THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 255,.No. 3. Issue of February 10. pp. 895-902, 1980 Printed in U.S.A.

Biosynthesis of the Yeast Cell Wall 11. REGULATION OF p-(1+ 3)GLUCAN SYNTHETASE BY ATP AND GTP*

(Received for publication, April 27, 1979)

Eleanor M. Shematek and Enrico CabibS From the Laboratory of Biochemistry and Metabolism, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205

Yeast @-(1+ 3)glucan synthetase, a membrane-bound enzyme, is stimulated by ATP or GTP. GTP activation (apparent K, = 1.6 X 10“ M) requires EDTA, occurs rapidly at O”C, and is maintained after the particulate enzyme is either incubated at 30°C and centrifuged or is treated with alkaline phosphatase. On the other hand, ATP activation is inhibited by EDTA, requires incubation at 30”C, and is partially or totally lost by prior incubation of the enzyme at 3OoC followed by centrifugation or by incubation of the particulate prep- aration with phosphatase. After either centrifugation or phosphatase treatment, the effect of ATP was re- stored by the addition of supernatant fluid from incu- bated and centrifuged membranes. Several GTP ana- logs which are structurally unable to transfer the y- phosphate or which lack that phosphate acted as stim- ulators of the synthetase, an indication that transfer of the y-phosphate is not involved in GTP action. GTP showed saturable binding to the particulate enzyme, with a binding constant of 8 to 10 X lo” M.

When membranes were obtained by lysis of proto- plasts in the presence of a high EDTA concentration, the resulting glucan synthetase was very active in the absence of nucleotide. Incubation in the presence of M&+ led to a decrease in activity, which was, however, restored by addition of ATP or GTP. Similar results were obtained with a preparation obtained at low EDTA, but subsequently treated with GTP.

The results are interpreted in terms of a relatively simple working hypothesis. GTP stimulation would oc- cur simply by binding to a site in the membrane. ATP, in an enzymatically catalyzed reaction, would convert a loosely bound phosphorylated substance (superna- tant factor) into a product tightly attached to the en- zyme and endowed with stimulatory properties similar to those of GTP. In the presence of Mg+, an endogenous hydrolytic enzyme converts the ATP product back into the ATP substrate, thus allowing for reversible acti- vation-inactivation of glucan synthetase. This mecha- nism may be involved in regulation of wall synthesis during the yeast cell cycle.

One approach toward understanding morphogenesis is to study the formation of substances that are, in themselves,

* A preliminary account of some of these findings was presented at the meeting of the American Society of Biological Chemists, Atlanta, Ga., June 6, 1978 (Shematek, E. M., Braatz, J. A,, and &bib, E. (1978) Fed. Proc. 37, 1394). 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.

dressed. $ To whom correspondence concerning this paper should be ad-

major structural components. It seems reasonable to expect that the regulation of synthesis of these materials should reflect, in some measure, the morphogenetic regulation of the corresponding structures. This concept was applied to chitin, the polysaccharide that forms the primary septum of budding yeasts (1) and, more recently, to p-(1+ S)glucan, the principal strueturd component of the yeast cell w d (2). In the case of chitin, the regulatory process appears to involve a localized conversion of the zymogenic form of chitin synthetase into an active enzyme (3). For glucan, the need for a cyclical and localized activation-inactivation of the corresponding synthe- tase was indicated by the finding that the enzyme is attached to the plasma membrane (2). Since the latter is in direct contact with the cell wall, it was argued that the synthetase should be active at sites at which the wall is growing and inactive where it is quiescent. In this report, we show that the activity of glucan synthetase can, indeed, be reversibly modi- fied by a number of conditions. The activating effects of ATP and of GTP and their differences are examined in detail.

EXPERIMENTAL PROCEDURES

Materials p(CHdppA,’ P(NH)PPA, Wppp.4, P(CHdppG, P(NH)PPG, and

(S)pppG were purchased from Boehnnger; ATP and GTP were obtained from Sigma and all other nucleotides from P-L Biochemicals. [8-’%]ATP (51.9 mCi/mmol), [y-”’PIATP (10 Ci/mmol), [y-32P]GTP (29 Ci/mmol), [8-“C]GTP (45.7 mCi/mmol), and [U-I4C]GTP (447 mCi/mmoI) were from New England Nuclear. Nucleoside-5‘-diphos- phate kinase was a product of Sigma and bacterial alkaline phospha- tase was from Worthington. Polyethyleneimine cellulose thin layer plates were purchased from Brinkmann. Commercial baker’s yeast was from Anheuser Busch. The sources of other products were the same as in the accompanying report (2).

Methods Many of the procedures have been described in an accompanying

paper (2). Those that pertain only to this study are described below. Preparation of Enzyme with Different Concentrations of EDTA-

The lysis of protoplasts was carried out as described (2), but the lysis buffer was 50 mM Tris/chloride, pH 8, either without additions or containing 1 m ~ , 5 m ~ , or 10 m~ EDTA. The same buffer used for the lysis was employed in each case to wash the particulate enzyme (2). The final pellets were resuspended in lysis buffer containing 33% glycerol.

’ The abbreviations used are: &CH,)ppA, adenosine-5‘-(P,y-meth- y1ene)triphosphate; p(NH)ppA, adenosine-5”@,y-imino)triphos- phate; (S)pppA, adenosine-5’-(y-thio)triphosphate; pppApp, adeno- sine-3’-pyrophosphate-5”triphosphate; p(CNa)ppG, guanosine-5” (/3,y-methylene)triphosphate; p(NH)ppG, guanosine-Y-@,y- iminoftriphosphate; (S)pppG, guanosine-5’-(y-thio)triphosphate; ppGpp, guanosine-3’,5‘-bispyrophosphate; pppGpp, guanosine-3’-py- rophosphate-5’-triphosphate; ppG > p. guanosine-2’,3’-monophos- phate-5’-pyrophosphate; pppG > p, guanosine-2’,3’-monophosphate- 5‘-triphosphate.

895

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896 Regulution of Glucan Synthetase

Except where noted otherwise, the enzyme prepared with 50 mM Tris containing 1 mM EDTA was used.

