263
Biochimica et Biophysica Acta, 454 (1976) 263--272 © Elsevier/North-Holland Biomedical Press
BBA 98754 CELL WALL SYNTHESIS R E G U L A T I O N IN S A C C H A R 0114 Y C E S C E R E V I S I A E . E F F E C T OF R N A AND PROTEIN INHIBITION
M. VICTORIA ELORZA, CARLOS M. LOSTAU, JULIO R. VILLANUEVA and RAFAEL SENTANDREU Departamento de Microbiologia, C.S.I.C. Facultad de Ciencias, Universidad de Salamanca (Spain)
(Received May 21st, 1976} Summary In this investigation the regulation of wall formation in S a c c h a r o m y c e s cerevisiae ts -136 (Hutchison, H.T., Hartwell, L.H. and McLaughlin, C.S. (1969) J. Bacteriol. 99, 807--814) was analyzed by following the inhibition of RNA and protein synthesis. Lomofungin, thiolutin and 8-hydroxyquinoline at the concentrations needed to inhibit R N A synthesis also produced inhibition of glucan and mannan synthetases. The synthesis of RNA was also blocked in S. cerevisiae ts -136 by incubation at the non-permissive temperature (37 ° C). Mannan formation decreased steadily b u t glucan synthesis remained after 4 to 5 h. After a few minutes of blocking protein synthesis with cycloheximide mannan synthesis was also blocked whereas glucan formation was unaffected by the presence of the drug. These results suggest a high degree of stability for glucan synthetases. S. cerevisiae ts -136 after 2 h of incubation at the non-permissive temperature (37°C) showed a preferential formation of wall materials (mannan and glucan) indicating that t h e R N A messengers which codify wall mannan peptides have a slower decay rate than those of the cytoplasmic proteins. The data presented indicate that the existence of stable glucan synthetases and RNA messengers of the wail mannan peptides of slow decay rate results in the continuous synthesis of glucans and mannoproteins of the yeast wail throughout the cell cycle. .4¢
Introduction The cell wail of S a c c h a r o m y c e s cerevisiae is mainly c o m p o s e d of ~-linked glucans, the structural polymers responsible of the cell shape and mechanical
264 strength, and a series of mannoproteins called "yeast mannan", the matrix material of the wall in which several enzymic activities are located. Although something is known a b o u t the biosynthetic mechanisms involved in the formation of both polymers, little is known about the regulation of their formation and their integration into cellular metabolism. Studies carried o u t in our laboratory showed that both glucan and mannan are synthesized continuously throughout the entire cell cycle [1]. Several hypotheses could explain the results and in the present paper we report studies on wall formation following the inhibition of RNA or protein synthesis. The former was inhibited with various drugs-lomofungin [2], thiolutin [3] and 8-hydroxyquinoline [4]. Incubation at the non-permissive temperature of S. cerevisiae ts -~36, a mutant whose RNA synthesis is inhibited at 37°C but is normal at 23°C was also used. Protein synthesis was brought to a halt by the antibiotic cycloheximide [ 5]. Our results showed that RNA messengers of the wall mannan peptides and glucan and mannan synthetases are very stable and suggest that the continuous synthesis of wall polymers during the cell cycle may be explained by this stability. Materials and Methods
Materials. [U-14C]Threonine (specific activity 10 Ci/mol), [U-~4C]uridine (specific activity, 60 Ci/mol) and [U-~4C]glucose (specific activity, 3 Ci/mol) were obtained from the Radiochemical Centre, Amersham, Buckinghamshire. Whenever possible other reagents used were of analytical grade. Organism and culture conditions. S. cerevisiae ts -~36, an adenine and uracil thermosensitive m u t a n t derived from strain A364A by treatment with Nmethyl-N-nitro-N-nitroso guanidine, was obtained from L.M. Hartwell, University of Washington, Seattle. It was maintained on slants of medium YM-1 : yeast extract, 5 g; peptone, 1 g; yeast nitrogen base (Difco), 6.7 g; adenine, 0.01 g; uracil, 0.01 g; succinic acid, 10 g; NaOIq, 6 g; sodium lactate, 30 g; pH 6.8; solidified with 2% agar. For preparation of batches of cells the yeast was propagated in liquid YM-1 medium. Erlenmeyer flasks containing 150 ml of medium were inoculated with 1.8 mg of cells (dry weight) and incubated with shaking at 23°C for 18--20 h (early exponential phase). Measurements o f R N A and protein synthesis. To measure RNA and protein synthesis, either [ U-~4C] uridine or [ U-~4C] threonine was added to cell cultures. Cells (20 mg dry weight) suspended in 5 0 ml of YM-1 medium were incubated under the conditions indicated in each experiment and then 0.5 pCi of either [U-~4C]uridine or [U-14C]threonine added. Samples (5 ml) were removed at indicated times to tubes containing 5 ml of ice-cold 10% trichloroacetic acid. After 30 min, at 0°C, the precipitated material was collected on Whatman GF/C filters and washed. Dried filters were counted, in a toluene-based scintillation fluid (3.5 g of 2,5-diphenyloxazole; 50 mg of 1,4-bis-(4-methyl-5-phenyloxazol-2-yl) benzene and 100 ml of toluene), with a Tri-Carb Scintillation Spectrometer {Packard, model 3220). The efficiency of counting was 75% and no corrections were introduced.
265
Mannan and glucan determination. Mannan and glucan synthesis was followed by adding D-[U-14C] glucose to the growth medium. Samples (20 ml) of the cell suspensions were removed at indicated times to tubes containing 20 ml of ice-cold 10% trichloroacetic acid. After 30 min at 0°C the suspension was centrifuged at 2000 × g for 10 min and the pellet transferred into ampoules containing 2 N NaOH (3 ml). Mannan and alkali- and acid-insoluble glucan were extracted following the procedure of Northcote and H o m e [6]. Breakage of cells and preparation of different subcellular fractions. Washed cells were suspended in 50 mM Tris/maleate buffer (pH 6.8)/5 mM Mn C12/ 1 mM mercaptoethanol and broken in a Braun Model MSK mechanical cell homogenizer (1 g wet weight cells/ml) for 45--60 s. The suspension was centrifuged at 2000 × g for 10 min and the pellet called P2. The supernatant spun at 10 000 × g for 10 min (P,0) and the resulting supernatant fluid centrifuged again at 40 000 × g for 45 min and the pellet (P40) separated from the supernatant ($40). Each pellet was treated with 1 vol. of 10% trichloroacetic acid and the radioactivity associated with the precipitates counted. Results
Turnover of RNA was initially studied by pulses of [U-14C]uridine followed by "chase" of the radioactivity incorporated. After the addition of the cold uridine, incorporation of radioactivity went on undisturbed for several minutes and was still going on for at least 20 min. Yeast, like other eukaryotic cells,
B A 10 x
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45
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60
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60
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Fig. 1. R N A s y n t h e s i s b y S. cerevisiae ts - 1 3 6 . E f f e c t of l o m o f u n g i n a n d t h i o l u t i n (A) a n d 8 - h y d r o x y q u i n o l i n e (B). T o a cell c u l t u r e ( 2 0 m g dry w e i g h t ) in 50 m l o f YM-1 m e d i u m 0.5/~Ci o f [ U - 1 4 C ] u r i d i n e ( s p e c . a c t 1 0 . 4 8 C i / m o l ) a n d a n t i m e t a b o H t c s w e r e a d d e d a n d i n c u b a t e d at 23~°C. ( A ) o o, 5 p.g/ml thiolutin; X X. 10 # g / m l t h i o l u t i n ; o o 20 # g l m l thiolutin~ A ,~, 50 # g l m i l o m o f u n g i n , • -~, no d r u g a d d e d ( c o n t r o l ) . (B) o . . . . . . o, 1 0 0 # g / m l 8 - h y d r o x y q u i n o l i n e ; A. . . . . . ~, 3 0 0 # g l m l 8-hydroxyquinoline and X...... X, 4 0 0 # g / m l h y d r o x y q u i n o l i n e . A c e t i c acid was a d d e d to t h e c o r responding controls ( ).
