Mechanism of control of adenylate cyclase activity in yeast by fermentable sugars and carbonyl cyanide m-chlorophenylhydrazon

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THE JOURNAL OF BIOLOGICAL CHEMISTRY

Q 1986 by The American Society of Biological Chemists, h e . Vol. 261, No. 19, Issue of July 6 , pp. 8744-8749,1986 Printed in U.S.A.

Mechanism

of Control

of

Adenylate Cyclase Activity in Yeast

by

Fermentable Sugars and

Carbonyl Cyanide

rn-Chlorophenylhydrazone*

(Received for publication, January 21, 1986) Claudio PurwinS, Klaas Nicolayt, W. Alexander Scheffersy, and Helmut HolzerSII

From the SBwchemisches Institut der Universitat Freiburg, Hermann-Herder-Strasse 7, 0-7800 Freiburg, West Germany, the §Institute of Molecular Biology, University of Utrecht, Padualaan 8, NL-3584 CH Utrecht, The Netherlands, the 7Department

of Microbiology, Delft University of Technology, Julianulaan 67a, NL-2628 BC Delft, The Netherlands, and the JJGesellschaft fur Strahlen-und Umweltforschung, Abteilung fur Enzymchemie, Ingolsthdter Landstrasse 1 , 0-8042 Neuherberg bei Miinchen,

West Germany

The phosphorylation of fructose- 1,6-bisphosphatase is preceded by a transient increase in the intracellular level of cyclic AMP which activates a cyclic AMP- dependent protein kinase (Pohlig, G . , and Holzer, H.

(1985)

J.

Bioi. Chem. 260, 13818-13823). Possible

mechanisms by which sugars or ionophores might activate adenylate cyclase and thereby Iead to an increase in cyclic AMP concentrations were studied. Studies with permeabilized yeast cells demonstrated that nei- ther sugar intermediates nor carbonyl cyanide m-chlorophenylhydrazone are able to increase adenylate cyclase activity. In the light of striking differences of the effects of fermentable sugars and of carbonyl cyanide m-chlorophenylhydrazone on parameters characterizing the membrane potential, it seems not reasonable that the activity of adenylate is under control of the membrane potential. Rapid quenching of 9-aminoac- ridine fluorescence after addition of fermentable sug- ars to starved yeast cells indicated an intracellular acidification. The 31P NMR technique showed a fast drop of the intracellular pH from 6.9 to 6.55 or 6.4 immediately after addition of glucose or carbonyl cy- anide m-chlorophenylhydrazone. The time course of the decrease of the cytosolic pH coincides with the transient increase of cyclic AMP concentration and the 50% inactivation of fructose-l,6-bisphosphatase under the conditions of the NMR experiments. Kinetic studies of adenylate cyclase activity showed an approximately %fold increase of activity when the pH was decreased from 7.0 to 6.5, which is the result of a decrease in the apparent K , for ATP with no change in V,,,. These

studies suggest that activation of adenylate cyclase by

decrease in the cytosolic pH starts a chain of events leading to accumulation of cyclic AMP and phosphorylation of fructose- 1,6-bisphosphatase.

Addition of glucose or other fermentable sugars to glucose- derepressed yeast cells causes repression of the synthesis of enzymes participating in gluconeogenesis (“catabolite repression”) (1) and moreover rapid inactivation of fructose-1,6- bisphosphatase and other key enzymes of gluconeogenesis (“catabolite inactivation”) (2). In the case of fructose-1,6- *This work was supported by Sonderforschungsbereich 206 (Deutsche Forschungsgemeinschaft) and Fonds der Chemischen In-

dustrie (Frankfurt). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18

U.S.C. Section 1734 solely to indicate this fact.

bisphosphatase, a 50% inactivation resulting from a phosphorylation of the enzyme is observed within 1-3 min after addition of glucose (3-6). There is evidence that the covalent modification of fructose-1,6-bisphosphatase by phosphoryla- tion marks the enzyme for selective proteolysis ( 5 , 7) which is complete after 1-2 h (4, 8 , 9 ) . Studies of the mechanism of catabolite inactivation have revealed that the cAMP concen- tration increases about 5-fold within 30 s after addition of

glucose (10, 11). This suggested that the phosphorylation of

fructose-1,6-bisphosphatase might be dependent on cAMP

(11, 12). In fact, in vitro experiments with purified fructose-

1,6-bisphosphatase (13) and purified CAMP-dependent protein kinase from yeast (14, 15) or from beef heart (16) dem- onstrated phosphorylation of fructose-1,6-bisphosphatase ac- companied by about 50% loss of activity of the enzyme. Moreover, it was shown that some of the kinetic properties of

fructose-l,6-bisphosphatase (pH/activity profile, dependence

of the reaction rate on the concentration of Mg2‘ or Mn2+) changed during in vivo phosphorylation after addition of glucose to starved yeast cells in the same way as during in vitro phosphorylation with the purified yeast enzymes (15). Similar to fermentable sugars, ionophores such as CCCP’ or

2,4-dinitrophenol cause a transient increase of the concentration of cAMP (17) and phosphorylation of fructose-1,6-bisphosphatase (18) in glucose-derepressed yeast cells. This increase in cAMP concentration after addition of fermentable sugars or ionophores might be due to activation of adenylate cycIase and/or inhibition of cAMP phosphodiesterase. The present studies were designed to explore possible mechanisms by which glucose or CCCP might activate adenylate cyclase.