Determination of GTP and ATP Stability during Incubation with Glucan Synthetase Preparations-In a total volume of 40 pl, the reaction mixture contained 150 pg of enzyme protein, 80 m~ Tris- chloride, pH 8, 5 mM UDP-glucose, 20 p~ ['4C]CTP, or 5 mM [14C]ATP (60,000 cpm of either one). When both GTP and ATP were added, only GTP was labeled. EDTA, when present, was 2.5 mM. After 20 min of incubation at 30°C, the reaction was stopped with 120 pl of absolute ethanol and the mixture centrifuged for 10 min at 12,000 X g. The supernatant fluid was removed and evaporated to dryness. The residue was taken up in 20 p1 of water and spotted on a polyeth- yleneimine cellulose thin layer plate. The chromatogram was devel- oped with 1.25 M LiC1. Nucleotides were located by autoradiography using Kodak X-Omat RP film with a 24-h exposure time.

Preparation of Stabilizing Agent for Glucan Synthetase-Com- mercial yeast (100 g) was suspended in 100 ml of water and boiled for 15 min. The mixture was centrifuged for 10 min at 12,000 X g. The supernatant liquid was passed three times through a column of AG 50W-X8 (Bio-Rad) cationic resin (bed volume 60 ml) in the ammo- nium form. The column was washed with water until the volume of the combined eluates reached 200 ml. This eluate was applied at 2°C to a DEAE-cellulose column, bed volume 60 ml, previously equili- brated with 50 mM Tris/chloride, pH 7. A linear NaCl gradient, with 150 ml of 0.4 M NaCl in 50 mM Tris/chloride at pH 8 in the reservoir and the same volume of Tris buffer in the mixing chamber, was applied to the column; fractions of 2.5 ml were collected. The active fractions, which emerged between 0.2 and 0.34 M NaCl, were pooled and frozen.

In the assay for stabilizing agent, the incubation mixtures contained particulate glucan synthetase (150 pg of protein), 80 mM Tris/chloride, pH 8, 1 mM EDTA, and variable amounts of column fractions in a total volume of 32 pl. After 10-min incubation at 30"C, the tubes were cooled in ice and the mixtures were completed with additions as needed to obtain the standard assay composition (2), but including also 20 p~ GTP. Glucan synthetase was measured as previously described (2). Controls contained untreated enzyme or enzyme incu- bated in the absence of stabilizer. The minimal amount of material that yielded maximal stabilization was used in subsequent experi- ments. This corresponded to approximately 0.75 nmol of phosphorus/ standard enzymatic assay.

Preparation of pppG > p-pppG > p was obtained by phospho- rylation of ppG > p with ATP in the presence of nucleoside diphos-

0.5 pmol of [y-"'P]ATP (3.6 X lo6 cprn), 0.05 M potassium phosphate, phate kinase. The reaction mixture contained 0.5 pmol of ppG > p,

pH 6.3, 5 mM MgC12, and 100 units of nucleoside diphosphate kinase in a total volume of 0.2 ml. "P-labeled ATP was used to facilitate detection of the product and to determine yield. After incubation at 30°C for 3 h, mixtures were placed in a boiling water bath for 1 min. The coagulated protein was removed by centrifugation; the superna- tant fluid was streaked onto a plastic-backed sheet of polyethylene- imine and subjected to thin layer chromatography with 1 M formic acid containing 1 M LiCl as solvent. By observation under ultraviolet light and by autoradiography, a slow running (about 40% the mobility of ppG > p), UV-absorbing, and radioactive band was detected. The corresponding area was scraped off the plate and eluted with 8 ml of 1 M NH,HC03. Most of the salt was removed by lyophilization and the rest by filtration through a Sephadex G-10 (0.9 X 85 cm) column eluted with water. The radioactive peak recovered from the column was evaporated to dryness, and the residue was dissolved in 0.2 ml of water. Judged by the radioactivity of the product, a yield of 9.8% was calculated.

Determination of GTP Binding to Membranes-The assay mix- ture contained 80 mM Tris/chloride, pH 8, 1.25 mM EDTA, 8% glycerol, 50 to 100 pg of particulate enzyme protein, and variable concentrations of [Y-~'P]GTP, containing 1 X lo6 cpm (obtained by suitable dilutions with cold carrier), in a total volume of 0.4 ml. After 5-min incubation at 0°C 1.5 ml of Tris/chloride, pH 8, containing 1.25 mM EDTA were added and the suspension was filtered through a 0.45-pm Millipore filter. The filter was washed twice with 1-ml por- tions of the Tris/EDTA buffer, dried under an infrared lamp, placed in a scintillation vial, and counted after addition of 10 ml of Aquasol.

Incubation times of 1 min were insufficient to reach equilibrium,

value. The bound radioactivity was proportional to the amount of whereas IO-min incubations resulted in little increase over the 5-min

membranes added, although dispersion of the data was considerable.

were centrifuged for 20 min at 100,000 X g after addition of the Trk/ To analyze the material bound to the particles, incubated mixtures

EDTA buffer. The pellet was washed once with the' same buffer, resuspended in water, and precipitated with cold trichloroacetic acid at a final concentration of 10%. After centrifugation, trichloroacetic acid was extracted from the supernatant fluid with ether. The aqueous phase was neutralized, concentrated by evaporation under reduced pressure, and transferred to a polyethyleneimine plate. The chromat- ogram was developed with 1.5 M KHzPOl and subjected to autora- diography as outlined in a previous section.

RESULTS

Glucan Synthetase Activation by ATP and GTP: the Effect of EDTA-In an accompanying report (2), it was shown that both ATP and GTP increase the activity of glucan synthetase. The reaction product made in the presence of ATP was shown to be a linear ,5-(1 + 3)glucan. The same conclusion is valid for GTP, in that the product was completely resistant to a- and b-amylase and totally hydrolyzed by a specific exo-p-(l + 3)glucanase (not shown). GTP was effective as an activator at much lower concentrations than ATP (Fig. lA). The acti- vation constant, K,, was 1.6 X M, 2 orders of magnitude smaller than that of ATP (2). For maximal activation by GTP, it was necessary to add EDTA to the reaction mixture (Fig. 2). In contrast, the stimulation by ATP was inhibited by high concentrations of EDTA (Fig. 2). The activity of the enzyme, as measured in the absence of nucleotides, was usually but not always stimulated 1.5- to 2-fold by addition of EDTA (Fig. 2).

