Subcellular localization and some properties of the adenylate cyclase activity of the yeast, Saccharomyces cerevisiae

15

Biochimica et Biophysica Acta, 3 7 2 ( 1 9 7 4 ) 1 5 - - 2 2 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27507

SUBCELLULAR LOCALIZATION AND SOME PROPERTIES OF THE ADENYLATE CYCLASE ACTIVITY OF THE YEAST, SACCHAROMYCES CERE VISIAE

G R A H A M E. W H E E L E R a , *, A N G E L O SCHIBECIa. **, R I C H A R D M. E P A N D a , *** J A M E S B.M. R A T T R A Y a a n d D E N I S K. K I D B Y b, ~

Departments of aChemistry and bMicrobiology, University of Guelph, Guelph, Ontario N I G 2W1 (Canada) (Received M a r c h 2 5 t h , 1 9 7 4 )

Summary Saccharomyces cerevisiae was grown in the presence of 5% (w/v) glucose and converted to protoplasts. The total particulate material obtained from lysed protoplasts was fractionated by sucrose density gradient ultracentrifugation and the distribution of adenylate cyclase throughout the gradient determined. Adenylate cyclase activity was found to be largely associated with intracellular particulate fractions. Little activity was found in the plasma membrane-rich fraction. The adenylate cyclase activity was found to be inhibited by F-, pyrophosphate and aminophylline, whereas glucagon, 5-hydroxytryptamine and concanavalin A were without effect. The enzymic activity appeared to be modulated by "catabolite repressors" (glucose, fructose and ~-methylglucoside) as well as by acetate. A possible role for adenylate cyclase in regulating the levels of cyclic AMP in the cell during glucose repression is suggested.

Introduction

A variety of cellular functions, including catabolite repression, differentiation and hormonal response has been shown to be under the control of cyclic AMP [1,2]. In mammalian tissues adenylate cyclase, the enzyme responsible for the synthesis of cyclic AMP, is largely associated with the plasma membrane *Present address: School o f Veterinary Science, University of Bristol, Bristol, England. ** Present address: Kzesge Hearing Institute, University of Michigan, A n n Arbor, Mich. 48104, U,S.A. *** Present address: D e p a r t m e n t of Biochemistry, Health Science Centre, McMaster University, Hamilton, Ontario, Canada. § To whom reprint requests should be addressed.

16 [3,4] or plasma membrane-related systems [5,6] and is considered to be a good marker enzyme for the cell surface [7]. In prokaryotes this enzyme has been shown to be both soluble [8] and particulate [9]. Adenylate cyclase has been shown to occur in eukaryotic microorganisms [10--13] and reported to be associated with the plasma membrane of Saccharomyces cerevisiae [10], Saccharomyces fragilis [11] and Neurospora crassa [12], but is absent in the surface membranes of Acanthamoeba palestinensis [13] and Acanthamoeba castellanii [ 14]. In this communication we wish to present evidence for the intracellular localization of particle-associated adenylate cyclase in S. cerevisiae as well as some presumptive evidence suggesting a possible control of this enzyme activity by "catabolite" repressors. Materials and Methods

Cell culture S. cerevisiae, strain Y95, was grown statically at 30°C in the medium of Davies et al. [15] supplemented with 5% (w/v) glucose, and harvested in the log phase of growth. Preparation of protoplasts and subcellular fractionation The conversion of yeast cells to protoplasts and the subcellular fractionation of the total particulate material obtained from lysed protoplasts by sucrose density gradient centrifugation were carried out as previously described [16]. Gradient fractions were collected by means of a J-shaped pipette and washed once with 0.1 M sodium potassium phosphate buffer, pH 7.0, containing 10 mM MgC12. Adenylate cyclase assay The conversion of radioactive ATP into cyclic AMP, which was separated by paper chromatography, was a modification of the procedure of Londesborough and Nurminen [10]. The particulate preparation (250--500 pg of protein) was incubated at 37°C for 20 min in a final volume of 0.2 ml contalning: 200 mM potassium piperazine-N,N'-bis-2-ethane sulfonate, pH 6.0, 10 mM MnCI:, 2 mM MgC12, 80 mM phosphoenolpyruvate, 250 pg/ml pyruvate kinase (EC 2.7.1.40), 1 mM cyclic AMP, 0.2 mM GTP, 1 mM dithiothreitol, and 0.55 mM [8-14C]ATP (sodium salt, Schwartz-Mann, 275 000 cpm/ 200 pl assay mixture). After incubation, 10 pl of 2.5 mM cyclic [U -3 H] AMP (approx. 150 000 cpm) were added and the tubes placed in a boiling water bath for 3 min to stop the reaction. A control tube contained all components except the particulate preparation. Particulate material was removed by centrifugation. Separation by chromatography of labelled nucleotides in the supernatant and determination of radioactivity were carried out as described by Drummond and Duncan [ 17]. Protein was determined by the method of Miller [18] using bovine serum albumin as standard. Enzyme units have been expressed as picomoles of product (cyclic AMP) formed per minute, under the conditions of assay. Specific activities of adenylate cyclase have been reported as units per milligram of