MATERIALS AND METHODS~ RESULTS

The rapid transient increase in cAMP level after addition of fermentable sugars or ionophores to starved intact yeast The abbreviations used are: CCCP, carbonyl cyanide m-chloro- phenylhydrazone; Hepes, 4-(,2-hydroxyethyl)-l-piperazineethanesul- fonic acid Mes, 2-(N-morpholino)ethanesulfonic acid; TPP+, tetra- phenylphosphonium; Pipes, 1,4-piperazinediethanesulfonic acid; GMP-PNP, guanyl-5‘-yl (0,yimino)diphosphate.

*

Portions of this paper (including “Materials and Methods,” Table I, and Figs. 1, 3, and 5) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biolog- ical Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-0196, cite the authors, and include a check or money order for $3.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

at TU DELFT, on April 15, 2010

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Control of Adenylate Cyclase Actiuity in

Yeast

0745

cells suggested the possibility of activation of adenylate cyclase by glucose itself or by its metabolites. Accumulation of phosphorylated sugar derivatives after addition of glucose to starved yeast has been demonstrated previously (11, 33). It is shown in Fig. 1 that CCCP under the conditions used to demonstrate a transient increase of cAMP and inactivation of fructose-l,6-bisphosphatase also causes a transient accumulation of glucose 6-phosphate. Therefore, a direct demonstration of the effects of these sugars and of CCCP on adenylate cyclase activity in permeabilized cells was attempted. As shown in Table I, adenylate cyclase activity in yeast cells permeabilized by chitosan or toluene treatment was not sig- nificantly changed by addition of glucose or phosphorylation products of glucose. Also 2-dGlc and 6-dGlc as well as 2-dGlc- 6-phosphate showed no significant effect. Of a variety of possible effectors tested a significant effect on adenylate cyclase activity was seen with inorganic phosphate. Addition of 5 mM Pi results in an apparently 50% increase in enzyme activity. This cannot explain the transient increase in CAMP, since it is known that the level of inorganic phosphate decreases rapidly after addition of glucose to starved yeast cells (34). Of interest is, however, the inhibition (approximately

40%) of adenylate cyclase activity by 1 mM AMP. It has been

shown by von Herrath (35) in our laboratory that after addition of glucose to yeast under the conditions used in the experiments described here, the AMP concentration decreases in 1 min from 0.8 mM to about 0.2 mM. Therefore, a decrease of the AMP concentration after addition of glucose to starved yeast cells may activate adenylate cyclase by a deinhibition mechanism. The uncoupler CCCP, which, similar to glucose and other fermentable sugars, causes a transient increase in cAMP in intact cells (17) and also leads to a phosphorylation of fructose-1,6-bisphosphatase in intact cells (18) shows no significant direct effect on adenylate cyclase activity in permeabilized cells (Table I). Because 0.2 mM CCCP increases the AMP level 3-fold in starved yeast (data not shown) this ionophore in contrast to glucose cannot affect adenylate cyclase activity by release of AMP inhibition. In agreement with previous studies by Jaynes et al. (36) we found adenylate cyclase unaffected by 4 PM GMP-PNP, 1 mM KF, and 0.1 mM CCCP. This is in contrast to Casperson et al. (37) who

described a stimulation of adenylate cyclase by GMP-PNP or GTP. Because the concentration of GTP does not increase immediately after addition of glucose or CCCP t o starved yeast, a short term stimulation of adenylate cyclase by GTP might not be a reasonable explanation for the observed transient increase in CAMP.

As a next possibility for control of adenylate cyclase activity, dependence on the potential of the cell membrane was studied (cf. Ref. 38). As an indicator of the membrane poten-

tial the concentration of TPP' in the medium was measured with a TPP+-selective electrode (27). Addition of fermentable sugars immediately and drastically decreases the extracellular

TPP' concentration, i.e. increases the potential of the cell membrane (Fig. 2 A ) . Ethanol, 2-dGlc, or 6-dGlc show no such effect (Fig. 2B). Measurements of the fluorescence of rhoda- mine, 6 G, as an indicator of the membrane potential (39) after addition of glucose produced completely parallel results (data not shown). The effects of the fermentable sugars, 2-dGlc, 6-dGlc, and ethanol, on the membrane potential parallel the effects on increase of the cAMP level (Ref. 11 and data not shown). However, 0.2 mM CCCP, which behaves like fermentable sugars with respect to the effect on increase of cAMP (upper part of Fig. 3), and on the decrease of catalytic activity of fructose-l,6-bisphosphatase as well as on phospho- rylation of the 40-kDa subunits of fructose-1,6-bisphospha-

A B 6 0

-

5 . 0

-

4 . 0

-

3 0 -

-

4

a n ~ 2 . 0 3 1 5 -

-

k

-

_a 0 LI

2

1 . 0

-

Y 6o

tl

3 0 - 50mM €SOH 2 0

-

1 5 - IO

-

w .-

0751

I 0 5 IO 15 0 5 IO I5 time (rnin)

FIG. 2. Effect of fermentable sugars (Glc, Man, Fru), non-

fermentable sugars (2-dGlc, g-dGlc), and ethanol on the ex-

tracellular concentration of TPP+ (added as bromide) deter-

mined with a TPP+ selective electrode. Ethanol grown yeast

suspensions (2.5% wet weight/v) were incubated in 25 mM Hepes/ Tris, pH 7.0, at 30 "C (total volume 9 ml). The experiment was started by adding 9 pl of 6 mM TPP+ (final concentration 6 p ~ ) . As indicated by the uertical arrow, different sugars or ethanol were added from 100 times concentrated stock solutions.

k

\

e

I

a

4 ' Q - A , ~ 25mM Glc I I I 10 20 30 tlme (mlnl

FIG. 4. Effect of CCCP and glucose on extracellular TPP+

concentration. Ethanol grown, stationary yeast cells were incubated

at 5% suspension (wet weight/v) in 50 mM Hepes/Tris, pH 7.0, at

30 'C. At zero time, 6 PM [3H]TPP' was added. At the indicated times, 400-pl samples were withdrawn, centrifuged for 10 s, and 200 p1 of supernatant counted. At 20 min, CCCP dissolved in methanol (final concentration 0.2 mM) or glucose (final concentrations 25 mM) were added.

tase (lower part of Fig. 3) produces a different effect on membrane potential. As shown in Fig. 4, CCCP addition

results in a slow increase of the extracellular [3H]TPP+, i.e. depolarization of the cell membrane. Since CCCP interferes with the assay of TPP' using the selective electrode, the concentration of 3H-labeled TPP' in the medium was measured. After addition of glucose, the [3H]TPP+ showed a similar rapid decrease of the TPP' concentration in the medium (Fig.