As will be shown below, the presence of EDTA is apparently required only until GTP is bound. Removal of the chelating agent had no effect on an enzyme that had already bound GTP. The stimulation of the GTP effect by EDTA is probably due to protection against phosphatases. When 14C-labeled GTP was incubated with enzyme at the usual concentration of 20 p~ and under standard assay conditions, i.e. with 0.25 mM EDTA, almost all of the nucleotide disappeared, as judged by thin layer chromatography on polyethyleneimine cellulose, followed by autoradiography. The products seemed to be GDP and/or GMP (not resolved by the solvent used). Addi- tion of 2.5 mM EDTA to the incubation mixture resulted in conservation of most of the GTP. It is interesting to note that [I4C]ATP at 5 mM, under standard assay conditions, was also partially decomposed. Although EDTA, at 2.5 mM, completely protected the ATP from decomposition, at this concentration it abolished the activating effect of the nucleotide (Fig. 2).

Because of the difference in optimal conditions, it is difficult to determine directly whether the effects of ATP and GTP are additive (see, however, experiment of Table 111, below).

Stabilization of Glucan Synthetase Activity-Experiments on the mechanism of activation of glucan synthetase by ATP and GTP were hindered by the rapid loss of enzymatic activity upon incubation at 30°C in the absence of substrate. It was found, however, that most of the loss could be prevented by addition of a boiled extract from commercial yeast.* The stabilizing substance(s) could be purified by filtration through a cationic exchange resin, followed by chromatography on a DEAE-cellulose column (see "Methods"). In the presence of the stabilizer, the loss of enzymatic activity in 10 min of incubation at 30°C could be reduced from 30 to 70% to 20% or less. There was no interference of the protecting agent with the reversible activation by nucleotides.

The active material appears to be of fairly small molecular weight, in that it is partially retained by a Sephadex G-10 column (not shown). Although heat-stable at neutral or al- kaline pH, it was destroyed by boiling for 10 min in 0.1 M sulfuric acid. Its ability to stabilize the enzyme was also

Similar extracts, prepared from strain GS-1-36, contained not Only protecting but also activating material, probably GTP. (E. M. She- matek, H. Kawai, and E. Cabib, unpublished experiments).

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Regulation of Glucan Synthetase 897

0 9 3x10-* 10" 10" 10- 10" loa

GTP (MI

FIG. 1. Effect of GTP, GDP, and ppG > p on glucan synthe- tase activity. The assay mixture included 80 m~ Tris/chloride, pH 8, 0.8% bovine serum albumin, 5.3 n" UDP-["C]glucose (2 X IO5 cpm/pmol), 1 mM EDTA, enzyme (usually about 100 pg of protein), and the indicated nucleotide in a total volume of 40 pl. The final concentration of glycerol, contained in the enzyme solution, was 8% (v/v). Incubation and determination of reaction product were as

I I I 1 2 3

EDTA (mMI

FIG. 2. Effect of EDTA on ATP and GTP activation. The assay was as in Fig. 1, except for the nucleotide that was added as indicated below and for the concentration of EDTA that was varied as shown. The lowest concentration of the chelating agent was not zero because of the EDTA contained in the enzyme preparation. 0"-3, no nucleotide added; A-A, 1 mM ATP; M, 10 p~ GTP.

drastically reduced by incubation with alkaline phosphatase (not shown). From the method of purification used and the above mentioned properties, the stabilizing agent appears to consist of one (or perhaps more) negatively charged substance of small molecular weight containing phosphate in monoester linkage. Further investigation of its structure was deferred because of the very small amounts available and of the lack of an efficient assay.

The following substances did not appreciably stabilize the enzymatic activity at 30'C when added at the concentrations shown in parentheses: heparin (1 mg/ml), yeast-soluble ribo- nucleic acid (4.3 mg/ml), potassium fluoride (87 m ~ ) , mag- nesium sulfate (3 and 10 m ~ ) , spermine (0.3 m ~ ) , phenyl- methylsulfonyl fluoride (0.2 m ~ ) , sodium glycerophosphate (2.5 mM), fructose 1,6-bisphosphate (1.25 mM), glucose 1,6- bisphosphate (1.25 ITIM), phytic acid (0.5 m ~ ) , linear tripoly- phosphate (0.8 m ~ ) , ADP (1.25 m ~ ) , UDP-GlcNAc (3.3 w), guanosine 2',3'-monophosphate (25 and 100 PM), and guano- sine 3'-phosphate-5'-pyrophosphate (25 and 100 PM). ATP, GTP, glycerol, and bovine serum albumin, all activators of glucan synthetase (2), did not afford protection.

Activation of Glucan Synthetase by ATP-The availability of a stabilizing agent allowed us to investigate whether acti- vation of the enzyme by ATP required the presence of the nucleotide throughout the incubation or could occur in a step previous to the actual synthesis of glucan. For this purpose,

10- 10" lo4 10- GDP (01 or ppG>p (*I (MI

described (2). The incorporation of glucose into glucan observed in the absence of nucleotide was subtracted in each case. In A, this incorporation was 34 nmol; in B, 22.5 nmol. In E , at 10 p~ GTP, a total incorporation of 95 nmol (72.5 nmol net) was found. In A, only some of the values could be used for the inverse plot of the inset because of the very extensive range of ATP concentrations employed.

