fructose-1,6-diphosphate (FDP), adenosine 5'-triphosphate(ATP), and citrate (13, 18). Furthermore, it hasbeen suggested that these effectors, particu-larly FDP, may regulate the activity of pyru-vate kinase in vivo (4).One function of glycolysis is to generate the

cellular energy needed for anabolic processes.The adenine nucleotides adenosine 5'-mono-phosphate (AMP), adenosine 5'-diphosphate

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YEAST PYRUVATE KINASE MUTANT 233

(ADP), and adenosine 5'-triphosphate (ATP)stoichiometrically couple catabolic and ana-bolic processes, and a measure of the energystored in the adenine nucleotide pool is theadenylate energy charge, defined as [(ATP) +1/2(ADP)]/[(ATP) + (ADP) + (AMP)] (1). It hasbeen shown that the adenine nucleotide pooland energy charge values are relatively con-stant for yeast cells growing under a variety ofconditions, and these values are maintainedwhen yeast cells are subjected to stress condi-tions such as carbon and energy source starva-tion and nitrogen source starvation (3). In theseexperiments, cells starved for energy were nec-essarily starved for carbon source as well. How-ever, the mutant deficient in pyruvate kinaseactivity permits analysis of the response ofyeast cells to energy starvation alone since themutant cells can be provided with carbonsources from which net energy production is notpossible. Thus, adenine nucleotide pools andenergy charge values were measured whencells were subjected to a variety of growth andstarvation conditions, and the implications ofthese results regarding the regulation of glyco-lytic enzymes will be discussed.

(This work was part of a dissertation pre-sented by the author to the faculty of YaleUniversity in partial fulfillment of the require-ments for the Ph.D. degree.)

MATERIALS AND METHODSYeast strains. The strains of S. cerevisiae used in

this study are listed in Table 1.

TABLE 1. Yeast strains

Strain Genotype" Source

A364A a adel ade2 ural L. H. Hartwellhis7 tyrl lys7gall

D286-2A a adel hisl F. ShermanD273-llA a adel hisl G. R. FinkD517-4B a ade2 lys9 F. ShermanD603-1A a ade2 his7 G. R. FinkJB143 a ade2 leu2 P. T. MageeM2 a trp ilv2 P. T. MageeM15 a hisl ilv2 P. T. MageeXC38-74B a leu2 ura3 ade2 P. T. Magee

his4 gal2S8 pykl, other This paper

markers as instrain A364A

S29 pykl, other This papermarkers as instrain A364A

G7-4A, B, C, D Single-ascus seg- This paperregants of S8 xM15

a Genetic symbols are as described by Mortimerand Hawthorne (21).

Media and growth conditions. The minimal me-dium of Wickerham (30) was used (S medium). S-Nmedium lacked (NH4)2SO4. L-Amino acids and nu-cleotide bases were supplemented as needed at 20,ug/ml (adenine, uracil, histidine), 30 jag/ml (tyro-sine, lysine, leucine, isoleucine), or 150 ,ug/ml (va-line). SC medium, in addition to the nutrients listedabove, contained: methionine, arginine, tryptophan(all at 20 ,ug/ml); phenylalanine (50 ,ug/ml); glu-tamic acid (100 ,ug/ml); and serine (375 ,ug/ml). Scaamedium contained 0.1% vitamin-free CasaminoAcids. YEP medium (1% yeast extract and 2% pep-tone [Difco], supplemented with adenine and uracilas above) was used for genetic manipulations. Thesporulation regimen utilized presporulation me-dium (0.8% yeast extract, 0.3% peptone [Difco], and10% dextrose) and sporulation medium (1% potas-sium acetate, 0.1% yeast extract, and 0.05% dex-trose). Carbon sources (lactate, glycerol, or dex-trose) were added to 2%. Solid media contained 2%agar.

Liquid cultures were grown aerobically withshaking (250 rpm) in a gyratory water bath. Growthwas followed in a Klett-Summerson colorimeter at540 nm (green filter) (1 Klett unit 2 x 105 cells/ml). All experiments were performed while the cellswere in the logarithmic phase ofgrowth. The growthtemperature was 30°C.

