JOURNAL OF BACTERIOLOGY, Sept. 1971, p. 759-769 Vol. 107, No. 3Copyright © 1971 American Society for Microbiology Printed in U.S.A.

Glucose and Fructose Metabolism in aPhosphoglucoisomeraseless Mutant of

Saccharomyces cerevisiaeP. K. MAITRA

Molecular Biology Unit, Tata Institute of Fundamental Research, Bombay 5, India

Received for publication 5 May 1971

A mutant of Saccharomyces cerevisiae deficient in phosphoglucoisomerase (EC5.3.1.9) is described. It does not grow on glucose or sucrose but does grow on ga-lactose or maltose. Addition of glucose to cultures growing on fructose, mannose,or acetate arrests further growth without altering viability; removal of glucosepermits resumption of growth. Glucose causes accumulation of nearly 30 Mmoles ofglucose-6-phosphate per g (wet weight) of cells and suppresses synthesis of ribonu-cleic acid. Inhibition of growth by glucose does not appear to be due to a loss ofadenosine triphosphate or inorganic orthophosphate. The mutant, however, utilizesglucose-6-phosphate produced intracellularly. Release of carbon dioxide from spe-cifically labeled glucose suggests a C-l preferential cleavage. The kinetics of glu-cose-6-phosphate accumulation during glucose utilization in the mutant is not con-sistent with the notion that the utilization of glucose is controlled by glucose-6-phosphate.

Mutations affecting phosphoglucoisomerase(EC 5.3.1.9) have been described in enteric bac-teria (12, 13, 15). Loss of this enzyme seems tohave marginal effects on the physiological be-havior of such mutants, the most notable onebeing a reduction in growth rate on glucose.While studying the induction of glycolytic en-zymes in yeasts, we came across a mutant ofSaccharomyces cerevisiae lacking phosphoglu-coisomerase. This was characterized by its ina-bility to form colonies not only on glucose-min-eral medium but on any medium with a permis-sible source of carbon to which glucose was in-corporated. Inhibition of growth after accumula-tion of nonmetabolizable phosphate compoundsis a well-documented phenomenon (1, 2, 6, 11,16, 25). Despite the generality of this effect, themetabolic basis for such growth inhibition hasbeen traced to diverse causes (1, 11, 16). Norhave we been able to offer a general explanationfor such growth inhibition. However, a numberof interesting facts has emerged in the course ofthese studies that throw some light on the controlof glucose and fructose metabolism in yeasts.This paper reports some of those experiments.

MATERIALS AND METHODSMicrobiological. A wild-type haploid strain of S.

cerevisiae was used in these experiments. It was grownin a yeast extract-peptone medium supplemented with

extra carbon sources such as sugars or acetate. A salt-vitamin medium was also used. These have been de-scribed elsewhere (18).The following procedure was employed in isolating

the phosphoglucoisomeraseless mutant. An overnightculture of S. cerevisiae in salt-vitamin-glucose was re-suspended in fresh medium lacking glucose; mutagen-esis was initiated by adding to the suspension 0.1 mg ofN-methyl-N'-nitro-N-nitrosoguanidine per ml and in-cubating for 20 min. The mutagen was washed off bycentrifugation and the culture was allowed to growovernight in a number of separate tubes containingsalt-vitamin-fructose medium. Washed cells from eachtube were plated in a number of petri dishes containingthe salt-vitamin-fructose medium to yield nearly 100colonies per plate. After 5 days the colonies were repli-cated (17) on salt-vitamin-fructose and salt-vitamin-glucose media. Among nearly 8,500 colonies examined,approximately 0.4% did not grow on glucose plates.After purification by restreaking on the fructose me-dium, the glucose-negative clones were tested for phos-phoglucoisomerase activity after growth in liquid yeastextract-peptone-fructose. One clone, labeled 9520, wasfound to contain less than 1% of phosphoglucoiso-merase activity of the wild type and was selected forthese studies.

The concentration of yeast in suspensions was deter-mined by filtering a sample over membrane filters andwashing with 2 ml of distilled water. Filters wereweighed after the adherent water was removed.

Assay of enzynes and substrates. Most of these pro-cedures have been described (18, 19). Enzymes wereassayed generally in toluene lysates; cell extracts pre-

759

J. BACTERIOL.

pared with a French pressure cell were also used insome experiments. Fluorometric methods were em-ployed for assay of enzymes (9). The reaction mixturefor phosphoglucoisomerase assay contained 1 mm fruc-tose-6-phosphate free of glucose-6-phosphate, 0.05 mmnicotinamide adenine dinucleotide phosphate(NADP+), and 0.3 unit of glucose-6-phosphate dehy-drogenase. The enzyme activity of the mutant was al-ways checked for reduced NADP (NADPH) oxidaseactivity. By using a concentration of 10 to 20 uMNADPH, no significant oxidase activity could be de-tected. Linearity of the assays was also established bymixing the mutant and wild-type extracts; the resultantrates were essentially additive in the range of concen-trations used in these experiments. A unit of enzymeactivity referred to 1 Mmole of substrate converted permin at 22 C.

Nucleotides and substrates were assayed fluorome-trically by using a set up described earlier (18). Sugarswere measured enzymatically by using the fluorometer;glucose was also assayed by glucose oxidase-peroxidase-coupled assay (J. D. Teller, 1956. 130th Meeting Amer.Chem. Soc. Abstr., p. 69 C). Fructose-1,6-diphosphateand triose phosphates were measured together as trioses.Inorganic phosphate was estimated by the procedure ofFiske and SubbaRow (10). Respiration was measured bythe Clark oxygen electrode (7) in 50 mm potassiumphosphate buffer, pH 7.4, by using 100 mm ethanol as asubstrate.

