Eur. J. Biochem. 101,455-460 (1979)

Inactivation of Gluconeogenic Enzymes in Glycolytic Mutants of Saccharomyces cerevisiae Juana M . GANCEDO and Carlos GANCEDO Instituto de Enzimologia del Consejo Superior de Investigaciones Cientificas, Facultad de Medicina de la Universidad Autonoma, Madrid (Received May 17, 1979)

Yeast mutants blocked at different steps of the glycolytic pathway have been used to study the inactivation of several gluconeogenic enzymes upon addition of sugars. While phosphorylation of the sugars appears a requisite for the inactivation of fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, malate dehydrogenase is inactivated by fructose in mutants lacking hexokinase. The normal inactivation elicited by glucose in a mutant lacking phosphofructokinase indicates that the process does not require metabolism of the sugar beyond hexose monophosphates. A possible role for ATP in the inactivation process is suggested.

During the last decade evidence has accumulated showing that selective inactivation of certain enzymes is a relatively wide-spread mechanism for regulation of enzyme levels in microorganisms [l]. In yeast addition of glucose or related sugars to cultures growing on non-carbohydrate carbon sources initiates the inactivation of several enzymes (for a review see [2]). Existing evidence suggests that this inactivation is produced by a proteolysis of the enzyme [3,4]. However in spite of much work of different laboratories nothing is known about the intimate mechanism of the process. It appears of interest to determine to what extent the sugars need to be metabolized to trigger the inactivation and how the energy state of the cell influences it. To this aim we have employed yeast mutants specifically blocked at different steps in the glycolytic pathway to study the inactivation of fructose bisphosphatase, phosphoenolpyruvate carboxykinase and malate dehydrogenase. Our rationale was that if inactivation were different in the various mutants this could point to a particular metabolite as the signal for the physiological inactivation of the enzymes. The results do not make it possible to pinpoint a particular metabolite as the signal involved Abbreviations. Glucose-6-P or Glc6P, glucose 6-phosphate; fructose-6-P or FruhP, fructose 6-phosphate; fructose-l,6,Pz or Fru(l,6)P~,fructose 1,6-bisphosphate; h x k - , lacking hexokinase; p g i - , lacking glucosephosphate isomerase; p f k - , lacking phosphofructokinase; pyk-, lacking pyruvate kinase. Enzymes. Hexokinase (EC 2.7.1.1); glucosephosphate isomerase (EC 5.3.1.9); phosphofructokinase (EC 2.7.1.11); pyruvate kinase (EC 2.7.1.40); fructosebisphosphatase (EC 3.1.3.11); malate dehydrogenase (EC 1.1.1.37); phosphoenolpyruvate carboxykinase (EC 4.1.1.49).

in the inactivation and suggest that the phenomenon is a complex one. Phosphorylation of the sugars appears necessary for the inactivation of fructose bisphosphatase and phosphoenolpyruvate carboxykinase while malate dehydrogenase is inactivated by fructose in mutants lacking hexokinase. A preliminary account of these results was presented at the special FEBS meeting on Enzymes, Dubrovnik-Cavtat (Yugoslavia) 1979.

MATERIALS AND METHODS The strains of Saccharom-yces cerevisiae listed in Table 1 were used. They were grown with aeration at 30 "C in media containing 1 % yeast extract, 2 % peptone and 2 % pyruvate (strains of the D F Y series) or 2 % ethanol (other strains). The yeasts were harvested by centrifugation during the exponential phase of growth, washed with water, and frozen until used (no more than a day). No loss of the enzymatic activities studied was found in this time. Inactivation in vivo of the enzymes under study was achieved unless otherwise stated by addition of 1 % glucose to the cultures during the time indicated in each particular experiment. Extracts were obtained by grinding the cells with three times their weight of alumina and extracting the paste with three volumes of 40 mM 2-(N-morpholino)ethanesulfonic acid, 0.1 M KCl, 1 mM MgC12, 5 mM mercaptoethanol pH 6.4. The slurry was centrifuged at 10000 x g for 15 min and the supernatant used for enzyme assays.