17 protein. Relative specific activity refers to the specific activity of the studied fraction relative to the specific activity of the homogenate. The distribution of adenylate cyclase in the sucrose density gradient fractions is illustrated in the manner suggested by de Duve et al. [19]. Results

The specific activity of adenylate cyclase observed in the total particulate preparation obtained from lysed protoplasts derived from glucose-repressed cells of S. cerevisiae was 25--30 pmoles/min per mg protein. A similar value was obtained by Londesborough and Nurminen [10]. This value compares well with specific activities obtained for adenylate cyclase from various mammalian sources (cf. ref. 7), and is higher than that obtained for S. fragilis [11], but lower than N. crassa [12]. Under the conditions of assay, adenylate cyclase activity was observed to be linear for at least 40 min (Fig. 1). The activity was significantly inhibited (25%) by the presence of 10 mM NaF (Table I). This observation is in contrast to the case in mammalian tissues [20] where F- stimulates adenylate cyclase. The anion has been reported to be weakly stimulatory in S. cerevisiae [10] and S. fragilis [11], inhibitory in Escherichia coli [21], and without effect in N. crassa [ 12 ]. The cyclic AMP phosphodiesterase inhibitor, aminophylline [22], gave small but reproducible inhibition of enzyme activity and was, therefore, omitted from the assay system. Instead, a pool of cold cyclic AMP was used to dilute cyclic [8J 4 C] AMP and thereby limited significant degradation of radioactive product by hydrolytic enzymes [17]. The addition of 1 mM dithiothreitol to the assay medium produced a small stimulation (10%) of activity and therefore, was used routinely. This apparent stimulation may indicate stabilization of enzyme activity as is apparently the case with N. crassa enzyme in the presence of mercaptoethanol [12]. Marked inhibition {83%) of enzyme activity was observed in the presence of added product of reaction (2 mM MgC12 plus 2 mM sodium pyrophosphate), whereas acetate stimulated the activity by up to 30% of the control value (Table I).

~ 1.0 ~: 0.8

"~ 0.4 v ~ 0.2 .5

10

15 20 25 T i m e ( rain )

3o

35

40

Fig. 1. F o r m a t i o n o f cy'cHc A M P as a f u n c t i o n o f t i m e . C o n d i t i o n s for c y c l i c A M P f o r m a t i o n w e r e as d e s c r i b e d in Materials and M e t h o d s , e x c e p t t h a t 0 . 6 8 m g o f p r o t e i n w e r e used in triplicate d e t e r m i n a t i o n s .

18 TABLE I E F F E C T O F V A R I O U S A G E N T S ON T H E A C T I V I T Y O F A D E N Y L A T E C Y C L A S E O F M E M B R A N O U S P R E P A R A T I O N S F R O M S. C E R E V I S I A E C o n d i t i o n s o f assay w e r e as d e s c r i b e d in Materials a n d M e t h o d s . T h e p a r t i c u l a t e p r e p a r a t i o n s (0.6 m g p r o t e i n / a s s a y ) w e r e i n c u b a t e d at 3 7 ° C for 20 min. T h e results are q u o t e d as t h e a v e r a g e of t r i p l i c a t e determinations. A d d i t i o n to the assay s y s t e m

E n z y m e units ( p m o l e s . rain -1. m g -1)

Percent o f control

None 10 m M N a F 2.65 m M a m i n o p h y l l i n e 10 m M N a F + 2 . 6 5 raM a m i n o p h y l l i n e G l u c a g o n (5 ~ g / m l ) Concanavalin A (50/~g/ml) 5 - H y d r o x y t r y p t a m i n e (50 #g/ml) 2 m M MgCI 2 + 2 m M s o d i u m p y r o p h o s p h a t e 50 m M a c e t a t e 125 mM acetate

30.7 23.6 27.4 17.2 33.9 30.1 30.6 5.0 34.2 39.2

100 77 89 56 110 98 100 16 111 128

Sensitivity of the adenylate cyclase was tested by the addition of various hormones known to be stimulators of this enzyme in other organisms. Glucagon, 5-hydroxytryptamine and concanavalin A had no significant effect on activity (Table I).

