Molec. gen. Genet. 158, 81-91 (1977)
MGG © by Springer-Verlag 1977
Changes in Regulation of Ribosome Synthesis During Different Stages of the Life Cycle of Saccharomyces cerevisiae Nancy J. Pearson and James E. Haber Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02154, USA
Summary. Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna3, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34 ° C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34°C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34 ° C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34 ° C.
Introduction Sporulation in Saccharomyces cerevisiae is induced in a/c~ diploids by nitrogen starvation in the presence of an oxidative carbon source such as acetate. Under these conditions sporulation takes approximately twenty-four hours culminating in the production of four haploid ascospores. Despite nitrogen starvation conditions, macromolecular synthesis continues, facilFor oJ]prints contact." N.J. Pearson
itated by an increase in both RNA and protein turnover not characteristic of vegetative growth. During sporulation the total number of ribosomes does not change significantly, but the combination of turnover and new ribosome biosynthesis is such that approximately half of the ribosomes at the end of sporulation were synthesized during sporulation (Wejksnora and Haber, 1974). It is not known whether the synthesis of ribosomes is regulated by the same set of controls during sporulation as in growth; however, there are clear differences in ribosome synthesis between these stages of the yeast life cycle which may reflect changes in regulation. Unlike growth, the synthesis of rRNA is not continuous during sporulation but appears to be restricted to approximately the first twelve hours (Hopper, Magee, Friedman and Hall, 1974). Furthermore, it is not clear whether the turnover of ribosomes during sporulation is necessary simply to provide sufficient precursors for RNA synthesis or whether the newly synthesized ribosomes are in some way different from those made during vegetative growth. In an attempt to answer these questions we used five recessive temperature sensitive mutations that specifically block ribosome biosynthesis during vegetative growth. These mutant strains fall into 5 different complementation groups; all have a similar phenotype. In all of these mutants processing of ribosomal RNA (rRNA) is defective at the restrictive temperature, although some 35S ribosomal RNA precursor continues to be synthesized (Hartwell, McLaughlin and Warner, 1970). Several of these mutants have been examined in detail under vegetative conditions (Warner and Udem, 1971). Gorenstein and Warner (1976) found that there is a nearly complete inhibition of ribosomal protein synthesis at 34 ° C, whereas total protein synthesis is not severely affected. From the kinetics of inhibition of ribosomal protein synthesis,
82
N.J. Pearson and J.E. Haber : Changes in Regulation of Ribosome Synthesis in Yeast
G o r e n s t e i n a n d W a r n e r suggested that these r n a m u t a t i o n s m a y be alterations in regulatory genes which c o o r d i n a t e the t r a n s c r i p t i o n of messenger R N A from r i b o s o m a l p r o t e i n genes. Recently, H e r e f o r d a n d Rosbash (1977) have f o u n d that m o r e t h a n 100 different m o d e r a t e l y a b u n d a n t messenger R N A s are n o t transcribed when a n r n a 2 h a p l o i d is g r o w n at the restrictive temperature. The n u m b e r of messages a n d their frequency within the cell suggests that messages for r i b o s o m a l proteins a n d possibly other gene products are n o t t r a n s c r i b e d in the r n a 2 m u t a n t at 34 ° C. F u r t h e r m o r e , W a r n e r a n d G o r e n s t e i n (1977) have dem o n s t r a t e d in vitro, utilizing a wheat germ extract system, that m R N A for r i b o s o m a l proteins is n o t present in r n a 2 c o n t a i n i n g h a p l o i d after i n c u b a t i o n at the restrictive temperature. As only a / e diploid strains will sporulate, we used diploid strains each h o m o z y g o u s for one of the temperature sensitive r n a m u t a n t s . I n a p r e l i m i n a r y acc o u n t of this work (Haber, 1975), we f o u n d that sporu l a t i o n in these strains was t e m p e r a t u r e sensitive, as might be expected if new r i b o s o m e p r o d u c t i o n were essential. However, when m a c r o m o l e c u l a r synthesis was m o n i t o r e d , we f o u n d that there was n o a p p a r e n t differences between the synthesis of r R N A at the permissive a n d the restrictive temperatures. T h e results presented here d e m o n s t r a t e that u n d e r the nitrogen starvation c o n d i t i o n s used to induce sporulation, the r n a m u t a t i o n s fail to affect r i b o s o m e synthesis in the well d o c u m e n t e d fashion observed in vegetatively growing cells. The fact that these diploids are t e m p e r a t u r e sensitive d u r i n g s p o r u l a t i o n even t h o u g h they appear to c o n t i n u e to synthesize ribosomes suggests that these m u t a t i o n s are involved in the c o n t r o l of activity of other genes in a d d i t i o n to those c o o r d i n a t i n g ribosome biosynthesis a n d that the control of some or all of these functions is altered by the physiological or d e v e l o p m e n t a l state of the cells.
Materials and Methods Strains
Temperature sensitive haploid strains ts368 (rna2), ts125 (rna3), ts339 (rna4), ts219 (rna8) and ts166 (rna6) supplied by C. McLaughlin were used to construct diploid strains used in these investigations. In addition to the temperature sensitive allele, these strains each have the genotype a adel ade2 ural his7 lys2 tyrl gall. Each was mated with a nontemperature sensitive strain. Diploids were selected, sporulated and dissected by the method of Mortimer and Hawthorne (1969). The temperature sensitive alleles in the rna2, rna3, rna4 and rna8 heterozygous diploids segregated 2:2. Two temperature sensitive segregants with complementing nutritional markers were selected to make homozygous diploids. The genotypes of these strains, as well as other strains constructed, are shown in Table 1.
Table 1. Diploid strains rna2 tyrl rna2
+
ural +
rna6 his7 cysl rna6
+
rna3 ural rna3
+ +
+
adel
+
+
+
adel ade2 tyrl +
+
+
ural tyrl
rna8 adel ade2 ural tyrl rna8
+
+
rna2 adel thr4 rna2 c~
+
+
+
+
+
rna6 adel his7
+
+
+
+
his7 lys2 leul +
+
+
his7 lys2 leul +
+
+
lys2
+
+
+
tyrl ural his7
rna2 adel ade2 ural his7 lys2 tyrl + + + + + + +
a c~
+
+
+
+
rna4 adel ade2 rna4
+
adel ade2 lys2 his7 thr4
gall
+
+
thr4
cys 1 +
The temperature sensitive phenotype in strain ts166 (rna6) heterozygote did not segregate as a single gene. Segregations of one wild type to three temperature sensitive (ts) appeared in 8 of 25 tetrads. Further genetic analysis revealed at least two complementing ts markers. One of these segregated 2:2 in 120 out of 122 tetrads and retained the same general phenotype as rna6 with the exception that this new segregant was not cold sensitive. An rna6 homozygote was then constructed from these ts segregants. An rna2/rna2 c~/e diploid was made by force mating two c~ haploid strains with several complementing nutritional markers. The fact that these strains were c~/~diploids was confirmed by mating with a known a/a diploid. The resulting polyploid strain, when sporulated, gave close to 100% spore viability and segregating patterns of mating type characteristic of a tetraploid.
Media
For sporulation, cells were grown to stationary phase in YEPD, or mid log phase in acetate growth media, washed in 1% potassium acetate (KAC) pH 7, then resuspended again in 1% KAC at a 1:2 dilution for sporulation. Percent sporulation was determined by microscopic observation. Cells were grown vegetatively in YEPD medium (1% yeast extract, 2% peptone, 2% dextrose) or in minimal medium (0.67% yeast nitrogen base without amino acids, 2% glucose) or acetate growth medium (Roth and Halvorson, 1969). Cell growth was monitored by optical density in a Klett-Summerson colorimeter (red filter).
Total R N A and Protein Synthesis
Total RNA synthesis was measured as 3H-adenine incorporation into cold 10% trichloroacetic acid (TCA) precipitable counts (Rodenberg, Steinberg, Piper, Nickerson, Vary, Epstein and Halvorson, 1968). Total protein synthesiswas measured by 35S_methionine incorporation into hot TCA precipitable counts (Rodenberg et al., 1968). One ml samples were precipitated onto glass fiber filters, dried and counted in 5 ml of Econofluor (New England Nuclear) in a Beckman liquid scintillation counter. All incorporation measurements were corrected for differences in uptake. To measure
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast uptake of radioactive compound, 1 ml sample of cells were washed on filters with distilled water containing either 1% methionine or adenine, dried and counted as described.
Ribosomal Protein and Ribosomal RNA Synthesis In general, ribosomal protein synthesis and ribosomal RNA synthesis were measured as the amount of 35S-methionine and 3Hadenine label in 80S ribosomes after a long term labeling period.
a) Vegetative Growth. Cells were grown to mid logarithmic growth at 25 ° C in minimal medium. The culture was then split in half. One half was shifted to 34 ° C, the other remained at 25 ° C. A half hour after the temperature shift, a~S-methionine (2/~c/ml) and 3H-adenine (3 pc/ml) were added to each culture. Cells were harvested two hours later. The harvested cells were washed twice in low salt buffer (0.05 M sodium cacodylate, pH 6.8, 0.05 M KCL, 5mM Mg acetate) (Zeijst, Kool and Bloemers, 1972) containing 1% cold methionine and 1% cold adenine and broken in a Braun homogenizer. Debris was removed by low speed centrifugation. One ml samples of the supernatant were layered on I 5 4 0 % sucrose gradients prepared in the same buffer and centrifuged 14 h at 26,000 rpm in a Beckman SW 27 rotor. Gradients were monitored at 260 nm in a Beckman Model 25 spectrophotometer and fractionated. A 0.1 ml aliquot of each fraction was mixed in 5 ml of Aquasol (New England Nuclear) and counted in a Beckman liquid scintillation counter.