Mutagenesis. A haploid strain of yeast was muta-genized with N-methyl-N'-nitro-N-nitrosoguanidine(NTG) as described by Hartwell (14). The mutagen-ized cells were washed by centrifugation, suspendedin S lactate medium, and allowed to grow overnight.The culture was then passed through a nystatinenrichment procedure as follows (27). The overnightculture was diluted and growth was permitted forone or two generations. The culture was thenwashed three times with S-N medium by centrifu-gation and resuspended in S-N medium. After 8 to24 h of nitrogen starvation, the culture was pelletedby centrifugation and resuspended in S glycerol me-dium. When growth resumed, the antibiotic nysta-tin was added to 10 ,ug/ml. After 1 h of nystatintreatment, the culture was plated onto solid S lac-tate medium at a dilution sufficient to yield 100 to200 colonies per petri plate. The colonies were trans-ferred by replica-plating to S glycerol medium. Colo-nies that grew on S lactate medium but not on Sglycerol medium were purified by single-colony iso-lation on S lactate medium, and their phenotypewas retested. Those that grew on S lactate mediumbut not on S glycerol or S dextrose medium wereconsidered presumptive pyruvate kinase mutants,and they were characterized further by growth testsand finally by enzymatic assay.

Preparation of crude enzyme extracts. Cultures(100 ml) of strains to be assayed were grown in Scaamedium supplemented with the appropriate carbonsource. While still in the logaiithmic phase ofgrowth, the cells were harvested by centrifugation,washed twice with 50 mM sodium phosphate buffer,pH 6.5, and resuspended in 4.0 ml of this buffer. Thecells were ruptured by passage twice through aFrench pressure cell at 18,000 lb/in2 (American In-strument Co., Silver Spring, Md.). The homogenate

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234 SPRAGUE

was centrifuged (14,000 x g for 20 min), and thesupernatant fluid was used as the enzyme source.Enzyme assays. Pyruvate kinase was assayed es-

sentially by the procedure of Hunsley and Suelter(17). The assay mixture contained 20 mM MgCl2, 0.5to 5.0 mM trichyclohexylammonium PEP, 10 mMADP, 0.1 mM nicotinamide adenine dinucleotide,reduced form (NADH), 1.5 mM FDP (when added),100 mM KCl (when added), 7.0 U of rabbit musclelactate dehydrogenase per ml, cell extract (0.002 to0.02 mg of protein/reaction), and 50 mM sodiumphosphate, pH 6.5. The oxidation of NADH wasmonitored spectrophotometrically at 340 nm, usinga Gilford recording spectrophotometer (model 240).One unit of enzyme oxidized 1 ,umol of NADH permin.

Protein concentrations were determined by thebiuret method (19), using bovine serum albumin asstandard.

Perchloric acid extraction of growing andstarved cells. The adenine nucleotide concentrationsofparental and mutant strains were compared whenthe cells were subjected to various conditions ofgrowth and starvation. Before experimental manip-ulation, cells were grown in Scaa lactate mediumcontaining [8-14C]adenine (specific activity, 20 IACi/100 ,ug; 10 ,ug of adenine/ml of cell culture) for atleast four generations. While the cells were stillgrowing exponentially, they were harvested bymembrane filtration, washed three times on thefilter with Scaa medium, and then resuspended inScaa medium containing the appropriate carbonsource-lactate, glycerol, dextrose, or no carbonsource-and containing [8-14C]adenine at the samespecific activity. (For experiments in which dextrosewas to serve as carbon source, dextrose was simplyadded to the cells growing on Scaa lactate medium.)At the indicated times, 0.5-ml aliquots were re-moved from the cultures and rapidly pipetted intotest tubes containing 0.1 ml of 35% perchloric acid.The tubes were kept on ice for 20 min, and then theextracts were neutralized by adding 0.3 ml of 2.5 NKOH (3). The neutralized extracts were stored at-20°C until analyzed.Determination of adenine nucleotide concentra-

tions. The neutralized perchloric acid extracts werethawed, and the precipitated KC104 was removed bycentrifugation. An aliquot of the superantant fluidwas applied to polyethyleneimine-cellulose (PEI)thin-layer plates. The PEI plates were then washedtwice with water in shallow plastic pans and dried.The chromatograms were developed by ascendingchromatography in 0.75 M sodium phosphate, pH3.4, containing 1.0 mM ethylenediaminetetraaceticacid (6). The adenine nucleotides were visualized byautoradiography, the appropriate portions of thePEI plates were cut out, and the radioactivity wasdetermined by liquid scintillation counting, usingAquasol I liquid scintillation cocktail.The concentrations of ATP, ADP, and AMP that

were obtained were used to calculate energy chargevalues (1) according to the formula: [(ATP) + 1/2(ADP)]/[(ATP) + (ADP) + (AMP)].