1. i

NO PREINC NO PREINC PREINC PREINC + ATP + ATP

FIG. 3. Effect of incubation with ATP on glucan synthetase. The particulate enzyme was prepared and washed with 50 mM Tris, pH 8 (see "Methods"). Enzyme (1.5 mg of protein) was incubated at 30°C for 10 min with 80 m~ Tris/chloride, pH 8, 50 yl of stabilizing agent, and with or without 5 mM ATP as indicated, in a final volume of 0.4 ml. Incubation was stopped by addition of 1.5 ml of ice-cold 50 mM Tris/chloride, pH 8. The mixture was centrifuged for 20 min at 150,000 X g and pellets were suspended in 50 m~ Tris/chloride, pH 8, to a final volume of 100 pl. Glucan synthetase activity was assayed in a mixture containing 10 p1 of treated enzyme, 80 mM Tris/chloride, pH 8, 0.8% bovine serum albumin, 9.7% (v/v) glycerol, and 5.3 mM UDP-['4C]glucose (2 X lo5 cpm/pmol), in a total volume of 40 pi. Solid burs, no ATP in assay mixture. Empty burs, 5 mM ATP in assay mixture. Incubation and determination of product were as described (2). PREINC, preincubated. In the experiment Iabeled NO PREINC + ATP, addition of ATP was immediately followed by dilution with buffer and centrifugation.

the particulate glucan synthetase was incubated with ATP and stabilizer, diluted and centrifuged to remove the ATP, and finally resuspended in buffer and assayed. When the preincubation with ATP was carried out at 30°C, a dramatic change in the properties of the enzyme ensued (Fig. 3). The synthetase became much more active in the absence of nu- cleotides and addition of ATP to the assay mixture had little

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898 Regulation of Glucan Synthetase

effect. Appropriate controls showed that this change did not occur at 0°C and that the stabilizing agent by itself had no effect (Fig. 3). The lack of activation after incubating a t 0°C also shows that the results cannot be attributed to carryover of ATP from the fiist to the second incubation.

Activation by ATP was also time dependent. Maximal activation was attained after 5 to 10 min (Fig. 4) and was followed by some decline.

Whereas several enzyme preparations behaved as illus- trated in Figs. 3 and 4, others lost most or all of their ability to be activated by ATP merely by centrifugation or by incu- bation at 0°C followed by centrifugation (Table I, lines 1 and 5). In these cases, however, the capacity for activation could be restored by adding supernatant fluid from an enzyme that had been centrifuged after incubation at 30°C (Table I, lines 2 and 6).

5 t n

10 P 30

PREINCUBATION TIME (rnin.1 FIG. 4. Effect of time of incubation on ATP activation of

glucan synthetase. Conditions and assay as in the experiment of Fig. 3, except that the time of incubation with ATP at 30°C was varied as indicated. Solid bars, assayed without ATP; empty bars, assayed with 5 mM ATP.

TABLE I Effect of supernatant fluid on ATP activation of incubated and

centrifuged glucun synthetase Particulate enzyme was incubated with ATP, either at 0°C or a t

30”C, diluted with buffer, centrifuged, and resuspended as described under Fig. 3. The control corresponds to an enzyme that was subjected to the same treatment but with the omission of ATP and of incuba- tion. The conditions for enzymatic assay were the same as under Fig. 1, except that EDTA, ATP, and GTP were present only where indicated below. The assay mixtures ,also contained, where indicated, 20 pl of supernatant fluid (see below). Where missing, the supernatant fluid was substituted with a mixture containing 25 mM Tris/chloride, pH 8, 0.5 mM EDTA, and 4.3% (v/v) glycerol. Supernatant fluid was prepared by incubating for 10 min at 30°C particulate enzyme diluted with an equal volume of water, followed by centrifugation for 20 min a t 150,OOO X g . The supernatant liquid was used as such or after heating for 2 min at 100°C. Treatment of particulate Activity of glucan synthetase after following ad-

enzyme ditions to assay mixture

Incubation ,“,u,”,”fl”uri conditions in assay

EDTA ATP EDTA ( 1 . 5 m ~ )

None ( 5 m ) ( 1 . 5 m ~ ) + G T P (20 MM)

nmol glucose incorporated +ATP,O”C - 23 (0.9)“ 27 (1.1) 15 (0.6) 83 (3.3) +ATP, 0°C + 20 (0.81 67 (2.7) 28 (1.1) 86 (3.4)

+ATP, 30°C + 55 (2.2) 76 (3) 49 (2) 86 (3.4) Control - 25 (1) 37 (1.5) 20 (0.8) 90 (3.6) Control + 17 (0.7) 82 (3.3) 29 (1.2) 91 (3.6)

Numbers in parentheses are relative values, calculated by assign-

+ATP, 30°C - 47 (1.9) 49 (2) 36 (1.4) 87 (3.5)

ing a value of 1 to the control, as measured without additions.

TABLE I1 Effect of supernatant fluid on alkalinephosphatase-treated glucan

synthetase Enzyme (1.5 mg of protein) was incubated for 10 min a t 3OoC with

10 pg of bacterial alkaline phosphatase in 80 mM Tris/chloride, pH 8, in a total volume of 200 pl. The mixture was diluted with 1.5 ml of 50 mM Tris/chloride, pH 8, and centrifuged for 20 min a t 100,OOO X g. The supernatant fluid was discarded and the pellet was resuspended in 50 mM Tris/cNoride, pH 8, to a final volume of 100 pl. The control enzyme was neither incubated nor centrifuged. The glucan synthetase assay was carried out as in Table 11.

Treatment of particulate enztme

None Alkaline phosphatase Same, but supernatant

fluid in assay

Activity of glucan synthetase after following additions to assay mixture

ATP EDTA (1.25 r n M ) EDTA

None (5 m) (1.25 m) + GTP (20 PM)

nmol glucose incorporated 24 50 19 75 13 17 7 71

8 53 18 76

TABLE I11 Effect of incubating glucan synthetase with ATP or GTP at 0°C or

30” C Particulate enzyme was incubated with ATP at 0°C or 3OoC,

diluted with buffer, centrifuged, and resuspended as described under Fig. 3. Incubation with GTP included 80 mM Tris, pH 8, 1.25 mM EDTA, and 20 p~ GTP. Dilution, centrifugation, and resuspension were performed as with the ATP-incubated enzyme. Glucan synthe- tase was assayed as in Fig. 3, but ATP, GTP, and EDTA were added only where indicated below.