Experiments with labeled compounds. The release ofradioactive carbon dioxide from labeled glucose wasfollowed by doing the experiment in Warburg flasks.Alkali in the central cup absorbed the carbon dioxide,and radioactivity of a suitable sample was measuredwith a liquid scintillation spectrometer. The incorpora-tion of labeled precursors into macromolecules wasmonitored by withdrawing samples of cell suspensionsin a final concentration of 10% trichloroacetic acid at 0C and filtering and washing on membrane filters (3).These were counted in a gas-flow counter.

Chemicals. Sugars used in experiments on growth ofthe mutant were freed from contaminating glucose bytreatment with glucose oxidase as described elsewhere(20). Fructose and galactose were from Sigma Chem-ical Co., mannose was from British Drug Houses, andmaltose was from Difco Laboratories, Inc. Other sub-strates and enzymes were from Boehringer. Radioac-tive chemicals were obtained from the Bhabha AtomicResearch Centre, Bombay.

RESULTSCharacterization of the mutant. When the

mutant strain 9520 was plated on either the salt-vitamin or yeast extract-peptone media supple-mented with fructose, two types of colonies wereobserved. One of these, called 9520b, yielded rel-atively large-sized colonies, measuring about Imm in diameter after 5 days of growth. Theother colony type, called 9520s constitutingnearly 90% of the total colonies, was small, aver-aging 0.3 mm in diameter. Whereas the latter

was a stable mutant, the former, 9520b, segre-gated in the same ratio as 9520, giving consist-ently 90% of type 9520s and 10% of its own type.This behavior was maintained in repeated trans-fers with purified single clones. This property,together with the growth behavior of the twocolony types, suggested that 9520s was a petitemutation (24). The type 9520b grew on acetateat a very slow rate. The type 9520s, however,neither grew nor reverted on acetate as a carbonsource; the reversion frequency was less than 10-9.Measurements of respiration showed that 9520shad negligible respiration compared to the wildtype, whereas 9520b retained nearly a third of therespiratory capacity of wild-type parent. Both ofthese mutants, however, were deficient in phos-phoglucoisomerase. Compared to the wild typewhich had, in one experiment, 1,200 milliunits ofphosphoglucoisomerase activity per mg of pro-tein, 9520b had 10 and 9520s 7 milliunits per mgof protein. Both the colony types 9520b and9520s therefore had lost, respectively, 99.2 and99.4% of the phosphoglucoisomerase activity ofthe wild type. Except for this enzyme, the mu-tant 9520b possessed all the other glycolytic en-zymes in amounts comparable to the wild-typeparent (20). Unless otherwise stated, we used themutant 9520b in the experiments described here.When the mutant 9520b was plated on either

the salt-vitamin or yeast extract-peptone mediacontaining glucose, on an average about 2 out ofl07 cells gave rise to a colony. Examination of anumber of these purified glucose revertantsshowed partial recovery of phosphoglucoi-somerase activity in all of these to the extent of10 to 30% of wild-type activity. When 9520b wasreverted on glucose plates containing N-methyl-N'-nitro-N-nitrosoguanidine, as much as half ofthe wild-type phosphoglucoisomerase activitycould be recovered. The growth rates of all therevertants on glucose were, however, slower thanthat of the wild type. The reversion frequency of9520s on glucose was quite similar to that of9520b.To decide whether the mutant 9520b was a

structural or a regulator gene mutant, we hadexamined the thermal stability of the residualphosphoglucoisomerase activity of the mutant9520b. One of the spontaneous glucose revertantswas also examined in this regard. Results indi-cated in Fig. I showed that the wild-type enzymewas fairly stable to heat, losing half of the initialactivity in 19 min of exposure to 60 C. The ac-tivity from the mutant, however, was very sensi-tive to this tempeature as shown by its half-time(to.5) of 25 sec. The enzyme activity in the rever-tant was even more sensitive, to., being 10 sec.

760 MAITRA

PHOSPHOGLUCOISOMERASELESS MUTANT OF YEAST

MINUTES AT 60

FIG. 1. Thermal inactivation of phosphoglucoi-somerase from S. cerevisiae. Cultures of the wild type,the mutant 9520b, and its glucose-revertant 9520bR13were grown aerobically in yeast extract-peptone-fruc-tose for 24, 48, and 80 hr, respectively; washed cellswere extracted in 50 mm potassium phosphate buffer,pH 7.4, 2 mm ,3-mercaptoethanol, and 2 mm ethylene-diaminetetraacetic acid with a French press. The super-natants obtained by centrifuging the clarified homoge-nates at 20,000 x g for 10 min were heated at 60 C forthe periods indicated, chilled immediately, and assayedfor phosphoglucoisomerase in i-mi final volume. Thespecific activities of the unheated extracts from thewild type, the revertant, and the mutant were, respec-tively, 1.60, 0.24, and 0.01 units per mg ofprotein. Theresults were expressed as percentage of activities of therespective extracts before heating. The numbers withinparentheses indicate the time taken to reach half of theinitial activities. The inset shows the inactivation ki-netics of the wild type in a compressed time scale. Theordinates for both the figures refer to the percentageactivity remaining and are expressed in exponentialscale.

Both the forward and the back mutations, there-fore, seemed to have altered the polypeptidechain, enhancing thermosensitivity. The simplestinterpretation of this observation is that the mu-tation 9520b has affected the structural gene ofphosphoglucoisomerase.Growth behavior of 9520b. Unlike in Esche-

richia coli (12), the loss of phosphoglucoi-somerase in yeast resulted in complete absence ofgrowth on media containing glucose either as asole source of carbon or as a supplement to oth-erwise permissible media. Addition of 2 mm glu-cose to a culture growing on yeast extract-pep-tone containing 50 mm fructose inhibited growthas long as glucose was present. Smaller amountsof glucose had the same effect except that theinterval of inhibited growth was proportionatelyshorter. Glucose continued to be utilized duringgrowth inhibition; complete disappearance ofglucose from the medium led to resumption of

growth. Such an experiment, illustrating the ef-fect of adding 10 mm glucose to an exponentiallygrowing culture of the mutant, is shown in Fig.2. The results show the kinetics of growth inhibi-tion measured by turbidity as well as by viablecounts. The parallelism of the curves B and Cindicated that glucose did not decrease the via-bility of the culture. It, however, caused agradual arrest of growth. The control culture,curve A, with fructose alone continued to growexponentially. When glucose was removed aftergrowth had completely stopped, the culture re-sumed exponential growth after a lag period of 2hr. The onset of growth inhibition was found tobe sharper when higher concentrations of glucosewere used.