Genetic analyses. Standard yeast genetic tech-niques were used for genetic analysis of the mutant

strains except that it was necessary to substitutelactate for dextrose both for mating strains and forgerminating spores. Spore germination was poor onYEP lactate (YEPL) plates, but satisfactory germi-nation (>75%) was obtained by supplementingYEPL medium with carbon source levels of ethanoland low levels of glycerol (0.1%).

Matings were performed by mixing strains onYEPL agar plates. Diploids were isolated by proto-troph selection and sporulated by allowing growthon presporulation agar plates for 1 day followed byincubation on sporulation agar plates for 3 to 5 days.The sporulated culture was suspended in sterile wa-ter and treated with glusulase. Tetrads were dis-sected on an agar slab using a de Fonbrune micro-manipulator.Revertant strains were selected by applying a

stationary-phase culture of a mutant strain onto SCdextrose agar plates. Colonies that formed werepurified by single-colony isolation on SC dextroseagar plates, their phenotype was tested, and finallythey were assayed for pyruvate kinase activity. At-tempts at direct selection on SC glycerol mediumwere unsuccessful, suggesting the revertants arecounterselected under these conditions.

Chemicals and other supplies. NTG was fromAldrich Chemical Co., Milwaukee, Wis. Rabbitmuscle lactate dehydrogenase (type II), NADH,ATP, ADP, AMP, FDP, nystatin, and PEP were allfrom Sigma Chemical Co., St. Louis, Mo. [8-"4C]adenine and Aquasol I liquid scintillation cock-tail were from New England Nuclear Corp., Boston,Mass. PEI thin-layer plates were from BrinkmannInstruments, Inc., Westbury, N.Y. Glusulase wasfrom Endo Laboratories, Garden City, N.Y. Theagar was Special Agar-Noble from Difco Laborato-ries, Detroit, Mich. All other chemicals were re-agent grade.

RESULTSIsolation of pyruvate kinase mutants. Mu-

tants deficient in pyruvate kinase activityshould not be capable of growth when suppliedwith glycerol, dextrose, or other fermentablecarbon and energy sources. However, such mu-tants should be capable of growth when nonfer-mentable substrates such as lactate or ethanolare supplied as carbon and energy sources (Fig.1). The mutant isolation procedure describedabove was based on these assumptions. Whenthe isolation procedure was performed, severalpresumptive pyruvate kinase mutants were ob-tained. After purification, the growth charac-teristics of the strains were tested. Thosestrains capable of growth when supplied withlactate, but not glycerol or dextrose, as carbonsource were selected for biochemical analysis.Biochemical characterization of a pyruvate

kinase mutant. Since the mutant strains werecapable ofgrowth only when supplied with non-fermentable carbon sources, it was necessary todemonstrate that the parental strain possessed

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significant levels of pyruvate kinase activitywhen grown under these conditions. Extracts oflactate-grown strain A364A were prepared, andthe pyruvate kinase activity was determined.Significant levels of activity were found, al-though the specific activity was about twofoldless than that obtained when extracts of dex-trose-grown cells were assayed (Table 2).The kinetic properties of the pyruvate kinase

activity obtained from lactate-grown cells wereexamined. Typical Michaelis-Menten kineticswere observed with respect to the substrate,ADP, saturation being obtained at about 5 mMADP (Fig. 2). However, a sigmoidal relation-ship between initial reaction velocity and PEPconcentration was observed. This relationshipwas abolished by adding FDP to the reactionmixture (Fig. 2), and, simultaneously, the ap-parent Km for PEP was dramatically low-ered. Finally, the initial reaction velocity waslinear with respect to extract protein concentra-tion in the range used in typical assays (Fig. 2).Hence, the pyruvate kinase activity in crudeextracts prepared from lactate-grown cells pos-sesses kinetic properties similar to those ob-served with purified pyruvate kinase preparedfrom commercial baker's yeast (18).One presumptive pyruvate kinase mutant,

Glucose

IGlucos;-6- P04

Fructose-6-P04%b.