Glucan synthetase activity after following addi- tions to assay mixture

Treatment of particu- late enzyme EDTA

ATP EDTA ( 1 . 5 m ~ ) None (5 m) (1.5 n m ) + G T P

(20 PM) nmol glucose incorporated

ATP, 0 min,” 0°C 10 (1)’ 40 (4) 17 (1.7) 60 (6) ATP, 10 min, 30°C 36 (3.6) 40 (4) 32 (3.2) 65 (6.5) GTP, 0 min,” 0°C 60 (6) 65 (6.5) 53 (5.3) 75 (7.5) GTP, 10 min, 3OoC 60 (6) 60 (6) 57 (5.7) 72 (7.2)

a Mixture diluted with cold Tris buffer and centrifuged immediately after adding the nucleotide. ’ Numbers in parentheses are relative values, calculated by assign- ing a value of 1 to the ATP 0°C control, as assayed without additions.

I t should be remarked that although the ability of ATP to stimulate was lost in centrifugation, the enzyme that had already been activated by ATP maintained its activity after centrifugation and was only slightly affected by addition of supernatant fluid (Table I, lines 3 and 4).

The supernatant fluid material was stable to boiling and was lost after dialysis through a cellulose membrane (not shown). I t seems that phosphate groups are somehow involved in its action because incubation of the particulate enzyme with alkaline phosphatase before centrifugation strongly de- pressed the enzymatic activity, but addition of supernatant fluid restored the ATP-supplemented enzyme to the original value (Table 11).

The following substances did not replace the supernatant fluid factor($: NAD, pyridoxal, thiamin, FAD, glucose, aU at 1 mM; GMP, GDP, or ppG > p at 0.1 to 0.5 p ~ . Similarly, the stabilizing agent failed to substitute for the supernatant fluid factor($.

The variability of enzyme preparations with respect to the effect of incubation and centrifugation contrasted with the very good reproducibility in the specific activity of untreated preparations and in their sensitivity to ATP and GTP, pro-

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Regulation of Glucan Synthetase 899

vided the concentration of EDTA in the lysing buffer was maintained constant (see below).

The Effect of GTP-In contrast with ATP activation, the effect of GTP does not require measurable incubation time or a high temperature (Table 111). Brief contact with GTP at 0°C followed by centrifugation resulted in a completely active enzyme (Table 111). Although required initially, EDTA did not seem to be needed after GTP had interacted with the enzyme. Thus, in the experiment of Table I11 (compare fist column, lines 3 and 4, with third column, same lines), it can be seen that omission of EDTA from the final incubation mixture made little difference.

EDTA is also without effect on the ATP-preactivated en- zyme (Table 111, line 2, columns 1 and 3) although it inhibits the process of ATP activation (Fig. 2). This fact can be used to investigate the additivity of ATP and GTP stimulation under similar conditions, i.e. in the presence of EDTA. The values obtained with GTP + EDTA (last column of Table 111) are approximately the same whether or not the enzyme was preactivated with ATP, thus suggesting that the two nucleo- tides are acting on the same site.

Activation by GTP appeared to be a much more stable property of the enzyme than stimulation by ATP. Conditions that led to almost complete loss of ATP activation, such as centrifugation of some preparations of enzyme or alkaline phosphatase treatment of the particles followed by eentrifu- gation, resulted in conservation of the capacity to be activated by GTP (Tables I and 11). On the other hand, incubation of glucan synthetase at 30°C in the absence of stabilizing agents resulted in loss of both ATP- and GTP-stimulated activities.

In an effort to define the structural requirements for acti- vation, a number of analogs of GTP were assayed as possible effectors of glucan synthetase. As mentioned in the accom- panying paper (2), other nucleoside triphosphates, such as UTP and CTP, had no effect on the enzymatic activity at 25 ~ L M concentration. On the other hand, ITP was found to be as effective as GTP with regard to both activation constant and maximal velocity (not shown). Thus, it appears probable that the oxygen in position 6 of the purine ring is important for binding of the nucleotide to the enzyme.

Substances that include the structure of GTP in their molecule, such as guanosine 5”tetraphosphate and guanosine 5’-pentaphosphate, were almost as active as GTP. It is not known, however, whether their stimulatory effect was intrinsic to these compounds or due to release of GTP by the action of contaminating phosphatases. The triphosphate at the 5’ po- sition does not seem to be an absolute requirement for activity. Thus, GDP was an activator, although poorer than GTP (Fig. 1B). The K, was about M, 6 times greater than that of GDP, and the V,,, was about one-half that of the triphos- phate. The compound ppG > p also was effective, with about the same K, as GDP and a VmaX about 70% that of GTP (Fig. 1B). The corresponding triphosphate, pppG > p, was even

TABLE IV Effect of GTP analogs of glucan synthetase

The incubation mixture described under Fig. 1 was used, but with the additions of nucleotide and EDTA as indicated.

Glucose incorporated Nucleotide added Concentration No

EDTA EDTA 1 mM

m.u nmol

GTP None

0.02 9.8 16.2

p(CHn)ppG 39.4 73.5

0.02 44.4 43.4 P(NH)PPG 0.02 55.9 52.4 (S)PPPG ATP

0.02 65.8 81.2 1.0 46.4 17.1

0 I I

2 4 6 E 10

FIG. 5. Binc see “Methods.”

1 0 0

20

li1

GTP (Mx106) lg of GTP to particulate enzyme. For techniques

No Addition

0 EDTA + GTP n n c

0

n I I I I

EDTA h M 1 FIG. 6. Effect of EDTA concentration of protoplast lysis

buffer on activity of glucan synthetase. For the preparation of enzymes at different levels of EDTA, see “Methods.” Assays were carried out with the reaction mixture of Fig. 1, except for the addition, where indicated, of 20 PM GTP and 2.5 mM EDTA (empty bars).

more efficient, with K , and maximum velocity identical with those of GTP (not shown).

ppGpp, a compound implicated in the stringent response of bacteria to protein starvation (4) and also present in yeast (5), was ineffective, but the corresponding 5‘-triphosphate, pppGpp, gave 75% of the maximal stimulation obtained with GTP at 25 ~ L M and only slightly less at 10 p ~ . The correspond- ing adenosine compound, pppApp, had little effect a t those concentrations, but a t 1 l l l ~ yielded about two-thirds of the GTP activation. I t is assumed that these compounds were acting as GTP analogs because in every case, including that of the adenine nucleotide, EDTA increased their effect.