b) Sporulation. Cells were grown in YEPD to stationary phase at 25 ° C. Cells were then washed and diluted 1:2 in 1% KAC at 25 ° C or 1% KAC prewarmed to 34 ° C. After 2 h, 3 #c/ml of 3H-adenine and 2 #c/ml of 35S-methionine were added to each culture for a labeling period of 4 h (Y 2 T6). Cells were then harvested and 80S ribosomes were isolated as described above. This procedure resulted in very well resolved monosome peaks on gradients. However, with this buffer system a high specific activity 35S peak appeared in 40S region near the top of the gradient. This peak was especially prevalent in samples from sporulating cells. If cells were labeled with 3sS methionine for a half hour and then incubated with a vast excess of unlabeled methionine for 4 h, this material did not become incorporated into monosomes. When 80S monosomes were layered on a second gradient to isolate ribosomal subunits as described below, the true 40S ribosomal sabunit did not have an abnormally high 35S specific activity. The same abnormal peak appeared in diploid strains both heterozygous and homozygous for rna mutations as well as in other laboratory strains. We concluded that this labeled material was unrelated to the rna mutants under investigation, and might represent an aggregation of smaller protein molecules. Subunits were isolated by collecting the 80S monosome peak fractions from a 5-20% sucrose gradient centrifuged in a SW 27.1 rotor for 5 h at 26,000 rpm. This fraction was resuspended in an equal volume of 2 x concentrated high salt buffer (0.05 M sodium cacodylate, pH 6.8, 0.5 M KCL, 5 mM Mg acetate) (Zeijst, Kool and Bloemers, 1972), and immediately layered on 15-40% sucrose gradients in high salt buffer. These gradients were centrifuged for 14 h at 26,000 rpm in a SW 27.1 rotor at 4 ° C and then fractionated. Polysomes were isolated by the following procedure. Cycloheximide (200 #g/ml final concentration) was added to the sporulating cells 10min before harvesting. Cells were then harvested and washed once in cold McLaughlin buffer (0.01 M Tris, pH 7.4, 0.03 M MgC12, 0.1 M NaCI) containing 200 #g/m1 cycloheximide and 200 #g/ml heparin. Cells were broken in a Braun homogenizer and debris was removed by low speed centrifugation. The supernataut was layered onto a 5 20% sucrose gradient prepared in the same buffer and centrifuged at 4°C for 2 h at 26,000 rpms in a Beckman SW27.1 rotor.
83
Results Synthesis o f Total R N A and Total Protein in Diploid Mutants T h e h o m o z y g o u s d i p l o i d s o f rna2, rna3, rna4, rna6 a n d rna8 all h a v e t e m p e r a t u r e s e n s i t i v e v e g e t a t i v e growth phenotypes similar to haploid strains carrying t h e s e m u t a t i o n s . W h e n c u l t u r e s g r o w i n g a t 25 ° C a r e s h i f t e d t o 34 ° C, g r o w t h c o n t i n u e s e x p o n e n t i a l l y f o r about one generation and then stops. The diploids were screened for their ability to synthesize RNA and protein at permissive and restrictive temperatures d u r i n g v e g e t a t i v e g r o w t h a n d s p o r u l a t i o n ( T a b l e 2). D i p l o i d s h e t e r o z y g o u s f o r rna2 a n d rna6 w h i c h a r e not temperature sensitive for sporulation or growth w e r e u s e d as c o n t r o l s . W h e n v e g e t a t i v e l y g r o w i n g cells h o m o z y g o u s f o r o n e o f t h e rna m u t a t i o n s w e r e raised to the restrictive temperature there was a m a r k e d d i f f e r e n c e i n t h e a m o u n t o f t o t a l R N A synt h e s i s ( t h e b u l k o f w h i c h is r i b o s o m a l ) b u t little e f f e c t on total protein synthesis. These results are essentially identical to those previously obtained for haploid s t r a i n s c a r r y i n g t h e s a m e rna m u t a t i o n s ( H a r t w e l l , M c L a u g h l i n a n d W a r n e r , 1970). H o w e v e r , d u r i n g s p o r u l a t i o n , t o t a l R N A s y n t h e s i s as w e l l as t o t a l protein synthesis was approximately the same during the interval measured at both temperatures. This lack of inhibition of RNA synthesis was found whether t h e cells w e r e l a b e l e d a f t e r o n l y o n e h o u r i n s p o r u l a t i o n m e d i u m o r a f t e r six h o u r s . E v e n a t 3 7 ° C n o inhibition of RNA synthesis was seen during sporulation. Even though no major difference in synthesis o f R N A a n d p r o t e i n is d e t e c t e d a t t h e r e s t r i c t i v e t e m Table 2. Ratio of synthesis of RNA and protein (34 ° C/25 ° C) during vegetative growth and sporulation
rna2/+ rna6/+ rna2/rna2 rna3/rna3 rna4/rna4 rna6/rna6 rna8/rna8
Vegetative growth
Sporulation
% Sporulation
RNA
Protein
RNA
Protein
25C
34C
1.10 0.71 0.16 0.27 0.29 0.12 0.19
0.80 0.60 0.93 1.02 1.00 1.20 0.98
1.00 1.20 1.35 1.07 0.82 1.32 0.97
1.15 1.00 0.89 1.10 1.27 0.95 1.17
80 75 80 65 70 75 60
80 80 0 0 0 0 0
RNA synthesis in vegetative cells was measured as incorporation of 3H-adenine (1.0 gc/ml) into cold TCA precipitable counts one half hour after logarithmically growing cells in YEPD were shifted from 25 ° C to 34 ° C. The labeling period was 1 h. Protein synthesis was measured as incorporation of 35S-methionine (0.5 gc/ml) into hot TCA precipitable counts during the same period. Labeling during sporulation was carried out for 1 h in the same manner, after cells had been in sporulation medium for 4 h (T4-Ts). Ascus formation was measured 48 h after placing cells in 1% KAC
84
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast
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8. 34°C 2.0
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1600
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,.~ 0 2.0
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36
Froction Fig. 1A-D. Comparison of newly synthesized r R N A and ribosomal protein appearing in 80S ribosomes during vegetative growth of rna2/rna2 and rna2/+ diploids at 2 5 ° C and 34 ° C. One-half of a log phase culture growing in minimal media at 2 5 ° C was shifted to 34 ° C, the other half left at 25 ° C. After 1 h incubation 3 gc/ml of 3H-adenine and 2 gc/ml of 35S-methionine were added to each culture, for a labeling period of 2 h. Cells were then harvested and ribosomes were isolated, as described in Materials and Methods, by sucrose gradient centrifugation. Gradients were monitored in a Beckman spectrophotometer at OD/60, and fractionated. These results are also summarized in Table 3 along with other strains tested. A rna2/rna2 at 25 ° C; B rna2/rna2 at 34 ° C; C rna2/+ at 25 ° C; and D rna2/+ at 34 ° C. Symbols used are: ( e ) ODz60; (o) 3H-adenine; (zx) 3SS-methionine
perature under sporulation conditions, these cells are still temperature sensitive for the ability to complete sporulation.
Ribosome Synthesis During Sporulation and Vegetative Growth From the results presented above, it seemed that rRNA synthesis was not severely inhibited at 34°C during sporulation even though the same strains are inhibited for RNA synthesis under vegetative conditions. However, these results do not indicate whether all ribosome components are being made and assembled. We, therefore, examined in greater detail ribosome synthesis during sporulation for both rna2/rna2 and rna6/rna6 diploids. The incorporation of radioactive precursors into both rRNA and ribosomal proteins was measured after purifying ribosomes by sucrose gradient sedimentation (see Materials and Methods). Figure 1 describes the results of incorporation of radioactively labeled adenine and methionine into ri-
bosomes of rna2/rna2 and rna2/+ diploids at the restrictive and permissive temperatures during vegetative growth. The heterozygote is used as a nontemperature sensitive control. The results for the diploid homozygous for rna2 are displayed in the top half of the figure (Fig. 1A-B). At the restrictive temperature, a large decrease in specific activity of labeled rRNA and ribosomal protein is seen as compared to that at the permissive temperature. No such difference is seen in the specific activity of rna2/+ diploid when labeled at 25°C and 34°C (Fig. 1C D). Therefore, in vegetatively growing cells, synthesis of ribosomes is severely inhibited at the restrictive temperature in rna2 homozygous diploids as would be expected. The same is true of rna6 homozygous diploids. During sporulation, however, there is no inhibition of ribosome synthesis at 34° C. Figure 2 describes the results of incorporation of radioactively labeled adenine and methionine into ribosomes of sporulating rna2/rna2 and rna2/+ diploids. The 4 h period after sporulating for 2 h ( T z - T 6 ) w a s chosen as the long term labeling period because that is when most RNA and protein synthesis occurs (Hopper, Magee, Fried-
85
N.J. Pearson and J.E. Haber : Changes in Regulation of Ribosome Synthesis in Yeast A. 25"C
8, 34"C
I000
2.0
I0,000
800
8000
600
6000 1,0
400
4000
200
2000
v v
g
o
C. 25"C
E
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6
E
D. 34°C
IO,O00
2.0
800
8000
600
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400
4000
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2000
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36
o Fraction
18
man and Hall, 1974). However, similar results were obtained with labeling from TI-T3 and T6 T12. The top half of Figure 2A-B displays results of rna2 homozygote. Unlike vegetative growth, no difference in the specific activity of labeled ribosomal protein and ribosomal RNA is seen. The synthesis of rRNA and ribosomal protein is the same in the rna2 heterozygore also (Fig. 1C D). Therefore, synthesis of ribosomes apparently continues during sporulation at both restrictive and permissive temperatures. Differences in the incorporation of radioactive isotopes between rna2/rna2 and rna2/+ diploid strains are probably due to different pool sizes of adenine and methionine in different strains. In experiments with rna6 strains differences also occurred but were not correlated with the homozygosity or heterozygosity of the temperature sensitive mutation. Table 3 summarizes the results of experiments displayed graphically in Figures 1 and 2 as well as similar experiments for other strains tested. Even though rRNA accumulation was not inhibited during sporulation, it was possible that ribosomal proteins might not be synthesized. The new RNA could be incorporated into ribosomes by association with a pre-existing pool of ribosomal proteins or by turnover of vegetative ribosomes. However, we found that ribosomal protein synthesis is not inhibited at
E u
Fig. 2 A - D . Comparison of newly synthesized r R N A and ribosomal protein appearing in 80S ribosomes during sporulation of rna2/rna2 and rna2/+ diploids at 25°C and 34 ° C. Cells were grown in Y E P D to stationary phase at 25 ° C. Cells were then washed and diluted 1:2 in 1% K A C at 25 ° C or 1% K A C prewarmed to 34 °C. After 2 h, 3 ~tc/ml of 3H-adenine and 2 gc/ml of ~SS-methionine were added to each culture for a labeling period of 4 h (T2 T6). Cells were harvested and ribosomes were isolated as described in Materials and Methods, These results are also found in Table 3. A rna2/rna2 at 25°C; B rna2/rna2 at 34°C; C rna2/+ at 25°C; and D rna2/+ at 34°C. Symbols used are: ( e ) OD260; (0) 3H-adenine; (~) 3SS-methionine
36
3. Ratios (34 ° C/25 ° C) of specific activity of newly synthesized ribosomal protein and R N A in 80S ribosomes during spornlation and vegetative growth Table
rna6/rna6 rna2/rna2 rna2/+ rna6/+ a rna2 c~/c~rna2/rna2
Vegetative Growth
Sporulation (T2-T6)
Ribosomal protein
rRNA
Ribosomal protein
rRNA
0.18 0.17 0.80 0.68
0.13 0.12 1.10 0.71
0.80 0.96 0.98 0.93 0.91 0.92
0.64 0.80 0.85 0.82 1.11 0.74
Labeled m o n o s o m e s were isolated and fractionated as described in Materials and Methods. The results for rna2/rna2 and rna2/+ are displayed graphically in Figures 1 and 2. Specific activity of newly synthesized ribosomal protein and r R N A was determined by cutting and weighing graphed m o n o s o m e peaks for 3SS-methionine, 3H-adenine, and OD26o. In a l l experiments corrections for differences in uptake were made
the restrictive temperature. Labeling of proteins in separated ribosomal subunits appears to be the same under both permissive and restrictive conditions during sporulation. Sporulating cells were labeled from T2 T6 at both temperatures and ribosomal subunits were isolated as described in Materials and Methods.
