loss of the pasteur effect in phosphofructokinase ...

Inter. Jour. of Mod. Biotech. Vol.2 (1) 2012 pp.213-220

RESEARCH PAPER

LOSS OF THE PASTEUR EFFECT IN PHOSPHOFRUCTOKINASE DISRUPTANTS OF SACCHAROMYCES CEREVISIAE : DIRECT DEMONSTRATION BY MEMBRANE INLET MASS SPECTROMETRY _________________________________________________________ ARUNA K1 and DAVID LLOYD2 1 2

Department of Microbiology, Wilson college, Mumbai, India 400007.

School of Biosciences, Cardiff University, Main Building, Museum Avenue, Cathays Park, Cardiff CF10 3AT, Wales, UK E.mail: [email protected]

Abstract Respiration (O2 consumption) and fermentation (CO2 and ethanol production) were monitored continuously by mass spectrometry in washed cell suspensions of Saccharomyces cerevisiae EG103 (wild type), and in strains disrupted with respect to PFK1 or to PFK2 genes. In the wild type strain, the transition from aerobic conditions to anaerobiosis led to activation of glycolysis i.e. increased CO2 output and ethanol production known as the Pasteur effect. Neither of the disruption mutants showed increased fermentation rates i.e. Pasteur effect when O2 was depleted. Further evidence of loss of allosteric control in both disrupted strains was obtained by demonstration of a metabolic block at the phosphofructokinase step in non-proliferating organisms. This has been shown by measuring the rates of glucose utilization as well as by measuring the pool sizes of glucose 6-phosphate and fructose 1, 6 bis-phosphate in glucosesupplemented organisms. Studies on the controls implicated in the related Warburg Effect in tumour cells, whereby metabolism is switched to an anaerobic mode, could be facilitated by the continuous monitoring methods developed here for yeast. Keywords: Yeast, Saccharomyces cerevisiae, phosphofructokinase, Pasteur effect, membrane inlet mass spectrometer.

Introduction The inhibition of carbohydrate utilization by O2 has been observed in many cell-types (Krebs, 1972), and since the original discovery of this effect by Pasteur (1861) different mechanisms have been proposed. Energy demand in many types of yeast may be satisfied either by highly efficient mitochondrial energy conservation, or by the much less efficient glycolytic generation of ATP. Transition from the anaerobic to the aerobic state leads to a suppression of glycolytic flux, observable as a decreased rate of CO2 evolution; the more efficient ATP-generating system suppresses the less efficient one. The effect is reversible, so that an increased rate of production of CO2 is observed going from aerobiosis to the anaerobic state (Lloyd et al. 1983). In the yeast Saccharomyces cerevisiae, two phosphofructokinases have been identified as key control points in glycolysis (Ramaiah et al. 1964; Salas et al. 1965). Allosteric properties partly explain the mechanism whereby the

Inter. Jour. of Mod. Biotech.2(1)213-220(2012)

concentrations of many glycolytic intermediates may vary periodically over a time in many cell types. These glycolytic oscillations are conveniently studied in yeast (Higgins 1964; Chance et al. 1964, 1967). The critical role of phosphofructokinase in the Pasteur effect is universally accepted (Ramaiah 1974). The only point of control in the cases where the Pasteur effect occurs is at phosphofructokinase level since a crossover occurs only between hexosemonophosphates and fructose 1, 6-bisphosphate. No such crossover is observed when the Pasteur effect is absent (Lagunas and Gancedo 1983). Changes from variety of effectors like fructose-2,6 bis- phosphate, fructose 1,6 bis-phosphate, ATP, AMP, Pi and pH in going from aerobic to anaerobic conditions contributed to the changes in the rate of PFK observed during the Pasteur effect (Reibstein et al.1986) Genetic determinants for the phosphofructokinase enzymes have