In subsequent experiments we attempted todetermine the amount of glucose-6-phosphateaccumulated in the cell during growth on anumber of carbon sources. For this purpose, cellsfrom exponentially growing cultures were con-centrated by centrifuging and resuspending thecells in the same medium. The concentrated cell

Io

HOU R S

FIG. 2. Inhibition by glucose of growth of a mutant(9520b) of S. cerevisiae lacking phosphoglucoi-somerase. An overnight culture of 9520b in yeast ex-tract-peptone-fructose (50 mM) was diluted in freshmedium. Glucose (10 mM) was added to one-half ofthe culture after one generation. Nearly 8 hr after theaddition of glucose, the sugar was removed by centrifu-gation and washing with sterile glucose-free medium.The washed cells were resuspended in yeast extract-pep-tone-fructose and allowed to grow. Periodic sampleswere examined for growth by measuring the opticaldensity at 650 nm (E,,50) and also viable counts on thefructose medium. Curves A and B represent the growthcurves of the culture containing fructose alone andfructose plus glucose, respectively. In curve C the vi-able count of the culture B is plotted. Ordinates are inexponential scale. Glucose addition is indicated by thethin arrow and removal by the thicker arrow.

761VOL. 107. 1971

J. BACTERIOL.

suspension was incubated in a stream of oxygencontaining 5% carbon dioxide for I hr to approx-imate the aerobic steady state of the dilute sus-pension of the growing culture. Separate experi-ments indicated that oxygenation in presence of5% carbon dioxide did not alter the profile ofglycolytic metabolites as obtained during aera-tion. Perchloric acid was added to a concentra-tion of 0.7 N. Glucose-6-phosphate was deter-mined in the neutralized supematants (Table 1).The decreased rate of growth of the mutant inthe acetate medium compared to the wild typemight perhaps be traced to its decreased respira-tory capacity. On fructose and mannose, how-ever, the growth rates were comparable. An un-expected feature of these results was that themutant was capable of growing on galactose andmaltose both of which are metabolized throughglucose-6-phosphate. However, the level of intra-cellular glucose-6-phosphate during growth onthese two sugars was much less than when glu-cose was present in the growth medium. Glucose,when added to the culture growing in presence ofany of the other sugars listed in Table 1, inhib-ited growth. Growth of the mutant on untreatedcommercial mannose, for example, was asso-ciated with an unusually long lag period whichincreased with increasing concentration of man-nose; the delay could not be repaired by prioradaptation of the cells on this sugar. This wastraced to the presence of contaminating glucosein mannose. Treatment of mannose with glucoseoxidase eliminated the lag period. Unlike mal-tose, sucrose did not support growth of the mu-tant. Growth response of the mutant in salt-vi-

TABLE 1. Growth rates of a phosphoglucoisomeraselessmutant ofSaccharomyces cerevisiae on a number of

sugars and glucose-6-phosphate levels thereona

Glucose-6-phosphateDoubling time (hr) [gmole/g (wet

Sugars weight) of yeast]

Wild type 9520b Wild type 9520b

None ...... 4.9 16 0.2 0.01Glucose ... 1.8 > 24 0.8 5.8Fructose ... 1.9 2.6 0.9 0.03Mannose .. 1.9 3.2 0.9 0.1Galactose .. 2.2 12 0.6 0.2Maltose ... 3.7 8 1.0 1.2

a Cultures of the mutant 9520b and the wild-typeparent were grown in yeast extract-peptone-acetate me-dium containing the indicated additional carbonsources. The initial concentration of glucose was 10mM; maltose, 25 mm, and all others, 50 mm. Cultureswere shaken in air in water both at 30 C. For the deter-mination of glucose-6-phosphate. concentrated suspen-sions were incubated for I hr in a stream of oxygencontaining 5% carbon dioxide as described in the text.

tamin medium was quite similar to that in yeastextract-peptone medium.

Metabolism of glucose and fructose. Kesults inFig. 3 indicate the pattern of fluorescencechanges associated with the metabolism of glu-cose and fructose by intact cells of the mutant.The characteristic changes in fluorescence emis-sion under these conditions were contributedprimarily by reduced nicotinamide adenine dinu-cleotide (NADH) and NADPH as shown byChance (5) as also by the correspondence be-

90SECONDS 3 nMOLES

NADH4

w /t FRUCTOSE GLUCOSE

0

7UA.

W4

FRUCTOSE GLUCOSE

TIME

FIG. 3. Sugar-induced changes in reduced nicotina-mide adenine dinucleotide (NADH) and reduced nico-tinamide adenine dinucleotide phosphate levels of intactcells of the phosphoglucoisomeraseless mutant and thewild type as measured by fluorescence. Cell suspensionsin 50 mM triethanolamine buffer, pH 7.4, were irradi-ated with an actinic light beam (366 nm) and theemitted fluorescence signal was measured after passagethrough a filter passing 450 nm and higher wavelengths(9). An upward deflection of the traces indicates a riseof fluorescence corresponding to reduction of nicotin-amide adenine dinucleotide (NA D+) and NA D+ phos-phate; time flows from left to right. The insets indicatethe scales for both. The mutant 9520b, shown in curvesI and 2, was present at a concentration of 46 mg (wetweight) of cells per ml and the wild type (curves 3 and 4)at 21 mg per ml. The first arrow under each curverepresents addition of 10 mm sugars and the secondarrow indicates the instant at which the suspension be-came anaerobic, as indicated in separate polarographicrecords. The cells for these experiments were grown toend of stationary phase and possessed high respiratoryactivity.