Fructose-1,6-di P04

Dihydroxyacetone- P04

Glycerol -3- P04

Glycerol

GI yceraldehyde -3- P04

Jrt

4r'Phosphoenol pyruvote

c

Oxoloocetate

-Pyruvate d.

Lactate

FIG. 1. Pathways ofglycolysis and gluconeogene-sis and ofglycerol catabolism . The enzymes catalyz-ing the steps indicated by the letters a through e are:

(a) phosphofructokinase; (b) FDPase; (c) pyruvatekinase; (d) pyruvate carboxylase; (e) PEP carboxy-kinase.


TABLE 2. Specific activity ofpyruvate kinase in theparental strain, A364A

No. of ex- Sp acta (U/Mg OfCarbon source tracts a- protein)

sayedLactate.... 8 1.3 + 0.4Dextrose .... 3 2.7 + 0.5

a Data are mean ± range about the mean.

strain S8, was initially selected for biochemicalanalysis. Extracts of this strain and the paren-tal strain were prepared, and pyruvate kinaseactivity was determined. The level of pyruvatekinase activity found in strain S8 was greatlyreduced (to about 2%; see Table 3). The additionof FDP or KCI to the reaction mixture resultedin little or no increase in activity, indicatingthat the low level of activity was not simply dueto altered kinetic parameters (Table 3). Fur-thermore, the low level of activity was not dueto the presence of an inhibitor in the mutantstrain, since upon mixing extracts of strains S8and A364A, the expected amount of enzymeactivity was obtained (Table 3).The hypothesis that the low level ofpyruvate

kinase activity found in the mutant strain wasresponsible for the growth phenotype was sub-stantiated by isolating revertants. Revertantcolonies capable of growth when supplied withdextrose as carbon source were isolated as de-scribed in Materials and Methods and found tobe capable of growth with either dextrose orglycerol as carbon source. Furthermore, thelevel of pyruvate kinase activity found in twoindependent revertants was nonnal, and theactivities showed sensitivity to FDP activationat low PEP concentrations (Table 3).The reversion frequency of strains S8 (-10-7)

indicates that a single mutational event is re-sponsible for the phenotype. The genetic sym-bol pykl (for pyruvate kinase) is proposed forthis mutant. Strains with Pyk- phenotype arecapable of growth with lactate as a carbonsource, but not with glycerol or dextrose.Genetic analyses. To determine the genetic

basis of the phenotype of strain S8, this strainwas mated with a multiply marked haploidstrain of the opposite mating type, strain M15.Diploids were isolated by prototroph selection.Thepykl allele was shown to be recessive to thewild-type allele since heterozygous diploidswere capable of growth when supplied witheither glycerol or dextrose as the carbon source.

Diploids isolated from the cross strain S8 xstrain M15 were sporulated, and the resultingasci were dissected by micromanipulation. Atotal of 28 asci, all containing four spores, weredissected; 18 asci had 100% germination and 10


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236 SPRAGUE

*'1 11<10E

0 2 4 6 S 10 0 2 4 6 1000 0.005 0000 .OLO1ADP (mM) PEP (mM) Proteln(mg/re.ction)

FIG. 2. Determination of optimal assay conditions for pyruvate kinase activity. (a) Assay conditions werethose described in Materials and Methods except that the ADP concentration was varied. (b) Assay conditionswere those described in Materials and Methods except that the PEP concentration was varied. FDP was addedto the assay mixture as indicated in the figure. (c) Pyruvate kinase activity was determined for a number ofextract protein concentrations.