266 have large nucleotide pools so the "chase" of radioactive precursors does not seem to be a useful method in measuring R N A turnover. Addition of 5 #g/ml of lomofungin to a culture of S. cerevisiae Y166 has been reported to produce a rapid inhibition of RNA synthesis [2]. This concentration produced no effect in S. cerevisiae ts -~36. Higher concentrations (50 pg/ml) resulted in a 40% inhibition of [U-~4C]uridine incorporation into high molecular weight products after 15 min. This concentration of lomofungin was fairly large and the inhibition of R N A synthesis was far from complete (Fig. 1). It has been reported that thiolutin (2--4 gg/ml) produced a rapid and complete R N A synthesis inhibition in S. cerevisiae A 3 6 4 A [3]. The same concentrations produced no inhibition in the mutant ts -~36. Concentration of 10 pg/ml resulted in 80% inhibition of R N A synthesis after 15 min (Fig. 1). Higher concentrations of the drug (20--50 pg/ml) produced rapid and complete inhibition. 8-Hydroxyquinoline has also been described as an effective inhibitor of RNA synthesis in yeast [4]. In S. cerevisiae ts -~36, to obtain blockage of RNA synthesis the concentration of the drug present in the culture media must be at least 200 pg/ml (Fig. 1B). Solubilization of the 8-hydroxyquinoline is brought about with acetic acid [4] but the acid at the concentration required to solubi-
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120 180 240 300 Time ( rain )
Fig. 2. S y n t h e s i s o f R N A T o t w o cell c u l t u r e s ( 2 0 (spec. act. 10.48 Ci/mol) suspensions were divided o t h e r at t h e n o n p e r m i s s i v e
37"C
0
30
60
.-
_ 90
120
180
23"C
Time (rain)
a n d p r o t e i n b y S. cerevisiae ts - 1 3 6 . E f f e c t o f the n o n - p e r m i s s i v e t e m p e r a t u r e . m g d r y w e i g h t ) in 5 0 m l o f Y M - 1 m e d i u m e i t h e r 0 . 5 #Ci o f [ U - 1 4 C ] u r i d i n e o r 0 . 5 pCi o f [ U - 1 4 C ] t h r e o n i n e ( s p e c . a c t . 0 . 2 4 C i / m o l ) w e r e a d d e d • T h e cell in t w o parts; o n e w a s i n c u b a t e d at t h e p e r m i s s i v e t e m p e r a t u r e ( 2 3 ° C ) a n d t h e t e m p e r a t u r e ( 3 7 ° C ) P r o t e i n (© o ) , R N A (:~. ~).
Fig. 3. S y n t h e s i s o f R N A a n d p r o t e i n b y S. cerevisiae ts - 1 3 6 at 2 3 ~ C a f t e r i n c u b a t i o n at t h e n o n - p e r m i s sive t e m p e r a t u r e • T o a cell c u l t u r e ( 2 0 m g dry w e i g h t ) k e p t at 3 7 ° C f o r 2 . 5 h . [ U - 1 4 C ] u r i d i n e ( s p e c . a c t •10.48 Ci/mol) or [U-14C]threonine ( s p e c . a c t . 0 . 2 4 C i / m o l ) w a s a d d e d a n d a f t e r 3 0 rain at 3 7 ° C w e r e i n c u b a t e d at 2 3 ° C . C y c l o h e x i m i d e ( 1 0 0 p g / m l ) w a s a d d e d t o d u p l i c a t e cell c u l t u r e s 3 0 m i n b e f o r e t h e t r a n s f e r at 2 3 ° C . S a m p l e s w e r e t a k e n e v e r y 3 0 m i n a n d t h e r a d i o a c t i v i t y d e t e r m i n e d as i n d i c a t e d in Materials a n d M e t h o d s . o o threonine; • • threonine + cycloheximide; ~ ---~, u r i d i n e ; • A uridine + cycloheximide.
267 lize the drug, necessary for the inhibition of R N A synthesis in the mutant, produced secondary effects (Fig. 1B). To overcome this, in the other experiments reported in this paper, the drug was solubilized in ethanol, which at the necessary concentrations did n o t interfere with cellular metabolism. RNA synthesis was also inhibited in S. cerevisiae ts -136 by incubation at the non-permissive temperature (37°C). The inhibition was rapid a b o u t 63% after 5 min of incubation at 37°C and from then on, the synthesis of R N A practically blocked {Fig. 2). The slow residual incorporation of [UJ4C]uracil can be accounted for by the synthesis of mitochondrial R N A which is n o t altered at the non-permissive temperature [ 7]. Protein synthesis went on for some time and it was completely inhibited only after long periods of incubation at 37 ° C. Viability of S. cerevisiae tS -136 at the non-permissive temperature. Once it was known that the mutant could be used in determining the turnover of RNA, it was of interest to learn more about its metabolic behaviour and viability at the non-permissive temperature. Cells of S. cerevisiae t s -136 incubated at 37°C for 1.5 and 3 h and transferred to 23°C were able to initiate growth again after a lag period. After 4.5 h at 37°C, they were unable to grow again, although they did have an undisrupted plasmalemma as measured by the methylene blue m e t h o d of Arnold [8]. The stain did not penetrate the cells incubated at 37°C for 4.5 h suggesting that the permeability barrier apparently had n o t been destroyed. Recovery of the cells kept at 37°C for 3 h was studied by following formation of RNA and proteins. After transfer of cells at the permissive temperature
/
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Time (rain) Fig. 4. R N A s y n t h e s i s by S. eerevisiae ts T M at 23°C after short intervals at the non-permissive t e m p e r a t u r e . T o t w o cell c u l t u r e s ( 2 0 rag, d r y w e i g h t ) k e p t a t 3 7 ° C f o r 15 a n d 3 0 vain. 0 . 5 p C i o f [ U - 1 4 C ] u r i d i n e ( s p e c . a c t . 1 0 . 4 8 C i / m o l ) w a s a d d e d a n d i n c u b a t i o n c o n t i n u e d at 2 3 ° C . S a m p l e s w e r e t a k e n a n d radioactivity determined, o c, c o n t r o l at 2 3 ° C ; ~ ' -~, 15 rain a t 3 7 ° C ; A A, 30 m i n at o 37°C; • e, c o n t r o l at 37 C.