Subcellular distribution of adenylate cyclase activity On fractionation of the total particulate preparation, adenylate cyclase activity was found to be largely associated with low density fractions. Up to

9; 8

.>_ u =5

5

.>o_3

~2 !

o

2~

4b

Percent protein

6'o

~

loJo

Fig. 2. D i s t r i b u t i o n 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 o b t a i n e d o n u l t r a c e n t r i f u g a t i o n o f t h e t o t a l p a r t i c u l a t e m a t e r i a l f r o m l y s e d p r o t o p l a s t s . T h e d i s c o n t i n u o u s s u c r o s e d e n s i t y g r a d i e n t w i t h steps of 6 0 , 4 0 , 36, 32 a n d 25% ( w / v ) s u c r o s e w a s c e n t r i f u g e d in a S p i n c o SW40 r o t o r at 1 8 9 0 0 0 X g (av) f o r 2.5 h at 4 ° C . T h e d e s i g n a t e d f r a c t i o n s b a n d e d a t t h e i n t e r f a c e s o f t h e v a r i o u s d e n s i t y s t e p s a n d h a d b u o y a n t densities as follows: F r a c t i o n I , d < 1 . 0 5 ; 2, d 1 , 0 5 - - 1 . 1 0 ; 3, d I . I O - - I . I I ; 4, d 1 . 1 1 - - 1 . 1 2 ; 5, d 1 : 1 2 - - 1 . 1 4 ; 6, d 1 . 1 4 - - 1 . 1 5 ; 7, d 1 . 1 5 - - 1 . 2 3 . R e l a t i v e specific a c t i v i t y r e f e r s t o t h e specific a c t i v i t y o f t h e g r a d i e n t f r a c t i o n s relative to t h e specific a c t i v i t y of t h e h o m o g e n a t e (=1.0). T h e p e r c e n t a g e o f t h e t o t a l p r o t e i n in e a c h f r a c t i o n is p l o t t e d o n t h e abscissa.

19 76% of the total enzyme activity was recovered in Fractions 2--5 and only 8% in Fraction 7, which has previously been shown by our laboratory to contain the plasma membrane using radioactive tags specific for the plasma membrane [16,23]. Highest specific activity of the enzyme was observed in Fractions 3 and 4, banding at d 1.10--1.11 and d 1.11--1.12, respectively (Fig. 2). In N. crassa [12] peak activity of adenylate cyclase was also found to be associated with a fraction of low density banding at d 1.04--1.07 in a discontinuous sucrose gradient.

Effect o f carbohydrates on adenylate cyclase activity Several mono- and di-saccharides were investigated for their influence on adenylate cyclase activity. Addition of glucose, fructose, a-methylglucoside as well as glycerol to the assay system caused a significant reduction in the production of cyclic AMP, whereas galactose and maltose revealed a smaller inhibition of enzymatic activity (Table II). Sorbitol did not have any appreciable effect. Whereas the effect of glucose, fructose and acetate on the activity appeared to be concentration dependent, the inhibition by galactose and maltose was not. Sucrose added to the assay system appeared to stimulate activity at the lower (100 mM), but had no effect at the higher (250 mM) concentration. Determination of the effect of glucose and glycerol, at a concentration of 0.2 M, on adenylate cyclase activity revealed a 19% inhibition by glucose and a 98% stimulation by glycerol in a wild strain of E. coli [24]. Whereas glucose stimulated the same activity in a mutant strain insensitive to glucose repression [24].

T A B L E II E F F E C T OF V A R I O U S C A R B O H Y D R A T E S ON A D E N Y L A T E CYCLASE A C T I V I T Y IN T H E C R U D E M E M B R A N O U S P R E P A R A T I O N S O B T A I N E D F R O M S. C E R E V I S I A E

Conditions of assay w e r e as d e s c r i b e d in T a b l e I. Addition to assay s y s t e m

E n z y m e units ( p m o l e s - rain -1. m g -1 )

Percent o f control

None

30. 7 24.0 20.2 25.0 19.4 21.3 25.9 26.0 29.2 30.3 22.4 26.6 24.9 40.7 30.3

100 78 66 81 63 69 84 85 95 99 73 87 81 133 99

Glucose, 100 mM Glucose, 250 m M Fructose. 1 0 0 m M Fructose, 2 5 0 m M ~ - M e t h y l g l u c o s i d e , 2 5 0 rnM Galactose, 100 mM Galactose, 2 5 0 m M Sorbitol, 1 0 0 m M Sorbitol, 250 mM Glycerol 200 mM Maltose, 1 0 0 m M Maltose, 2 5 0 m M Sucrose, 1 0 0 m M Sucrose, 250 mM