TABLE 3. Pyruvate kinase activities in parental andmutant strains

Pyruvate kinase activitya (U/mg ofprotein)

Source of ex-tract 5 mM PEP 0.5 mM PEP

-FDP +FDP +KCI -FDP +FDP

A364A 1.2 1.5 1.6 0.06 1.0S8 0.02 0.03 0.03 - -

S8 + A364A 0.63 - - - -

(mixed)bS8rl 1.5 - - 0.1 1.3S8r2 1.4 - - 0.05 1.1

a Data of a typical experiment. A dash (-) indi-cates that the experiment was not done.

b Equal amounts ofextract protein were mixed.

asci had 75% germination. The Pyk phenotypeofthe spore progeny showed a 2+:2- segregationfor 16 of these asci and either 2+:1- or 1+:2- forthe 10 asci with three viable spores. The sporeprogeny from two asci displayed a 3+:1- segre-gation for the Pyk phenotype. (It is likely thatthis latter result is the manifestation of geneconversion events at the pykl locus, since occa-sional 3:1 segregations were observed for someof the auxotrophic markers that were includedin the cross.) These results clearly indicate thatpykl arose by mutation in the nuclear genome.

Nine genetic markers representing sevenchromosome arms were included in this cross.The segregation of one of these markers, adel,with pykl resulted in only parental ditype andtetratype asci (Table 4). The pykl and adel lociappear to be linked (X2 analysis, P < 0.005) andabout 30 centimorgans apart. Normal segrega-tion of pykl and the other eight markers wasobserved (Table 4).

All four segregants from a single ascus wereassayed for pyruvate kinase activity. For thisascus, cosegregation of the growth phenotype(Pyk+/Pyk-) and the biochemical phenotype(presence or absence of pyruvate kinase activ-ity) was observed, supporting the hypothesisthat the enzymological defect is responsible forthe growth phenotype (Table 5).The hypothesis that the enzymological defect

is responsible for the growth phenotype wasfurther supported by the isolation of a second,apparently independent mutant strain, S29,that displayed the. Pyk- phenotype and wasalso deficient in pyruvate kinase activity. Ge-netic analysis demonstrated that the phenotypeof strain S29 was due to a single, nuclear muta-tional event. Furthermore, this mutation didnot complement the pykl mutation of strain S8(data not shown).Growth characteristics of strain S8. The

growth characteristics of strain S8 describedthus far have been based on growth compari-sons on solid minimal media. A more detaileddescription of the growth properties of thisstrain was obtained by monitoring growth inliquid culture. Strain S8 grew as well as itsparent, strain A364A, on S lactate or Scaa lac-tate liquid medium (Fig. 3a). However, whenglycerol or dextrose replaced lactate as the car-bon source, growth ceased immediately (Fig.3b). Furthermore, when carbon source levels ofdextrose were added to a culture of strain S8growing in Scaa lactate medium, growth ceasedimmediately (Fig. 4a). Upon removal of dex-trose, growth resumed (after a short lag) at arate identical to that observed before the addi-tion of dextrose, indicating that the presence ofdextrose had caused growth stasis rather thancell death (Fig. 4a) (the lag that was observed

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TABLE 4. Linkage' test ofpykl with various genetic markers

Marker

Tetradailv2 tyri Iys2 ural Mating adel ade2 hisl his7

PD 1 5 4 7 5 11 4 5 4NPD 7 5 6 3 2 0 2 6 6T 18 16 16 16 19 15 20 15 16

a Abbreviations: PD, parental ditype; NPD, nonparental ditype; T, tetratype.

TABLE 5. Cosegregation ofPyk phenotype andenzymological defect

Pyruvate kinaseStrain Pyk phenotype activitya (U/mg

of protein)

G7-4A + 0.90G7-4B - 0.013G7-4C + 1.15G7-4D - 0.008

a Data of a typical experiment.

was similar to that observed for a control cul-ture washed identically, but to which dextrosewas not added). Lower concentrations of dex-trose also caused growth stasis, although thetime required to produce stasis increased as theconcentration was lowered (Fig. 4b). Growthstasis was also observed when either fructose ormannose was added to a growing culture ofstrain S8. However, the addition of glycerol didnot inhibit growth (data not shown).Adenine nucleotide metabolism in parental

and mutant strains. Pyruvate kinase catalyzesthe terminal and net energy-producing reactionof glycolysis. Hence, it is possible to partiallydissociate carbon supply and energy productionin the mutant strain. For this reason, I decidedto monitor the metabolism of adenine nucleo-tides (and the derivative parameter, energycharge) in the parental and mutant strainswhen they were subjected to various growthand starvation conditions.