4) in agreement with the data using the TPP'-selective electrode (Fig. 2). Opposite effects of glucose and CCCP were also

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8746

Control

of

Adenylate Cyclase Activity in Yeast

obtained studying changes in the concentration of K+ in the medium. As shown in Fig. 5, glucose causes a rapid decrease of the extracellular K' concentration, analogous to the decrease of the TPP' concentration shown in Figs. 2 and 4. In contrast, 0.2 mM CCCP, a concentration which increases CAMP, causes not a decrease but a slow increase in the extracellular K+ concentration parallel to the slow release of [3H]TPP+ shown in Fig. 4. When CCCP is added after K+

has reached the low plateau, it rapidly reverses the K+ de- creasing effect of glucose (Fig. 5). Because of the striking differences between the effects of fermentable sugars and CCCP on parameters characterizing the membrane potential, a control of adenylate cyclase activity by the membrane potential as an explanation for the mechanism of increase of cAMP initiated by fermentable sugars as well as by CCCP appears unlikely. The idea of control of adenylate cyclase activity by the intracellular pH was raised by results of Nicolay et al. (32). These authors used NMR spectra of inorganic phosphate for measurement of the cytosolic pH value of the yeast Zygosacchuromyces bailii. After addition of glucose to the starved yeast cells, a transient decrease of about

0.2 pH units was observed. Similar results have been reported by Den Hollander et al. (40) for Saccharomyces cereuisiae.

To check if under the conditions used in the present work addition of fermentable sugars also causes a decrease in the intracellular pH, changes of the fluorescence of 9-aminoacridine as an indicator of the intracellular p H (29, 30) were measured. As shown in Fig. 6, addition of fermentable sugars to starved yeast cells under the conditions where cAMP increases and fructose-1,6-bisphosphatase is phosphorylated causes distinct changes in fluorescence. 2-dGlc or 6-dGlc show no effects (CCCP cannot be studied because of heavy inter- ference with the fluorescence measurements). With the con- tinuous recording of the fluorescence it could be shown that

FIG. 6. Effect of fermentable (Glc, Fru, Man) or nonfer-

mentable (a-dGlc, 6-dGlc) sugars or ethanol on the intracel-

lular pH. Glucose grown yeast cells (30 mg wet weight) suspended

in 3 ml of 50 mM Hepes/Tris, pH 7.0, were incubated at room temperature in the presence of 10 PM 9-aminoacridine for about 5 min until the decrease of the fluorescence was slow and linear. After this preincubation period, 25 mM fermentable or nonfermentable deoxy sugars were added from 2.5 M stock solutions.

the changes in intracellular pH are at least as rapid as the changes of the concentrations of glycolytic metabolites observed after addition of fermentable sugars (11, 33). The findings suggest that the activity of adenylate cyclase may be under control of the intracellular pH. There is controversy about whether changes of fluorescence of the dye actually measures a pH change or a membrane polarization effect. Therefore, a more direct measurement of changes of intracellular pH was done by applying the NMR technique used in the above-mentioned measurements of cytosolic pH values in Z. bailii (32). As shown in Table 11, decrease of pH up to 0.35

units is observed after addition of glucose. In this experiment for technical reasons the concentration of yeast was raised from 2.5% or 5% wet weight/v used in the experiments shown in Figs. 1-6 to 40%. As depicted in Table I1 under exactly the same conditions of the NMR experiment with 40% yeast,

inactivation of fructose-1,6-bisphosphatase and a transient increase of cAMP were observed as described for the experi- ments with 2.5% yeast suspensions. It is evident that under the nonphysiological conditions of 40% cell suspension the phenomenons being under study (transient increase of cAMP and inactivation of fructose-l,6-bisphosphatase) are observed similar to the 2.5-5% cell suspensions. With the NMR tech- nique it was also possible to study the effect of CCCP on intracellular pH. Because of the high concentration of yeast

(40%) necessary for the short term NMR experiments, instead

of 0.2 mM CCCP ( c f . Figs. 1, 4, and 5) 2 mM CCCP (final concentration) were added to the starved yeast. A decrease of

0.5 p H units within 1 min after addition of CCCP was observed (cf. Table 11). Controls with addition of the solubilizer for CCCP (dimethyl sulfoxide) exhibited no change in the cytosolic pH. The effects of CCCP on fructose-1,6-bisphosphatase activity and cAMP concentration are shown in Table

11. The results summarized in Table I1 demonstrate clearly that both CCCP and glucose decrease the cytosolic pH.

Activity of adenylate cyclase in permeabilized yeast cells at different pH values of the suspension medium was tested. It is shown in Fig. 7 that at pH 7, i.e. the cytosolic pH of starved yeast cells (32), a low activity of adenylate cyclase is observed. With both methods of permeabilization, chitosan or toluene treatment, a decrease of the pH below 7.0 causes distinct increases in the adenylate cyclase activity. Measurements of

TABLE I1

Time course of the levels of fructose-1,6-bisphosphutase activity, p H

in the cytosol, and CAMP in a 40% (wet weightlv) yeast suspension after addition of 2 mM CCCP or 2% Glc.