Aruna and Llyod ,2012

two been

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characterized (Lobo and Maitra 1982 a, b, 1983; Nadkarni et al. 1984).The soluble allosteric enzyme (PFKI) is encoded by two genes (PFKI and PFK2), which respectively specify the catalytic () and regulatory () subunits (Gayatri and Maitra 1991). It is an octamer of 4α- and 4β-subunits (Taucher et al. 1975; Kopperschlager et al. 1976). PFK2 also specifies the catalytic subunit of PFKII, while other genes (PFK3, 4, 5, 6) may also have regulatory functions for this particulate activity (Maitra et al. 1987). The soluble enzyme, PFK I, can account for both the catalytic and control functions associated with the glycolytic pathway. Transcriptional control of phosphofructokinase genes at non-coding sequences has been elucidated. Regulatory elements found in the promoters of other glycolytic genes, although present; do not seem to play a major role in PFK gene expression (Heinisch et al. 1991). Mutagenesis of heterooctameric yeast phosphofructokinase modified the allosteric properties of it and thus phosphofructokinase plays an important role in regulation of the glycolytic flux through its activation by fructose 2, 6-bisphosphate (Heinisch et al. 1996). The crystal structure of this enzyme has recently been published (Benaszak et al. 2011). A second enzyme PFK II has been reported in mutants lacking PFK I that are still able to grow on glucose (Lobo and Maitra 1982). This enzyme activity is particulate (membrane-bound) (Lobo and Maitra 1983; Nadkarni et al. 1984; Maitra et al. 1987). This PFKII is present only in cultures grown on glucose, and permits anaerobic growth only after pre-induction aerobically (Nadkarni et al. 1982). Synthesis of PFKII occurs transiently and by the time the growth of the culture is completed, the membrane-bound PFK II is undetectable. The non-sense mutant pfk1-1 produces this PFKII enzyme during glucose supported growth (Nadkarni et al. 1982) but it is absent in ethanolgrown cells (Lobo and Maitra 1982). A glucosenegative strain (pfk1 pfk2) has been transformed with

multiple copies of PFK2 (Lobo et al. 1995). This strain overexpresses the particulate enzyme independently of the dosage of the PFKI encoded -subunit, the enhanced enzyme activity correlates directly with the “particulate’ association of the  subunit. The characteristics of respiration and fermentation of glucose in the wild-type and disruptants (pfk1 and pfk2) of yeasts can be conveniently studied by the use of a membrane-inlet mass spectrometer (Lloyd and Scott 1983; Lloyd et al. 1985; Degn et al. 1985). This device enables simultaneous and continuous recording of O2 and CO2 dissolved in yeast suspensions (Lloyd et al. 1983a, b); ethanol can also be monitored (Lloyd and James 1987; Cox 1987). A disruptant pfk1, completely lacking PFKI activity, and a disruptant pfk2, lacking PFKII activity facilitate studies of the roles of these two enzymes (Heinisch 1986). These disruptant mutants are extremely unlikely to show any leaky phenotype as shown by mutants isolated in classical mutant screenings (Aguilera and Zimmermann 1986). Current studies show the simultaneous and continuous monitoring of respiration and fermentation directly in suspensions of wild type and pfk disruptants of Saccharomyces cerevisiae by the use of membrane-inlet mass spectrometry. We also show here that the controls necessary for the expression of the Pasteur effect are lost when either PFKI or PFK2 is deleted. Further evidence of loss of allosteric control in both disrupted strains is obtained by demonstration of a metabolic block at the phosphofructokinase step in non-proliferating organisms. This has been shown by measuring the rates of glucose utilization as well as by measuring the pool sizes of glucose 6-phosphate and fructose 1, 6 bis-phosphates in glucose-supplemented organisms.

Material and Methods Strains

Estimation of profile of cellular metabolites during glycolysis in cells of EG103, pfk1 and pfk2

The wild type strain of Saccharomyces cerevisiae- EG 103 (mata trp1 leu2 ura3) haploid cells containing PFK1 and PFK2 and disruptants pfk1 lacking soluble PFK1 and pfk2 lacking the particulate PFK2 (gift from Dr. J. Heinisch) were used in this study.