762 MAITRA

PHOSPHOGLUCOISOMERASELESS MUTANT OF YEAST

tween the rise of the fluorescent signal and dropof nicotinamide adenine dinucleotide (NAD+)and NADP+ (not shown). The reduction of thesenucleotides after sugar addition, as shown in Fig.3, was quite rapid in the wild type; fructose addi-tion to the mutant also elicited comparable speedof reduction. The metabolism of glucose in themutant, however, was slow, as suggested by thereduced velocity of increase in fluorescence(trace 2, Fig. 3). The steady-state oxidation alsowas lower compared to the traces 1, 3, and 4,reflecting presumably a decreased concentrationof glycolytic metabolites which serve to acceptelectrons from these reduced n ucleotides. Theincreased duration of the aerobic phase duringglucose metabolism by the mutant compared tothat during fructose metabolism also confirmedthe decreased respiration with glucose as sub-strate. In a separate experiment with 50 mm po-tassium phosphate buffer, pH 7.4, the mutant9520b respired in the presence of fructose andglucose at rates of 2.3 and 1.2 (endogenous, 0.3)timoles of oxygen per min per g (wet weight) ofcells. A discernible stimulation of respiratoryrate by glucose took as much as 20 sec, whereasfructose oxidation started within 5 sec of fructoseaddition. With the wild-type parent, no distinc-tion could be made between the kinetics of fruc-tose and glucose oxidation; the respiratory ratewas nearly 8 ,umoles of oxygen per min per g(wet weight) of cells with either substrate. En-zymatic assay during aerobic glucose metabolismin the mutant showed a faster reduction ofNADP+ than that of NAD+. The percentage ofreduction of NADP+ was also higher than in thewild type. The level of NADP+ was reducedfrom 20 nmoles per g (wet weight) of yeast inabsence of sugar to nearly 7 nmoles per g whenthe mutant was treated with glucose. In the wildtype only marginal decrease of NADP+ levelcould be seen. The maximal extent of reductionof NAD+, however, was similar in the two cases,although in the mutant this took place with adelay.Addition of glucose or fructose to a culture of

the wild-type yeast or of fructose to a culture ofthe mutant 9520b growing on acetate resulted inan immediate increase in growth rate. As indi-cated earlier, addition of glucose to the mutantculture arrested growth. Since this might lead toan abortive overproduction of glucose-6-phos-phate, we examined if glucose addition caused adrainage of the adenosine triphosphate (ATP)pool in the mutant. We show in Fig. 4 the ade-nine nucleotide and inorganic orthophosphate(Pi) analyses of nongrowing cultures of the mu-tant during glucose and fructose metabolism.Parallel experiments with the wild-type parentare also shown for purposes of comparison. The

results show that in the wild type the kinetics ofchanges in levels of adenine nucleotides and Piafter glucose or fructose addition were indistin-guishable and bore resemblance with those in themutant culture treated with fructose. In all ofthese cases, the addition of sugars led to rapidrise of ATP and a drop of adenosine diphosphate(ADP), adenosine monophosphate (AMP), andP1. Addition of glucose to the mutant, however,led to the same end result, although the kineticswere different. There was a lag of nearly 4 minbefore the drop of Pi and AMP or the rise ofATP was evident. This was surprising, because inthe mutant it was the utilization of fructoserather than that of glucose which showed a lag.This has been verified in a number of other ex-periments where early kinetics of sugar utiliza-tion had been examined. The behavior of ADPwas the most striking in this respect. Clearly, thetime to turn over the adenine nucleotide pool wasmuch longer with glucose than with fructose. Atany rate, the addition of glucose led to an in-crease of ATP level in the mutant. One otherfeature of these experiments was that in the wildtype the rate of glucose utilization was fasterthan the rate of fructose utilization, whereas thereverse was the case with the mutant.

Results in Fig. 5 further illustrate the differ-ences in the profile of the early glycolytic inter-mediates during glucose metabolism by the mu-tant and the wild type. So that the early kineticsof glucose utilization in the mutant was not lim-ited by the low ATP level of these cells, we havepreincubated the cell suspension aerobically for10 min in presence of 20 mm ethanol. Separateexperiments indicated that this pretreatment in-creased ATP level without affecting the levels ofglycolytic intermediates measured in this experi-ment. In the wild-type parent the concentrationsof glucose-l-phosphate and fructose-6-phosphatebore a fixed relationship to those of glucose-6-phosphate within limits of experimental error,whereas in the mutant this constant ratio wasmaintained only between glucose-l-phosphateand glucose-6-phosphate. Despite the markedrise in the level of glucose-6-phosphate, that offructose-6-phosphate was nearly zero throughoutthe course of incubation. The metabolism of glu-cose in the mutant was further characterized bythe feature that the overshoot in the concentra-tion of glucose-6-phosphate, seen in the wildtype, was absent. One other feature of these andrelated experiments was that the rate of glucoseutilization in the mutant was constant in the faceof a marked rise in the intracellular concentra-tion of glucose-6-phosphate. Nevertheless, therate of glucose utilization in the mutant wasnever more than a third of that of the wild-typeS. cerevisiae. Results in Fig. 6 showing the accu-

763VOL. 107. 1971

MAITRA

16-j

NW 12-J0

< 20

wO3.(D 6

0

- 04~-04cn

W 0 2

H 003ao(3IN

J0

3,k

M U T A N T0 10 20 30~~~~~Glu

0_-O-- ISUGARS|

'v Fru

03- AT D Fru[A2TG I u

0-2 - --"GlI~~~~~~~~I

I.~0 10 20

M N U T E S

W L D T Y P E

14 -J

Nw1-J

12 02

10

10HU)

5 Fw

IN3W

-J0

[ -I],FuI

8\ Fru LADPJ -06GIu 03

.. - 0001-

-2-4LL-24 u.