Determination of fructose-l,6-bisphophatase activity and assays of cAMP were done in simultaneous parallel experiments outside the magnet.

Addition

Glc CCCP

Specific activity of fructose-1,6-bisphosphatase (%)'

5 min after addition 53 49

Cytosolic pH

Before addition 6.90 6.90

1 min after addition 6.71 6.40 2 min after addition 6.62 6.40 3 min after addition 6.55 6.40 cAMP (nmol/g wet weight)

Before addition 0.65 0.62

40 s after addition 1.2 0.88

80 s after addition 0.78 1.17

5 min after addition 0.82 0.98

The reference value is that before addition of glucose or CCCP.

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Control

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Adenylate Cyclase Activity in Yeast

8747 3000

1 I

i?

5

1000

-

chltosan- u) 0

-

E,

0 ’ I I P 5.0 6.0 7 . 0 8.0 PH

FIG. 7. pH dependence of adenylate cyclase activity of glu-

cose grown yeast cells permeabilized with toluene or chitosan.

The assay mixture contained 0.5 mM ATP, 0.4 mM CAMP, 5 mM MnC& and was buffered in the case of toluene-treated cells with 0.1

M Mes/KOH, pH 5.1, 0.1 M Pipes/KOH, pH 6.2, 0.1 M Pipes/KOH, pH 7.0, or 0.1 M Hepes/KOH, pH 8.0, and in the case of chitosan- treated cells with 50 mM Mes adjusted with imidazole for the pH values 5.0-6.5 and 50 mM imidazole adjusted with Mes for the pH values 7.0 and 7.5. The reaction was started by addition of permeabilized yeast (0.2-0.5 mg of protein/ml).

TABLE I11

ATP concentrations for half-maximal velocity (apparent K,,, values) of adenylute cyclase activity as a function of the p H value Glucose grown yeast cells were permeabilized with chitosan. The composition of assay mixtures was as described in the legend to Fig. 7 and the reaction was started by addition of permeabilized yeast (0.2 mg of protein/ml). The maximal velocity extrapolated from Line- weaver and Burk plots was at all pH values, 2400 pmol cAMP X h” X me”.

pH _{half-maximal velocity}Concentration for

5.5 6.0 6.5 7.0 7.5 m M 0.4 0.6 1.2 4.0 5.0

the dependence of adenylate cyclase activity of chitosan- permeabilized cells on the ATP concentration at different pH values showed that between p H 7.5 and 5.5 the apparent K, values, but not the Vmax values are dependent on the pH (Table 111). The transient decrease of the cytosolic pH value after addition of glucose to starved yeast cells may therefore cause a n activation of adenylate cyclase by increasing the affinity of the enzyme for ATP. This then, might account for the observed transient increase of CAMP.

DISCUSSION

In the present paper, evidence is accumulated in support of the hypothesis that a fast decrease of the intracellular p H after addition of glucose or of CCCP was responsible for in

vivo activation of the adenylate cyclase. A similar hypothesis was mentioned by Caspani et al. (41) and by Valle et al. (42) for regulation by p H of the glucose induced, cAMP mediated activation of trehalase in S. cereuisiue. The NMR experiments summarized in Table I1 demonstrated definitively a decrease of the cytosolic p H after addition of fermentable sugars. Relatively high concentrations of CCCP are necessary to induce a cAMP increase, in contrast to low CCCP concentrations for uncoupling. T h e high CCCP concentrations increase the vacuolar pH to the level of the cytosolic pH, i.e. they

destroy the compartmentation between vacuoles and cytosol

Glc, Man, Fru, Protonophores

$.

cH+

k

Adenylate-Cyclase

k

cyclic AMP

4

CAMP-Proteinklnase

+

Fru-1,6-P2ase-P

+

Proteolysis

FIG. 8. Proposed sequence of events after addition of fer- mentable sugars or CCCP to starved yeast.

(data not shown). This is evidence that CCCP when stimulating adenylate cyclase acts as a “protonophore.”

To investigate the p H dependence of adenylate cyclase activity we used permeabilized yeast cells for closer physiological conditions compared to membrane preparations obtained from broken cells. Fig. 7 demonstrates the pH depend- ence of toluene- and chitosan-treated yeast cells at 0.5 mM ATP. Within the range of the physiological pH a considerable increase of the adenylate cyclase activity from pH 7 to 6 is observed. As calculated from the Lineweaver-Burk plot for chitosan-treated cells (data not shown), Vmax was unaffected between pH 5.5 and 7.5, whereas the ATP concentration for 50% activity (“apparent K,”) showed a minimum of 0.4 mM A T P a t p H 5.5 and increased to 5 mM at pH 7.5. These findings are quite similar to the experimental values for

toluene/ethanol-permeabilized cells reported by Varimo and Londesborough (43). Detailed pH kinetic studies of glucose- 6-phosphatase, a membrane-bound enzyme intensively inves- tigated in rat liver, revealed a very similar situation as with yeast adenylate cyclase, i.e. a marked dependence of the K,,,

value but no modulation of VmaX as a consequence of changes in the pH of the assay mixture (44).

At first sight protons as effectors in metabolic regulation seems improbable because most enzymes and many other catalysts in a variety of different metabolic pathways would respond at the same time to a change of the proton concentration. This would be in contrast to the pathway specific actions known for many regulatory effectors. Nevertheless, in recent years more and more control mechanisms affected by changes in the pH have been described (for a recent summary see Ref. 45). It may be that membrane-bound enzymes, such as liver glucose-6-phosphate phosphatase (44) or yeast adenylate cyclase are preferred candidates for regulation by proton concentration.