268 (23°C) formation of both polymers was slow initially but after 60 min, it increased significantly (Fig. 3). Recovery of S. cerevisiae ts -~36 from the thermal shock requires apparently a lag period. When the recovery was carried out under conditions in which protein synthesis was inhibited by adding cycloheximide 30 min before transfer at the permissive temperature an almost total inhibition of R N A formation was detected. When cycloheximide was added at the m o m e n t of transfer at 23°C, synthesis after 150 min was only 32% of the R N A formed by control cells. When the antibiotic was added after 60 min of incubation at the permissive temperature, R N A synthesis was normal. It was also of interest to study cell recovery after shorter periods of incubation at 37°C. After 15 and 30 min of incubation at 37°C, initiation of RNA synthesis at the permissive temperature also needed a lag period (Fig. 4). This period was similar to that required after 3 h at 37°C (Fig. 3). These results suggested that the effect of the non-permissive temperature on the cells was rapid and apparently independent of the time kept at 37 ° C. Effect o f drugs and the non-permissive temperature on glucan and mannan synthesis. Addition of thiolutin to a cell culture produced a 70% inhibition of mannan synthesis after 30 min but total inhibition was n o t obtained under the experimental conditions (Fig. 5B). Glucan synthesis was also inhibited (Fig. 5A). 8-Hydroxyquinoline interfered strongly with mannan formation; after 120 min the residual mannan synthesis was a b o u t 7%. Glucan formation was also arrested; its inhibition accounted for a 67% and a 71% after 60 and 120 min, respectively (Fig. 5B and A). 25
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Fig. 5. G l u c a n ( A ) a n d m a n n a n (B) s y n t h e s i s b y S. cerevisiae t s - 1 3 6 . E f f e c t of a n t i m e t a b o l i t e s a n d t e m p e r a t u r e . T o cell c u l t u r e s ( 2 0 rag, d r y w e i g h t ) in 50 m l , 5 #Ci of [ U A 4 C ] - g l u c o s e spec. act. 2 C i / m o l ) a n d d i f f e r e n t a n t i m e t a b o l i t e s w e r e a d d e d a n d i n c u b a t e d a t 2 3 ° C . A n o t h e r cell c u l t u r e s u p p l e m e n t e d w i t h r a d i o a c t i v e g l u c o s e o n l y was i n c u b a t e d a t 3 7 ° C . S a m p l e s w e r e t a k e n a n d g l u c a n a n d m a n n a n synt h e s i z e d d e t e r m i n e d as i n d i c a t e d in Materials a n d M e t h o d s . ~ •, t h i o l u t i n , 20 ~tg/ml; ~ D 8 - h y d r o x y q u i n o l i n e , 4 0 0 Mg/ml; X X, c y c l o h e x i m i d e , 1 0 0 # g / m l ; Q ~ , c o n t r o l at 2 3 ° C ; e-• c o n t r o l at 3 7 ° C .
269
The kinetics of glucan formation at 37°C (at this temperature there is no transport of newly synthesized R N A into the cytoplasm [18]) suggested that its synthesis may initially be independent of new messenger RNA. It would only be dependent on the turnover of the enzymes involved. Mannan formation would be independent of new messenger R N A at least initially; afterwards it would be dependent on new m R N A and probably on nucleic acids which code for proteins acting as mannose acceptors. To check this point and to gain information in the way in which thiolutin and 8-hydroxyquinoline interfere with glucan synthetase activities, it was of interest to study glucan and mannan synthesis under conditions in which protein formation had been blocked by cycloheximide. Effect of cycloheximide on glucan and mannan synthesis. Glucan synthesis was not affected in the presence of the antibiotic for at least 5 h according to Fig. 5A. Mannan synthesis was severely inhibited 87% after 120 min {Fig. 5B) These results indicate that mannan formation is blocked by cycloheximide more rapidly than glucan synthesis because of its requirement of protein acceptors. Cytological location of polymers synthesized by S. cerevisiae ts -136 after incubation at the non-permissive temperature. The experiment described in Fig. 5 showed that at 37°C the mutant went on synthesizing both glucan and mannan polymers. These results did not establish the fate of the wall polymers formed under those conditions and if they were actually incorporated into the wall. To learn about this point, cells incubated at 37°C and at 23°C for t w o hours were pulse-labelled with either [U-14C]glucose or [U-14C]threonine and after 30 min, fractionated as indicated in Methods. Threonine incorporated by cells incubated at 37°C was mainly found in Fraction P: {mostly cells walls). When the cells were labelled at 23 ° C, fraction $40 (soluble proteins and ribosomes) was the most radioactive one {Table I). Distribution of radioactivity incorporated from [U-14C]glucose was also significant. The radioactivity found in the walls from cells labelled at 37°C was 12-fold the radioactivity found in fraction $40. In cells labelled at 23°C
TABLE I CYTOLOGICAL LOCATION OF[U -14C]THREONINE EFFECT OF TEMPERATURE
INCORPORATED
BY S. C E R E V I S I A E ts - 1 3 6 :
T w o cell c u l t u r e s ( 3 5 m g , d r y w e i g h t ) in 4 0 m | i n c u b a t e d w i t h 0 . 5 ~Ci r a d i o a c t i v e t h r e o n i n e ( s p e c . a c t . 2 C i / r n o l ) d e s c r i b e d in M e t h o d s . Each p e l l e t (P2, P I 0 a n d P 4 0 ) and w i t h an e q u a l v o l u m e o f 1 0 % t r i c h l o r o a c e t i c acid and the
at 3 7 ° C and at 2 3 ° C for 2 h w e r e p u l s e d - l a b e l l e d f o r 3 0 r a i n . T h e ceils t h e n w e r e f r a e t i o n a t e d as an a l i q u o t o f t h e s u p e r n a t a n t ( $ 4 0 ) w e r e t r e a t e d radioactivity determined.
Ternpera-
[ U - 1 4 C ] T h z e o n i n e i n c o r p o r a t e d i n t o t h e t r i c h l o r o a c e t i c acid p r e c i p i t a t e s
ture (oC)
P2
23 37
P 10
P40
$40
(cp i0 rain)
%
(cp 10 rain)
%
(cp I0 rain)
%
(cp I0 rain)
%
24 920 1 580
24 55
20 110 390
19.1 13.6
13 940 480
13.1 16.6
45 090 400
43 14
270 T A B L E II C Y T O L O G I C A L L O C A T I O N OF [ U - 1 4 C ] G L U C O S E I N C O R P O R A T E D E F F E C T OF T E M P E R A T U R E
BY S. C E R E V I S I A E
ts-136:
T w o cell c u l t u r e s ( 3 5 m g d r y w e i g h t ) in 4 0 ml i n c u b a t e d at 3 7 ° C a n d at 2 3 ° C for 2 h w e r e pulse-labelled w i t h 5 pCi o f r a d i o a c t i v e glucose (spec. act. 2 C i / m o l ) for 30 m i n . T h e cells w e r e f r a c t i o n a t e d as d e s c r i b e d in M e t h o d s and the r a d i o a c t i v i t y d e t e r m i n e d in each f r a c t i o n . Ternperature (° C)
23 37
[ U - 1 4 C ] G l u c o s e i n c o r p o r a t e d i n t o t h e t r i c h l o r o a c e t i c acid p r e c i p i t a t e s P2
PIO
P40
$40
(cp 10 m i n )
%
(cp 10 m i n )
%
(cp 10 m i n )
%
(ep 10 m i n )
%
5381 1386
63.8 70.6
912 325
10.8 16.5
607 142
7.2 7.2
1527 110
18.1 5.6
the corresponding radioactivity incorporated in fraction P2 (walls) was only 5-fold (Table II). Therefore it seemed that at 37°C S. cerevisiae ts -136 synthesized and incorporated preferentially wall specific substances. This also suggests that the mRNA that codify wall glycoproteins are more stable than those of cytoplasmic proteins.
