Phosphorylation in living yeast cells

PHOSPHORYLATION I N LIVING YEAST CELLS

ASER ROTHSTEIN Department of Eiology, University of Rochester and Department of Physiology

of the University of Rochester, School of Medicine and Dentistry, Xochester, New P o r k

INTRODUCTIOX

In recent years, a great deal of evidence has accumulated indicating that carbohydrate metabolism involves phosphorylated intermediaries (see reviews of Lipmann, '41 ; Kalcker, '42; and Cori, '42). Most of this evidence has been adduced from the study of tissue extracts and relatively simple sys- tpms of enzymes and substrates. Phosphorylation in intact cells is more difficult to demonstrate because steady states may exist whereby phosphorylated compounds are decom- posed almost as rapidly as they are synthesized, with little net change in concentration.

Changes in the concentrations of various phosphate fractions have been demonstrated in the living yeast cells, during anaerobic metabolism of sugar (Levitov, '36; Ostern et al., '38 ; Kruyk and Klingmuller, '39 ; I\lcFar.lane, '39 ; and bIirslci and Wertheimer, '39).

I n this paper further evidence will be presented concerning the relationship of phosphorylation to carbohydrate metabolism in the living yeast cell, with special reference to aerobic metabolism.

METHODS

Fresh Baker's yeast (Standard Brands Inc.) was used for all experiments. The yeast was washed twice and suspended in the experimental medium. During washing, the centrifuga-

l Present Address : Biochemistry and Pharmacology Department, School of Mectirinr and Dentistry, T!niversit>7 of Rochester, Rochester, N. Y.

221

222 ASER ROTHSTEIN

tion was sufficient to throw down only 90% of the cells and the rest were discarded with wash water. In this way, relatively uniform yeast suspensions were obtained. About 60 ml of 10% yeast suspension were placed in a 100ml graduated cylinder in a constant temperature bath at 25°C. The suspension was aerated violently at a constant rate through a sintered glass aerator, ensuring adequate mixing and uniform samples.

Aeration proceeded for several hours before a control sample of yeast was taken. This allowed the yeast to reach the constant phase of endogenous metabolism (Stier and Stannard, '37). The substrate was then added, and samples of yeast were taken at intervals thereafter. When poisons were used, these were added to the yeast suspension, 15 minutes before the substrate. I n anaerobic experiments, N, (purified over hot Cu) was bubbled through the yeast suspension continuously, starting 23 miniites before the addition of substrate.

All saniples of yeast were immediately centrifuged for 3 minutes. This time was sufficient to separate out 99% of the cells. The medium was decanted and the cells were ex- tracted with cold 10% trichloracetic acid for 30 minutes. This extract was then analyzed for phosphates by the method of Fiske and Subbarrow ( '29)' using a photo-electric colorimeter. One aliquot was analyzed immediately for inorganic phosphate. A second aliquot was analyzed after hydrolysis for 15 minutes in I N H C 1 at 100°C. A third aliquot was analyzed for total acid extractable phosphate after boiling in concentrated H,SO,, and digestion with 30% H,O,. The easily hydrolysable (labile) , and the stable phosphate fractions were calculated by difference.

Yeast concentrations were determined densitometrically with a photo-electric colorimeter calibrated against dry weight and cell volumes (measured by centrifugation in calibrated tubes).

PHOSPHORPLATION I N YEAST 223

THE DISTRIEUTION O F PHOSPHATE I N ENDOGENOUS YEAST

The total phosphate content of yeast previously aerated for 2 to 3 hours in -067 A 1 KH,PO,, ranged from 114 to 140 millimols per liter of cells, with an average of 124 millimols per liter (4 determinations). Of this total, about 40 millimols per liter or approximately 31% was extractable in trichloracetic acid.

The total trichloracetic acid extractable phosphate was divided by the method of analysis into inorganic, labile and stable fractions. I n twenty determinations on different batches of yeast, the mean concentration in millimols per liter of cells, and the range (highest and lowest values) were as follows: inorganic phosphate 12.3 to 17.6 with a mean of 15.8; labile phosphate 4.8 to 8.9 with a mean of 7.0; stable phosphate 11.7 to 26.0 with a mean of 16.9; total extractable phosphate 32.5 to 46.2 with a mean of 39.7.

Some of the individual data summarized above are repre- sented by the control values ir i table 1. Although these data indicated that considerable variation existed between different samples of yeast, it was observed that the concentration of phosphates in a given sample remained constant for at least 2 hours in the absence of substrate. Changes that occurred within the first hour after the addition of a substrate were therefore attributed to the onset of exogenous metabolism.