-16 0

0

3O C 10 Z0

M N U T E S

FIG. 4. Time-course of changes in the concentrations of adenine nucleotides and P, during glucose and fructosemetabolism by the phosphoglucoisomeraseless mutant and the wild-type parent. Washed cells from overnight cul-tures from yeast extract-peptone-fructose were suspended in 0.1 M triethanolamine buffer, pH 7.3 and kept aeratedby passing a stream ofoxygen containing 5% carbon dioxide. Samples were withdrawn at indicated intervals in 0.7N perchloric acid and the neutralized supernatants analyzed. Glucose was measured by glucose oxidase and fruc-tose by a fluorometric procedure involving the use of hexokinase, phosphoglucoisomerase and glucose-6-phosphatedehydrogenase. For the assay of adenosine triphosphate in glucose-treated mutant samples, the fluorometric pro-cedure with the 3-phosphoglycerate kinase-glyceraldehyde-3-phosphate dehydrogenase couple was used. All otherprocedures were the same as in Materials and Methods. Solid lines indicate experiments with glucose (Glu) andbroken lines with fructose (Fru). Figures on the left hand column refer to experiments with the mutant and thoseon the right to the wild-type parent. Note that in the figures showing sugar disappearance the ordinates do notbegin at 0 and that the sugar concentrations are expressed as micromoles per milliliter of the suspension. Additionof glucose or fructose was made at time 0. Concentration of cells in the incubation mixture in milligrams of cells(wet weight) per ml were as: wild type-glucose 21; fructose 19; mutant-glucose 21.5; fructose 17.5.

mulation of the aldolase metabolites in the mu-tant during glucose and fructose metabolism alsosuggested the decreased flow of glucose carbonalong the glycolytic pathway. Surprisingly, no

gluconate-6-phosphate could be detected in theacid extracts when the mutant accumulated glu-cose-6-phosphate; the estimated level was lessthan 5 nmoles per g.When the release of carbon dioxide from spe-

cifically labeled glucose was examined, the mu-

tant was found to contribute a much larger share

of glucose carbon-l than glucose carbon-6 in theexpired gas. These results are shown in Table 2.This experiment indicated that, compared to thewild type, the loss of phosphoglucoisomerase inthe mutant had caused the major bulk of glucosecatabolism to be channeled through the oxida-tive pentose phosphate pathway. However, a sub-stantial part of glucose-6-phosphate was con-served as shown by the accumulation of radioac-tivity in the cell during metabolism of glucose-l-14C. We have not followed the fate of the con-

764 J. BACTERIOL.

9.-Fru E

0

Glu

b,-( ------ |- GlAMPu

° ----FGlu

|"w FADP0------- - F Glu

,~oFru. .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

HU)4

w

H:

!- U-C -- - -w k; 1U- Fru

rATP]

2:31

VOL. 107, 1971 PHOSPHOGLUCOISOMERASELESS MUTANT OF YEAST

4

2

w

8

w

4z0

0

w

0-i

O-J

2

12 w

-i

0uD

en8 aJI

M N U T ES

FIG. 5. Kinetics of hexose monophosphates during aerobic glucose metabolism by the phosphoglucoisomerase-deficient mutant and the wild-type S. cerevisiae. Stationary phase cultures from yeast extract-peptone-fructosewere washed and resuspended in 50 mm triethanolamine buffer containing 5 mm magnesium chloride at pH 7.4.The suspensions were incubated aerobically with 20 mm ethanol 10 min before 10 mm glucose (Glu) addition attime 0. Other details were as in FiR. 4. GJP, G6P, and F6P indicate, respectively, glucose-i-phosphate, glucose-6-phosphate, and fructose-6-phosphate. The incubation mixtures for the mutant and wild type contained, respec-

tively, 46 and 1S mg (wet weight) of cells per ml.

served glucose. When washed cells were incu-bated with glucose or fructose in phosphatebuffer, no appreciable difference could be foundbetween the effect of these two sugars on thecontent of glucose in cell hydrolysates.

Synthesis of macromolecules during growth in-hibition by glucose. We have indicated elsewhere(20) that the mutant 9520b synthesized glycolyticenzymes at a faster rate in presence of smallconcentrations (2 to 5 mM) of glucose than in itsabsence, although growth continued to be inhib-ited. At higher concentrations, however, additionof glucose brought about net decay of basal syn-thesis. Figure 7 shows the results of such an ex-

periment. The steady state rate of differentialsynthesis of phosphoglycerate kinase was notonly inhibited by the addition of 50 mm glucose,but there ensued a drop of the total enzyme ac-

tivity. The differential rate of this decay seemedto increase with time. It has not been determinedwhether the decrease of enzyme activity in thecells was due to its release into the medium. Not

every glycolytic enzyme behaved in this manner.Hexokinase activity, for example, continued toincrease after glucose addition at a rate slightlylower than the basal rate. Incubation of restingcultures in buffered-sugar solutions indicatedthat the rate of release of 260 nm-absorbing ma-

terial into the medium was only marginallyhigher in the mutant in presence of glucose thanin presence of fructose.

Results in Fig. 8 describe experiments thatsuggest a possible clue to the mechanism ofgrowth inhibition by glucose. The synthesis ofribonucleic acid (RNA) during growth of themutant was studied by following the incorpora-tion of 14C_uracil into trichloroacetic acid-insol-uble material (3). The results indicated that fruc-tose stimulated RNA synthesis after a lag pe-riod, whereas glucose brought about an inhibi-tion. Higher concentrations of glucose stoppedRNA synthesis completely within 3 min. Sepa-rate experiments, not shown here, suggested thatthis inhibition was reversible. After removal of

765

G6P

G I u

F6PG IP

J. BACTERIOL.

15 FRUCTOSE

lo-

w~~

* >10_ O SEX1

w

(1)

w

_j50

GLUCOSE x 10

0~~~~~

0 4 8 12

MINUTESFIG. 6. Accumulation of aldolase metabolites in the

phosphoglucoisomeraseless mutant during aerobic me-

tabolism ofglucose and fructose. Two separate culturesin the stationary phase were used in 50 mm potassiumphosphate buffer, pH 7.4. Fructose-], 6-diphosphate,and triose phosphates were measured together and theresults were expressed in units of total triose phos-phates; those of glucose were multiplied by 10. Cellconcentrations for fructose and glucose were, respec-

tively, 26.5 and 192 mg (wet weight) of yeast per ml.All other details were the same as in Fig. 5.

glucose, incorporation of "4C-uracil into RNAresumed at the uninhibited rate.