86
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast 600
70,000
A. 2 5 °C 25C
500
60,000
400
E ¢3. o
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200 15,000
30,000
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ff
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32
Fig. 3A and B. Comparison of the distribution of radioactivity in ribosomal subunits of rna2/rna2 diploids labeled during sporulation at 25 ° C (A) and 34 ° C (B). Cells were labeled with 2 gc/ml of ~SS-methionine and subunits were isolated by sucrose gradient centrifugation as described in Materials and Methods
Figure 3 shows the results for the rna2/rna2 diploid. The specific activity of 35S methionine incorporated into subunits is the same at both temperatures. We considered the possibility that ribosomes synthesized at the restrictive temperature were in fact not functional. This idea seems unlikely in view of the observation that the ribosomes made during sporulation at both 25 ° C and 34 ° C are found in polyribosomes (Fig. 4). Sporulating rna2/rna2 cells were labeled with 32p-phosphoric acid at either 25°C or 34 ° C for 10 h before the cells were broken and polyribosomes separated by sucrose gradient centrifugation. Ribosomes made at the restrictive temperature were as likely as those synthesized at the permissive temperature to be in polyribosomes. Although this observation cannot assess the fidelity of proteins synthesized at 34 ° C, it does show that ribosomes made at 34 ° C are able to carry out active protein synthesis. A temperature sensitive c~/c~rna2/rna2 diploid and an rna2 haploid (which do not sporulate) were tested to see if they also made ribosomal components at the restrictive temperature under sporulating condi-
30,000
,0,000
20,000
5000
I0,000
lO
20
30
40
Fraction
50
60
70
Fig. 4. Comparison of polyribosomes from diploids homozygous for rna2 during sporulation at the permissive and restrictive temperatures. Vegetatively growing cells at 25°C were labeled overnight with 14C-adenine (2 gc/ml) and then transferred to sporulation media at 25 ° C or prewarmed to 34 ° C. The sporulating rna2/rna2 diploids were labeled with 32p-phosphoric acid (3 gc/ml) at each temperature for 10 h. Cells were then broken and polyribosomes were separated by sucrose gradient centrifugation
tions (Table 3). As with cells able to complete sporulation, these strains also continued to synthesize ribosomes under sporulating conditions although they were temperature sensitive under growing conditions. Thus, the absence of temperature sensitive ribosome biosynthesis is not a sporulation-specific phenomenon but a response to the nitrogen starvation conditions necessary for sporulation. This is not surprising, for it is known that R N A metabolism is similar in a/c~ and c~/c~diploids when placed under sporulating conditions (Hopper, Magee, Friedman and Hall, 1974).
Reversion Studies Diploids homozygous for any of the mutations studied do not sporulate at 34 ° C while heterozygotes
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast
sporulate well at both temperatures. To verify that the temperature sensitive sporulation phenotype was caused by the same mutation as the temperature sensitivity for growth, spontaneous revertants were selected from rna2 and rna6 homozygous diploids. These revertants were then tested for sporulation. These results are summarized in Table 4. Of 29 rna2 revertants, 3 sporulated to nearly the same extent at both temperatures, 22 partially regained the ability to sporulate at the restrictive temperature, and 2 still did not sporulate at the restrictive temperature. Of 7 rna6 revertants for growth, 6 diploids regained their ability to sporulate at either temperature. We examined genetically two revertants of the rna2 diploid and two revertants of the rna6 diploid, all four of which could sporulate well at both 25 ° C and 34 ° C. At least 10 meiotic tetrads for each revertant were analyzed. In each case the ts growth phenotype segregated 2:2, indicating that a single site mutation caused the reversion. We conclude that temperature sensitivity for growth and sporulation were caused by the same mutation.
Effect of rna Mutants on Sporulation Specific Events Diploids homozygous for rna mutations are blocked late in sporulation. Meiotic D N A synthesis occurs in both rna6 and rna2 homozygous diploids at permissive and restrictive temperatures. The data for the rna6 homozygous diploid is shown in Figure 5. Table 4. Percent sporulation of rna6/rna6 and rna2/rna2 revertants for growth at 34 ° C Sporulation 25°C
34°C
80% 70 % 60 % 70%
0% 65 % 0% 70%
6
60 80%
60-80%
2 6 5 2 3 6 3
60 80% 60-80% 60 80% 60-80% 40-60 % 40-60% 40-60%
60-80% 20 30% 10 20% 0% 40 60 % 20-30% 10 20%
87
100
z
75
o/
50
$
a.
25
0
I
0
3
6
9
12 hr.
24
Fig. 5. D N A synthesis during sporulation of strain homozygous for rna6. Stationary-phase cells were transferred to sporulation medium at 25 ° C. At 6 h (arrow), the culture was divided in half and the one flask was transferred to 34 ° C. The sporulation of cells kept at 25°C (o) was 53% after 48 h; no sporulation was found in the culture transferred to 34 ° C (©). (Haber, 1975)
To determine at which stage of nuclear division these mutant strains are arrested during meiosis, the nuclear stain mithramycin was employed. Diploid strains homozygous for rna2 and rna6 were incubated in sporulation media for 18 h at both 25 ° C and 34 ° C. These cells were then stained with mithramycin as described by Slater (1976), and examined in a Zeiss fluorescence microscope. After 18 h of sporulation at 25 ° C both strains contained cells which were bi-, tri- and tetranueleate. Many tetranucleate cells showed evidence of spore wall formation when viewed under visible light source. In cultures held at the restrictive temperature, bi-, tri- and tetranucleate cells were als0 seen. However, even cells which were tetranucleate did not show evidence of spore wall formation. It seems that rna diploids can complete the second meiotic nuclear division at the restrictive temperature but do not form mature ascospores.
I. Control strains a
rna2/rna2 rna2/+ rna6/rna6 rna6/+ 2. rna6/rna6 # of revertants
3. rna2/rna2 # of revertants
See Table 1 for complete genotype
Time of Temperature Sensitivity During Sporulation Temperature shift experiments demonstrated that the temperature sensitive period occurs during the first 12 15h of sporulation (Fig. 6). If shifted to the restrictive temperature after 15 h, the cells sporulate normally. Cells shifted to the restrictive temperature for more than six hours during the sensitive period gradually lost their ability to sporulate as the time at 34°C was increased. Cells left at 34°C for 6 h or less during the sensitive period can recover their ability to sporulate if left at the permissive temperature for the remainder of time it takes to sporulate. Figure 7 shows that this 6 h period at 34°C does not have to be the first 6 h during sporulation but that cells shifted from 25°C to 34°C during T6 T12
88
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast
I00
340C
BO
25°C
-
60
60 34oC
4O
25oc
"
60
I
,o
.~ 20 o 34oC
m
25oc
go IO0
g o
80
340C
x 5 0 ~o c
250C 40
g_
340C 2O 250C 0
3
F i g . 6. T e m p e r a t u r e
6
9
12 Hours
dependence of
15
2
;4 340C
rna2/rna2 ( t o p )
and
rna6/rna6
(bottom) diploids during sporulation. Cells were grown to stationary phase in YEPD, washed and diluted into 1% KAC. At intervals cultures were shifted from 34° C to 25° C. (e) or from 25° C to 34°C (©). Percent sporulation was determined at 48 h. Similar results were obtained when cells were grown in acetate growth medium
250C
15
34=C
0
25oc
-----i
0
I
4
I
,
8
70
I
I
12
48
Hours
will also regain their a b i l i t y to s p o r u l a t e if shifted b a c k to the permissive t e m p e r a t u r e . It c a n be seen, however, t h a t if these cultures are n o t shifted b a c k to 25 ° C after 12 h t h a t r e c o v e r y does n o t occur. Thus, these d a t a s h o w t h a t the t e m p e r a t u r e sensitive interval is s p r e a d over the first 12-15 h o f s p o r u l a t i o n a n d t h a t the ts effect is c u m m u l a t i v e . T h e i n a b i l i t y to recover if cells are held for m o r e t h a n 6 houl:s d u r i n g the sensitive p e r i o d m a y be related to the fact t h a t the m a i n synthetic activity o f s p o r u l a t i n g cells also occurs d u r i n g the first 12 h (at b o t h t e m p e r a t u r e s ) . A f t e r 12 h, the synthetic c a p a c i t y o f cells m a y be insufficient to b r i n g a b o u t c o m p l e t e r e c o v e r y f r o m the t e m p e r a t u r e sensitive effect.