The following compounds were essentially without effect on glucan synthetase at concentrations up to 25 p: guanosine- 2‘-P, guanosine-3’-P, guanosine-ti’-P, guanosine 2‘,3‘-mono- phosphate, guanosine 3’,5’-monophosphate, guanosine 2‘,5‘- diphosphate, guanosine 3‘,5’-diphosphate, guanosine 2’,3” cyclic phosphate-5’-phosphate, and guanosine 3’-phosphate- 5‘-pyrophosphate.

The Effect of Nonphosphorylating Analogs of G T P a n d ATP-In order to investigate whether the mechanism of GTP or ATP activation involved phosphorylation by the y-phos-

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Regulation of Glucan Synthetase

No Addition 0 ATP 8 E D T A + GTP

A

No Incubation Incubated Incubated + Mg'*

FIG. 7. Inactivation of glucan synthetase by incubation with M&+. A, a particulate preparation was obtained by lysis of protoplasts in 50 mM Tris/chloride, pH 8, containing 10 mM EDTA, as described under "Methods." The mixtures for enzyme treatment included 10 pl of enzyme, 5 pl of stabilizing agent, and 2.5 mM MgCb, where added, in a total volume of 32 pl. After incubation for 10 min at 30"C, the tubes were cooled in ice. To the mixture containing MgC12, EDTA was added to a final concentration of 2.5 mM to chelate Mgz+. Then other additions were made to complete the reaction mixture of Fig. 1, with the following changes. In some tubes (solid burs), no nucleotide was added; in others (empty burs), 5 mM ATP was included; and,

phate of the nucleotides, analogs were used which are deemed unable to function as phosphate donors. All of the GTP analogs, at concentrations similar to those found effective for GTP, led to activation of glucan synthetase (Table IV). EDTA had little effect on stimulation by the GTP analogs, presum- ably because these substances were immune to the action of phosphatases. Indeed, no formation of GDP from the analogs could be detected (6) under the conditions of incubation even in the absence of the chelating agent. It may be concluded that transfer of the terminal phosphate is not involved in stimulation by the GTP analogs and consequently by GTP itself.

The corresponding ATP analogs were almost without effect at the concentrations used for GTP (10 to 20 PM) but showed a 3- to 5-fold stimulation at 4 m~ concentration (not shown). This effect was only partially inhibited by increasing concen- trations of EDTA.

Binding of GTP to Particulate Enzyme-The fact that preactivation of glucan synthetase by GTP proceeded very rapidly at 0°C and that the effect of the nucleotide could be mimicked by presumably nonmetabolizable analogs suggested that GTP was acting simply by binding. I t was, therefore, of interest to measure binding of the nucleotide to the particulate enzyme. This was done by a fitration assay (see "Methods") with the use of [Y-~~PIGTP. Saturable binding by GTP was detected (Fig. 5). With two different preparations of the enzyme, the dissociation constant was 8 X M and 10 X

M, respectively. The amount of GTP bound at saturation was 7, 14, and 17 pmol/mg of protein with three different preparations of the enzyme. When calculated as picomoles/ milliunit of glucan synthetase, the corresponding values were 0.27, 0.19, and 0.35.

No addition 0 ATP

0 EDTA + GTP

B

I No incubation Incubated Incubated

+Mg2+ q finally, in those symbolized by cross-hatched bars, 2.5 m~ EDTA

and 20 p~ GTP were added. B, an enzyme prepared with 1 m~ EDTA was activated with GTP at 0°C and centrifuged as in the experiment of Table IV. The resuspended particles (100 pl) were incubated for 10 min at 30°C in a mixture that included 50 pl of stabilizing agent and 2.5 mM MgClz, where added, in a total volume of 0.4 ml. After incubation, the mixture was diluted with 1.5 ml of 50 mM Tris/ chloride, pH 8, and centrifuged for 20 min at 150,000 X g. Pellets were resuspended in Tris buffer and assayed under the same conditions outlined under A.

When [8-14C]GTP was used for these experiments, the calculated amount bound from the radioactivity measure- ments was about 4 times higher.3 Since in this case decom- position products of GTP, such as GDP and GMP, would also be counted, the material bound was examined by polyethyl- eneimine thin layer chromatography and autoradiography after extraction from the membranes with 10% trichloroacetic acid. Radioactive spots corresponding to GTP and GDP were observed, the GDP spot being the stronger of the two. Thus, it appears that part of the GTP bound is transformed into GDP which remains attached to the membranes. In similar experiments with [Y-~~PIGTP, only a spot corresponding to this nucleotide and a weaker inorganic phosphate spot were detected.

Preparation of Glucan Synthetase at Different Levels of Activation and Inactivation of the Enzyme by M$+-It was observed that lysis of protoplasts and washing of the particu- late preparation in the absence of EDTA led to an enzyme with very low activity in the absence of ATP or GTP. This type of preparation was used to advantage when a very low baseline was desired (experiments of Figs. 3 and 4). It seemed interesting to investigate the effect of increasing the concen- tration of EDTA in the lysis above the usual 1 mM level. The surprising result was that increasing levels of the chelating agents gave rise to preparations with higher and higher activ- ities when measured in the absence of nucleotides (Fig. 6). Addition of GTP brought all of the preparations to about the same activity.

Because of the relatively low specific activity of [8-"C]GTP, these experiments could only be carried out at saturating concentrations of the nucleotide. For the same reason, the amount added was calculated from the value for specific activity provided by the manufacturer.

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Regulation of Glucan Synthetase 901

Incubation of a highly active preparation (obtained with 10 mM EDTA) in the presence of Mg2+ led to a drastic loss in activity (Fig. 7A). The resulting enzyme behaved as did those prepared in low EDTA since it could be stimulated back to the original value by ATP or GTP (Fig. 7A).