DISCUSSIONThe partial loss of respiratory activity in the

mutant clone 9520b appeared to be due to a ge-netic lesion additional to the one causing phos-phoglucoisomerase deficiency. This was sug-

gested by the observation that in 9520s, a deriva-tive of 9520b, respiratory activity was furtherreduced without much alteration of the phos-phoglucoisomerase activity. The reversion ofboth these clones on glucose led to reappearanceof only phosphoglucoisomerase, leaving respira-tory activity unchanged. No revertant from9520s could be isolated that grew on acetate. Webelieve the loss of respiration in 9520s wascaused by genetic deletion of some cytoplasmicrespiratory determinant (24). Although we havenot mapped the phosphoglucoisomerase muta-tion, the instability of the mutant and revertantenzymes to heat suggested a structural defect.Furthermore, the occurrence of a mutant lackingmore than 99% of the wild-type activity in a

single-step isolation procedure indicated a singlegene specifying the structure of phosphoglu-coisomerase. This was also consistent with themonophasic heat-inactivation kinetics of thewild-type enzyme.Since the studies reported here have been car-

ried out with a mutant continuously segregatinginto respiratory-deficient clones, the possibilityremains that some of the biochemical propertiesof the mutant derive from its respiratory lesion.Two lines of evidence, however, suggest this tobe unlikely. A partial loss of respiration has littleeffect on the metabolism of fructose in the mu-tant as shown by the similarity in its features offructose metabolism with those of the wild typemetabolizing either glucose or fructose (Fig. 3and 4). The features of glucose metabolism inthe mutant, however, are distinctly different.Secondly, the profiles of glycolytic intermediatesand nucleotides during metabolism of either

TABLE 2. Release of radioactive carbon dioxide fromglucose-l-14C and glucose6-1-4C by cultures ofthe

phosphoglucoisomeraseless mutantand its wild-type parenta

14C°2Total '4C (I4CO 2/ from glu-

Yeast Labeled in CO, '4C-glU- icose-- 14C/glucose (counts cose) 14CO

per min) x 100 from glu-cose-6-'4C

Mutant 9520b 1-14C 10,000 0.426-14C 863 0.03 14.0

Wild type 1_14C 9,500 0.40 2.56-14C 3,940 0.16

a Stationary phase cultures from yeast extract-pep-tone medium containing glucose (wild type) or fructose(mutant) were washed and diluted freshly in yeast ex-tract-peptone and incubated for 3 hr at 30 C. The ex-periment was conducted aerobically in Warburg flaskscontaining the following: 2 ml of the diluted culture inyeast extract-peptone; 0.15 ml of 20% potassium hy-droxide in the central cup; 0.1 ml of 10 N sulfuric acidin a side arm; 0.2 ml of 100 mm radioactive glucosecontaining 2 jsCi (2.4 x 106 counts per min for glu-cose-I-14C and 2.5 x 106 counts per min for glucose-6-14C) in the other side arm. After 30 min of equilibra-tion in the bath at 30 C, glucose was tipped in and in-cubation was continued for 15 min. Shaking was con-tinued throughout. At the end of incubation, the acidwas tipped in from the side arm and a shaking periodof I hr was allowed for absorption of carbon dioxideby the alkali. Samples corresponding to 25 Mliters ofthe alkali in the central cup were counted in Bray'sscintillation fluid (4). An identical sample of alkali, notcontaining any added radioactivity, served as the con-trol. The concentration of cells in the reaction mixturewas such as to give the following extinction values at650 nm: wild type, 5.2; mutant, 10.2.

766 MAITRA

VOL. 107, 1971 PHOSPHOGLUCOISOMERASELESS MUTANT OF YEAST

,o /

//

00 1000

dilte in02rseim Smlso 0 mlwrewt-0

drawn for enzyme assay (20) after resumption of expo-nential growth. A t the time the second sample wastaken out, 50 mm glucose was added to one-half of theculture. E,,,, refers to the optical density of the cultureat 650 nm.

sugar by 9520b were indistinguishable fromthose by 9520s, the petite variant. It is reasona-ble, therefore, to conclude that we are studyinghere primarily the effects of the loss of phos-phoglucoisomerase rather than those due to thepartial loss of respiratory activity.One of the major differences in the properties

of phosphoglucoisomerase-negative mutants de-scribed so far in bacteria and the one describedhere lies in their growth response on glucose (12,13). The yeast mutant turned out to be exclu-sively glucose-negative in growth unlike the bac-terial mutants. At first sight this appeared to bedue to the limited capacity of the pentose-phos-phate pathway to produce fructose-6-phosphate.However, the ability of maltose and galactose tosupport growth, although slowly, suggested thatthis pathway could indeed sustain the requiredflux of carbon. The inability of glucose in thisregard was therefore not due to questions of in-suff'icient rate of carbon flow or changes in cel-lular ATP or Pi pools (Fig. 4). Results in TableI on the levels of glucose-6-phosphate duringgrowth indicated that unlike glucose, both galac-tose and maltose allowed accumulation of rea-sonably small levels of this intermediate. Therate of maltose fermentation by intact yeast cellsis known to be comparable to that of glucosefermentation and is limited by the activity of

maltose permease rather than by maltase (8).The maintenance of controlled levels of glucose-6-phosphate in the mutant during growth on

maltose indicated to us operation of a feedbackregulation of maltose transport. We had ob-served that the ability of a maltose-grown cellsuspension of the mutant in buffer to oxidizemaltose fell off very rapidly. This, together withthe earlier observations on the preferential inacti-vation by glucose of maltose permease ratherthan of a-glucosidase activity in S. cerevisiae(14, 22), suggested that glucose-6-phosphatemight be the signal responsible for inactivation.Any rise of glucose-6-phosphate concentration as

a result of the block in phosphoglucoisomerasewould prevent further formation of glucose-6-phosphate controlled by the rate-limiting maltosepermease. Maltose metabolism in the mutant,therefore, would be a delicately poised steadystate between the synthesis or activation of mal-tose permease and the rate of glucose-6-phos-

9000

w

0-.1.