20 Discussion

It has been reported that adenylate cyclase is localized in the plasma membrane of yeasts, S. fragilis [11] and S. cerevisiae [10], and the mold, N. crassa [12]. In glucose-repressed S. cerevisiae, strain Y95, however, adenylate cyclase activity was found to be primarily associated with intracellular membranes with buoyant densities less than 1.14 g • cm -3 presumably derived from the intracellular vesicle population of the yeast, including endoplasmic reticulum (d 1.13) [25]. Electron microscopic observations and enzyme composition studies of these fractions are consistent with this suggestion [26]. In N. crassa [12] peak activity of adenylate cyclase was also found to be associated with a fraction of low density banding at d 1.05--1.07. The activity of adenylate cyclase in the soil amoeba, A. palestinensis [13] was highest in the microsomal fraction and was found to be most concentrated in the rough endoplasmic reticulum whilst only low specific activities of the enzyme was found in enriched fractions of plasma membrane and mitochondria. Recently, Tu and Malhotra [27] have attempted to localize adenylate cyclase activity histochemically in the fungus Phycomyces blakesleeanus. These workers reported finding enzymatic activity in various cellular organelles including cytoplasmic, nuclear and inner-mitochondrial membranes. Their conclusion, however, rests on the assumption that no other ATP-hydrolyzing enzymes are present. Phosphatidyl inositol kinase activity (EC 2.7.1.--), which occurs predominantly on the surface membrane of animal cells [28,29], has been shown in S. cerevisiae, NCYC 366, to have a similar distribution to that of adenylate cyclase in the present study, viz. in low-density subcellular elements (ref. 30 and Wheeler, G.E., Michell, R.H. and Rose, A.H., unpublished). It is contended, therefore, that both enzymes are associated with intracellular membranes and not with the surface membrane. Glucose has been shown to control the intracellular levels of cyclic AMP in various yeast strains [11,31] and E. coli [2]. Derepression of glucoserepressed cells is associated with an increase in the intracellular level of cyclic AMP. Addition of exogenous cyclic AMP reverses repression of the respiratory adaption of anaerobically grown S. cerevisiae [31]. The activity of adenylate cyclase, has been observed to be affected by the concentration of glucose in the growth medium of S. fragilis [11]. Almost no adenylate cyclase activity could be detected when cells were grown on 10% (w/v) glucose, and cyclic AMP levels were low under these conditions [11]. The data in the present communication reveal inhibition of adenylate cyclase by glucose, fructose and ~-methylglucoside. While this inhibition was not dramatically large, it did exhibit a high degree of specificity. The degree of repression of respiratory ability in S. cerevisiae is inversely proportional to the fermentative capacity of the yeast cells: glucose > maltose > galactose [32], and compares closely with the observed degree of inhibition of adenylate cyclase activity (Table II): glucose ~ fructose > ~-methylglucoside > maltose ~ galactose. Simple sugars affect adenylate cyclase activity of mammalian tissues in vitro. Glucose stimulates adenylate cyclase in the islets of Langerhans [33];

21 and other simple sugars, particularly erythrose, inhibit intestinal adenylate cyclase [34]. The concentrations of sugars used in our experiments, while high, do correspond to the levels of carbohydrate in the culture medium required to repress S. cerevisiae and are of a similar concentration range to that shown to influence adenylate cyclase activity o f E. coli [24]. The negative modulation of adenylate cyclase activity by carbohydrates is one possible control mechanism in "catabolite repression". Other control mechanisms are, no doubt, simultaneously exercised in the cell. For example, glucose-stimulated secretion of cyclic AMP into the culture medium has been observed in E. coli [35]. Cyclic AMP phosphodiesterase might be positively modulated by glucose, but this has not been observed so far. Furthermore, glucose may repress inducible enzymes by means other than by lowering cyclic AMP levels [36]. The significance of the localization of adenylate cyclase of S. cerevisiae in intracellular organelles is not clear; but, a possible role for this enzyme in the control of various metabolic activities, including synthesis and secretion of enzymes [37] may be suggested. The observed modulation of adenylate cyclase activity by various metabolites might implicate this enzyme as a site of control of metabolic activity. Since the completion of this work Schultz (unpublished observations in ref. 38) reports that phosphoenolpyruvate, a part of the essential ATP-regenerating system in the assay system for adenylate cyclase, causes inhibition of adenylate cyclase activity in rat liver; it is not known if this observation is also true for the adenylate cyclase of this yeast.

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