THE DISTBIEUTION O F PHOSPHATES DURING EXOGENOUS MET-4EOLISM

A redistribution of cellular phosphates took place within 4 minutes after dextrose mas added to yeast suspended in distilled water. Section A, table 1, indicates that the redistribution involved a decrease of inorganic phosphate and of total trichloracetic acid-extractable phosphate, and an increase in labile phosphate. The decrease in total aeid-extractable phosphate of approximately 5.0 millimols per liter was not due to leakage of phosphate out of the cells, because under the conditions of the experiment only a trace of phosphate appeared in the medium, accounting for less than 0.1 milli-

224 ASER ROTHSTEIX

TABLE 1

Changes zn the dzstrzbutzon of trzclzloracetie acid ext i uctable pliosphates in yeast, associated with nietabolism.

The control values are expressed in milliiiaols per liter of cells. The ezppa'mental valves are expressed as the change from the control value. All values

repesent the average for the stated number of experzments. ~ _________ ~ - -- __ ~~ _ _ -

CELLULAR PHOSPHATES EXPERI- NO. OF TIME AFTER SECTION TREATMENT MEXTAL ESPERI- ADDITION O F Total acid

Stab'e extractable x E D I u M KENTS SUBSTRATB Inorganic Labile

A Control H,O 3 0 16.9 8.2 15.3 39.8

Dextrose H,O 3 4 - 9.1 + 5.1 + 0.3 - 3.5 (1%) 8 - 10.1 + 3.0 + 1.4 - 3.7

20 - 7.2 + 2.9 - 1.0 - 5.3 13 -8.0 + 2.3 + 0.6 - 5.1

30 -5.2 + 3.2 - 2.1 - 4.1 ~

~~

-- ~~ ~- __ _ _ ~ ~ ~- - ~ - ~

B Control .033 M KCI 3 0 1.5 4 7.9 15.9 39.2

Dextrose .033M 3 4 - 3.7 + 2.5 - 1.8 - 5 0 (1%) KCl 8 -G.0 + 1.3 - 2.8 - 7.5

13 - 6.0 + 1.3 - 3.0 - 7.7 20 -5.0 + 1.8 - 3.8 - 6.5 30 -5.7 + 2.2 - 3.8 - 7.3

_ _ _ _ _ _ _ _ _ ~ ~ - - __-___

_ _ _ _ - -~ ~-~ ~ ~ ~ ~ ~ ~- ~ ~ - - __ -~ _ _ _ ~ ~ ~ ~ _ _ _ _ _ _ _ _ ~ _ _ _ ~~~~ - -

C Control .067 M KH,PO, 8 0 1.5.8 6.0 15.6 38.2

Dextiose .067 M KH,PO, 8 30 + 1.T + 3.9 - 5.9 + 6.6

~- ~ - ~ _________ -

~ ~ ~ - -~ (1%)

~~ -~ - ~~ ~- ~

D Control ,067 M KH,PO, 3 0 16.7 5.9 14.3 36.9

Aimerobic .067 M NO dextrose KH,PO, 3 40 -0.9 + 1.9 + 0.1 + 0.9

Anaerohic Dextrose .067 IT

(1 %ib) KH,PO, 3 40 + 2.n + 2.4 - 0.6 + 3.8

Aerobic Dextrose .067 M

KH,PO, 3 30 + 0.3 + 6.4 - 0.4 + 6.2 ___-. ~- ~ - ~ ~ _ _ _ ( I %)

- -. -~ -

PHOSPHORYLATION I N YEAST 225

TABLE 1 (Continued) -__ __ __ - -__

CELLULAR PHOSPHATES EXPERI- NO OF TIMEAFTER -

MEDIUM XENTS SUBSTRATE Inorqanic Labile Total acid S ~ b l e extractable

F C T I O X TRDATMENT MENTAI, EXPERI ADDITION OF

________ _.___. - ___ -~ ~ _ _ _ __ - _. ____ . manutps m X I 1 mN/Z m H / l m M / l

E Control .067 M KH,PO, 1 0 15.6 7.7 17.4 40.7

Alcohol .067 M (1%) KHIPO, 1 46 + 5.0 + 5.1 + 1.2 + 14.3

Alcohol .067 M KH,PO, 1 46 + 1.9 + 4.3 - 0 6 + 5.8

____ (.25%)

(1%) KH,PO, 1 46 - 0.9 + 10.8 + 0.0 + 9.9 Dextrose .OF7 M

- - ____ - _ _ ~- ~- - ~ _ ._ - - - -

F Control .067 M KH9P0 , 1 0 12.9 7 1 18.2 38.2

NaN, .067 M (5X 10-4M) KH,PO, No dextrose 1 45 + 0.7 + 0.2 - 0.8 + 0.1

NaN, (5 X 104M) Dextrose .067 M (1%) - 0.4 - 1.4 KH,PO, 1 45 - 1.3 + 0.3

Dextrose (1%) .067 M NoNaN, EH,PO, 1 45 + 6.5 + 6.8 + 0.9 + 14.2

G Control .06i M KH2P0 , 1 0 16.7 i .2 22.3 4F.2

N a F .067 ?LI ( .02M) KHIPO, 1 40 - 7.9 - 7.9 - 0.3 - 2.6 No dextrose 100 -6.1 + 2.1 + 7.7 + 3.7

K a F .067 M (.02 M) KHJ’O, 1 30 -8.6 + 3.4 + 7.9 + 2.7 Dextrose (1%) 100 - 13.0 + 4.3 + 11.7 + 3.0

~ ~~ ~~

Dextrose (1%) .067 M N o N a F KHJ’O, 1 40 + 1.7 + 2.8 - 0.1 + 4.4

---______-. - __ ._ .- - .. .