Inactivation of Enzymes in Glycolytic Mutants of Yeast

456

Table 1. The strains of S. cerevisiae which were used DFY 22 is the parental strain of DFY 64, DFY 34 and DFY 70. S288Cmet is the parental strain of CJM-pk2 and CJM-pk14. Enzymes were assayed as described in Materials and Methods. Figures in brackets indicate the activities of the deficient enzymes in the parental strains ~

Strains of S. cerevisiae

Relevant enzymatic defect

Specific activity of the deficient enzyme

Source/reference

mU/mg protein none hexokinase glucosephosphate isomerase phosphofructokinase none pyruvate kinase pyruvate kinase

DFY 22 DFY 64 DFY 34 DFY 70 S288C met CJM-pk2 CJM-pk 14

D. G . Fraenkel [5] D. G. Fraenkel [5] D. G. Fraenkel [5] D. G. Fraenkel [5] Cold Spring Harbor Yeast Course this laboratory (unpublished results) this laboratory (unpublished results)

50 (1800) 10 (1500) 5 (140)

70 (1400) 50 (1400)

Table 2. Concentration i m M ) ojseveral metabolites in an hxk- strain and in the wild type after addition of glucose The yeasts were grown on pyruvate as described in Materials and Methods. While in the exponential phase of growth glucose (1 2)was added to the cultures. Samples were taken at the times indicated and treated as described in Material and Methods. Figures are mean values of three different samples. For calculations it has been assumed that 1 g wet yeast contains 0.6 ml of cell sap [14] Concentration in DFY 22 (wild type) after

Concentration in DFY 64 ( h x k - ) after

Metabolites

~

0 min

30min

~

60min

90min

2.9 3.2

4.7 5.9

~~~

0 min

30min

60min

90min

0.4 0.2

1.2 2.9

1.5 3.6

1.8 6.4

mM ~~~

+

Glucose-6-P fructose-6-P Fructose-1,&Pz

0.5 0.4