The regulation of adenylate cyclase activity by the proton concentration fills a gap in the chain of events leading from application of fermentable sugars or protonophores to prote- olysis of fructose-1,6-bisphosphatase (Fig. 8). In this process, which is part of “catabolite inactivation” in yeast (2), a rapid phosphorylation of fructose-1,6-bisphosphatase ( 5 , 6) pre-

ceded by a transient increase of cAMP (11) had been demonstrated previously in intact yeast cells. A CAMP- and fruc- tose 2,6-bisphosphate-dependent phosphorylation of fructose- 1,6-bisphosphatase has been demonstrated with purified enzymes from yeast (14, 15). Dependence of proteolysis of fructose-1,6-bisphosphatase on CAMP-dependent phosphorylation of the enzyme was demonstrated with adenylate cyclase- deficient mutants of S. cereuisiae ( 7 ) . The question how a

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rapid increase of cAMP is affected by application of fermentable sugars or protonophores to starved yeast may now be answered by the demonstration of a rapid decrease of the cytosolic pH which causes activation of adenylate cyclase. These studies do not explain the transiency of the increase of

the cAMP level. It is possible that a change in the activity of cAMP phosphodiesterase also participates in the control of the level of CAMP.

Acknowledgments-We are grateful to Dr. Alan Peterkofsky (NIH, Bethesda) for stimulating discussions and suggestions. Thanks are due to Dr. Ernst Freese (NIH, Bethesda) for useful discussions and for the suggestion to include the possibility of pH control of adenylate cyclase activity in our studies. We also thank Dr. Milan Hofer (Bonn) for helpful discussions. Facilities for NMR work were kindly made available to us by Dr. R. Kaptein and Klaas Dijkstra, State University Groningen (The Netherlands), Institute for Physical Chemistry. We also thank Wolfgang Fritz and Ulrike Eitel for help with the figures and for typing the manuscript. .

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45. Busa, W. B., and Nuccitelli, R. (1984) Am. J. Physiol. 2 4 6 , R409- R438 M e c h a n i s m o f c o n t r o l o f a d e n y l a t e c y c l a s e a c t i v i t y I " y e a s t b y d e s c r l b e d b y fliiller a n d H o l z e r ( 5 ) . T h e a n t i s e r u m n a s k l n d l y p r o v l d e d b y f e r m e n t a b l e s u g a r s a n d c a r b o n y l c y a n r d e m - c h l o r o p h e n y l h y d r a z o n e Dr. T . N o d a ( 1 3 ) . P h o s p h o r y l a t l o n o f f r u c t o s e 1 . 6 - b l s p h o s p h a t a s e i n t h e C l a u d i o P u r u i n . K l a a s N i c o l a y , Y. A l e x a n d e r S c h e f f e r s a n d H e l m u t H o l z e r X A R - 5 1 o f s l a b g e l s w i t h a L K B 2202 u l t r o s c a n l a s e r d e n s i t o m e t e r . G e l I m m u n o p r e c i p i t a t e s w a s m e a s u r e d b y s c a n n l n g t h e a u t o r a d i o g r a m s ( K o d a k F i l m e l e c t r o p h o r e s l s w a s p e r f o r m e d i n g r a d l e n t s o f 10.20% p o l y a c r y l a m i d e u s i n g t h e p r o c e d u r e o f Laemrnl, ( 2 4 ) a n d p r o t e l n s w e r e s t a l n e d w i t h C o o m a s s i e H a t e r i a l s a n d M e t h o d s Brilliant B l u e R250. F o r a i l e x p e r l m e n t s t h e d l p l o i d s t r a l n M I o f S a c c h a r o m c e s c e r e v i s i a e . k i n d l y p r o v l d e d b y P r o f . L i n n a n e ( M o n a s h U n i v e r s d ,Australla), w a s u s e d . T h e y e a s t c e l l s w e r e c u l t i v a t e d o n Y E P O - m e d l u m ( 1 % E a c t o y e a s t e x t r a c t . 2% B a c t o o e D t o n e a n d 2% a l u c o s e l o r E t h a n o l - m e d i u m 1 0 . 6 7 4 Y e n q t N l t r o g e i B a s e Y / O A m i n o a c i d s , 0.52 e t h a n o l - a n d 50 mM M e S ~ i K O H ~ ~ i H 5.6I-and h a r v e s t e d a f t e r 2 4 h o r 48 h , r e s p e c t i v e l y E n z y m e a s s a y s a n d i n v i v o p h o s p h o r y l a t l o n o f f r U c t o S e - l , 6 - b i s p h o s p h a t a s e - T U C o s e - - isp o s p a a s e w a s a s s a y e s p e c r o o 0 o m e r l c a y wi a modi-