10 mMSUGAR

FRUCTOSE

_-D CONTROL

IGLUCOSE

30001F

0

0 20 40 60

MI N U TES

FIG. 8. Stimulation ofRNA synthesis in the mutant9520b by fructose and its inhibition by glucose. A 96-hrculture from yeast extract-peptone-acetate was dilutedin fresh medium and incubated for an hour. At time 0,I MCi carrier-free "4C-uracil was added and periodicsamples of 0.5 ml were added to a final concentrationof 10% trichloroacetic acid at 0 C. At the instant de-noted by the arrowhead, 10 mm glucose or fructose was

added to two separate samples from the same culture.Sampling was continued as indicated. After I hr thecells were filtered on membrane filters and washed with25 ml of 10% cold trichloroacetic acid. Filters were

dried before counting on a gas-flow counter. The op-tical density of all the cultures at 650 nm (E,,50) was

1.3 throughout except that containing fructose. At theend of the incubation the suspension containing fruc-tose recorded an extinction of 1.5. At intermediatepoints the specific activity was calculated by assumingexponential growth throughout. The curve labeled con-

trol refers to the culture containing only acetate as theextra carbon source.

767

J. BACTERIOL.

phate expenditure. Although no such control ofgalactose metabolism is known at the level ofgalactose transport, the low glucose-6-phosphatelevel during growth of the mutant on galactosesuggests similar feedback controls.The inhibition of growth of the mutant by glu-

cose differed from the effect of 2-deoxyglucoseon the growth of Schizosaccharomyces pombe(21) in that glucose did not alter the titer ofviable cells (Fig. 2). Since glucose inhibitedgrowth of the mutant on fructose as well, inhibi-tion of fructose- 1, 6-diphosphatase reaction byglucose-6-phosphate (11) could not be the mech-anism of growth inhibition; we have observed thepresence of significant amounts of fructose-6-phosphate inside the cell under conditions indi-cated in Fig. 2. Although high concentrations ofglucose-6-phosphate have been found to be aninhibitor of E. coli fructose-1 , 6-diphosphatase(11), it is perhaps easier to visualize that the lossof phosphate potential (5) caused by the abortiveformation of glucose-6-phosphate in the doublemutant would lead to accumulation of AMP,which would keep this gluconeogenic enzyme inan inhibited state. In the mutant described here,our experiments suggested (Fig. 8) inhibition ofRNA synthesis as one of the expressions of theinhibitory effect of glucose. High concentrationsof glucose-6-phosphate might inhibit some stepsin RNA synthesis by competing, for example,with certain phosphorylated-nucleotide precur-sors. However, we have not determined whetherthe synthesis of other macromolecules is also in-hibited by glucose. Addition of glucose broughtabout a decay of total activity of phosphoglyc-erate kinase, although hexokinase was not soaffected. Release of 260 nm-absorbing materialsalso was not particularly marked. It appears,therefore, that if some permeability changes wereinvolved in the growth inhibition, such effectswere of a specific nature.

Intact cells of the mutant did not produce anydetectable amounts of either fructose-6-phos-phate or glucose-6-phosphate when treated withglucose and fructose, respectively, indicating thatphosphoglucoisomerase activity was not signifi-cant in vivo. The catabolism of glucose thereforetook place, as results in Table 2 indicated,mainly via the pentose-phosphate pathway. Thisrestricted the rate of formation of glycolytic in-termediates as also ATP synthesis from glucose.In the experiment shown in Fig. 6, the rate offormation of alcohol in the mutant with glucoseas the substrate was 1.0 umole per min per g(wet weight) of yeast, whereas with fructose thiswas nearly 10 times as much. In a wild-typeyeast harvested in the exponential phase ofgrowth, such as to have respiratory activity com-

parable to that of the mutant 9520b, the rate ofaerobic alcohol formation from glucose wasnearly 15 ,moles per min per g (wet weight) ofyeast. The contribution of the pentose-phosphatepathway in glucose catabolism under these condi-tions in S. cerevisiae was, therefore, around 7%of the total glycolytic flux. The reduced flowthrough glycolysis in the mutant was also re-flected in the difference between glucose andfructose in the initial kinetics of changes in cel-lular ADP, AMP, P,, and ATP levels (Fig. 4).In absence of sugars the ATP-ADP ratio of themutant was very much lower compared to thewild type; the level of P, also was nearly fourtimes as much.The maximum rate of intracellular consump-

tion of glucose-6-phosphate in the mutant, foundby first incubating a cell suspension with glucoseand then quickly removing the extracellular glu-cose by filtration and washing, was 3 umoles perg (wet weight) of cells per min. A substantialpart of glucose-6-phosphate metabolized wastherefore diverted to extraglycolytic products,perhaps polysaccharides. The synthesis of suchcompounds was expected to be stimulated by theaccumulated glucose-6-phosphate (23). However,we have not investigated this. The sustained ac-cumulation of glucose-l-phosphate in Fig. 5 indi-cated that the rate of polysaccharide synthesiswas slower than those of hexokinase and phos-phoglucomutase. The yeast mutant differed fromthe Escherichia coli mutant (12) in one otherfeature; growth of the latter in absence of glu-cose yielded cells in which hydrolyzable glucosewas very much reduced compared to cells grownon glucose. With the yeast mutant, however, cellsgrown on fructose plus small amounts of glucosehad 112 and those grown on fructose alone had87 qmoles of hydrolyzable glucose residue per g(wet weight). Whether the synthesis of glucosefrom fructose in the mutant was through a leakyphosphoglucoisomerase or through any otherunknown reaction has not been determined.The accumulation of glucose-6-phosphate

during the metabolism of glucose in this mutantafforded an opportunity of studying the effect ofglucose-6-phosphate on the rate of glucose utili-zation. The results in Fig. 5 on the kinetics ofglucose-6-phosphate accumulation during glucoseutilization showed that the rate of glucose utili-zation was independent of the rising concentra-tion of glucose-6-phosphate in the range of 0 to27 mm (,umoles per g (wet weight) of cells). Thelack of kinetic correlation between the glucose-6-phosphate level and rate of glucose consumptionsuggested strongly that other determinants wereresponsible for the control of sugar utilization inyeast cells.