Effect o f p H During Sporulation on rna2 A t the b e g i n n i n g o f s p o r u l a t i o n in 1% K A C there is a r a p i d rise in p H f r o m p H 7 to a b o u t p H 9 (Mills, 1972), whereas g r o w i n g cells are in m e d i a with a p H b e l o w 6. To d e t e r m i n e w h e t h e r this c h a n g e in p H is r e s p o n s i b l e for the c h a n g e in the p h e n o t y p e o f rna m u t a n t s , we s p o r u l a t e d a n rna2 d i p l o i d in 1% K A C buffered to p H 6.5 with m o r p h o l i n o p r o p a n e sulfonic acid ( M O P S ) in which yeast cells s p o r u l a t e n o r m a l l y ( M c C u s k e r a n d H a b e r , s u b m i t t e d for p u b l i c a t i o n ) . T h e i n c o r p o r a t i o n o f 3 H - a d e n i n e into T C A - p r e c i p i t a ble c o u n t s was m e a s u r e d as d e s c r i b e d in T a b l e 2. T h e r a t i o o f a d e n i n e i n c o r p o r a t i o n o f 3 4 ° C to 2 5 ° C
Fig. 7. Temperature dependence at various times during sporulation of rna2/rna2. Cells were pregrown in YEPD, washed and diluted into 1% KAC. Cultures were then shifted from 34° C to 25° C or 25° C to 34° C for intervals of time displayed. The results for cells maintained at 25° C or 34° C throughout sporulation are shown at the bottom of the figure. The dotted lines indicate that cells were incubated for an additional 36 h after 12 h (total 48) at the indicated temperature before percent sporulation was measured was 0.99. C o n s e q u e n t l y , a c h a n g e in p H a c c o m p a n y ing s p o r u l a t i o n c a n n o t a c c o u n t for the lack o f rna phenotype.
Expression of rna Mutants in Cells Committed to Sporulation The lack o f i n h i b i t i o n o f r i b o s o m e biosynthesis in rna2 d i p l o i d s d u r i n g s p o r u l a t i o n is n o t m e r e l y f o u n d when cells are u n d e r the n i t r o g e n s t a r v a t i o n c o n d i tions used to induce cells to sporulate. B e y o n d a certain p o i n t in the sequence o f events leading to ascospore f o r m a t i o n , d i p l o i d cells b e c o m e c o m m i t t e d to sporulate, even if r e t u r n e d to vegetative g r o w t h med i u m ( S h e r m a n a n d R o m a n , 1963). C o m m i t m e n t occurs a b o u t the time o f m e i o t i c D N A synthesis, or after 10 h with the strains a n d s p o r u l a t i o n c o n d i t i o n s used in this s t u d y ( d a t a n o t shown). W h e n cells are t r a n s f e r r e d f r o m 1% p o t a s s i u m acetate to Y E P D g r o w t h m e d i u m after the time o f c o m m i t m e n t , m o s t o f the cells f o r m e d ascospores, while some (those t h a t
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast
most likely would not sporulate if left in 1% KAC acetate) begin to bud. We have used diploids homozygous for rna2 to see if temperature sensitive ribosome biosynthesis would soon appear if cells committed to sporulation were transferred back to growth medium. For this experiment cells homozygous for rna2 were sporulated for 16 h at 25 ° C, the permissive temperature. Cells were then washed and transferred to four different media: sporulation medium (1% KAC) at both 25 ° C and 34° C and growth medium (YEPD) at both 25°C and 34 ° C. After 1 h in the new media, the cells were examined microscopically and incubated with 3H-adenine for 30 min to measure RNA synthesis as described above. The percent sporulation and the amount of vegetative growth were monitored until 36 h after the beginning of sporulation. All of the cultures sporulated to about the same extent. At 16 h, when the cells were transferred, approximately 5% of the cells had sporulated. By 36 h about 70% of each culture had sporulated, and the spores in YEPD at 25°C also began to germinate and grow. Neither spores formed in YEPD at 34° C nor spores formed in sporulation medium at 25°C germinated in YEPD at the restrictive temperature. However, all of the cells that did not form spores did begin to grow within 1 h after transfer to YEPD at 34° C. These cells grew for nearly two doublings before arresting, as measured by haemocytometer counts. When RNA synthesis was analyzed under these different conditions, we found that there was no inhibition of incorporation of 3H-adenine into TCA-precipitable counts at 34°C as compared to the 25°C cultures in the same media (Table 5). There is significantly more incorporation of 3H-adenine into RNA Table 5. Effect of rna2 on R N A synthesis in cells committed to sporulation but incubated in YEPD growth medium a Cells transferred to
3H-adenine incorporation cpm/10: cells b
KAC, 25 ° C KAC, 34 ° C YEPD, 25 ° C YEPD, 34 ° C
2957 2711 7866 8214
Ratio 25°~C 34 ° C
1.09 0.96
" After 16 h in sporulation medium (KAC) at 25 ° C, a diploid strain homzygous for rna2 was transferred to the media and temperatures indicated. At this time approximately 5% of the cells had sporulated. After 1 h in the new media, the cells were labeled with 3 gc/ml 3H-adenine, and allowed to incorporate label for 30 min. Growth and sporulation was followed for 36 h by which time 70% of the cells in all four cultures had sporulated b Incorporation of radioactivity into TCA precipitable counts after 30 min incubation was measured as described in Materials and Methods
89
in the YEPD cultures. This is to be expected, in view of the less efficient uptake of 3H-adenine into cells in sporulation medium (Mills, 1972), but may also reflect a more active rate of ribosome biosynthesis. Nevertheless, it is clear that, unlike cells simply growing in YEPD, cells committed to sporulation (but in the same growth medium) are not affected by the rna2 mutation one hour after transfer to 34° C. We conclude from this experiment that cells committed to sporulation are unable to respond to the temperature sensitive defect in rna2, even when placed in a nitrogen-rich growth medium.
Discussion
Temperature sensitive rna mutations that inhibit ribosome synthesis in vegetative yeast cells do not do so under sporulation conditions. Even when cells committed to complete sporulation are returned to growth medium, these rna diploids continue to synthesize ribosomes at the restrictive temperature. If the rna mutations are indeed mutations in regulatory genes, as has been suggested (Gorenstein and Warner, 1976), then these results demonstrate that the regulation of ribosome synthesis during sporulation is different from that during vegetative growth. Sporulation in yeast occurs under nitrogen starvation conditions that do not support growth. RNA synthesis during sporulation is not continuous as it is in growing cells, but is quite low at the very beginning of sporulation and then rises to a high level until the time of DNA synthesis, when it declines (Hopper, Magee, Welch, Friedman and Hall, 1974). Recently, Rhaese, Hoch and Groscurth (1977), have found that highly phosphorylated adenine compounds appear at the beginning of sporulation in yeast. Phosphorylation of adenine or guanine appear to be important in the regulation of ribosome synthesis in bacteria (Cashel and Gallant, 1974) and may also reflect changes in the regulation of ribosome synthesis in yeast. It is possible that some components of the ribosomes synthesized during sporulation differ from those in growing cells. In Bacillus subtilis, there is growing evidence that there are a number of changes in proteins associated with ribosomes when cells begin sporulation (Rhaese, Groscurth and Scheckel, 1977; Kobayashi and Domoto, 1977). There is extensive turnover of vegetative ribosomes and synthesis of new ribosomes when yeasts are transferred to sporulation medium, as there is in other organisms undergoing gametogenesis (Martin, Chiang, and Goodenough, 1976; Dickinson and Heslop, 1977). However, there is no evidence in any of these eucaryotes that any
90
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast
ribosomal proteins in newly synthesized ribosomes differ from those made in vegetative cells. Our data do not rule out the possibility that a small class of sporulation-specific ribosomes are not made at 34 ° C in the r n a diploids so that sporulation is not completed. The data do show that the vast majority of ribosomes made during sporulation are not regulated by the r n a mutants. The role of the r n a genes in wild-type cells is not known. All 10 complementation groups appear to have a common mutant phenotype in vegetative cells- a cessation of ribosomal protein synthesis at 34 ° C leading to an inhibition of ribosome synthesis. All of the r n a gene products may interact with a common regulatory mechanism that regulates ribosomal protein synthesis, perhaps by monitoring the availability of nutrients in the cell. Somehow, the regulation associated with each of the 5 r n a mutants we studied is bypassed in sporulating cells. If the r n a mutants somehow signal that the cell is starving, when the temperature is raised to 34 ° C, such information may be " i g n o r e d " by a cell that is induced to differentiate precisely because of nitrogen starvation. The mutations are not completely bypassed, however, as cells are unable to sporulate even though ribosome synthesis is unaffected. If the r n a mutants are bypassed in the regulation of ribosome synthesis, why do the cells not sporulate? One explanation is that the r n a gene products have two roles, one directly involved in metabolism and the other regulatory. For example, such a situation occurs in bacteria. When a single species of tRNA isoacceptor is mostly uncharged within the cell, either due to starvation of a particular amino acid or mutation, there is a specific regulation of transcription of ribosomal protein and ribosomal R N A (Cashel and Gallant, 1974). However, even without its regulatory function, lack of aminoacylation eventually inhibits protein synthesis by directly interfering with translation. An analogous situation may occur in yeast. R N A genes may have two functions, one being the regulation of ribosome synthesis. This regulatory function may be overridden during sporulation, yet cells carrying an r n a mutation could be prevented from completing ascus formation by some direct effect of the mutation on cellular metabolism. Another alternative is that the r n a mutants turn off the transcription of other genes besides ribosomal proteins and that these additional sequences are still regulated during sporulation. Hereford and Rosbash (1977) showed that when a r n a 2 strain was raised to 34 ° C, more than 100 different moderately abundant messages and possibly many less frequent messages were not transcribed. More messages are
affected than can be accounted for by ribosomal proteins, so that other messages necessary for both sporulation and growth may still be regulated. Acknowledgements. We are grateful for the critical reading of this manuscript by Bob Schleif, Jim Hopper and Lynna Hereford. We would also like to thank Peter Wejksnora for many helpful suggestions. N. Pearson was supported by NIH Training Grant GM-1586 in microbiology. This research was supported by USPHS grant GM-20056.