Wild type EG103 and pfk1 and pfk2 were -1 grown at 30C with shaking at 200 rev. min (100ml in 250ml conical flasks) on a medium containing (w/v) 0.3% yeast extract, 1% peptone and 1% glucose until the stationary phase. Organisms were centrifuged at 1000g for 2 min then washed twice and resuspended in 20 mM of Na citrate buffer (pH4.2)


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For aerobic incubation the suspension was bubbled with a gas mixture containing 95% O2 and 5% CO2 with stirring in air. The anaerobic incubation was done in 50-ml test tubes with a slow stream of gas (95%N2 and 5% CO2) while gently agitating the suspension. Glucose (10 mM) was added at zero time. Samples (1 ml) were withdrawn at designated time intervals, stopped by adding 15% HClO4 and rapidly chilled. After neutralization with KOH to pH 7.4, the pool size of the intermediate metabolites, fructose 1, 6- bis-phosphate and glucose 6- phosphate were assayed in the supernatants. This was achieved by using fluorimetric determinations (Maitra and Lobo 1971; Gayatri and Maitra 1991). Residual glucose was estimated by glucose oxidase-peroxidase method (Bergmeyer and Bernt 1974). Measurement of O2, CO2 and ethanol A quadrupole mass spectrometer (Hiden Analytical, Gemini Business Park, Warrington, Cheshire WA5 5TN, U.K.) was used to measure O2 consumption, CO2 and ethanol production simultaneously and continuously. The membrane inlet probe consisted of a quartz tube (3.5 mm o.d.) sealed at one end and having a 0.25 mm diameter orifice, 5mm from its sealed end. A silicone rubber sleeve (o.d. 0.19 mm, i.d. 0.15 mm) was stretched into place over this orifice. Measurements were performed in a closed 2-ml tube (1-ml working volume), with the mass spectrometer inlet submerged in reaction mixture.Ethanol was monitored at m/z = 31: determination at other m/z values (29, 45 and 46) were less sensitive and less

specific (Lloyd et al. 1985). High pressure liquid chromatography (HPLC) analyses of culture filtrates indicated that some other volatiles detectable at m/z = 31 (e.g. isopropanol) were not produced by the strains of yeasts used in this work. At the stage where ethanol was maximally accumulated in cultures of the wild type (at the end of the glucose-repressed phase), its concentration reached 200mM. At this stage about 5mM-acetate was also present and this compound also gives a fragment ion at m/z = 31. Other fermentation products detected (citrate, glycerol, succinate and malate) were all non-volatile. Solubility of O2 was taken as being 258 M in air-saturated buffer (Wilhelm et al. 1977); CO2 and ethanol calibrations employed additions of standard solutions to the reaction mixture at pH 4.0. Biomass determinations were made using values of A600mm of washed cell suspensions (Lloyd et al. 1992). Calculation of Pasteur quotients Rates of CO2 and ethanol production from glucose under anaerobic conditions were direct measures of rates of glycolysis. Aerobic glycolysis cannot be assessed directly. For CO2 production, calculation of the glycolytic component necessitates subtraction of the contribution of the respiratory CO2 from total CO2 evolution. This was performed by assuming a 1: 1 stoichiometry between respiratory CO2 output and O2 uptake. Pasteur quotients are therefore presented as the ratio of anaerobic CO2 production to aerobic glycolytic CO2 production; aerobic glycolytic CO2 production is the measured total CO, evolution rate minus the contribution of respiratory CO2.

Result and Discussion Mass spectrometric continuous monitoring of metabolism by intact cells Figure 1 shows the uptake of O2, production CO2 and ethanol by washed suspensions of intact yeast strains. The Fig 1 (top panel) showed the responses observed in a suspension of wild-type strain (PFK1, PFK2). Addition of 10mM glucose gave no evident stimulation of either the O2 consumption or the CO2 production rates in this strain. This is a consequence of the unusually high levels of endogenous respiration by comparison with previously documented yeasts. Depletion of dissolved O2 from closed system led to the gradual transition to anaerobiosis after about 6 mins. The transition from the aerobic to anaerobic state markedly stimulated both CO2 output and ethanol formation due to the operation of the Pasteur effect. The Pasteur Quotient (ratio of anaerobic to aerobic CO2 output rates) was in Inter. Jour. of Mod. Biotech.2(1)213-220(2012)