~~~

.~

~~~

1.9 2.2

Fructose bisphosphatase was assayed as described in [6], malate dehydrogenase as in [7] and phosphoenolpyruvate carboxykinase as in [S]. Glycolytic enzymes were assayed as described in [9]. For determination of metabolites the yeast was treated as described by Saez and Lagunas [lo] and metabolites tested as described in [ll]. Protein was assayed by the method of Lowry et al. [12]. Yeast extract and peptone were from Difco. Auxiliary enzymes and coenzymes were from Sigma (St Louis, U.S.A.) or Boehringer (Mannheim, F.R.G.). All other reagents were of analytical grade. RESULTS Inactivation in an hxk- Mutant Glucose and related sugars may trigger inactivation of gluconeogenic enzymes either directly or indirectly by increasing the levels of certain glycolytic intermediates. A mutant lacking both hexokinase A and B [13] would make possible in principle to distinguish between these possibilities. In such a mutant fructose is not phosphorylated and therefore not metabolized while glucose can be utilized via glucokinase. The rate of phosphorylation of glucose in extracts of this mutant is a third of that measured in the wild type, and

the growth rate on glucose is similar for both strains [5]. The changes in concentration of hexose phosphates upon glucose addition are also similar in the wild type and in the mutant as shown in Table 2. Table 3 shows the effect of glucose and fructose in the hxk- mutant. As it can be seen glucose inactivates the enzymes to the same extent as it does in the wild type while fructose is ineffective except in the case of malate dehydrogenase. Effect ojsugars on a pgi- Mutant To determine whether phosphorylation of glucose to glucose-6-P is a sufficient signal for inactivation or if further metabolism is necessary a mutant lacking phosphoglucose isomerase was used. The results obtained when glucose or fructose were added to pyruvate growing cultures are shown in Table 3. Glucose still inactivates fructose bisphosphatase and phosphoenolpyruvate carboxykinase although to a much lesser extent than in the wild type while in the case of malate dehydrogenase inactivation is similar to that of the wild type. The changes in concentration of several metabolites upon glucose addition to the mutant are shown in Table 4. As it can be seen there is a great increase in the concentration of glucose-6-P while fructose-6-P decreases and fructose-l,6-P2 does not show measurable variations. It is noteworthy that

J. M. Gancedo and C. Gancedo

457

Table 3. Inactivation of gluconeogenic enzymes in hxk- and pgi- yeast mutants Inactivation of the gluconeogenic enzymes was carried out with 1 % glucose or fructose as described in Materials and Methods. Figures are mean values of at least two separate experiments Enzyme

Strain

Enzyme defect

Relative activity after addition of _

-

glucose

_

fructose -

Omin

60min

90min

Omin

60min

90 min

100 100 100

10 19 58

5 16 36

100 100 100

12 80 71

11 81 51

43 37 79

21 30 70

100 100 100

39 76 91

34 85 91

68 48 53

58 44 51

100 100 100

58 67 87

58 68 86

% Fructose bisphosphatase

DFY22 DFY 64 DFY 34

Phosphoenolpyruvate carboxykinase

Malate dehydrogenase

none

hxkpgi-

DFY 22 DFY64 DFY 34

none pgi-

100 100 100

DFY22 DFY64 DFY 34

none hxkpgi-

100 100 100

hxk-

Table 4. Concentration ( m M ) of several metabolites in a pgi- strain after addition of glucose or fructose The yeast was grown on pyruvate as described in Materials and Methods. While in the exponential phase of growth glucose (1 %) or fructose (1 %) was added to the cultures. Samples were taken at the times indicated and treated as described in Materials and Methods. Figures are mean values of three different samples. For calculations it has been assumed that 1 g wet yeast contains 0.6 ml of cell sap [I41 ~~

Time after addition

Concentration after addition of __ glucose __ Glc6P Fru6P Fru(l,6)P2

__

~

min

mM

0 30 60 90

< 0.1 40 50 60

fructose ATP

energy charge

Glc6P

Fru6P

Fru(l,6)P2

ATP

energy charge

0.7 1 1 0.9

0.3 11 12 13

1.7 0.3 0.2 0.1

0.71 0.45 0.38 0.32

mM 0.7

< 0.1 < 0.1 < 0.1

0.2 0.2 0.2 0.2

2.2 1.6 1.6 3.2

the concentration of ATP remains unchanged although ATP is being used for the phosphorylation of glucose and cannot be regenerated by glycolysis. Fructose is very inefficient in promoting inactivation in the pgi- mutant (Table 3). A mixture of 1 % fructose and 0.01 % or 0.5 % glucose was more effective than glucose alone although for fructose bisphosphatase and phosphoenolpyruvate carboxykinase the extent of inactivation remained lower than in the wild type. (In 60 min fructose bisphosphatase and malate dehydrogenase were inactivated up to 60 %, phosphoenolpyruvate carboxykinase up to 40 %). Sucrose was also no more effective than glucose alone although it has been reported [15] to inactivate fructose bisphosphatase in a mutant where glucose or fructose were unable to do it. As seen in Table 4 addition of fructose resulted in a slight increase of fmctose-6-P and in a drop of ATP with a concomitant decrease of the energy charge. A depletion of the adenosinnucleotide

0.63 0.55 0.49 0.62

< 0.1 < 0.1 < 0.1 < 0.1

phosphate pool was also observed. (In 90 min the sum of ATP + ADP + AMP dropped from about 3 mM to less than 1 mM). Inactivation in a pfl- Mutant

The use of a p j l - mutant would make possible to decide if metabolism of the sugar needs to proceed beyond fructose-6-P or if phosphorylation to the stage of hexose monophosphate is sufficient. Fig. 1 shows the profile of hexose monophosphates and fructose1,6-P2 after addition of glucose to these mutants. As expected there is a great accumulation of hexose monophosphates while fructose-1,6-P~remains at the same level as when the yeast is grown on pyruvate. Table 5 shows the behaviour of the gluconeogenic enzymes. There are inactivated to the same extent as in the wild type. ATP concentration and energy charge (Fig. 2) behave also like in the wild type.

~

ii

Inactivation of Enzymes in Glycolytic Mutants of Yeast

458

’;I’ I : I il

6-

B A

12

52 -

10

I

E

E

-

.

a

0.5

4 c

1-

5n wildtype pfk-

pyK

pyk-

wildtype pfk-

tl

i n n” DFY 2 2 D F Y 70CJMpkZCJMpk14” OFY 22DFY70CJMpk2CJMpk 14

pyk-

wildtype pfk-

pyk-

Fig. 1 . Concentratiori of 1ie.vo.w phosphates qftci glirc,ose aclilition in several yeast glycolytic rnutants. (A) Glucose-6-P plus fructose6-P; (B) fructose-1,6-Pz. Yeasts were grown on pyruvate or ethanol as carbon sources as described in Materials and Methods. While in the exponential phase of growth glucose to a final concentration of 1 was added and samples taken as described in Materials and Methods. Results are mean values of three determinations. For calculations it has been assumed that 1 g wet yeast contains 0.6 ml of cell sap [14]. The columns for each metabolite are arranged in the sequence 0, 30, 60, 90 min

pyk-

pyk-

wildtype pfk-

pyk-

pyk-

Fig. 2. Concentrution ojATP ( A ) and values ofthe energy charge ( B ) in several yeast glycolytic mutants. Treatment of the yeasts and other conditions as in Fig. 1. The columns for each strain are arranged in the order 0, 30, 60, 90 min