Discussion The two main polymers of S. cerevisiae walls (13-glucans and a series of glycoproteins whose carbohydrate moiety is known as "yeast m a n n a n " ) are synthesized continuously t h r o u g h o u t the cell cycle [1]. Halvorson et al. [9] proposed that the genes of each chromosome are transcribed in an orderly and linear way. If this is true, the continuous synthesis of wall polymers could be due to: (a) Presence of several duplicated copies of the genes involved distributed t h r o u g h o u t the cell genome. (b) The genes being present in small number but transcribed constantly. In this case the "linear reading" of at least some of the genes does not take place. (c) The RNA messengers and/or the glucan and mannan synthetases show slow turnover. To decide between these hypotheses we wanted to determine the turnover of the RNA and synthetases involved in wall formation. In the study of mRNA decay, as in the case of other macromolecules, it is normal practice to chase radioactive precursors with non-radioactive ones. In S. cerevisiae the mRNA decay can n o t proceed in this way in spite of the addition of cold precursors because to the large nucleotide pool, the incorporation of label into the polymer continues. This result indicates t h a t the pool of RNA precursors is quite large as has also been described for other eukaryotic cells. Actinomycin D and cordicepin are routinely used to inhibit RNA synthesis in eukaryotic cells but these compounds are not taken up by S. cerevisiae. Moreover Actinomycin D is now known to affect both protein synthesis and RNA stability [ 10--11 ].
271 Lomofungin has also been described as an inhibitor of R N A synthesis in yeast b u t at the concentration described [2] it produced no effect in S. cerevisiae ts-13% Higher concentrations of lomofungin produced inhibition b u t it was far from complete. Moreover, the high concentration used could interfere with other cell processes. This inhibitor chelates divalent cations [12] and both Mn 2÷ and Mg 2÷ are required by the glucan [13] and mannan [14--15] synthetases. It is quite probable that synthesis of the wall polymers in the presence of the drug might be inhibited at the level of the synthetases. 8-Hydroxyquinoline strongly inhibited mannan synthesis and as it also acts by cation chelation [4] this inhibition might in part be due, as in the case of lomofungin, to deprivation of Mn 2+ of Mg 2+. This hypothesis was confirmed when glucan synthesis was studied in the presence of the drug. Glucan and mannan formation were inhibited similary after 60 min and from then on glucan synthesis continued at the same partially inhibited rate, as did mannan, though at a consistently lower rate. This result suggests that the drug inhibited the glucan synthetases. This was finally established when found that in the presence of cycloheximide, in the absence of protein synthesis, glucan formation was n o t inhibited and went on for long periods of time (Fig. 5A). Thiolutin also inhibits RNA synthesis. When glucan and mannan formation was studied the latter was most severely affected. This inhibition of both glucan and mannan formation suggested that the effect of thiolutin is n o t only the result of an interference at the level of RNA polymerases [3] but also at the level of other cell processes. That is to say, the effect of this antibiotic is similar to that of 8-hydroxyquinoline. We have also employed a novel m e t h o d to study glucan and mannan metabolism. In this method we take advantage of the mutant, S. cerevisiae ts -136 whose genetic defect is a blockage of RNA transport from the nucleus to the cytoplasm at the non-permissive temperature [ 7,16--17]. Temperature sensitive mutants normally present a point mutation and the rest of the cell genome is expressed normally. Moreover, the non-permissive temperature is low enough (37 ° C) to allow the development of an ordinary metabolism. The parent strain, S. cerevisiae A 364 A grows normally at 37°C and thus it seems that the RNA messengers present in the cytoplasm of S. cerevisiae ts -136 at the m o m e n t of transfer to 37°C are functional and will be translated into proteins. What happens from then. on will depend on the progressive degradation of the RNA messengers. At the non-permissive temperature the rate of mannan formation was the same as that at 23°C for the first hour. On the other hand, the rate of glucan synthesis at 37°C was initially greater than at 23 ° C. This was probably due to the increase in the catalytic activity of metabolic reactions when temperature is raised. Just a similar effect was found in the parent strain S. cerevisiae A 364A (results to be published elsewhere). The amounts of glucan synthesized at 23°C and at 37°C were practically identical after 3 h (Fig. 5A). This suggests that inhibition of RNA messenger synthesis apparently did not interfere with biosynthetic cell activities. Cycloheximide produced an immediate and complete blockage of protein synthesis and interfere rapidly and strongly with mannan synthesis. Glucan formation was unaffected. Mannan synthesis requires, as has already been
272 shown, a continuous synthesis of protein [5]. In the case of glucan it seems evident t h a t its continued synthesis depends on the slow decay of the glucan synthetases since in the presence of cycloheximide, the rate of synthesis remains constant for at least 5 h. Subcellular location of the threonine and glucose in S. cerevisiae ts -'3~' after exposure to 37°C showed that they were preferentially incorporated in wall materials. It was not a mere redistribution of preexisting substances but an orderly and preferential synthesis of wall substances. It is also suggested that the RNA messengers for wall glycoproteins show a half life which is longer than the average RNA messenger for soluble proteins. The actual decay of the mRNA involved in the synthesis of wall macromolecules has been obtained in a computer Univac 1106 with a program specifically devised for this purpose (results to be published elsewhere). These results are consistent with the existence of relatively long-lived mRNAs which codify invertase and other extracellular enzymic glycoproteins [19,20]. The findings reported in this paper indicate that independently of any control at the transcription level, the existence of RNA messengers for mannoproteins and synthetases of low decay results in the continuous synthesis of both glucan and mannan t h r o u g h o u t the entire cell cycle. Acknowledgement We thank Prof. H.J. Phaff of California University for helpful discussions and critical reading of the manuscript.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Sierra, J . M . , S e n t a n d r e u , R. a n d V i l l a n u e v a , J . R . ( 1 9 7 3 ) FEBS L e t t . 34, 2 8 5 - - 2 9 0 C a n o n , M., Davies, J . E . a n d J i m e n e z , A. ( 1 9 7 3 ) FEBS L e t t . 32, 2 7 7 - - 2 8 0 T i p p e r , J . D . ( 1 9 7 3 ) J. Bacteriot. 1 1 6 , 2 4 5 - - 2 5 6 F r a s e r , S.S.R. a n d C r e a n o r , J. ( 1 9 7 4 ) Eur. J. B i o c h e m . 