Cells of strain A364A growing exponentiallyin Scaa lactate medium were transferred toScaa medium containing lactate, glycerol, or nocarbon source. Initially, the energy chargevalue was about 0.8. However, as growth re-sumed in the cultures supplied with lactate or

glycerol, the energy charge quickly rose to 0.85to 0.90. Energy charge was maintained in thisrange throughout the exponential growthphase (Fig. 5a,b). Furthermore, cells growingon lactate or glycerol contained similar levels ofATP and total adenine nucleotides. Cells in theculture without added carbon source main-tained the energy charge at the initial 0.8 value

throughout the incubation period, and the ATPand total adenine nucleotide levels increasedonly slightly (Fig. 5c).An identical experiment was performed with

the pykl strain, S8. The initial energy chargewas 0.75, although it increased to 0.85 to 0.90 asgrowth resumed in the culture supplied withlactate as carbon source (Fig. 6a). In addition,ATP and total adenine nucleotide levels compa-rable to those of strain A364A were observed.Strain S8 cultures supplied with glycerol andthose to which no carbon source was addedmaintained the energy charge near the initialvalue, although the ATP and total adenine nu-cleotide levels fell to 60 to 80%o of the initiallevels (Fig. 6b,c).The effect of the addition of dextrose to cul-

tures of strains A364A and S8 growing expo-nentially in Scaa lactate liquid media uponenergy charge and adenine nucleotide levelswas examined. Strain A364A maintained theenergy charge near 0.9, and the adenine nu-cleotide levels increased with growth (Fig. 7a)(note change in time scale). In marked con-trast, the ATP level in strain S8 fell to 50% ofthe initial level 1 min after dextrose additionand to 10 to 20% of the initial level after 1 h. Adecrease in total adenine nucleotides to 30% ofthe initial level was observed after 1 h (Fig. 7b).(A slight increase in ADP + AMP levels wasseen). The energy charge dropped from 0.9 to0.6 and then stabilized at this value (Fig. 7b).(Similar results were obtained if the lactatewas removed by membrane filtration before theaddition of dextrose).

DISCUSSIONI have described the isolation of a mutant of

S. cerevisiae that is not capable of growth whensupplied with any of several fermentable sugarsor glycerol as a carbon source, and I have dem-onstrated that this phenotype is the result ofthe loss of pyruvate kinase activity. These re-sults provide strong evidence in support of twoconclusions.

First, there is a single pyruvate kinase activ-

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238 SPRAGUE

100 /0/

50-Gyeo

01-0 400 600 1200 0 400 600 1200

Time (minutes)

FIG. 3. (a) Growth curves of strains A364A (0) and S8 (a). The growth of these strains in Scaa lactatemedium was monitored as described in Materials and Methods. (b) Growth curves ofstrain S8 incubated withdifferent carbon sources. A culture of this strain was grown in Scaa lactate medium. At time zero, the culturewas divided into three aliquots and each aliquot was washed on membrane filters with Scaa medium. Onealiquot was resuspended in Scaa lactate medium (a), the second aliquot was resuspended in Scaa glycerolmedium (0), and the third aliquot was resuspended in Scaa dextrose medium (U).

200 L a. * b.Dextrose

None

100 I z f

0.02*L ~~~~~~~~~~~~~~~~~~~~0.22 0--0- 2.0

XI

Time (minutes)

FIG. 4. (a) Inhibition of the growth of strain S8 by dextrose. At the time indicated by the first arrow,dextrose was added (final concentration, 2.0%) to a culture ofstrain S8 growing in Scaa lactate medium. Atthe time indicated by the second arrow, the dextrose was removed by washing the cells on membrane flterswith Scaa medium. The cells were then resuspended in Scaa lactate medium. (b) At the time indicated by thearrow, a culture of strain S8 growing in Scaa lactate medium was divided into four aliquots. Dextrose wasadded to one aliquot to a final concentration of2.0%, (O), to the second aliquot to a final concentration of0.2%(0), and to the third aliquot to a final concentration of0.02% (A). Dextrose was not added to the fourth aliquot(0).

ity in this yeast; there does not appear to be analternate pathway that can be used to bypassthis reaction when cells are supplied with gly-colytic substrates as the carbon source.