References Cashel, M., Gallant, J. : Cellular regulation of guanosine tetraphosphate and guanosine pentaphosphate, pp. 733-745. In: Ribosomes. Nomura, M., Tissieres, A. and Lengyel, P., ed. New York: Cold Spring Harbor Laboratory 1974 Dickinson, H.G., Heslop-Harrison, J. : Ribosomes, membranes and organelles during meiosis in angiosperms. Phil. Trans. 277, 327-342 (1977) Gorenstein, C., Warner, J.R.: Coordinate regulation of the synthesis of eukaryotic ribosomal proteins. Proc. nat. Acad. Sci. (Wash.) 73, 1547-1551 (1976) Haber, J.E. : Requirement for expression of genes controlling synthesis of ribosomal ribonucleic acid during sporulation of Saccharomyces cerevisiae cells. Spores, Vol. VI, pp. 158-164, Gerhardt, P., Gostilow, R., Sadoff, H., ed. Washington, DC: ASM 1975 Hartwell, L.H., McLaughlin, C.S., Warner, J.R.: Indentification of ten genes that control ribosome formation in yeast. Molec. gen. Genet. 109, 42-55 (1970) Hereford, L.M., Rosbash, M.: Regulation of a set of abundant mRNA sequences. Cell 10, 463-467 (1977) Hopper, A.K., Magee, P.T., Welch, S.K., Friedman, M., Hall, B.D.: Macromolecular synthesis and breakdown in relation to sporulation and meiosis in yeast. J. Bact. 119, 619 628 (1974) Kobayashi, Y., Domoto, T. : Role of ribosomes in bacterial sporulation. In : Growth and Differentiation in Microorganisms. Ishikawa, T., Maruyama, H., ed. Tokyo: University of Tokyo Press 1977 Martin, N.C., Chiang, K., Goodenough, U.W. : Turnover of chloroplast and cytoplasmic ribosomes during gametogenesis in Cblamydomonas reinhardi. Develop. Biol. 51, 190-201 (1976) Mills, D. : Effect of pH on adenine and amino acid uptake during sporulation in Saccharomyees cerevisiae. J. Bact. 112, 519-526 (1972) Mortimer, R.K., Hawthorne, D.C.: Yeast genetics, pp. 386 460. In: TheYeasts, Vol. I. New York: Academic Press, Inc. 1969 Rhaese, H.J., Groscurth, R., Scheckel, R.: Correlation of Ascus Formation and Synthesis of Highly Phosphorylated Nucleotides in Saccharomyces cerevisiae. Abstract No. 64 American Society for Microbiology, 1977 Annual Meeting, 165 (1977) Rhaese, H.J., Hoch, J.A., Groscurth, R.: Studies on the control of development : isolation of Bacillus subtilis mutants blocked early in sporulation and defective in synthesis of highly phosphorylated nucleotides. Proc. nat. Acad. Sci. 74, 1125-1129 (1977) Rodenberg, S., Steinberg, W., Piper, J., Nickerson, K., Vary, J., Epstein, R., Halvorson, H.O.: Relationship between protein and ribonucleic acid synthesis during outgrowth of spores of Bacillus cereus. J. Bact. 96, 492-500 (1968)
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast Roth, R., Halvorson, H.O. : Sporulation of yeast harvested during logarithmic growth. J. Bact. 98, 831-832 (1969) Sebastian, J., Mian, F., Halvorson, H.O.: Effect of the growth rate on the level of DNA-dependent RNA polymerase in Saccharomyces cerevisiae. FEBS Lett. 34, 159-162 (1974) Sherman, F., Roman, H. : Evidence for two types of allelic recombination in yeast. Genetics 48, 255 (1963) Slater, M.L.: Rapid nuclear staining method for Saccharomyces cerevisiae. J. Bact. 126, 1339 134I (1970) Warner, J.R., Gorenstein, C.: The synthesis of eucaryotic ribosomal proteins in vitro. Cell 11,201 212 (1977) Warner, J.R., Udem, S.A. : Temperature sensitive mutations affecting ribosome synthesis in Saccharomyces cerevisiae. J. molec. Biol. 65, 243357 (1972)
91
Wejksnora, P.J., Haber, J.E.: Methionine dependent synthesis of ribosomal ribonucleic acid during sporulation and vegetative growth of Saccharomyces cerevisiae. J. Bact. 120, 1344-1355 (1974) Zeijst, B.A.M., van der, Kool, A.J., Bloemers, H.P.J.: Isolation of active ribosomal subunits from yeast. Europ. J. Biochem. 30, 15-25 (1972)
Communicated by F. Kaudewitz Received July 18, 1977
MGG © by Springer-Verlag 1977
Changes in Regulation of Ribosome Synthesis During Different Stages of the Life Cycle of Saccharomyces cerevisiae Nancy J. Pearson and James E. Haber Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02154, USA
Summary. Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna3, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34 ° C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34°C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34 ° C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34 ° C.
Introduction Sporulation in Saccharomyces cerevisiae is induced in a/c~ diploids by nitrogen starvation in the presence of an oxidative carbon source such as acetate. Under these conditions sporulation takes approximately twenty-four hours culminating in the production of four haploid ascospores. Despite nitrogen starvation conditions, macromolecular synthesis continues, facilFor oJ]prints contact." N.J. Pearson
itated by an increase in both RNA and protein turnover not characteristic of vegetative growth. During sporulation the total number of ribosomes does not change significantly, but the combination of turnover and new ribosome biosynthesis is such that approximately half of the ribosomes at the end of sporulation were synthesized during sporulation (Wejksnora and Haber, 1974). It is not known whether the synthesis of ribosomes is regulated by the same set of controls during sporulation as in growth; however, there are clear differences in ribosome synthesis between these stages of the yeast life cycle which may reflect changes in regulation. Unlike growth, the synthesis of rRNA is not continuous during sporulation but appears to be restricted to approximately the first twelve hours (Hopper, Magee, Friedman and Hall, 1974). Furthermore, it is not clear whether the turnover of ribosomes during sporulation is necessary simply to provide sufficient precursors for RNA synthesis or whether the newly synthesized ribosomes are in some way different from those made during vegetative growth. In an attempt to answer these questions we used five recessive temperature sensitive mutations that specifically block ribosome biosynthesis during vegetative growth. These mutant strains fall into 5 different complementation groups; all have a similar phenotype. In all of these mutants processing of ribosomal RNA (rRNA) is defective at the restrictive temperature, although some 35S ribosomal RNA precursor continues to be synthesized (Hartwell, McLaughlin and Warner, 1970). Several of these mutants have been examined in detail under vegetative conditions (Warner and Udem, 1971). Gorenstein and Warner (1976) found that there is a nearly complete inhibition of ribosomal protein synthesis at 34 ° C, whereas total protein synthesis is not severely affected. From the kinetics of inhibition of ribosomal protein synthesis,
82
N.J. Pearson and J.E. Haber : Changes in Regulation of Ribosome Synthesis in Yeast
G o r e n s t e i n a n d W a r n e r suggested that these r n a m u t a t i o n s m a y be alterations in regulatory genes which c o o r d i n a t e the t r a n s c r i p t i o n of messenger R N A from r i b o s o m a l p r o t e i n genes. Recently, H e r e f o r d a n d Rosbash (1977) have f o u n d that m o r e t h a n 100 different m o d e r a t e l y a b u n d a n t messenger R N A s are n o t transcribed when a n r n a 2 h a p l o i d is g r o w n at the restrictive temperature. The n u m b e r of messages a n d their frequency within the cell suggests that messages for r i b o s o m a l proteins a n d possibly other gene products are n o t t r a n s c r i b e d in the r n a 2 m u t a n t at 34 ° C. F u r t h e r m o r e , W a r n e r a n d G o r e n s t e i n (1977) have dem o n s t r a t e d in vitro, utilizing a wheat germ extract system, that m R N A for r i b o s o m a l proteins is n o t present in r n a 2 c o n t a i n i n g h a p l o i d after i n c u b a t i o n at the restrictive temperature. As only a / e diploid strains will sporulate, we used diploid strains each h o m o z y g o u s for one of the temperature sensitive r n a m u t a n t s . I n a p r e l i m i n a r y acc o u n t of this work (Haber, 1975), we f o u n d that sporu l a t i o n in these strains was t e m p e r a t u r e sensitive, as might be expected if new r i b o s o m e p r o d u c t i o n were essential. However, when m a c r o m o l e c u l a r synthesis was m o n i t o r e d , we f o u n d that there was n o a p p a r e n t differences between the synthesis of r R N A at the permissive a n d the restrictive temperatures. T h e results presented here d e m o n s t r a t e that u n d e r the nitrogen starvation c o n d i t i o n s used to induce sporulation, the r n a m u t a t i o n s fail to affect r i b o s o m e synthesis in the well d o c u m e n t e d fashion observed in vegetatively growing cells. The fact that these diploids are t e m p e r a t u r e sensitive d u r i n g s p o r u l a t i o n even t h o u g h they appear to c o n t i n u e to synthesize ribosomes suggests that these m u t a t i o n s are involved in the c o n t r o l of activity of other genes in a d d i t i o n to those c o o r d i n a t i n g ribosome biosynthesis a n d that the control of some or all of these functions is altered by the physiological or d e v e l o p m e n t a l state of the cells.
Materials and Methods Strains
Temperature sensitive haploid strains ts368 (rna2), ts125 (rna3), ts339 (rna4), ts219 (rna8) and ts166 (rna6) supplied by C. McLaughlin were used to construct diploid strains used in these investigations. In addition to the temperature sensitive allele, these strains each have the genotype a adel ade2 ural his7 lys2 tyrl gall. Each was mated with a nontemperature sensitive strain. Diploids were selected, sporulated and dissected by the method of Mortimer and Hawthorne (1969). The temperature sensitive alleles in the rna2, rna3, rna4 and rna8 heterozygous diploids segregated 2:2. Two temperature sensitive segregants with complementing nutritional markers were selected to make homozygous diploids. The genotypes of these strains, as well as other strains constructed, are shown in Table 1.
Table 1. Diploid strains rna2 tyrl rna2
+
ural +
rna6 his7 cysl rna6
+
rna3 ural rna3
+ +
+
adel
+
+
+
adel ade2 tyrl +
+
+
ural tyrl
rna8 adel ade2 ural tyrl rna8
+
+
rna2 adel thr4 rna2 c~
+
+
+
+
+
rna6 adel his7
+
+
+
+
his7 lys2 leul +
+
+
his7 lys2 leul +
+
+
lys2
+
+
+
tyrl ural his7
rna2 adel ade2 ural his7 lys2 tyrl + + + + + + +
a c~
+
+
+
+
rna4 adel ade2 rna4
+
adel ade2 lys2 his7 thr4
gall
+
+
thr4
cys 1 +
The temperature sensitive phenotype in strain ts166 (rna6) heterozygote did not segregate as a single gene. Segregations of one wild type to three temperature sensitive (ts) appeared in 8 of 25 tetrads. Further genetic analysis revealed at least two complementing ts markers. One of these segregated 2:2 in 120 out of 122 tetrads and retained the same general phenotype as rna6 with the exception that this new segregant was not cold sensitive. An rna6 homozygote was then constructed from these ts segregants. An rna2/rna2 c~/e diploid was made by force mating two c~ haploid strains with several complementing nutritional markers. The fact that these strains were c~/~diploids was confirmed by mating with a known a/a diploid. The resulting polyploid strain, when sporulated, gave close to 100% spore viability and segregating patterns of mating type characteristic of a tetraploid.