46, 6 7 - - 7 3 S e n t a n d r e u , R. a n d L a m p e n , J . O . ( 1 9 7 0 ) FEBS L e t t . 11, 9 5 - - 9 9 N o r t h c o t e , D.H. a n d H o m e , R.D. ( 1 9 5 2 ) B i o c h e m . J. 51, 2 3 2 - - 2 3 6 S h i o k a w a , K. a n d P o g o , A.O. ( 1 9 7 4 ) Proc. Natl. A c a d . Sci. U.S. 71, 2 6 5 8 - - 2 6 6 2 A r n o l d , W.N. ( 1 9 7 2 ) J. Biol. C h e m . 247, 1 1 6 1 - - 1 1 6 9 H a l v o r s o n , H . O . , C a r t e r , B . L . A . a n d T a u r o , P. ( 1 9 7 1 ) In: A d v a n c e s in Microbial P h y s i o l o g y ( R o s e , A . H . a n d W i l k i n s o n , F.J., c d s . ) , Vol. 6, p p . 4 7 - - 1 0 6 , A c a d e m i c Press, L o n d o n Singer, R . H . a n d P e n m a n , S. ( 1 9 7 2 ) N a t u r e 2 4 0 , 1 0 0 - - 1 0 2 Singer, R . H . a n d P e n m a n , S. ( 1 9 7 3 ) J. Mol. Biol. 7 2 , 3 2 1 - - 3 3 4 P a v l e t i c h , K., K u o , S.C. a n d L a m p e n , J.O. ( 1 9 7 4 ) B i o c h e m . Bioph.vs. Res. C o m m u n . 6 0 , 9 4 2 - 9 5 0 S e n t a n d r e u , R., E l o r z a , M.V. a n d V i l l a n u e v a , J . R . ( 1 9 7 5 ) J. G e n . Microbiol. 9 0 , 1 3 - - 2 0 B e h r e n s , N . H . a n d C a b i b , E. ( 1 9 6 8 ) J. Biol. C h e m . 2 4 3 , 5 0 2 - - 5 0 9 L e h l e , L. a n d T a n n e r , W. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 5 0 , 2 2 5 - - 2 3 5 H a r t w e l l , L . H . ( 1 9 6 7 ) J. Bacteriol. 93, 1 6 6 2 - - 1 6 7 0 H u t c h i s o n , H . T . , H a r t w e l l , L . H . a n d M c L a u g h l i n , C.S. ( 1 9 6 9 ) J. Bacteriol. 99, 8 0 7 - - 8 1 4 Brafias, L. a n d P o g o , A.O. ( 1 9 7 5 ) Eur. J. B i o c h e m . 5 4 , 3 1 7 - - 3 2 8 K u o , S.C., C a n o , F.R. a n d L a m p e n , J . O . ( 1 9 7 3 ) A n t i m i c r o b . Ag. C h e m o t c r . 3, 7 2 3 - - 7 2 8 C r e a n o r , J., M a y , J.W. a n d M i t c h i s o n , J.M. ( 1 9 7 5 ) Eur. J. B i o c h e m . 6 0 , 4 8 7 - - 4 9 3
Biochimica et Biophysica Acta, 454 (1976) 263--272 © Elsevier/North-Holland Biomedical Press
BBA 98754 CELL WALL SYNTHESIS R E G U L A T I O N IN S A C C H A R 0114 Y C E S C E R E V I S I A E . E F F E C T OF R N A AND PROTEIN INHIBITION
M. VICTORIA ELORZA, CARLOS M. LOSTAU, JULIO R. VILLANUEVA and RAFAEL SENTANDREU Departamento de Microbiologia, C.S.I.C. Facultad de Ciencias, Universidad de Salamanca (Spain)
(Received May 21st, 1976} Summary In this investigation the regulation of wall formation in S a c c h a r o m y c e s cerevisiae ts -136 (Hutchison, H.T., Hartwell, L.H. and McLaughlin, C.S. (1969) J. Bacteriol. 99, 807--814) was analyzed by following the inhibition of RNA and protein synthesis. Lomofungin, thiolutin and 8-hydroxyquinoline at the concentrations needed to inhibit R N A synthesis also produced inhibition of glucan and mannan synthetases. The synthesis of RNA was also blocked in S. cerevisiae ts -136 by incubation at the non-permissive temperature (37 ° C). Mannan formation decreased steadily b u t glucan synthesis remained after 4 to 5 h. After a few minutes of blocking protein synthesis with cycloheximide mannan synthesis was also blocked whereas glucan formation was unaffected by the presence of the drug. These results suggest a high degree of stability for glucan synthetases. S. cerevisiae ts -136 after 2 h of incubation at the non-permissive temperature (37°C) showed a preferential formation of wall materials (mannan and glucan) indicating that t h e R N A messengers which codify wall mannan peptides have a slower decay rate than those of the cytoplasmic proteins. The data presented indicate that the existence of stable glucan synthetases and RNA messengers of the wail mannan peptides of slow decay rate results in the continuous synthesis of glucans and mannoproteins of the yeast wail throughout the cell cycle. .4¢
Introduction The cell wail of S a c c h a r o m y c e s cerevisiae is mainly c o m p o s e d of ~-linked glucans, the structural polymers responsible of the cell shape and mechanical
264 strength, and a series of mannoproteins called "yeast mannan", the matrix material of the wall in which several enzymic activities are located. Although something is known a b o u t the biosynthetic mechanisms involved in the formation of both polymers, little is known about the regulation of their formation and their integration into cellular metabolism. Studies carried o u t in our laboratory showed that both glucan and mannan are synthesized continuously throughout the entire cell cycle [1]. Several hypotheses could explain the results and in the present paper we report studies on wall formation following the inhibition of RNA or protein synthesis. The former was inhibited with various drugs-lomofungin [2], thiolutin [3] and 8-hydroxyquinoline [4]. Incubation at the non-permissive temperature of S. cerevisiae ts -~36, a mutant whose RNA synthesis is inhibited at 37°C but is normal at 23°C was also used. Protein synthesis was brought to a halt by the antibiotic cycloheximide [ 5]. Our results showed that RNA messengers of the wall mannan peptides and glucan and mannan synthetases are very stable and suggest that the continuous synthesis of wall polymers during the cell cycle may be explained by this stability. Materials and Methods
Materials. [U-14C]Threonine (specific activity 10 Ci/mol), [U-~4C]uridine (specific activity, 60 Ci/mol) and [U-~4C]glucose (specific activity, 3 Ci/mol) were obtained from the Radiochemical Centre, Amersham, Buckinghamshire. Whenever possible other reagents used were of analytical grade. Organism and culture conditions. S. cerevisiae ts -~36, an adenine and uracil thermosensitive m u t a n t derived from strain A364A by treatment with Nmethyl-N-nitro-N-nitroso guanidine, was obtained from L.M. Hartwell, University of Washington, Seattle. It was maintained on slants of medium YM-1 : yeast extract, 5 g; peptone, 1 g; yeast nitrogen base (Difco), 6.7 g; adenine, 0.01 g; uracil, 0.01 g; succinic acid, 10 g; NaOIq, 6 g; sodium lactate, 30 g; pH 6.8; solidified with 2% agar. For preparation of batches of cells the yeast was propagated in liquid YM-1 medium. Erlenmeyer flasks containing 150 ml of medium were inoculated with 1.8 mg of cells (dry weight) and incubated with shaking at 23°C for 18--20 h (early exponential phase). Measurements o f R N A and protein synthesis. To measure RNA and protein synthesis, either [ U-~4C] uridine or [ U-~4C] threonine was added to cell cultures. Cells (20 mg dry weight) suspended in 5 0 ml of YM-1 medium were incubated under the conditions indicated in each experiment and then 0.5 pCi of either [U-~4C]uridine or [U-14C]threonine added. Samples (5 ml) were removed at indicated times to tubes containing 5 ml of ice-cold 10% trichloroacetic acid. After 30 min, at 0°C, the precipitated material was collected on Whatman GF/C filters and washed. Dried filters were counted, in a toluene-based scintillation fluid (3.5 g of 2,5-diphenyloxazole; 50 mg of 1,4-bis-(4-methyl-5-phenyloxazol-2-yl) benzene and 100 ml of toluene), with a Tri-Carb Scintillation Spectrometer {Packard, model 3220). The efficiency of counting was 75% and no corrections were introduced.