Second, pyruvate kinase need not participatein PEP synthesis during gluconeogenesis inthis organism. In contrast, it has been proposedthat pyruvate kinase fulfills this function inrabbit skeletal muscle (9), a tissue that does notpossess pyruvate carboxylase activity (23). In-

deed, it was shown recently that pyruvate ki-nase can catalyze the synthesis of PEP frompyruvate in vitro at a rate sufficient to accountfor the observed rate of gluconeogenic flux inrabbit skeletal muscle (9).Adenine nucleotide levels were determined

in parental and mutant strains. In good agree-ment with previously reported values for S.cerevisiae (3, 11), the ATP concentration was2.0 + 0.3 mM and the total adenine nucleotide

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concentration was 2.5 + 0.4 mM in exponen-tially growing cells. The energy charge of expo-nentially growing cells varied between 0.80 and0.93, and the average value was 0.87. Further-

200 a. Lactate b. Glycerol

O Energy charge .....*,_ OI..100 ZAaP

Zi F*I~~~~~~~'Turbidity

c 30 Oe.--CD~41

more, it had been reported (3) that when cells ofS. cerevisiae were deprived of a carbon andenergy source, the energy charge fell onlyslightly and the adenine nucleotide pool re-

c. No carbon sourceorgy chare

/ Energy charge 0.6AiP

-,T/ ZAiP0.6___.__t_ ___ _

,Turbidity n0.46

%-C

Time (hours)

FIG. 5. (a) Energy charge values of strain A364A growing in Scaa lactate medium. Strain A364A waspregrown in Scaa lactate medium containing ['4C]adenine as described in Materials and Methods. At timezero, the cells were harvested and washed by membrane filtration and then resuspended in Scaa lactatemedium containing ['4C]adenine at the same specific activity. Adenine nucleotide concentrations in theneutralized perchloric acid extracts and energy charge values were determined as described in Materials andMethods. Relative adenine nucleotide concentrations and culture turbidity are indicated by the left ordinate;energy charge values are indicated by the right ordinate. Symbols: (0) Relative total adenine nucleotideconcentration (I A x P = ATP + ADP + AMP); (0) relative ATP concentration; (U) energy charge; (A)turbidity. (b) Energy charge values ofstrain A364A growing in Scaa glycerol medium. Cells were processedas described in (a), except that the cells were resuspended in Scaa glycerol medium. Symbol identification isthe same as (a). (c) Energy charge values ofstrain A364A incubated in Scaa medium. Cells were processed asdescribed in (a), except that the cells were resuspended in Scaa medium without added carbon source. Symbolidentification is the same as in (a).

200 0. Lactate b. Glycerol l c. No carbon source-1.0

Energy 4charge

ATP Energy chargeEngycae0.+~~~~~~~~~~~~. . . . . . . ..... .. ..

_-_0- . - 0.6 °

.- 0_ TurbiubidttTurbidity - . TriIt

0.4 0;ac30~ ~ ~ ~ rZAiP ZAxPZ0 A~~~~~~~~~~~~~~~~

. c

Time (hours)

FIG. 6. (a) Energy charge values ofstrain S8 growing in Scaa lactate medium. Cells ofstrain S8 growingin Scaa lactate medium containing ['4C]adenine were processed as described for cells ofstrain A364A in Fig.5a and resuspended in Scaa lactate medium containing ['4CJadenine at the same specific activity. Symbols:(A) Turbidity; (0) relative total adenine nucleotide concentration; (a) relative ATP concentration; (U) energycharge. (b) Energy charge values of strain S8 incubated in Scaa glycerol medium. Cells were processed asdescribed in (a), except that the cells were resuspended in Scaa glycerol medium. Symbol identification is thesame as in (a). (c) Energy charge values of strain S8 incubated in Scaa medium. Cells were processed asdescribed in (a), except that the cells were resuspended in Scaa medium. Symbol identification is the same asin (a).