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w a s m e a s u r e d n a l m e t h o d 20 t o o f 0.4 mM c o n c e r n i n g k o f s k y ( 2 2 ) . l a b e l e d C A M P a n d B l o - R a d t e s t p h a t a s e a n d M e t a b o l i t e s d e t e r m l n e d ( 1 1 ) . C e l l p e m e t h o d f o r c l o s e r t o s u s p e n s i o n 6.21 . w a s d e s c r l b e d g a t i o n ty. F o r c o u l d b e c e l l s w e r e slightly m o d i f l e d O f t h e y e a s t a n d s t i r r e d O n l y f r e s h l y a n a b o u t B e n t s . at TU DELFT, on April 15, 2010 www.jbc.org Downloaded from a c c o r d i n g t o S a l o m o n e t a l . ( 2 0 ) . l a t e c y c l a s e a c t i v i t y in t o l u e n e - t r e a t k d c e i i s a n d c h i t o s a n - t r e a t e d c e l i s a m o d i f l c a t i o n o f t h e o r l g i - o f K r l s h n a e t a l . ( 2 1 ) . R e a c t i o n r a t e s a t 3 0 ° C w e r e l i n e a r f o r 3 0 m i n w i t h o u t a n A T P - r e a e n e r a t i n a S v s t e m . w h e r e a s without a d d i t i o n C A M P a significant i o w e r a c t i v l i y was m e a s u r e d . F O P a i l s t h e t e r m l n a t i o n o f t h e e n z y m e 3 r e a c t l o n , s e p a r a t i o n o f d3'P - P r o t e i n WdS d e t e r m l n e d a c c o r d i n g t o S r a d f o r d ( 2 3 ) u s i n g t h e r e c o v e r y C o n t r o l s w i t h H C A M P , s e e H a r v o o d a n d P e t e r - s o l u t i o n . I n v i v o p h o s p h o r y l a t i o n o f f r u c t o s e - 1 . 6 - b i s p h o s - i m m u n o p r e c l p i f a m o f t h e c r u d e e x t r a c t w a s c a r r i e d o u t a s - C O n C e n t P a t l O n S o f g l u c o s e - 6 - p h o s p h a t e , A T P a n d c A M P w e r e a f t e r e x t r a c t i o n w l t h p e r c h l o r i c a c i d a s p r e v i o u s l y d e s c r i b e d r m e a b l l i z a t i o n - T o l u e n e t r e a t m e n t w a s e x p e c t e d t o b e a s u l t a b l e m e a s u r l n g a d e n y l a t e c y c l a s e a c t l v i t y e x h i b i t i n g C h a r a c t e r i s t i c s t h e i n v i v o condition t h a n m e m b r a n e p r e p a r a t l o n s ( 2 2 ) . T h e y e a s t ( Im e d y e a s t c e l l s s u s p e n d e d i n 4 m I 0.1 M P i p e s I K O H . p H s h a k e n in t h e p r e s e n c e o f 16 m l t o l u e n e f o r 5 m i " a t 4 0 ° C a s b y M u r a k a m i e t a l . ( 2 5 ) . T h e c e l l s w e r e w a s h e d t n l c e b y c e n t r i f u - a t 10.000 xg f o r 10 m i n a n d r e s u s p e n d e d in t h e s a m e b u f f e r . T h e y c h i t o s a n t r e a t m e n t , t h e p r o c e d u r e o f J a s p e r s e t al. ( 2 6 ) w a s s t o r e d a t 4 ° C f o r s e v e r a l d a y s w l t h o u t S l g n l f l c a n t l o s s o f a C t l v 1 - s u s p e n d e d in 1 5 0 m l 2 5 mM H e p e s l l r i s p H 7.0 c o n t a i n i n g 50 mM K C 1 t o o b t a i n p e r m e a b i l l z a t i o n a t O'C. 0.75 g p a c k e d y e a s t c e l l s t h e s u p e r n a t a n t w a s a s p i r a t e d . A d e n y l a t e c y c l a s e S h o w e d f o r 5 h i n t h e p r e s e n c e o f 7.5 m g c h l t o s a n . A f t e r s e d l m e n t a t l o n p e r m e a b i i l z e d y e a s t c e l l s w e r e " s e a f o r t h e d e s c r i b e d e x p e r l - 40% d e c r e a s e in a c t i v l t y a f t e r t w o d a y s d u r i n g s t o r a g e a t 0°C.

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Membrane p o t e n t i a l and e x t r a c e l l u l a r p o t a s s i u m - TPP+ ( 2 7 ) and e x t r a c e l l u - l a r p o t a s s i u m ( 2 8 1 c o n c e n t r a t l o n s ( a s l n d l c a t o r s o f t h e membrane potential) w e r e m e a s u r e d w l t h i o n S e l e c t i v e e l e c t r o d e s . The AglAgC113 M KC1 - h a i f c e l l o f a c o m b i n a t i o n pH e l e c t r o d e EA 1 2 5 ( M e t r o h m , H e r i s a u . F . R . G . ) was u s e d a s r e f e r e n c e e l e c t r o d e . F o r t h e m e a s u r e m e n t o f p o t a s s i u m c o n c e n t r a - t l o n , t h e h a l f - c e l l c o n t a i n e d 3 M N a C l i n s t e a d o f 3 M K C I . The e l e c t r o d e p o t e n t l a l s w e r e m e a s u r e d w i t h pH m e t e r E 5 1 0 ( M e t r o h m , H e r I S a U . F.R.G.) and r e c o r d e d b y a m u l t l - p e n r e c o r d e r R103 ( R l k a d e n k i , F r e i b u r g , F.R.G.). Membrane p o t e n t i a l and e x t r a c e l l u l a r p o t a s s i u m - TPP' ( 2 7 ) and e x t r a c e l l u - l a r p o t a s s i u m ( 2 8 1 c o n c e n t r a t l o n s ( a s l n d l c a t o r s o f t h e membrane ~ o t e n t l a l l w e r e m e a s u r e d w l t h i o n S e l e c t i v e e l e c t r o d e s . The AglAgC113 M KC1 - h a i f c e l l o f a c o m b i n a t i o n pH e l e c t r o d e EA 1 2 5 ( M e t r o h m , H e r i s a u . F . R . G . ) was u s e d a s r e f e r e n c e e l e c t r o d e . F o r t h e m e a s u r e m e n t o f p o t a s s i u m c o n c e n t r a - t l o n . t h e h a l f - c e l l c o n t a i n e d 3 M N a C l in s t e a d o f 3 M K C I . The e l e c t r o d e p o t e h t l a l s w e r e m e a s u r e d w i t h pH m e t e r E S l O ~ ( M e t r o h m , H e r ; s a u . ~ F . R I G . ) and r e c o r d e d b y a m u l t l - p e n r e c o r d e r R103 ( R l k a d e n k i , F r e i b u r g , F.R.G.). l n t r a c e i l u l a r pH - A simple- f l u o r e s c e n c e a s s a y u s ~ n g 9 - a r n l n o a c r l d l n e