265
Mannan and glucan determination. Mannan and glucan synthesis was followed by adding D-[U-14C] glucose to the growth medium. Samples (20 ml) of the cell suspensions were removed at indicated times to tubes containing 20 ml of ice-cold 10% trichloroacetic acid. After 30 min at 0°C the suspension was centrifuged at 2000 × g for 10 min and the pellet transferred into ampoules containing 2 N NaOH (3 ml). Mannan and alkali- and acid-insoluble glucan were extracted following the procedure of Northcote and H o m e [6]. Breakage of cells and preparation of different subcellular fractions. Washed cells were suspended in 50 mM Tris/maleate buffer (pH 6.8)/5 mM Mn C12/ 1 mM mercaptoethanol and broken in a Braun Model MSK mechanical cell homogenizer (1 g wet weight cells/ml) for 45--60 s. The suspension was centrifuged at 2000 × g for 10 min and the pellet called P2. The supernatant spun at 10 000 × g for 10 min (P,0) and the resulting supernatant fluid centrifuged again at 40 000 × g for 45 min and the pellet (P40) separated from the supernatant ($40). Each pellet was treated with 1 vol. of 10% trichloroacetic acid and the radioactivity associated with the precipitates counted. Results
Turnover of RNA was initially studied by pulses of [U-14C]uridine followed by "chase" of the radioactivity incorporated. After the addition of the cold uridine, incorporation of radioactivity went on undisturbed for several minutes and was still going on for at least 20 min. Yeast, like other eukaryotic cells,
B A 10 x
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5
.9 n~
15
30
45
Time (rain)
60
15
30
45
60
Time (min)
Fig. 1. R N A s y n t h e s i s b y S. cerevisiae ts - 1 3 6 . E f f e c t of l o m o f u n g i n a n d t h i o l u t i n (A) a n d 8 - h y d r o x y q u i n o l i n e (B). T o a cell c u l t u r e ( 2 0 m g dry w e i g h t ) in 50 m l o f YM-1 m e d i u m 0.5/~Ci o f [ U - 1 4 C ] u r i d i n e ( s p e c . a c t 1 0 . 4 8 C i / m o l ) a n d a n t i m e t a b o H t c s w e r e a d d e d a n d i n c u b a t e d at 23~°C. ( A ) o o, 5 p.g/ml thiolutin; X X. 10 # g / m l t h i o l u t i n ; o o 20 # g l m l thiolutin~ A ,~, 50 # g l m i l o m o f u n g i n , • -~, no d r u g a d d e d ( c o n t r o l ) . (B) o . . . . . . o, 1 0 0 # g / m l 8 - h y d r o x y q u i n o l i n e ; A. . . . . . ~, 3 0 0 # g l m l 8-hydroxyquinoline and X...... X, 4 0 0 # g / m l h y d r o x y q u i n o l i n e . A c e t i c acid was a d d e d to t h e c o r responding controls ( ).
266 have large nucleotide pools so the "chase" of radioactive precursors does not seem to be a useful method in measuring R N A turnover. Addition of 5 #g/ml of lomofungin to a culture of S. cerevisiae Y166 has been reported to produce a rapid inhibition of RNA synthesis [2]. This concentration produced no effect in S. cerevisiae ts -~36. Higher concentrations (50 pg/ml) resulted in a 40% inhibition of [U-~4C]uridine incorporation into high molecular weight products after 15 min. This concentration of lomofungin was fairly large and the inhibition of R N A synthesis was far from complete (Fig. 1). It has been reported that thiolutin (2--4 gg/ml) produced a rapid and complete R N A synthesis inhibition in S. cerevisiae A 3 6 4 A [3]. The same concentrations produced no inhibition in the mutant ts -~36. Concentration of 10 pg/ml resulted in 80% inhibition of R N A synthesis after 15 min (Fig. 1). Higher concentrations of the drug (20--50 pg/ml) produced rapid and complete inhibition. 8-Hydroxyquinoline has also been described as an effective inhibitor of RNA synthesis in yeast [4]. In S. cerevisiae ts -~36, to obtain blockage of RNA synthesis the concentration of the drug present in the culture media must be at least 200 pg/ml (Fig. 1B). Solubilization of the 8-hydroxyquinoline is brought about with acetic acid [4] but the acid at the concentration required to solubi-
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Fig. 2. S y n t h e s i s o f R N A T o t w o cell c u l t u r e s ( 2 0 (spec. act. 10.48 Ci/mol) suspensions were divided o t h e r at t h e n o n p e r m i s s i v e
37"C
0
30
60
.-
_ 90
120
180
23"C
Time (rain)
a n d p r o t e i n b y S. cerevisiae ts - 1 3 6 . E f f e c t o f the n o n - p e r m i s s i v e t e m p e r a t u r e . m g d r y w e i g h t ) in 5 0 m l o f Y M - 1 m e d i u m e i t h e r 0 . 5 #Ci o f [ U - 1 4 C ] u r i d i n e o r 0 . 5 pCi o f [ U - 1 4 C ] t h r e o n i n e ( s p e c . a c t . 0 . 2 4 C i / m o l ) w e r e a d d e d • T h e cell in t w o parts; o n e w a s i n c u b a t e d at t h e p e r m i s s i v e t e m p e r a t u r e ( 2 3 ° C ) a n d t h e t e m p e r a t u r e ( 3 7 ° C ) P r o t e i n (© o ) , R N A (:~. ~).
Fig. 3. S y n t h e s i s o f R N A a n d p r o t e i n b y S. cerevisiae ts - 1 3 6 at 2 3 ~ C a f t e r i n c u b a t i o n at t h e n o n - p e r m i s sive t e m p e r a t u r e • T o a cell c u l t u r e ( 2 0 m g dry w e i g h t ) k e p t at 3 7 ° C f o r 2 . 5 h . [ U - 1 4 C ] u r i d i n e ( s p e c . a c t •10.48 Ci/mol) or [U-14C]threonine ( s p e c . a c t . 0 . 2 4 C i / m o l ) w a s a d d e d a n d a f t e r 3 0 rain at 3 7 ° C w e r e i n c u b a t e d at 2 3 ° C . C y c l o h e x i m i d e ( 1 0 0 p g / m l ) w a s a d d e d t o d u p l i c a t e cell c u l t u r e s 3 0 m i n b e f o r e t h e t r a n s f e r at 2 3 ° C . S a m p l e s w e r e t a k e n e v e r y 3 0 m i n a n d t h e r a d i o a c t i v i t y d e t e r m i n e d as i n d i c a t e d in Materials a n d M e t h o d s . o o threonine; • • threonine + cycloheximide; ~ ---~, u r i d i n e ; • A uridine + cycloheximide.