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240 SPRAGUE

0

a _,

._c 0, 0_ ._

T_I- ID

U

= c

A c,- ._

, 0

60 1200Time (minutes)

m0

40%C0

FIG. 7. (a) Energy charge values of strain A364A after dextrose addition. At time zero, dextrose (finalconcentration, 2.0%) was added to a culture of strain A364A growing in Scaa lactate medium containing[14C]adenine. Growth ofstrain A364A continued at a rate typical for lactate-grown cells for 1.5 to 2 h, when anew, faster growth rate typical for dextrose-grown cells was observed. Adenine nucleotide concentrations andenergy charge values were determined as described in Materials and Methods. Symibols: (A) Turbidity; (0)relative total adenine nucleotide concentrations; (0) relative ATP concentrations; (M) energy charge. (b)Energy charge values of strain S8 after dextrose addition. Growth of strain S8 ceased immediately upondextrose addition. The cells were processed as described in (a). Symbol identification is the same as in (a).

mained constant. This result was confirmed forthe parental strain, A364A. When the mutantstrain deficient in pyruvate kinase activity wasstarved for carbon and energy source, a slightdrop in energy charge was also observed. Incontrast to the parental strain, however, theadenine nucleotide pool of the mutant strainfell by about 30%, suggesting that endogenousmetabolism through the pyruvate kinase reac-tion is necessary to maintain the adenine nu-cleotide pool when cells are deprived of a carbonand energy source.The results described above indicate that en-

ergy charge is a tightly controlled parameterand that it is maintained under at least someconditions of stress, particularly carbon andenergy source starvation. However, carbon sup-ply and energy supply can be partially disso-ciated in the mutant strain lacking pyruvatekinase activity. Thus, when the mutant strainwas supplied with dextrose, it was effectivelystarved only for energy. The energy charge andATP levels fell dramatically when carbon andenergy supplies were dissociated in this man-ner. This result suggests that dextrose trans-port and phosphorylation are not the rate-limit-ing steps in glycolysis. If either of these reac-tions is rate limiting, the ATP levels should notdecrease because the production of ATP cata-lyzed by phosphoglycerate kinase will balancethe utilization ofATP by the early reactions ofglycolysis. The decrease in ATP and energy

charge levels further suggest that neither thetransport of dextrose or the phosphorylation ofdextrose by hexokinase is rapidly inhibited byfeedback controls. There is evidence that puri-fied yeast hexokinase can be inhibited by lowconcentrations of ADP (8) and that glucose-6phosphate can inhibit dextrose transport (2).However, neither of these control mechanismsappears to be effective, at least under the condi-tions of this experiment.A similar dramatic drop in energy charge

and ATP levels in Neurospora crassa cells towhich the glycolytic inhibitor 2-deoxyglucosewas added has been reported (26). Further-more, for both S. cerevisiae (this study) and N.crassa (26), a large decrease in the total ade-nine nucleotide pool has been observed whenthe complete metabolism of glycolytic sub-strates is prevented. The reason for this de-crease is not clear. In part, the decrease in thetotal adenine nucleotide pool may reflect theactivation of adenylate-degradative enzymes(7, 25), and, in part, it may reflect the contin-ued synthesis of nucleic acids. It is clear, how-ever, that the inhibition of glycolysis has simi-lar effects on adenine nucleotide metabolism intwo fungal species, S. cerevisiae and N. crassa.

ACKNOWLEDGMENTSI thank John Cronan for helpful discussions and for his

comments on the manuscript.This work was supported by research grant AI-10186

J. BACTERIOL.


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awarded to John E. Cronan, Jr., by the Public HealthService, National Institute of Allergy and Infectious Dis-eases. The author was a predoctoral trainee (GM-7223) ofthe Public Health Service, National Institute of GeneralMedical Sciences.

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19. Layne, E. 1957. Spectrophotometric and turbidimetricmethods for measuring proteins. II. Biuret method.Methods Enzymol. 3:450-451.

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23. Opie, L. H., and E. A. Newsholme. 1967. The activitiesof fructose-1,6-diphosphatase, phosphofructokinase,and phosphoenolpyruvate carboxykinase in whitemuscle and in red muscle. Biochem. J. 103:391-399.

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25. Schramm, V. L., and H. Leung. 1973. Regulation ofadenosine monophosphate levels as a function ofadenosine triphosphate and inorganic phosphate. Aproposed metabolic role for aden,sine monophosphatenucleosidase from Azotobacter vinelandii. J. Biol.Chem. 248:8313-8315.

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Isolation andCharacterization Saccharomyces Mutant ... · After 1 h of nystatin treatment, the culture wasplated onto solid S lac-tate mediumat a dilution sufficient to yield 100

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