T29,301 a s a pH Indicator was used. I n t r a c e l l u l a r a c l d t f l c a t l o n causes an dCCUnUlatlOn O f t h e d y e I n s l d Q t h e c e l l , b e c a u s e P r O t o n d t l O n O f t h e d y e d e c r e a s e s I t s a b l l l t y t o p a s s t h r o u g h t h e p l a s m a m e m b r a n e . F l u o r e s c e n c e O u e n c h l n o . o b s e r v e d when 9 - a m l n o a c r l d l n e I S t a k e n " 0 bv t h e Y e a s t c e l l s . wood. NJ, r e s c e n c e w a s m e a s u r e d w I t h U S A ) ( e x c l t a t l o n w a v e l e n g t h a s p e c t r o f l u o r o m e t e r R R S t o 0 0 ( S c h o e f f e l , 397 nm. e m l s s l a n w a v e l e n g t h 4 5 4 nm) u e s t - a n d r e c o r d e d w l t h an X Y f l a t b e d r e c o r d e r P M 8 1 2 5 ( P h l l l o s . E l n d h o v e n . 31P-NMR - A f t e r h a r v e s t i n g , t h e c e l l s w e r e washed t w i c e i n i c e - c o l d r e s u s - pension medlum c o n t a i n i n g 0.1 M H e p e s I T r i s , pH 7 . 0 , a n d re s u s p e n d e d i n t h e same medium. The. c e l l o e l l e t v o l u m e w a s u s u a l l v 3 0 - 4 0 4 o f t h e t o t a l s a m p l e v o l u m e . "P NMR s p e c t r a w e r e o b t a i n e d a t i4 5 . 8 M H i u s l n g a B r u k e r H X - 3 6 0 s p e c t r o m e t e r o p e r a t i n g i n t h e F o u r l e v - t r a n s f o r m mode. A c c u m u l a t i o n ~ . ~ . " was c a r r i e d o u t e m p l o y i n g 6 0 - p u I s e s and a 0.34 s r e p e t i t i o n t i m e . R o u t i n e - l y , t i m e p r o f l l e s w e r e o b t a i n e d b y s e q u e n t i a l l y s t o r i n g on d i s k f r e e I n d u c - t i o n d e c a v s . e a c h c o n s i s t i n a o f 100 t o 250 s c a n s . G l v c e r o o h o s D h o r v l c h o l i n e a t 0 . 4 9 .pbm r e l a t i v e t o 8 b % O r t h o P h o s D h o r i C a c i d w a s used as an- i n t e r n a l c h e m l c a l s h i f t m a r k e r . U n l e s s s t a t e d o t h e r w i s e , NMR e x p e r i m e n t s weve c a r - r i e d o u t a t 2 2 1'C. S a m p l e s c o n s i s t e d o f 3 m l o f th e y e a s t s u s p e n s l o n I n 10 mm t u b e s . A e r o b i c c o n d i t i o n s i n t h e NMR t u b e w e r e maintained b v b u b b l i n o P u r e 0, g a s t h r o u q h t h e d e n s e c e l l s u s o e n s i o n . I n a l l e x o e r i m e n t ; . id i ~ ; a ; b u b b l e 6 t h r o u g h a - g l a s s c a p i l l a r y ( 3 1 ) ' a t a r a t e o f 30 - 4 0 ml1mln.- Prior t o e a c h e x p e r i m e n t . 50 y I a n t i f o a m was added t o t h e c e l l s u s p e n s i o n . Sub- was a c h l e v e d b y i n t r o d u c l n g 1 5 0 u 1 5 0 % g l u c o s e s o l u t i o n in t o a b y p a s s o f s t r a t e s w e r e I n j e c t e d d l r e c t l y in t o t h e NMR t u b e in s i d e th e m a g n e t . T h i s t h e s i l l c o n e t u b i n g u s e d f o r t h e g a s s u p p l y . S u b s e q u e n t l y , t h e g a s f l o w was y e a s t s u s p e n s i o n ( 3 1 ) . CCCP ( 1 2 V I . 0.5 M s o l u t i o n in d i m e t h y l s u l f o x i d e ) d i r e c t e d th r o u g h th e b y p a s s , th u s p u s h l n g th e s u b s t r a t e s o l u t l o n in t o th e was added t o t h e NMR t u b e s o u t s i d e t h e m a g n e t . I n t r a c e l l u l a r pH - The pH o f t h e c y t o s o l a n d v a c u o l e was d e t e r m l n e d b y 3 1 P NMR f r o m t h e c h e m i c a l s h i f t s o f l n o r g a n l c p h o s p h a t e i n t h e s p e c l f l c com- p a r t m e n t s a s d e s c r i b e d e l s e w h e r e ( 3 2 ) . m