267 lize the drug, necessary for the inhibition of R N A synthesis in the mutant, produced secondary effects (Fig. 1B). To overcome this, in the other experiments reported in this paper, the drug was solubilized in ethanol, which at the necessary concentrations did n o t interfere with cellular metabolism. RNA synthesis was also inhibited in S. cerevisiae ts -136 by incubation at the non-permissive temperature (37°C). The inhibition was rapid a b o u t 63% after 5 min of incubation at 37°C and from then on, the synthesis of R N A practically blocked {Fig. 2). The slow residual incorporation of [UJ4C]uracil can be accounted for by the synthesis of mitochondrial R N A which is n o t altered at the non-permissive temperature [ 7]. Protein synthesis went on for some time and it was completely inhibited only after long periods of incubation at 37 ° C. Viability of S. cerevisiae tS -136 at the non-permissive temperature. Once it was known that the mutant could be used in determining the turnover of RNA, it was of interest to learn more about its metabolic behaviour and viability at the non-permissive temperature. Cells of S. cerevisiae t s -136 incubated at 37°C for 1.5 and 3 h and transferred to 23°C were able to initiate growth again after a lag period. After 4.5 h at 37°C, they were unable to grow again, although they did have an undisrupted plasmalemma as measured by the methylene blue m e t h o d of Arnold [8]. The stain did not penetrate the cells incubated at 37°C for 4.5 h suggesting that the permeability barrier apparently had n o t been destroyed. Recovery of the cells kept at 37°C for 3 h was studied by following formation of RNA and proteins. After transfer of cells at the permissive temperature
/
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Time (rain) Fig. 4. R N A s y n t h e s i s by S. eerevisiae ts T M at 23°C after short intervals at the non-permissive t e m p e r a t u r e . T o t w o cell c u l t u r e s ( 2 0 rag, d r y w e i g h t ) k e p t a t 3 7 ° C f o r 15 a n d 3 0 vain. 0 . 5 p C i o f [ U - 1 4 C ] u r i d i n e ( s p e c . a c t . 1 0 . 4 8 C i / m o l ) w a s a d d e d a n d i n c u b a t i o n c o n t i n u e d at 2 3 ° C . S a m p l e s w e r e t a k e n a n d radioactivity determined, o c, c o n t r o l at 2 3 ° C ; ~ ' -~, 15 rain a t 3 7 ° C ; A A, 30 m i n at o 37°C; • e, c o n t r o l at 37 C.
268 (23°C) formation of both polymers was slow initially but after 60 min, it increased significantly (Fig. 3). Recovery of S. cerevisiae ts -~36 from the thermal shock requires apparently a lag period. When the recovery was carried out under conditions in which protein synthesis was inhibited by adding cycloheximide 30 min before transfer at the permissive temperature an almost total inhibition of R N A formation was detected. When cycloheximide was added at the m o m e n t of transfer at 23°C, synthesis after 150 min was only 32% of the R N A formed by control cells. When the antibiotic was added after 60 min of incubation at the permissive temperature, R N A synthesis was normal. It was also of interest to study cell recovery after shorter periods of incubation at 37°C. After 15 and 30 min of incubation at 37°C, initiation of RNA synthesis at the permissive temperature also needed a lag period (Fig. 4). This period was similar to that required after 3 h at 37°C (Fig. 3). These results suggested that the effect of the non-permissive temperature on the cells was rapid and apparently independent of the time kept at 37 ° C. Effect o f drugs and the non-permissive temperature on glucan and mannan synthesis. Addition of thiolutin to a cell culture produced a 70% inhibition of mannan synthesis after 30 min but total inhibition was n o t obtained under the experimental conditions (Fig. 5B). Glucan synthesis was also inhibited (Fig. 5A). 8-Hydroxyquinoline interfered strongly with mannan formation; after 120 min the residual mannan synthesis was a b o u t 7%. Glucan formation was also arrested; its inhibition accounted for a 67% and a 71% after 60 and 120 min, respectively (Fig. 5B and A). 25
B
A
8
% 3(
U v
10
.9
5
i
1
2
3 Time
4 (h)
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Fig. 5. G l u c a n ( A ) a n d m a n n a n (B) s y n t h e s i s b y S. cerevisiae t s - 1 3 6 . E f f e c t of a n t i m e t a b o l i t e s a n d t e m p e r a t u r e . T o cell c u l t u r e s ( 2 0 rag, d r y w e i g h t ) in 50 m l , 5 #Ci of [ U A 4 C ] - g l u c o s e spec. act. 2 C i / m o l ) a n d d i f f e r e n t a n t i m e t a b o l i t e s w e r e a d d e d a n d i n c u b a t e d a t 2 3 ° C . A n o t h e r cell c u l t u r e s u p p l e m e n t e d w i t h r a d i o a c t i v e g l u c o s e o n l y was i n c u b a t e d a t 3 7 ° C . S a m p l e s w e r e t a k e n a n d g l u c a n a n d m a n n a n synt h e s i z e d d e t e r m i n e d as i n d i c a t e d in Materials a n d M e t h o d s . ~ •, t h i o l u t i n , 20 ~tg/ml; ~ D 8 - h y d r o x y q u i n o l i n e , 4 0 0 Mg/ml; X X, c y c l o h e x i m i d e , 1 0 0 # g / m l ; Q ~ , c o n t r o l at 2 3 ° C ; e-• c o n t r o l at 3 7 ° C .
269
The kinetics of glucan formation at 37°C (at this temperature there is no transport of newly synthesized R N A into the cytoplasm [18]) suggested that its synthesis may initially be independent of new messenger RNA. It would only be dependent on the turnover of the enzymes involved. Mannan formation would be independent of new messenger R N A at least initially; afterwards it would be dependent on new m R N A and probably on nucleic acids which code for proteins acting as mannose acceptors. To check this point and to gain information in the way in which thiolutin and 8-hydroxyquinoline interfere with glucan synthetase activities, it was of interest to study glucan and mannan synthesis under conditions in which protein formation had been blocked by cycloheximide. Effect of cycloheximide on glucan and mannan synthesis. Glucan synthesis was not affected in the presence of the antibiotic for at least 5 h according to Fig. 5A. Mannan synthesis was severely inhibited 87% after 120 min {Fig. 5B) These results indicate that mannan formation is blocked by cycloheximide more rapidly than glucan synthesis because of its requirement of protein acceptors. Cytological location of polymers synthesized by S. cerevisiae ts -136 after incubation at the non-permissive temperature. The experiment described in Fig. 5 showed that at 37°C the mutant went on synthesizing both glucan and mannan polymers. These results did not establish the fate of the wall polymers formed under those conditions and if they were actually incorporated into the wall. To learn about this point, cells incubated at 37°C and at 23°C for t w o hours were pulse-labelled with either [U-14C]glucose or [U-14C]threonine and after 30 min, fractionated as indicated in Methods. Threonine incorporated by cells incubated at 37°C was mainly found in Fraction P: {mostly cells walls). When the cells were labelled at 23 ° C, fraction $40 (soluble proteins and ribosomes) was the most radioactive one {Table I). Distribution of radioactivity incorporated from [U-14C]glucose was also significant. The radioactivity found in the walls from cells labelled at 37°C was 12-fold the radioactivity found in fraction $40. In cells labelled at 23°C
TABLE I CYTOLOGICAL LOCATION OF[U -14C]THREONINE EFFECT OF TEMPERATURE
INCORPORATED
BY S. C E R E V I S I A E ts - 1 3 6 :
T w o cell c u l t u r e s ( 3 5 m g , d r y w e i g h t ) in 4 0 m | i n c u b a t e d w i t h 0 . 5 ~Ci r a d i o a c t i v e t h r e o n i n e ( s p e c . a c t . 2 C i / r n o l ) d e s c r i b e d in M e t h o d s . Each p e l l e t (P2, P I 0 a n d P 4 0 ) and w i t h an e q u a l v o l u m e o f 1 0 % t r i c h l o r o a c e t i c acid and the
at 3 7 ° C and at 2 3 ° C for 2 h w e r e p u l s e d - l a b e l l e d f o r 3 0 r a i n . T h e ceils t h e n w e r e f r a e t i o n a t e d as an a l i q u o t o f t h e s u p e r n a t a n t ( $ 4 0 ) w e r e t r e a t e d radioactivity determined.