Media
For sporulation, cells were grown to stationary phase in YEPD, or mid log phase in acetate growth media, washed in 1% potassium acetate (KAC) pH 7, then resuspended again in 1% KAC at a 1:2 dilution for sporulation. Percent sporulation was determined by microscopic observation. Cells were grown vegetatively in YEPD medium (1% yeast extract, 2% peptone, 2% dextrose) or in minimal medium (0.67% yeast nitrogen base without amino acids, 2% glucose) or acetate growth medium (Roth and Halvorson, 1969). Cell growth was monitored by optical density in a Klett-Summerson colorimeter (red filter).
Total R N A and Protein Synthesis
Total RNA synthesis was measured as 3H-adenine incorporation into cold 10% trichloroacetic acid (TCA) precipitable counts (Rodenberg, Steinberg, Piper, Nickerson, Vary, Epstein and Halvorson, 1968). Total protein synthesiswas measured by 35S_methionine incorporation into hot TCA precipitable counts (Rodenberg et al., 1968). One ml samples were precipitated onto glass fiber filters, dried and counted in 5 ml of Econofluor (New England Nuclear) in a Beckman liquid scintillation counter. All incorporation measurements were corrected for differences in uptake. To measure
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast uptake of radioactive compound, 1 ml sample of cells were washed on filters with distilled water containing either 1% methionine or adenine, dried and counted as described.
Ribosomal Protein and Ribosomal RNA Synthesis In general, ribosomal protein synthesis and ribosomal RNA synthesis were measured as the amount of 35S-methionine and 3Hadenine label in 80S ribosomes after a long term labeling period.
a) Vegetative Growth. Cells were grown to mid logarithmic growth at 25 ° C in minimal medium. The culture was then split in half. One half was shifted to 34 ° C, the other remained at 25 ° C. A half hour after the temperature shift, a~S-methionine (2/~c/ml) and 3H-adenine (3 pc/ml) were added to each culture. Cells were harvested two hours later. The harvested cells were washed twice in low salt buffer (0.05 M sodium cacodylate, pH 6.8, 0.05 M KCL, 5mM Mg acetate) (Zeijst, Kool and Bloemers, 1972) containing 1% cold methionine and 1% cold adenine and broken in a Braun homogenizer. Debris was removed by low speed centrifugation. One ml samples of the supernatant were layered on I 5 4 0 % sucrose gradients prepared in the same buffer and centrifuged 14 h at 26,000 rpm in a Beckman SW 27 rotor. Gradients were monitored at 260 nm in a Beckman Model 25 spectrophotometer and fractionated. A 0.1 ml aliquot of each fraction was mixed in 5 ml of Aquasol (New England Nuclear) and counted in a Beckman liquid scintillation counter.
b) Sporulation. Cells were grown in YEPD to stationary phase at 25 ° C. Cells were then washed and diluted 1:2 in 1% KAC at 25 ° C or 1% KAC prewarmed to 34 ° C. After 2 h, 3 #c/ml of 3H-adenine and 2 #c/ml of 35S-methionine were added to each culture for a labeling period of 4 h (Y 2 T6). Cells were then harvested and 80S ribosomes were isolated as described above. This procedure resulted in very well resolved monosome peaks on gradients. However, with this buffer system a high specific activity 35S peak appeared in 40S region near the top of the gradient. This peak was especially prevalent in samples from sporulating cells. If cells were labeled with 3sS methionine for a half hour and then incubated with a vast excess of unlabeled methionine for 4 h, this material did not become incorporated into monosomes. When 80S monosomes were layered on a second gradient to isolate ribosomal subunits as described below, the true 40S ribosomal sabunit did not have an abnormally high 35S specific activity. The same abnormal peak appeared in diploid strains both heterozygous and homozygous for rna mutations as well as in other laboratory strains. We concluded that this labeled material was unrelated to the rna mutants under investigation, and might represent an aggregation of smaller protein molecules. Subunits were isolated by collecting the 80S monosome peak fractions from a 5-20% sucrose gradient centrifuged in a SW 27.1 rotor for 5 h at 26,000 rpm. This fraction was resuspended in an equal volume of 2 x concentrated high salt buffer (0.05 M sodium cacodylate, pH 6.8, 0.5 M KCL, 5 mM Mg acetate) (Zeijst, Kool and Bloemers, 1972), and immediately layered on 15-40% sucrose gradients in high salt buffer. These gradients were centrifuged for 14 h at 26,000 rpm in a SW 27.1 rotor at 4 ° C and then fractionated. Polysomes were isolated by the following procedure. Cycloheximide (200 #g/ml final concentration) was added to the sporulating cells 10min before harvesting. Cells were then harvested and washed once in cold McLaughlin buffer (0.01 M Tris, pH 7.4, 0.03 M MgC12, 0.1 M NaCI) containing 200 #g/m1 cycloheximide and 200 #g/ml heparin. Cells were broken in a Braun homogenizer and debris was removed by low speed centrifugation. The supernataut was layered onto a 5 20% sucrose gradient prepared in the same buffer and centrifuged at 4°C for 2 h at 26,000 rpms in a Beckman SW27.1 rotor.
83
Results Synthesis o f Total R N A and Total Protein in Diploid Mutants T h e h o m o z y g o u s d i p l o i d s o f rna2, rna3, rna4, rna6 a n d rna8 all h a v e t e m p e r a t u r e s e n s i t i v e v e g e t a t i v e growth phenotypes similar to haploid strains carrying t h e s e m u t a t i o n s . W h e n c u l t u r e s g r o w i n g a t 25 ° C a r e s h i f t e d t o 34 ° C, g r o w t h c o n t i n u e s e x p o n e n t i a l l y f o r about one generation and then stops. The diploids were screened for their ability to synthesize RNA and protein at permissive and restrictive temperatures d u r i n g v e g e t a t i v e g r o w t h a n d s p o r u l a t i o n ( T a b l e 2). D i p l o i d s h e t e r o z y g o u s f o r rna2 a n d rna6 w h i c h a r e not temperature sensitive for sporulation or growth w e r e u s e d as c o n t r o l s . W h e n v e g e t a t i v e l y g r o w i n g cells h o m o z y g o u s f o r o n e o f t h e rna m u t a t i o n s w e r e raised to the restrictive temperature there was a m a r k e d d i f f e r e n c e i n t h e a m o u n t o f t o t a l R N A synt h e s i s ( t h e b u l k o f w h i c h is r i b o s o m a l ) b u t little e f f e c t on total protein synthesis. These results are essentially identical to those previously obtained for haploid s t r a i n s c a r r y i n g t h e s a m e rna m u t a t i o n s ( H a r t w e l l , M c L a u g h l i n a n d W a r n e r , 1970). H o w e v e r , d u r i n g s p o r u l a t i o n , t o t a l R N A s y n t h e s i s as w e l l as t o t a l protein synthesis was approximately the same during the interval measured at both temperatures. This lack of inhibition of RNA synthesis was found whether t h e cells w e r e l a b e l e d a f t e r o n l y o n e h o u r i n s p o r u l a t i o n m e d i u m o r a f t e r six h o u r s . E v e n a t 3 7 ° C n o inhibition of RNA synthesis was seen during sporulation. Even though no major difference in synthesis o f R N A a n d p r o t e i n is d e t e c t e d a t t h e r e s t r i c t i v e t e m Table 2. Ratio of synthesis of RNA and protein (34 ° C/25 ° C) during vegetative growth and sporulation
rna2/+ rna6/+ rna2/rna2 rna3/rna3 rna4/rna4 rna6/rna6 rna8/rna8
Vegetative growth
Sporulation
% Sporulation
RNA
Protein
RNA
Protein
25C
34C
1.10 0.71 0.16 0.27 0.29 0.12 0.19
0.80 0.60 0.93 1.02 1.00 1.20 0.98
1.00 1.20 1.35 1.07 0.82 1.32 0.97
1.15 1.00 0.89 1.10 1.27 0.95 1.17
80 75 80 65 70 75 60
80 80 0 0 0 0 0
RNA synthesis in vegetative cells was measured as incorporation of 3H-adenine (1.0 gc/ml) into cold TCA precipitable counts one half hour after logarithmically growing cells in YEPD were shifted from 25 ° C to 34 ° C. The labeling period was 1 h. Protein synthesis was measured as incorporation of 35S-methionine (0.5 gc/ml) into hot TCA precipitable counts during the same period. Labeling during sporulation was carried out for 1 h in the same manner, after cells had been in sporulation medium for 4 h (T4-Ts). Ascus formation was measured 48 h after placing cells in 1% KAC
84
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast
A. 25°C
8. 34°C 2.0
ZOO0
I0,000
1600
8000
1200
6000 1.0
800
4000
400~._
2000
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=o
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,.~ 0 2.0
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8000
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400
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18
36
Froction Fig. 1A-D. Comparison of newly synthesized r R N A and ribosomal protein appearing in 80S ribosomes during vegetative growth of rna2/rna2 and rna2/+ diploids at 2 5 ° C and 34 ° C. One-half of a log phase culture growing in minimal media at 2 5 ° C was shifted to 34 ° C, the other half left at 25 ° C. After 1 h incubation 3 gc/ml of 3H-adenine and 2 gc/ml of 35S-methionine were added to each culture, for a labeling period of 2 h. Cells were then harvested and ribosomes were isolated, as described in Materials and Methods, by sucrose gradient centrifugation. Gradients were monitored in a Beckman spectrophotometer at OD/60, and fractionated. These results are also summarized in Table 3 along with other strains tested. A rna2/rna2 at 25 ° C; B rna2/rna2 at 34 ° C; C rna2/+ at 25 ° C; and D rna2/+ at 34 ° C. Symbols used are: ( e ) ODz60; (o) 3H-adenine; (zx) 3SS-methionine
perature under sporulation conditions, these cells are still temperature sensitive for the ability to complete sporulation.