i z

F i g . 1

Changes I n S P e C I f i c a c t l v l t y o f f r u c t o S e - 1 , 6 - b i s p h o s p h a t a s e (FBPase) and CCCP ( f i n a l concentration 0 . 1 m M ) t o a 2 . 5 % ( w e t w e i g h t p e r v o l u m e ) y e a s t C o n c e n t r a t l o n s o f CAMP, ATP a n d g l u c o s e - 6 - p h o s p h a t e ( G 6 P ) a f t e r a d d l t l o n o f Suspension i n 10 m M s o d l u m m a l e a t e pH 6.0 a t 3OPC. Inc"b..IIM to- <M"l T a b l e I A d e n y l a t e c y c l a s e a c t i v l t y i n c e l l s o f t h e y e a s t S a c c h a r o m y c e s c e r e v i s i a e M l p e r m e a b i l i z e d b y c h l t o s a n - t r e a t m e n t o r t o l u e n e - t r e a t m e n t . A d d l t i o n C h i t o s a n - t r e a t e d T o l u e n e - t r e a t e d C o n c e n t r a t i o n C o n c e n t r a t i o n A c t i v l t y A c t i v i t y o f a d d i t i o n o f a d d i t i o n L m M ) ( I ) ( m M ) ( X ) G l c 6 - d G l c 2-dGIC 2 - d G l c - 6 - P G I c - 6 - P G I C - 1 - P F r u - 6 - P G l c - 1 6 - P F r u - l ' 6 - P 2 F r u - 2 1 6 - P g P . AhP AMP GMPPNP KF CCCP ~~~~ P ! 5 5 5 5 5 n . t . 5 n . t . 5 0.005 5 15 0 . 1 1 n . t . n . t . 0.1 8 8 8 5 95 9 7 110 n . t . 97 110 n . t . 109 142 164 95 63 " . t . 8 1 n . t . n . t . n . t . n . t . n . t . n . t . n . t . n . t . n . t . 0.004 100 I n . t . 95 n . t . T a b l e I The y e a s t c e l l s w e r e c u l t i v a t e d o n YEPD-medlum. M i x t u r e s f o r a s s a y o f a d e n y l a t e c y c l a s e c o n t a i n e d i n t h e c a s e o f c h i t o s a n - p r e t r e a t m e n t ( f l n a l c o n c e n t r a t i o n ) : 50 mM l m l d a z o l e l M e s pH 7.0, 1.0 m M ATP, 5 mM MnCl 0 . 4 mM

CAMP and c e l l s ( 0 . 2 m g l m l p r o t e i n ) and i n t h e c a s e o f to l u e n e - p r e t ? ; a t m e n t : 100 mM MesIKOH pH 5 . 5 , 0 . 5 mM ATP, 0.5 nM MnCl 0 . 4 m M CAMP and c e l l s ( 0 . 6 m g l m l p r o t e i n ) . P r o t e l n c o n t e n t o f c e l l s was & a s u r e d I n a F r e n c h p r e s s e d s u s p e n s l o n . n . t . : n o t t e s t e d 1 ) a c t u a l v a l u e : 6 7 0 p m o l e s l h l m g 2 ) a c t u a i v a l u e : 1 1 0 0 p m o l e s l h l m g F i g . 3 E f f e c t o f i n c r e a s i n g c o n c e n t r a t l o n s o f CCCP o n t h e a c t l v l t y o f and 3 2 P CAMP. The y e a s t c e l l s cultivated on YEPO-medlum w e r e i n c u b a t e d a t a d e n s l t y

i n c o r p o r a t l o n I n t o f r u c t o s e - 1 . 6 - b i s p h o s p h a t a s e (FBPase) and o n l e v e l s o f o f 5 % w e t u e i g h t l v o l I n 50 mM H e p e s I T r l s , pH 7.0 a t 30°C. CCCP d i s s o l v e d i n

m e t h a n o l was a d d e d a f t e r 5 m l n p r e i n c u b a t l o n . S a m p l e s w e r e w i t h d r a w n f o r

d e t e r m l n a t l o n o f cAMP 45 sec a f t e r CCCP and f o r d e t e r r n l n a t i o n o f enzyme a c t i v l t y o r e n z y m e p h o S p h o P y l a t l O n 5 m i n a f t e r CCCP.

F i g . 3

E f f e c t o f i n c r e a s i n g c o n c e n t r a t l o n s o f CCCP o n t h e a c t l v l t y o f and 3 2 P i n C o r D o r a t l o n I n t o f r u c t o s e - 1 . 6 - b i s D h o s D h a t a s e (FBPase) and o n l e v e l s o f CAMP. The y e a s t c e l l s cultivated on YEPO-medlum w e r e i n c u b a t e d a t a d e n s l t y m e t h a n o l was a d d e d a f t e r 5 m l n p r e i n c u b a t l o n . S a m p l e s w e r e w i t h d r a w n f o r

o f 5 % w e t u e i g h t l v o l I n 50 mM H e p e s I T r l s , pH 7.0 a t 30°C. CCCP d i s s o l v e d i n

d e t e r m l n a t l o n o f cAMP 45 sec a f t e r CCCP and f o r d e t e r r n l n a t i o n o f enzyme a c t i v l t y o r e n z y m e p h o S p h o P y l a t l O n 5 m i n a f t e r CCCP. F i g . 5 g r o w n . s t a t l o n a r y y e a s t . Washed y e a s t c e l l s ( 2 5 mg w e t w e l g h t l m l ) w e r e E f f e c t o f g l u c o s e a n d CCCP o n t h e e x t r a c e l l u l a r K t c o n c e n t r a t i o n o f g l u c o s e d a y a n d r e s u s p e n d e d p r l o r t o t h e e x p e r l m e n t I n t h e same b u f f e r . As l n d i - S t o r e d overnight a t 0°C I n 50 mM H e p e s I T r l s . pH 7.0, c e n t r i f u g e d t h e n e x t CCCP ( d o t t e d i i n e s ) f r o m a 1 0 0 mM s t o c k solution I n m e t h a n o l w e r e a d d e d . c a t e d b y v e r t l c a l a r r o w 25 mM g l u c o s e f r o m a 2 . 5 M s t o c k S o l u t i o n O P 0 . 2 mM at TU DELFT, on April 15, 2010 www.jbc.org Downloaded from