the range 1.7 to 2.2 for five separate cultures. This direct observation of the Pasteur effect (Lloyd et al. 1983b; Lloyd and James 1987; Lloyd et al. 1992) indicates control of key reactions of glycolysis by metabolites that are present at high concentrations under aerobic conditions. One of the control points is the cytoplasmic phosphofructokinase (PFKI) which is sensitive to concentrations of fructose 6-phosphate, fructose 2, 6-bisphosphate, ATP, AMP, Pi and NH4 (Laurent et al.1984; Lloyd et al.1983a).Thus the overall rate of the glycolytic flux was greater under anaerobic conditions.


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Estimation of pools Glucose 6-phosphate and Fructose 1, 6 bis-phosphate Figure 2c and 2d show low steady state levels of glucose 6-phosphate under aerobic and anaerobic conditions in wild type. Whereas in pfk2, the level of glucose 6-phosphate was very low before addition of glucose and increased after addition of glucose, it remained high throughout indicating a block in further utilization of glucose 6-phosphate under the aerobic condition. However, the anaerobic condition showed a high level of glucose 6-phosphate throughout. Similar observations were made in pfk1. However, the basal level of glucose 6-phosphate was very low.

Figure 1. Mass spectrometric monitoring of cells. Top panel represents wild type and bottom panel pfk1 strain. The first arrow indicates () addition of yeast cells; the second () addition of glucose 10 mM to the cell suspension and the third arrow onset of anaerobiosis. The cell suspension taken was 1 mg/ml in both the cases. The topmost trace refers to O2, the middle to CO2 and the bottom trace ethanol.

Fig. 1(bottom panel) indicates that the deletion mutant pfk1, lacking PFK1 (Heinisch 1986) showed high respiratory activity of endogenously stored reserve materials and again added glucose gave no measurable increase. It also showed high CO2 production. However, the transition to anaerobiosis did not stimulate further CO2 evolution and no detectable ethanol was formed. Results obtained using strain pfk2 lacking PFK2 were similar to those seen from pfk1 i.e. neither of the phosphofructokinase disruption strains showed a Pasteur effect and neither strains produced ethanol at measurable rates. Table 1 summarizes the results obtained in several experiments with different strains. Utilization of Glucose Figure 2a and 2b shows that in the wild type strain glucose disappearance both aerobically and anaerobically proceeded rapidly, 3 mols and 5.5 mols glucose/min/g wet wt. of cells respectively. However, in pfk2, there was no glucose utilization under either aerobic or anaerobic conditions. The pfk1 strain showed a lower rate of glucose utilization by comparison with that of wild type under aerobic condition, but no measurable utilization under anaerobic conditions.


Figure 2e and 2f present the fructose 1, 6 bisphosphate levels in all the three strains mentioned above. Fructose 1, 6 bis-phosphate level remained high only in wild type indicating the presence of PFK activity, where fructose 1, 6 bis-phosphate level was very low in both pfk2 and in pfk1 strains under aerobic and anaerobic conditions. For the past 40 years it has been widely believed that 6-phosphofructo-l-kinase (PFKI, EC 2.7.1.11) is an enzyme with a significant flux control coefficient for glycolysis, although this view has occasionally been questioned (Boscar and Corredor 1984). Thus this enzyme was identified as the “primary oscillophore” responsible for the oscillatory glycolytic state sometimes observed in yeast (Hess and Bioteaux 1971). Effects of over-expression of glycolytic enzymes on glycolytic flux and growth rates have been studied. Four-fold over-expression of PFK1 hardly increased or decreased glycolytic flux or growth rate (Schaaff et al. 1989). Concomitantly with 4-fold over-expression of PFK, the activity of 6-phosphofructo-2-kinase and the concentration of fructose 2, 6 bis-phosphate decreased by a factor of 2 under anaerobic conditions, thus decreasing the in vivo activity of PFK (Davies and Brindle 1992). The measurements reported in this paper can explain why PFK does not have any control in anaerobically growing cells. The other effect is to increase the aerobic glycolytic flux up to the anaerobic level, and thereby abolish the Pasteur effect. In another study (Galazzo and Bailey 1990) the importance of control by glucose transport was emphasised (control coefficient 0.45 as compared with a value of only 0.2 for PFK). Lagunas and Gancedo (1983) have shown that the Pasteur effect is not always observed in growing yeast cells, although it can be demonstrated under special conditions (in cultures in chemostats with limiting sugar or under non-growing starvation Aruna and Llyod ,2012