226 ASER ROTHSTEIN

.)

mols of phosphate per liter of cells (two experiments). The decrease of total phosphate must therefore have been due to an increase in cellular phosphates insoluble in trichloracetic acid.

In experiments in which the medium contained phosphates, the yeast samples were washed three times with H,O in order to remove external phosphate. Such washing does not change the distribution of cellular phosphates, because in control experiments, yeast was washed as much as six times with no significant changes. Because washing was necessary, it was not feasible to obtain samples very soon after the addition of substrate.

Section C, table 1, indicates that 30 minutes after the addition of substrate to yeast suspended in .067 11 KH,PO, there was ail increase not only in labile phosphate, but in inorganic and total phosphate as well. The difference between these results and those obtained with yeast suspended in H,O (table 1, section A) seemed to be due largely to the fact that some of the phosphate in the medium diffused into the cells after dextrose was added. The phosphate concentration of the medium decreased by an amount equivalent to a gain by the cells of 6.0 millimols per liter (two experiments). The accuracy of the above determinations was limited because the phosphate leaving the medium was but a small fraction of the total.

In connection with other studies, some experiments were performed on yeast suspended in .033 M KCl (section B, table 1). The phosphate redistribution that took place in these experiments was somewhat different from that which occurred in yeast suspended in H,O (section A, table 1). No immediate explanation is obvious, although it will be shown in another paper that yeast metabolism is influenced by the presence of potassium in the medium.

The changes in phosphate distribution that occurred during exogenous metabolism were not transitory. They persisted for at least 5 hours (the longest time any of these experi-

PHOSPHORYLATION I N YEAST 227

iiieiits were carried out), whereas all of the sugar was utilized in 30 to 40 minutes.

ANAEROBIC METABOLISM O F DEXTROSE

During fermentation the changes in distribution of phosphates were similar to those that took place during aerobic metabolism, but were not nearly as marked. Section D, table 1, shows for example, that the increase in labile phosphate during anaerobiosis was only 38% of that during aerobiosis.

When yeast was made anaerobic in the absence of substrate there was a significant increase in labile phosphate of 1.9 millimols per liter (section D, table 1). This map signify that endogenous metabolism proceeds through phosphorylated intermediaries, which accumulate in the absence of 0,.

METABOLTSM O F ALCOHOL

Alcohol is a normal end product of yeast fermentation. In the presence of 0,, yeast can oxidize alcohol rapidly and con- vert a large percentage of it to glycogen (Briicke, '33).

Section E, table 1, indicates that when alcohol was the substrate, there were changes in phosphate distribution of considerable magnitude, and of the same general character as those that occurred when dextrose was the substrate. The work of Cori and his co-workers (see Colowick and Suther- land, '42) indicates that glycogen formation proceeds through phosphorylated intermediates. Possibly the changes in phosphate distribution during alcohol metabolism are related to its synthesis into glycogen.

SODIUM AZIDE POISONED YEAST

Sodium azide is a respiratory poison which inhibits the cytochrome s p t e m (Keilin, '36). At a concentration of 5 x

M, it prevents respiration in yeast, but allows fermentation to proceed (Winzler, '40). It also poisons the assimilation, preventing the formation of glycogen (Pickett and Clifton, '41).

228 ASER ROTHSTEIN

The effect of sodium azide on phosphorylation in living yeast is shown in section F, table 1. No change in the distribution of phosphates occurred either in yeast without or with a substrate (dextrose), in spite of the fact that the substrate was utilized at approximately 60% of the normal rate. The absence of changes in phosphate distribution did not necessarily indicate that metabolism was not proceeding through phosphorylated intermediates. It might indicate that under the condition of azide poisoning, the phosphate compounds were broken down as rapidly as they mere formed.

SODIUM FLUORIDE POISONED YEAST

I n yeast poisoned with NaF, in the presence of added dextrose, there was a marked increase in both labile and stable phosphate (section G, table 1), and a marked decrease in inorganic phosphate, The stable phosphate did not increase in any of the other experimenh.