768 MAITRA

ACKNOWLEDGMENTSI thank Zita Lobo for her excellent assistance.

LITERATURE CITED1. Bernheim, N. J., and W. J. Dobrogosz. 1970. Amino sugar

sensitivity in Escherichia coli mutants unable to grow onN-acetylglucosamine. J. Bacteriol. 101:384-391.

2. Bdck, A., and F. C. Neidhardt. 1966. Properties of a mu-tant of Escherichia coli with a temperature-sensitivefructose-l, 6-diphosphate aldolase. J. Bacteriol. 92:470-476.

3. Bollum, F. J. 1966. Filter paper disk techniques for assayingradioactive macromolecules, p. 296-300. In G. L. Can-toni and D. R. Davies (ed.), Procedures in nucleic acidresearch. Harper and Row, New York.

4. Bray, G. A. 1960. A simple efficient liquid scintillator forcounting aqueous solutions in a liquid scintillationcounter. Anal. Biochem. 1:279-285.

5. Chance, B. 1961. Energy-linked cytochrome oxidation inmitochondria. Nature (London) 189:719-725.

6. Cozzarelli, N. R., J. P. Koch, S. Hayashi, and E. C. C.Lin. 1965. Growth stasis by accumulated L-a-glycero-phosphate in Escherichia coli. J. Bacteriol. 90:1325-1329.

7. Davies, P. W. 1962. The oxygen cathode, p. 137-179. In W.L. Nastuk (ed.), Physical techniques in biological re-search, vol. 4. Academic Press Inc., New York.

8. De La Fuente, G., and A. Sols. 1962. Transport of sugarsin yeasts. 11. Mechanisms of utilization of disaccharidesand related glycosides. Biochim. Biophys. Acta 56:49-62.

9. Estabrook, R. W., and P. K. Maitra. 1962. A fluorimetricmethod for the quantitative microanalysis of adenineand pyridine nucleotides. Anal. Biochem. 3:369-382.

10. Fiske, C. H., and Y. SubbaRow, 1925. The colorimetricdetermination of phosphorus. J. Biol. Chem. 66:375-400.

11. Fraenkel, D. G. 1968. The accumulation of glucose 6-phosphate from glucose and its effect in an Escherichiacoli mutant lacking phosphoglucose isomerase and glu-cose 6-phosphate dehydrogenase. J. Biol. Chem. 243:6451-6457.

12. Fraenkel, D. G., and S. R. Levisohn. 1967. Glucose and

769

gluconate metabolism in an Escherichia coli mutantlacking phosphoglucose isomerase. J. Bacteriol. 93:1571 -1578.

13. Fraenkel, D., M. J. Osbom, B. L. Horecker, and S. M.Smith. 1963. Metabolism and cell wall structure of amutant of Salmonella typhimurium deficient in phos-phoglucose isomerase. Biochem. Biophys. Res.Commun. 11:423-428.

14. Gorts, C. P. M. 1969. Effect of glucose on the activity andthe kinetics of the maltose uptake system and of a-glu-cosidase in Saccharomyces cerevisiae. Biochim. Biophys.Acta 184:299-305.

15. Hattman, S. 1964. The functioning of T-even phages withunglucosylated DNA in restricting Escherichia coli hostcells. Virology 24:333-348.

16. Heredia, C. F., G. De La Fuente, and A. Sols. 1964. Met-abolic studies with 2-deoxyhexoses 1. Mechanism of in-hibition of growth and fermentation in baker's yeast.Biochim. Biophys. Acta 86:216-223.

17. Lederberg, J., and E. M. Lederberg. 1952. Replica platingand indirect selection of bacterial mutants. J. Bacteriol.63:399-406.

18. Maitra, P. K. 1970. A glucokinase from Saccharomycescerevisiae. J. Biol. Chem. 245:2423-2431.

19. Maitra, P. K., and Z. Lobo. 1971. A kinetic study of gly-colytic enzyme synthesis in yeast. J. Biol. Chem. 246:475-488.

20. Maitra, P. K., and Z. Lobo. 1971. Control of glycolyticenzyme synthesis in yeast by products of the hexokinasereaction. J. Biol. Chem. 246:489-499.

21. Megnet, R. 1965. Effect of 2-deoxyglucose on Schizosac-charomyces pombe. J. Bacteriol. 90:1032-1035.

22. Robertson, J. J., and H. 0. Halvorson. 1957. The compo-nents of maltozymase in yeast, and their behavior duringdeadaptation. J. Bacteriol. 73:186-198.

23. Rothman, L. B., and E. Cabib. 1969. Regulation of gly-cogen synthesis in the intact yeast cell. Biochemistry 8:3332-3341.

24. Winge, O., and C. Roberts. 1958. Yeast genetics, p. 128-156. In A. H. Cook (ed.), The chemistry and biology ofyeasts. Academic Press Inc., New York.

25. Yarmolinsky, M. B., H. Wiesmeyer, H. M. Kalckar, andE. Jordan. 1959. Hereditary defects in galactose metabo-lism in Escherichia coli mutants. 11. Galactose-inducedsensitivity. Proc. Nat. Acad. Sci. U.S.A. 45:1786-1791.

VOL. 107, 1971 PHOSPHOGLUCOISOMERASELESS MUTANT OF YEAST