A low EDTA enzyme, that had been activated with GTP and then centrifuged as in the experiment of Table 111, be- haved in the same manner as the high EDTA enzyme upon incubation with Mg‘+ (Fig. 7B).

DISCUSSION

As noted above and in the accompanying paper (2), the existence of a period of cell wall growth followed by a period of wall quiescence in the yeast cell cycle, together with the finding that glucan synthetase is localized at the plasma membrane (2), suggests that the synthetase activity is the subject of strict and localized regulation. It is deemed likely that the activation-inactivation effects reported in the present paper reflect aspects of this in vivo regulation.

Several important features of the ATP and GTP stimula- tion have been clarified. The effects of the two nucleotides have been found to differ, although, as discussed below, both substances may ultimately lead to a very similar stimulator - enzyme complex.

Properties of GTP Actiuation-Stimulation by GTP occurs very rapidly and at low temperatures (Table 111). The ability of GTP to stimulate is not lost after incubation and centrifu- gation of the particulate enzyme (Table I) or after treatment of the particles with alkaline phosphatase (Table 11). EDTA is required but can be removed after GTP has been in contact with the enzyme, without loss of activity. Since EDTA pro- tects GTP against phosphatases, it seems likely that the complex formed after GTP has reacted with the enzyme is resistant to the endogenous phosphatases.

GTP analogs, whose y-phosphate cannot be transferred because of the methylene, imino, or sulfur group to which it is attached, are strong activators of glucan synthetase; there- fore, these compounds do not act by phosphorylating an acceptor in the enzyme preparation. It is possible that GTP itself modified the enzyme covalently in other ways. Never- theless, because of the rapidity of the reaction and the lack of a temperature requirement, it seems more probable that it simply forms a complex with a component of glucan synthe- tase. The complex is a tight one, as judged by its ability to resist dissociation by centrifugation (Table 111). GTP was indeed found to bind to the particulate preparation with a binding constant of 8 to 10 X a value higher but not too dissimilar from the kinetic activation constant of 1.6 X These results, however, should be considered with great cau- tion because there are at present no criteria to establish whether the measured binding is related to the activation of glucan synthetase. Part, indeed most, of the bound GTP is converted into GDP. It is difficult to decide whether this conversion is related to the activity of the enzyme, as appears to be the case for mammalian adenylate cyclase ( 7 ) , or it is caused by unspecific phosphatases despite the presence of EDTA. This result would seem to be in partial contradiction with the stability of the GTP activation, even subsequent to elimination of EDTA (see above). Nonetheless, it is still possible that the GTP and GDP binding sites are not func- tionally related.

The minimum structure required,for stimulation appears to be that of GDP. A 5”triphosphate group, however, improves the stimulatory ability. Thus, GTP is a better activator than GDP, both in K, and in Vmax. A pyrophosphate group at position 3’ tends to decrease the activity. Thus, ppGpp is

inactive and pppGpp is not as good a stimulator as GTP. On the other hand, a cyclic phosphate group at the 2 ‘ 3 positions seems to enhance activity because ppG > p is more efficient than GDP. Nevertheless, a compound bearing both the tri- phosphate and the cyclic phosphate group, pppG > p, was not more active than GTP itself.

Properties of ATP Actiuation-In contrast with GTP, the effect of ATP requires time and a higher temperature (Figs. 3 and 4 and Table 111). Under conditions in which the acti- vation by GTP is maintained, such as incubation of the enzyme at 30°C followed by centrifugation (Table I) or treat- ment with alkaline phosphatase, the ability of ATP to stim- ulate is partially or totally lost. These results suggest that the action of ATP may require a covalent modification catalyzed by an enzyme present in the membrane preparation. This would explain the time and temperature requirement as well as the lability of the system.

The effect of ATP also differs from that of GTP in that it is inhibited, rather than stimulated, by EDTA. After ATP has acted on the enzyme, however, EDTA seems to have little effect (see Table I, lines 3 and 4 and Table 111, line 2).

In an attempt to determine whether the effect of ATP could be mediated through phosphorylation, the methylene, imino, and sulfur analogs of the nucleotide were used. The results were inconclusive. Whereas all of the analogs stimulated the synthetase to some extent, the inhibition by EDTA was only partial and stabilized at 1.25 mM concentration of the chelating agent. Therefore, it was not possible to decide whether the analogs were acting in the same way as ATP or in the fashion of GTP, as seems to be the case for pppApp when used at high concentrations. If the fiist alternative were correct, one would conclude that the action of ATP does not take place through phosphorylation; adenylylation would still be a pos- sibility.

The Effect of Supernatant Factor(s) and a Working Hy- pothesis for Glucan Synthetase Regulation-The loss of ATP stimulation, observed when glucan synthetase preparations were either centrifuged or treated with alkaline phosphatase, could be repaired by addition of supernatant fluid obtained after incubation and centrifugation of a concentrated particle preparation. The effect of supernatant fluid was especially striking after alkaline phosphatase treatment of the enzyme (Table 11). The material contained in the supernatant liquid

Endogenous Phosphatases

+ ATP “+ ADP + EDTA * + Alkaline Phosphatase

J

Inactive Form

Active Form

FIG. 8. A working hypothesis for the regulation of glucan synthetase. For explanations, see text. PI and PZ are phosphate residues; Pi, inorganic phosphate. The shapes indicated as “Active form” and “Inactive form” represent either glucan synthetase itself or a regulatory subunit. The binding site of the active form has been drawn nearly closed to symbolize the tight binding of GTP and PIXPB. The crossed arrow above EDTA stands for inhibition.

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902 Regulation of GLucan Synthetase

is heat-stable and appears to be of low molecular weight. On the basis of these findings and of those of the previous

sections, it is possible to draw a relatively simple scheme (Fig. 8). Although tentative, this scheme is in agreement with the results and may be useful as a framework for speculation as well as a source of ideas for future experiments. The working hypothesis assumes that ATP acts as a phosphorylating agent, but if another group, e.g. an adenylyl residue, were transferred, the basic features of the scheme would be unchanged.