Ribosome Synthesis During Sporulation and Vegetative Growth From the results presented above, it seemed that rRNA synthesis was not severely inhibited at 34°C during sporulation even though the same strains are inhibited for RNA synthesis under vegetative conditions. However, these results do not indicate whether all ribosome components are being made and assembled. We, therefore, examined in greater detail ribosome synthesis during sporulation for both rna2/rna2 and rna6/rna6 diploids. The incorporation of radioactive precursors into both rRNA and ribosomal proteins was measured after purifying ribosomes by sucrose gradient sedimentation (see Materials and Methods). Figure 1 describes the results of incorporation of radioactively labeled adenine and methionine into ri-
bosomes of rna2/rna2 and rna2/+ diploids at the restrictive and permissive temperatures during vegetative growth. The heterozygote is used as a nontemperature sensitive control. The results for the diploid homozygous for rna2 are displayed in the top half of the figure (Fig. 1A-B). At the restrictive temperature, a large decrease in specific activity of labeled rRNA and ribosomal protein is seen as compared to that at the permissive temperature. No such difference is seen in the specific activity of rna2/+ diploid when labeled at 25°C and 34°C (Fig. 1C D). Therefore, in vegetatively growing cells, synthesis of ribosomes is severely inhibited at the restrictive temperature in rna2 homozygous diploids as would be expected. The same is true of rna6 homozygous diploids. During sporulation, however, there is no inhibition of ribosome synthesis at 34° C. Figure 2 describes the results of incorporation of radioactively labeled adenine and methionine into ribosomes of sporulating rna2/rna2 and rna2/+ diploids. The 4 h period after sporulating for 2 h ( T z - T 6 ) w a s chosen as the long term labeling period because that is when most RNA and protein synthesis occurs (Hopper, Magee, Fried-
85
N.J. Pearson and J.E. Haber : Changes in Regulation of Ribosome Synthesis in Yeast A. 25"C
8, 34"C
I000
2.0
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800
8000
600
6000 1,0
400
4000
200
2000
v v
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o
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8000
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400
4000
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o Fraction
18
man and Hall, 1974). However, similar results were obtained with labeling from TI-T3 and T6 T12. The top half of Figure 2A-B displays results of rna2 homozygote. Unlike vegetative growth, no difference in the specific activity of labeled ribosomal protein and ribosomal RNA is seen. The synthesis of rRNA and ribosomal protein is the same in the rna2 heterozygore also (Fig. 1C D). Therefore, synthesis of ribosomes apparently continues during sporulation at both restrictive and permissive temperatures. Differences in the incorporation of radioactive isotopes between rna2/rna2 and rna2/+ diploid strains are probably due to different pool sizes of adenine and methionine in different strains. In experiments with rna6 strains differences also occurred but were not correlated with the homozygosity or heterozygosity of the temperature sensitive mutation. Table 3 summarizes the results of experiments displayed graphically in Figures 1 and 2 as well as similar experiments for other strains tested. Even though rRNA accumulation was not inhibited during sporulation, it was possible that ribosomal proteins might not be synthesized. The new RNA could be incorporated into ribosomes by association with a pre-existing pool of ribosomal proteins or by turnover of vegetative ribosomes. However, we found that ribosomal protein synthesis is not inhibited at
E u
Fig. 2 A - D . Comparison of newly synthesized r R N A and ribosomal protein appearing in 80S ribosomes during sporulation of rna2/rna2 and rna2/+ diploids at 25°C and 34 ° C. Cells were grown in Y E P D to stationary phase at 25 ° C. Cells were then washed and diluted 1:2 in 1% K A C at 25 ° C or 1% K A C prewarmed to 34 °C. After 2 h, 3 ~tc/ml of 3H-adenine and 2 gc/ml of ~SS-methionine were added to each culture for a labeling period of 4 h (T2 T6). Cells were harvested and ribosomes were isolated as described in Materials and Methods, These results are also found in Table 3. A rna2/rna2 at 25°C; B rna2/rna2 at 34°C; C rna2/+ at 25°C; and D rna2/+ at 34°C. Symbols used are: ( e ) OD260; (0) 3H-adenine; (~) 3SS-methionine
36
3. Ratios (34 ° C/25 ° C) of specific activity of newly synthesized ribosomal protein and R N A in 80S ribosomes during spornlation and vegetative growth Table
rna6/rna6 rna2/rna2 rna2/+ rna6/+ a rna2 c~/c~rna2/rna2
Vegetative Growth
Sporulation (T2-T6)
Ribosomal protein
rRNA
Ribosomal protein
rRNA
0.18 0.17 0.80 0.68
0.13 0.12 1.10 0.71
0.80 0.96 0.98 0.93 0.91 0.92
0.64 0.80 0.85 0.82 1.11 0.74
Labeled m o n o s o m e s were isolated and fractionated as described in Materials and Methods. The results for rna2/rna2 and rna2/+ are displayed graphically in Figures 1 and 2. Specific activity of newly synthesized ribosomal protein and r R N A was determined by cutting and weighing graphed m o n o s o m e peaks for 3SS-methionine, 3H-adenine, and OD26o. In a l l experiments corrections for differences in uptake were made
the restrictive temperature. Labeling of proteins in separated ribosomal subunits appears to be the same under both permissive and restrictive conditions during sporulation. Sporulating cells were labeled from T2 T6 at both temperatures and ribosomal subunits were isolated as described in Materials and Methods.
86
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast 600
70,000
A. 2 5 °C 25C
500
60,000
400
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300 20,000
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30,000
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50,000
20.000 l ~
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5,000 1
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24
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32
Fig. 3A and B. Comparison of the distribution of radioactivity in ribosomal subunits of rna2/rna2 diploids labeled during sporulation at 25 ° C (A) and 34 ° C (B). Cells were labeled with 2 gc/ml of ~SS-methionine and subunits were isolated by sucrose gradient centrifugation as described in Materials and Methods
Figure 3 shows the results for the rna2/rna2 diploid. The specific activity of 35S methionine incorporated into subunits is the same at both temperatures. We considered the possibility that ribosomes synthesized at the restrictive temperature were in fact not functional. This idea seems unlikely in view of the observation that the ribosomes made during sporulation at both 25 ° C and 34 ° C are found in polyribosomes (Fig. 4). Sporulating rna2/rna2 cells were labeled with 32p-phosphoric acid at either 25°C or 34 ° C for 10 h before the cells were broken and polyribosomes separated by sucrose gradient centrifugation. Ribosomes made at the restrictive temperature were as likely as those synthesized at the permissive temperature to be in polyribosomes. Although this observation cannot assess the fidelity of proteins synthesized at 34 ° C, it does show that ribosomes made at 34 ° C are able to carry out active protein synthesis. A temperature sensitive c~/c~rna2/rna2 diploid and an rna2 haploid (which do not sporulate) were tested to see if they also made ribosomal components at the restrictive temperature under sporulating condi-
30,000
,0,000
20,000
5000
I0,000
lO
20
30
40
Fraction
50
60
70
Fig. 4. Comparison of polyribosomes from diploids homozygous for rna2 during sporulation at the permissive and restrictive temperatures. Vegetatively growing cells at 25°C were labeled overnight with 14C-adenine (2 gc/ml) and then transferred to sporulation media at 25 ° C or prewarmed to 34 ° C. The sporulating rna2/rna2 diploids were labeled with 32p-phosphoric acid (3 gc/ml) at each temperature for 10 h. Cells were then broken and polyribosomes were separated by sucrose gradient centrifugation
tions (Table 3). As with cells able to complete sporulation, these strains also continued to synthesize ribosomes under sporulating conditions although they were temperature sensitive under growing conditions. Thus, the absence of temperature sensitive ribosome biosynthesis is not a sporulation-specific phenomenon but a response to the nitrogen starvation conditions necessary for sporulation. This is not surprising, for it is known that R N A metabolism is similar in a/c~ and c~/c~diploids when placed under sporulating conditions (Hopper, Magee, Friedman and Hall, 1974).
Reversion Studies Diploids homozygous for any of the mutations studied do not sporulate at 34 ° C while heterozygotes
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast
sporulate well at both temperatures. To verify that the temperature sensitive sporulation phenotype was caused by the same mutation as the temperature sensitivity for growth, spontaneous revertants were selected from rna2 and rna6 homozygous diploids. These revertants were then tested for sporulation. These results are summarized in Table 4. Of 29 rna2 revertants, 3 sporulated to nearly the same extent at both temperatures, 22 partially regained the ability to sporulate at the restrictive temperature, and 2 still did not sporulate at the restrictive temperature. Of 7 rna6 revertants for growth, 6 diploids regained their ability to sporulate at either temperature. We examined genetically two revertants of the rna2 diploid and two revertants of the rna6 diploid, all four of which could sporulate well at both 25 ° C and 34 ° C. At least 10 meiotic tetrads for each revertant were analyzed. In each case the ts growth phenotype segregated 2:2, indicating that a single site mutation caused the reversion. We conclude that temperature sensitivity for growth and sporulation were caused by the same mutation.
Effect of rna Mutants on Sporulation Specific Events Diploids homozygous for rna mutations are blocked late in sporulation. Meiotic D N A synthesis occurs in both rna6 and rna2 homozygous diploids at permissive and restrictive temperatures. The data for the rna6 homozygous diploid is shown in Figure 5. Table 4. Percent sporulation of rna6/rna6 and rna2/rna2 revertants for growth at 34 ° C Sporulation 25°C
34°C
80% 70 % 60 % 70%
0% 65 % 0% 70%
6
60 80%
60-80%
2 6 5 2 3 6 3
60 80% 60-80% 60 80% 60-80% 40-60 % 40-60% 40-60%
60-80% 20 30% 10 20% 0% 40 60 % 20-30% 10 20%
87
100
z
75
o/
50
$
a.
25
0
I
0
3
6
9
12 hr.
24
Fig. 5. D N A synthesis during sporulation of strain homozygous for rna6. Stationary-phase cells were transferred to sporulation medium at 25 ° C. At 6 h (arrow), the culture was divided in half and the one flask was transferred to 34 ° C. The sporulation of cells kept at 25°C (o) was 53% after 48 h; no sporulation was found in the culture transferred to 34 ° C (©). (Haber, 1975)
To determine at which stage of nuclear division these mutant strains are arrested during meiosis, the nuclear stain mithramycin was employed. Diploid strains homozygous for rna2 and rna6 were incubated in sporulation media for 18 h at both 25 ° C and 34 ° C. These cells were then stained with mithramycin as described by Slater (1976), and examined in a Zeiss fluorescence microscope. After 18 h of sporulation at 25 ° C both strains contained cells which were bi-, tri- and tetranueleate. Many tetranucleate cells showed evidence of spore wall formation when viewed under visible light source. In cultures held at the restrictive temperature, bi-, tri- and tetranucleate cells were als0 seen. However, even cells which were tetranucleate did not show evidence of spore wall formation. It seems that rna diploids can complete the second meiotic nuclear division at the restrictive temperature but do not form mature ascospores.