217

conditions). Under highly controlled conditions of limited growth, control was distributed with PFK playing a major role (Cortassa and Aon 1998).Glucose assimilation in washed cell suspension of wild type Saccharomyces cerevisiae was accelerated when changed from aerobic to anaerobic conditions. This was demonstrated by the increased rate of glucose disappearance and the increased rate of CO2 production as measured by mass spectrometry. Under aerobic conditions the wild-type S. cerevisiae PFKl PFK2 showed responses in respiratory and fermentative activities similar to those previously observed in other yeasts with a Pasteur effect (Lloyd et al. 1983b; Lloyd and James 1987). In wild type cells where Pasteur effect was noticeable, the pool size of glucose 6-phosphate was small and did not change under anaerobic conditions (Lobo and Maitra 1982). During the anaerobic phase, intracellular concentration of fructose 1, 6 bisphosphates was increased fourfold. In contrast, when the Pasteur effect is absent, anaerobiosis provoked a decrease in all glycolytic intermediates tested; this result is in agreement with the small but significant decrease in the glycolytic flux observed. No variations were found between anaerobiosis and aerobiosis when the Pasteur effect is not observed (Lagunas and Gancedo 1983). For both pfk1 and pfk2 addition of 10mM glucose gave about 30% inhibition of O2 consumption, but stimulated CO2 production was not observed with either strain. The PFK activities were not seen in both deletion strains under stationary phase conditions. Buffered suspensions of stationary cultures of pfkl strain utilized glucose aerobically, CO2 is produced but no alcohol was made. CO2 production -1 -1 was 77 nmol min (mg wet wt cells) as measured by mass spectrometry, and remained the same during anaerobic conditions (Fig. 1 lower panel and Table 1). These results were consistent with pfk1-1 PFK2 which was obtained through classical mutant screening (Lloyd et al.1992). pfk1 strain utilized sugar aerobically through HMP shunt and TCA cycle, and so produced CO2. The stationary cultures of pfk1 strain however, has a decreased level of particulate enzyme activity, as seen by its low rate of glucose utilization under aerobic conditions. Anaerobically, however, the situation was very different. pfkl strain neither fermented glucose nor produced ethanol during anaerobic incubation. Glucose metabolism was negligible and the kinetics of metabolites reflected blocked glycolysis. This result is consistent with rise of glucose 6-phosphate and the absence of fructose 1, 6 bis-phosphate level (Fig.2 b, d, and f). This shows that Inter. Jour. of Mod. Biotech.2(1)213-220(2012)

particulate activity (Phosphofructokinase II) is independent of the  subunit. Similar results were obtained with pfk1mutant which were obtained by classical mutant screening. The failure of stationary cultures of pfk1 mutants to produce alcohol was found to be strongly correlated with the absence of the particulate phosphofructokinase in such cultures. When the same experiment was repeated with cells in the exponential phase of growth the pfk1 mutant displayed a much higher glycolytic activity both in regard to the utilisation of glucose and formation of ethanol (Nadkarni et al. 1982). In pfk2 strain there was no utilization of sugar, suggesting PFK activity is negligible. Mutations in PFK2 confer regulatory changes in soluble phosphofructokinase clearly indicate a dual role for the gene PFK2: it acts both as a structural determinant for the particulate enzyme and confers allosteric properties on the soluble phosphofructokinase in wild type cells (Lobo and Maitra 1982b).This also confirms the absence of fructose 1, 6 bis-phosphate on addition of glucose in contrast to the wild type. The pool size of glucose 6-phosphate is tenfold higher in this strain due to limitation of phosphofructokinase step. As earlier reports claim, PFK activity is present when either  or  subunit is present (Heinisch 1986). However, this present data confirm our earlier scheme of essential catalytic role of  subunit in both PFK I and PFK II (Lobo et al. 1995). As previously shown, PFK II rises to its maximum during exponential growth, and falls to a level below detectability during stationary phase of growth (Nadkarni et al. 1982). Deletion of  subunit therefore leads to complete loss of glycolysis in stationary phase of growth, where both PFK I and PFK II disappear. In summary, deletion of either PFK1 or PFK2 makes the cells unable to carry out glycolysis in yeast cells at stationary growth phase. The situation during exponential growth, however, is different. In the experiments reported here we have demonstrated that the disruption strains had metabolic block at phosphofructokinase step at stationary growth phase which was shown by measuring the rate of glucose utilization as well as by estimating the pool size of glucose 6-phosphate and fructose 1, 6 bis-phosphates on addition of glucose to these cells. We have also shown that deletion of PFK1 or PFK2 genes from Saccharomyces cerevisiae leads to loss of the Pasteur effect demonstrable in washed cell suspensions as measured by rates of CO2 and ethanol production when O2 becomes exhausted with the help of membrane-inlet mass spectrometry.