The substance PIX (Fig. 8) , a phosphorylated compound, would be the supernatant factor. For the sake of economy, it was depicted with only one phosphate group, but it may contain others. PIX would be loosely bound to the particulate glucan synthetase and, thus, would be partially lost to the supernatant upon centrifugation. ATP would transform PIX into PIXP2 by an enzymatically catalyzed phosphorylation that is inhibited by EDTA. The product, P1XP2, would act as a strongly bound stimulator of glucan synthetase. The tight- ness of its binding would explain the stability of the ATP- preactivated enzyme to centrifugation. PlXP2 would in fact act as GTP, and the latter would be capable of displacing PIX from the enzyme. This ability of GTP to override the P2X- PlXP2 system explains the relative constancy of the activity as determined with GTP after different treatments of the enzyme and the lack of independence of ATP and GTP activation that is suggested by the results of Table TI1 (see “Results”). GTP would be able to occupy only the sites that are not filled by P,XP2 and would, thus, bring the system to the state of maximal activity.

Treatment of the particulate preparation with alkaline phosphatase would convert both PIX and PIXPZ into X. Whereas the GTP effect would be unchanged, as was indeed found, the ability of ATP to activate would be lost because its substrate would have been destroyed. Addition of PIX (su- pernatant fluid) could restore the ATP effect and does so.

As described under “Results,” the use of high concentra- tions of EDTA in the lysis of protoplasts results in prepara- tions of glucan synthetase that are almost fully active in the absence of ATP or GTP. Incubation of these preparations in the presence of Mg” results in loss of activity, which can, however, be restored by addition of ATP or GTP (Fig. 7A) . Our interpretation of these results is that high EDTA protects PIXPZ from degradation during preparation of the enzyme. Incubation with Mg’+ would lead to conversion of PlXP2 into PIX (Fig. 8). This reaction would form a closed loop with that of phosphorylation; in this way, the reversible activation of glucan synthetase can be achieved.

The scheme of Fig. 8 could be considerably simplified by assuming that PIX and P1XP2 are GDP (or GMP) and GTP, respectively. The similarity in the effect of Mg2+ on a high EDTA enzyme (Fig. 7A) or on a low EDTA enzyme that was previously activated with GTP (Fig. 7B) would support this hypothesis. On the other hand, the fact that GMP and GDP could not substitute the supernatant factor (PIX) is against this notion. A final decision will require isolation and identi- fication of the supernatant factor. The amount of this material cannot exceed the concentration of binding sites for GTP, z.e.

10 to 15 pmol/mg of protein at best, if it is assumed that all of the GTP bound to the membranes is attached to the glucan synthetase complex. Because of technical difficulties in iden- tifying such small amounts of substance, this point must await further in~estigation.~

The glucan synthetase system is associated with the plasma membrane (2) and forms with it a physiological unit. Despite its complexity, it is important to study it in its integrity in order to minimize the possibility of missing some of its essen- tial functions. An analogy may be made with the mammalian adenylate cyclase system, which shares some aspects with glucan synthetase, such as attachment to the plasma mem- brane and stimulation by GTP and its analogs (8). In that system, relatively mild treatments result in the loss of some of its paramount features, such as activation by hormones. Sim- ilarly, in glucan synthetase, ATP stimulation is easily lost. Nevertheless, fulI understanding of the regulatory mecha- nisms will depend on dissociation of the system, analysis of its components, and, finally, reconstitution of the complex. Al- though preliminary attempts to solubilize the enzymatic ac- tivity with detergents and chaotropic agents have so far met with failure, we hope that the information gathered about the partial reactions proposed in the scheme of Fig. 8 may in the future lead to the isolation of some of those steps.

Irrespective of the correct interpretation of our results, it is now clear that two systems involved in the synthesis of structural polysaccharides, i.e. glucan and chitin (I) , are sub- ject to complex regulatory mechanisms which are presumably involved in the process of morphogenesis of the corresponding structures. These results attest to the soundness of the chosen approach and suggest that similar mechanisms may be oper- ating at the enzymatic level in other morphogenetic processes.

Acknowledgments-We are indebted to Drs. G. Ashwell, J. A. Braatz, J . Correa, W . B. Jakoby, I. Polacheck, and M. L. Slater for useful discussions and criticism.

REFERENCES 1. Cabib, E. (1975) Annu. Reu. Microbiol. 29, 191-214 2. Shematek, E. M., Braatz, J. A., and Cabib, E. (1980) J. Biol.

3. Duran, A,, and Cabib, E. (1978) J. Biol. Chem. 253,4419-4425 4. Casbel, M. (1975) Annu. Rev. Microbiol. 29, 301-318 5. Pao, C. C., Paietta, J., and Gallant, J. A. (1977) Biochem. Biophys.

Res. Commun. 74,314-322 6. Jaworek, D., Gruber, W., and Bergmeyer, H. U. (1974) in Methods

of Enzymatic Analysis (Bergmeyer, H. U., ed), 2nd Ed, Vol. 4, pp. 2127-2129, Academic Press, New York

7. Cassel, D., and Selinger, 2. (1977) Proc. Natl. Acad. Sci. U. S. A.

8. Ross, E. M., and Gilman, A. G. (1977) Proc. Natl. Acad. Sci. U.

Chem. 255,888-894

74,3307-3311

S. A . 74, 3715-3719 ~_____._____c

Cabib, unpublished results), incubation of [y-32P]ATP but not of [8- In preliminary experiments (E. M. Shematek, H. Kawai, and E.

I4C]ATP with large amounts of enzyme resulted in the formation of radioactive membrane-bound material which moved in the GTP area upon polyethyleneimine cellulose chromatography. The study of this material is hampered by its extremely small amount and by the presence of substance(s) with a similar RF in the zero time control. Further investigations in this area are in progress.

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E M Shematek and E Cabibsynthetase by ATP and GTP.

Biosynthesis of the yeast cell wall. II. Regulation of beta-(1 leads to 3)glucan

1980, 255:895-902.J. Biol. Chem. 

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