Ternpera-
[ U - 1 4 C ] T h z e o n i n e i n c o r p o r a t e d i n t o t h e t r i c h l o r o a c e t i c acid p r e c i p i t a t e s
ture (oC)
P2
23 37
P 10
P40
$40
(cp i0 rain)
%
(cp 10 rain)
%
(cp I0 rain)
%
(cp I0 rain)
%
24 920 1 580
24 55
20 110 390
19.1 13.6
13 940 480
13.1 16.6
45 090 400
43 14
270 T A B L E II C Y T O L O G I C A L L O C A T I O N OF [ U - 1 4 C ] G L U C O S E I N C O R P O R A T E D E F F E C T OF T E M P E R A T U R E
BY S. C E R E V I S I A E
ts-136:
T w o cell c u l t u r e s ( 3 5 m g d r y w e i g h t ) in 4 0 ml i n c u b a t e d at 3 7 ° C a n d at 2 3 ° C for 2 h w e r e pulse-labelled w i t h 5 pCi o f r a d i o a c t i v e glucose (spec. act. 2 C i / m o l ) for 30 m i n . T h e cells w e r e f r a c t i o n a t e d as d e s c r i b e d in M e t h o d s and the r a d i o a c t i v i t y d e t e r m i n e d in each f r a c t i o n . Ternperature (° C)
23 37
[ U - 1 4 C ] G l u c o s e i n c o r p o r a t e d i n t o t h e t r i c h l o r o a c e t i c acid p r e c i p i t a t e s P2
PIO
P40
$40
(cp 10 m i n )
%
(cp 10 m i n )
%
(cp 10 m i n )
%
(ep 10 m i n )
%
5381 1386
63.8 70.6
912 325
10.8 16.5
607 142
7.2 7.2
1527 110
18.1 5.6
the corresponding radioactivity incorporated in fraction P2 (walls) was only 5-fold (Table II). Therefore it seemed that at 37°C S. cerevisiae ts -136 synthesized and incorporated preferentially wall specific substances. This also suggests that the mRNA that codify wall glycoproteins are more stable than those of cytoplasmic proteins.
Discussion The two main polymers of S. cerevisiae walls (13-glucans and a series of glycoproteins whose carbohydrate moiety is known as "yeast m a n n a n " ) are synthesized continuously t h r o u g h o u t the cell cycle [1]. Halvorson et al. [9] proposed that the genes of each chromosome are transcribed in an orderly and linear way. If this is true, the continuous synthesis of wall polymers could be due to: (a) Presence of several duplicated copies of the genes involved distributed t h r o u g h o u t the cell genome. (b) The genes being present in small number but transcribed constantly. In this case the "linear reading" of at least some of the genes does not take place. (c) The RNA messengers and/or the glucan and mannan synthetases show slow turnover. To decide between these hypotheses we wanted to determine the turnover of the RNA and synthetases involved in wall formation. In the study of mRNA decay, as in the case of other macromolecules, it is normal practice to chase radioactive precursors with non-radioactive ones. In S. cerevisiae the mRNA decay can n o t proceed in this way in spite of the addition of cold precursors because to the large nucleotide pool, the incorporation of label into the polymer continues. This result indicates t h a t the pool of RNA precursors is quite large as has also been described for other eukaryotic cells. Actinomycin D and cordicepin are routinely used to inhibit RNA synthesis in eukaryotic cells but these compounds are not taken up by S. cerevisiae. Moreover Actinomycin D is now known to affect both protein synthesis and RNA stability [ 10--11 ].
271 Lomofungin has also been described as an inhibitor of R N A synthesis in yeast b u t at the concentration described [2] it produced no effect in S. cerevisiae ts-13% Higher concentrations of lomofungin produced inhibition b u t it was far from complete. Moreover, the high concentration used could interfere with other cell processes. This inhibitor chelates divalent cations [12] and both Mn 2÷ and Mg 2÷ are required by the glucan [13] and mannan [14--15] synthetases. It is quite probable that synthesis of the wall polymers in the presence of the drug might be inhibited at the level of the synthetases. 8-Hydroxyquinoline strongly inhibited mannan synthesis and as it also acts by cation chelation [4] this inhibition might in part be due, as in the case of lomofungin, to deprivation of Mn 2+ of Mg 2+. This hypothesis was confirmed when glucan synthesis was studied in the presence of the drug. Glucan and mannan formation were inhibited similary after 60 min and from then on glucan synthesis continued at the same partially inhibited rate, as did mannan, though at a consistently lower rate. This result suggests that the drug inhibited the glucan synthetases. This was finally established when found that in the presence of cycloheximide, in the absence of protein synthesis, glucan formation was n o t inhibited and went on for long periods of time (Fig. 5A). Thiolutin also inhibits RNA synthesis. When glucan and mannan formation was studied the latter was most severely affected. This inhibition of both glucan and mannan formation suggested that the effect of thiolutin is n o t only the result of an interference at the level of RNA polymerases [3] but also at the level of other cell processes. That is to say, the effect of this antibiotic is similar to that of 8-hydroxyquinoline. We have also employed a novel m e t h o d to study glucan and mannan metabolism. In this method we take advantage of the mutant, S. cerevisiae ts -136 whose genetic defect is a blockage of RNA transport from the nucleus to the cytoplasm at the non-permissive temperature [ 7,16--17]. Temperature sensitive mutants normally present a point mutation and the rest of the cell genome is expressed normally. Moreover, the non-permissive temperature is low enough (37 ° C) to allow the development of an ordinary metabolism. The parent strain, S. cerevisiae A 364 A grows normally at 37°C and thus it seems that the RNA messengers present in the cytoplasm of S. cerevisiae ts -136 at the m o m e n t of transfer to 37°C are functional and will be translated into proteins. What happens from then. on will depend on the progressive degradation of the RNA messengers. At the non-permissive temperature the rate of mannan formation was the same as that at 23°C for the first hour. On the other hand, the rate of glucan synthesis at 37°C was initially greater than at 23 ° C. This was probably due to the increase in the catalytic activity of metabolic reactions when temperature is raised. Just a similar effect was found in the parent strain S. cerevisiae A 364A (results to be published elsewhere). The amounts of glucan synthesized at 23°C and at 37°C were practically identical after 3 h (Fig. 5A). This suggests that inhibition of RNA messenger synthesis apparently did not interfere with biosynthetic cell activities. Cycloheximide produced an immediate and complete blockage of protein synthesis and interfere rapidly and strongly with mannan synthesis. Glucan formation was unaffected. Mannan synthesis requires, as has already been
272 shown, a continuous synthesis of protein [5]. In the case of glucan it seems evident t h a t its continued synthesis depends on the slow decay of the glucan synthetases since in the presence of cycloheximide, the rate of synthesis remains constant for at least 5 h. Subcellular location of the threonine and glucose in S. cerevisiae ts -'3~' after exposure to 37°C showed that they were preferentially incorporated in wall materials. It was not a mere redistribution of preexisting substances but an orderly and preferential synthesis of wall substances. It is also suggested that the RNA messengers for wall glycoproteins show a half life which is longer than the average RNA messenger for soluble proteins. The actual decay of the mRNA involved in the synthesis of wall macromolecules has been obtained in a computer Univac 1106 with a program specifically devised for this purpose (results to be published elsewhere). These results are consistent with the existence of relatively long-lived mRNAs which codify invertase and other extracellular enzymic glycoproteins [19,20]. The findings reported in this paper indicate that independently of any control at the transcription level, the existence of RNA messengers for mannoproteins and synthetases of low decay results in the continuous synthesis of both glucan and mannan t h r o u g h o u t the entire cell cycle. Acknowledgement We thank Prof. H.J. Phaff of California University for helpful discussions and critical reading of the manuscript.
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