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Continuing interest in the mechanism of the Pasteur Effect not only in yeasts (Barnett and Entian 2005), but also in a wide diversity of cells and organisms revolves about the altered glucose metabolism in pathophysiological states (Diaz-Ruiz et

al. 2009, 2011). Thus the Warburg and Crabtree Effects, closely related to the phenomena reported here, may also be studied by continuous monitoring but under rigorously controlled conditions as described here for S. cerevisiae.

[b]

[a]

Aerobic

Anaerobic 30

Glucose µ moles / ml

Glucose µ moles /ml

30

20

10

20

10

0

0 0

10

20

30

0

10

Time, mins

20

30

Time, mins

[c]

[d] Aerobic

Anaerobic

3

G6P µ moles / gm

G6P µ moles / gm

3

2

1

2

1

0 0

0 0

10

20

10

[f ] Aerobic

Anaerobic

0.6

FDP µ moles /gm

FDP µmoles / gm

30

Time, mins

[e] 0.4

20

30

Time, mins

0.2

0.4

0.2

0

0 0

10

20

0

30

10

20

30

Time, mins

Time, mins

(FDP) level under aerobic and anaerobic condition of the two PFK disruption strains and of the wild type, (e) and (f) respectively. The wet weights of the cells suspensions were EG103 = 70mg/ml, Pfk1 = 55 mg/ml, pfk2 = 56 mg/ml., for aerobic conditions, and 52 mg/ml, 70 mg/ml, 53 mg/ml for anaerobic conditions respectively. EG103 ( ), pfk1 ( ) , pfk2 ( ).

Figure 2. Glucose utilization under aerobic and anaerobic condition of the two PFK disruption strains and of the wild type, (a) and (b) respectively. Glucose 6-phosphate (G6P) level under aerobic and anaerobic condition of the two PFK disruption strains and of the wild type, (c) and (d) respectively. Fructose 1, 6-bisphosphate

Table 1. Respiration and fermentation in washed cell suspensions of Saccharomyces cerevisiae strain EG103, pfk1 and pfk2 incubated with 10mM glucose Strain

wild type

Incubation conditions a O2 consumption

pfk1

Aerobic 34

Anaerobic --

Aerobic 25

b

44

75c PQ 1.7

77

d

72

138 c PQ 1.9

ND

CO2 production Ethanol production

a, b, d c

-1

pfk2

Anaerobic --

Aerobic 29

Anaerobic --

77

36

13

ND

ND

ND

-1

Specific activities expressed as nmol min (mg wet wt cells) , results typical of 5 experiments. PQ (Pasteur Quotient) = ratio of activities: anaerobic/aerobic. ND: not dectectable



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Acknowledgements This work was supported by the Department of Biotechnology, Government of India and Wellcome Trust. We are continually indebted to the Late Professor P.K. Maitra and Late Dr. Zita Lobo for their inspirational insights into the genetics and metabolism of yeasts and for conceiving the experiments performed in this paper.Dr. J. Heinisch kindly provided the yeast strains.

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