The increase in stable phosphate was probably due in part to the accumulation of phosphoglyceric acid which is known to occur in fluoride poisoned yeast during fermentation (McFar- lane, '39). Flouride has been shown by Lohman and Meyerhof ('34) to inhibit the enzyme associated with the conversion of phosphoglpceric acid to phosphopyruvic acid.

Not oilly did the stable phosphate of fluoride poisoned yeast fraction increase in the presence of a substrate (dextrose), but it also increased in its absence (section G, table 1). Thus, both endogenous and exogenous metabolism are associated with phosphorylated intermediaries, and hoth have fluoride sensitive pathways.

SUMMBRY

1. During aerobic metabolism of sugar by yeast, marked changes took place in the distribution of cellular phosphates. There was an increase in labile and in trichloracetic acid- insoluble phosphate and a decrease in inorganic, and tric1iloi.- acetic acid-soluble phosphate.

PHOSP€fOKPLATION IN YEAST 229

2. When potassium ions were present in the medium, the cellular redistribution of phosphate associated with sugar metabolism %?as somewhat different.

3. When phosphate was present in the medium, it diffused into the cells as soon as a substrate was added, somewhat obscuring the internal changes in phosphate distribution.

4. During fermentation of sugar, the internal changes in distribution of phosphates were less marked than during aerobic metabolism.

5. Sodium azide prevented changes in distribution of phosphates, even though the substiate was utilized.

6. The presence of sodium fluoride resulted in the accumulation of large quantities of stable phosphate within the cells.

7. Metabolism of alcohol resulted in clianges in distribution of phosphates similar to those caused by the metabolism of dextrose.

8. When yeast cells without substrate were made anaerobic, or were poisoned with fluoride, changes in internal phosphate distribution occurred which indicated that endogenous metabolism was proceeding through phosphorylated intermediates.

The author is grateful to W. 0. Fenn for his continued interest and advice.

LITERATURE CITED

BRUCKE, F. TH. 1933 Ubcr (~lycogenhildi~ng in Hefe. Biochem. Z., vol. 264, p. 157.

COLOWICK, S. P., AND E. W. SUTHEKLAND 1942 Polysaceharide synthesis from glucose by means of purified enzymes. J. Biol. Chem., vol. 144, p. 423.

CORI, C. F. 1942 Phosphorglation of carbohydrates. A Symposium on Respira- tory Enrymes. Univ. of Wisconsin Press, Madison, Wisconsin.

FISKE, C. H., AND Y. SUBBARROW 1029 Phosphocreatine. a. Biol. Chem., vol. 81, p. 629.

KALCKAR, H. M. The nature of energetic coupling in biological synthesis. Chem. Reviews, vol. 28, 11. 71.

R s ~ r ~ s , D. 1936 The action of sodium azide on cellular respiration and on some catalytic oxidation reactions. Proe. Roy. Soe., London, vol. 121, p. 163.

ISRUPK. K., AND V. KLINGMULLER 1939 Uber vorgange bei der alkoholischen Glrung der lebenden Hrfe. Biochem. Z., vol. 300, p. 343.

1942

230 ASER ROTHSTEIN

LEVITOV, M. M. Uber den Umsatz der ' ' Pyro phosphat " Fraktion in der Hefezelle. Bioehem. Z., vol. 284, p. 86.

LIPMANN, F. 1941 Metabolic generation and utilization of phosphate bond energy. Advances in Enzymology; vol. 1, p. 99. Interscience Pub- lishers, Inc., New York.

fiber die enzymatische umwandlung von Phosphoglycerinsaure in Breuztraubdure und Phosphorsaure. Bio- chem. Z., vol. 273, p. 60.

MCFARLANE, M. G. 1939 Phosphorylation of carbohydrate in living cells. Biochem. J., vol. 33, p. 565.

MIRSKI, A., AND E. WERTHEIMER 1939 Ziickerassimilatioii durch lebende Hefezelle. Enzymologia, vol. 7, p. 58.

OSTERN, P., T. BARANOWSKI, AND J. TERAZAKOWIC 1938 Uber die Phosphory- lierung des Adenosins durch Hefe and die Bedeutung dieses Vorgangs f u r die alkoholische Garung. 2. Physiol. Chem., vol. 251, p. 238.

Effect of selective poisons on utilization of glucose by yeast. Proc. SOC. Exptl. Biol. Med., vol. 46, pp. 443-445.

On the mechanism of carbohydrate dissimilation in Baker's yeast. J . Cell. Comp. Physiol., vol. 10,

WINZLER, R. J. 1940 The oxidation and assimilation of acetate by Baker's

1936

LOHMANN, K., AND 0. MEYERHOF 1934

PICKETT, M. H., AND C. E. CLIFTON 1941

STIER, T. J. B., AND J. N. STANNARD 1937

p. 79.

yeast. J. Ccll. Comp. Physiul., vol. 15, p. 343.

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Phosphorylation in living yeast cells