I. Control strains a
rna2/rna2 rna2/+ rna6/rna6 rna6/+ 2. rna6/rna6 # of revertants
3. rna2/rna2 # of revertants
See Table 1 for complete genotype
Time of Temperature Sensitivity During Sporulation Temperature shift experiments demonstrated that the temperature sensitive period occurs during the first 12 15h of sporulation (Fig. 6). If shifted to the restrictive temperature after 15 h, the cells sporulate normally. Cells shifted to the restrictive temperature for more than six hours during the sensitive period gradually lost their ability to sporulate as the time at 34°C was increased. Cells left at 34°C for 6 h or less during the sensitive period can recover their ability to sporulate if left at the permissive temperature for the remainder of time it takes to sporulate. Figure 7 shows that this 6 h period at 34°C does not have to be the first 6 h during sporulation but that cells shifted from 25°C to 34°C during T6 T12
88
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast
I00
340C
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6
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dependence of
15
2
;4 340C
rna2/rna2 ( t o p )
and
rna6/rna6
(bottom) diploids during sporulation. Cells were grown to stationary phase in YEPD, washed and diluted into 1% KAC. At intervals cultures were shifted from 34° C to 25° C. (e) or from 25° C to 34°C (©). Percent sporulation was determined at 48 h. Similar results were obtained when cells were grown in acetate growth medium
250C
15
34=C
0
25oc
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0
I
4
I
,
8
70
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I
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will also regain their a b i l i t y to s p o r u l a t e if shifted b a c k to the permissive t e m p e r a t u r e . It c a n be seen, however, t h a t if these cultures are n o t shifted b a c k to 25 ° C after 12 h t h a t r e c o v e r y does n o t occur. Thus, these d a t a s h o w t h a t the t e m p e r a t u r e sensitive interval is s p r e a d over the first 12-15 h o f s p o r u l a t i o n a n d t h a t the ts effect is c u m m u l a t i v e . T h e i n a b i l i t y to recover if cells are held for m o r e t h a n 6 houl:s d u r i n g the sensitive p e r i o d m a y be related to the fact t h a t the m a i n synthetic activity o f s p o r u l a t i n g cells also occurs d u r i n g the first 12 h (at b o t h t e m p e r a t u r e s ) . A f t e r 12 h, the synthetic c a p a c i t y o f cells m a y be insufficient to b r i n g a b o u t c o m p l e t e r e c o v e r y f r o m the t e m p e r a t u r e sensitive effect.
Effect o f p H During Sporulation on rna2 A t the b e g i n n i n g o f s p o r u l a t i o n in 1% K A C there is a r a p i d rise in p H f r o m p H 7 to a b o u t p H 9 (Mills, 1972), whereas g r o w i n g cells are in m e d i a with a p H b e l o w 6. To d e t e r m i n e w h e t h e r this c h a n g e in p H is r e s p o n s i b l e for the c h a n g e in the p h e n o t y p e o f rna m u t a n t s , we s p o r u l a t e d a n rna2 d i p l o i d in 1% K A C buffered to p H 6.5 with m o r p h o l i n o p r o p a n e sulfonic acid ( M O P S ) in which yeast cells s p o r u l a t e n o r m a l l y ( M c C u s k e r a n d H a b e r , s u b m i t t e d for p u b l i c a t i o n ) . T h e i n c o r p o r a t i o n o f 3 H - a d e n i n e into T C A - p r e c i p i t a ble c o u n t s was m e a s u r e d as d e s c r i b e d in T a b l e 2. T h e r a t i o o f a d e n i n e i n c o r p o r a t i o n o f 3 4 ° C to 2 5 ° C
Fig. 7. Temperature dependence at various times during sporulation of rna2/rna2. Cells were pregrown in YEPD, washed and diluted into 1% KAC. Cultures were then shifted from 34° C to 25° C or 25° C to 34° C for intervals of time displayed. The results for cells maintained at 25° C or 34° C throughout sporulation are shown at the bottom of the figure. The dotted lines indicate that cells were incubated for an additional 36 h after 12 h (total 48) at the indicated temperature before percent sporulation was measured was 0.99. C o n s e q u e n t l y , a c h a n g e in p H a c c o m p a n y ing s p o r u l a t i o n c a n n o t a c c o u n t for the lack o f rna phenotype.
Expression of rna Mutants in Cells Committed to Sporulation The lack o f i n h i b i t i o n o f r i b o s o m e biosynthesis in rna2 d i p l o i d s d u r i n g s p o r u l a t i o n is n o t m e r e l y f o u n d when cells are u n d e r the n i t r o g e n s t a r v a t i o n c o n d i tions used to induce cells to sporulate. B e y o n d a certain p o i n t in the sequence o f events leading to ascospore f o r m a t i o n , d i p l o i d cells b e c o m e c o m m i t t e d to sporulate, even if r e t u r n e d to vegetative g r o w t h med i u m ( S h e r m a n a n d R o m a n , 1963). C o m m i t m e n t occurs a b o u t the time o f m e i o t i c D N A synthesis, or after 10 h with the strains a n d s p o r u l a t i o n c o n d i t i o n s used in this s t u d y ( d a t a n o t shown). W h e n cells are t r a n s f e r r e d f r o m 1% p o t a s s i u m acetate to Y E P D g r o w t h m e d i u m after the time o f c o m m i t m e n t , m o s t o f the cells f o r m e d ascospores, while some (those t h a t
N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast
most likely would not sporulate if left in 1% KAC acetate) begin to bud. We have used diploids homozygous for rna2 to see if temperature sensitive ribosome biosynthesis would soon appear if cells committed to sporulation were transferred back to growth medium. For this experiment cells homozygous for rna2 were sporulated for 16 h at 25 ° C, the permissive temperature. Cells were then washed and transferred to four different media: sporulation medium (1% KAC) at both 25 ° C and 34° C and growth medium (YEPD) at both 25°C and 34 ° C. After 1 h in the new media, the cells were examined microscopically and incubated with 3H-adenine for 30 min to measure RNA synthesis as described above. The percent sporulation and the amount of vegetative growth were monitored until 36 h after the beginning of sporulation. All of the cultures sporulated to about the same extent. At 16 h, when the cells were transferred, approximately 5% of the cells had sporulated. By 36 h about 70% of each culture had sporulated, and the spores in YEPD at 25°C also began to germinate and grow. Neither spores formed in YEPD at 34° C nor spores formed in sporulation medium at 25°C germinated in YEPD at the restrictive temperature. However, all of the cells that did not form spores did begin to grow within 1 h after transfer to YEPD at 34° C. These cells grew for nearly two doublings before arresting, as measured by haemocytometer counts. When RNA synthesis was analyzed under these different conditions, we found that there was no inhibition of incorporation of 3H-adenine into TCA-precipitable counts at 34°C as compared to the 25°C cultures in the same media (Table 5). There is significantly more incorporation of 3H-adenine into RNA Table 5. Effect of rna2 on R N A synthesis in cells committed to sporulation but incubated in YEPD growth medium a Cells transferred to
3H-adenine incorporation cpm/10: cells b
KAC, 25 ° C KAC, 34 ° C YEPD, 25 ° C YEPD, 34 ° C
2957 2711 7866 8214
Ratio 25°~C 34 ° C
1.09 0.96
" After 16 h in sporulation medium (KAC) at 25 ° C, a diploid strain homzygous for rna2 was transferred to the media and temperatures indicated. At this time approximately 5% of the cells had sporulated. After 1 h in the new media, the cells were labeled with 3 gc/ml 3H-adenine, and allowed to incorporate label for 30 min. Growth and sporulation was followed for 36 h by which time 70% of the cells in all four cultures had sporulated b Incorporation of radioactivity into TCA precipitable counts after 30 min incubation was measured as described in Materials and Methods
89
in the YEPD cultures. This is to be expected, in view of the less efficient uptake of 3H-adenine into cells in sporulation medium (Mills, 1972), but may also reflect a more active rate of ribosome biosynthesis. Nevertheless, it is clear that, unlike cells simply growing in YEPD, cells committed to sporulation (but in the same growth medium) are not affected by the rna2 mutation one hour after transfer to 34° C. We conclude from this experiment that cells committed to sporulation are unable to respond to the temperature sensitive defect in rna2, even when placed in a nitrogen-rich growth medium.
Discussion
Temperature sensitive rna mutations that inhibit ribosome synthesis in vegetative yeast cells do not do so under sporulation conditions. Even when cells committed to complete sporulation are returned to growth medium, these rna diploids continue to synthesize ribosomes at the restrictive temperature. If the rna mutations are indeed mutations in regulatory genes, as has been suggested (Gorenstein and Warner, 1976), then these results demonstrate that the regulation of ribosome synthesis during sporulation is different from that during vegetative growth. Sporulation in yeast occurs under nitrogen starvation conditions that do not support growth. RNA synthesis during sporulation is not continuous as it is in growing cells, but is quite low at the very beginning of sporulation and then rises to a high level until the time of DNA synthesis, when it declines (Hopper, Magee, Welch, Friedman and Hall, 1974). Recently, Rhaese, Hoch and Groscurth (1977), have found that highly phosphorylated adenine compounds appear at the beginning of sporulation in yeast. Phosphorylation of adenine or guanine appear to be important in the regulation of ribosome synthesis in bacteria (Cashel and Gallant, 1974) and may also reflect changes in the regulation of ribosome synthesis in yeast. It is possible that some components of the ribosomes synthesized during sporulation differ from those in growing cells. In Bacillus subtilis, there is growing evidence that there are a number of changes in proteins associated with ribosomes when cells begin sporulation (Rhaese, Groscurth and Scheckel, 1977; Kobayashi and Domoto, 1977). There is extensive turnover of vegetative ribosomes and synthesis of new ribosomes when yeasts are transferred to sporulation medium, as there is in other organisms undergoing gametogenesis (Martin, Chiang, and Goodenough, 1976; Dickinson and Heslop, 1977). However, there is no evidence in any of these eucaryotes that any
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N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast
ribosomal proteins in newly synthesized ribosomes differ from those made in vegetative cells. Our data do not rule out the possibility that a small class of sporulation-specific ribosomes are not made at 34 ° C in the r n a diploids so that sporulation is not completed. The data do show that the vast majority of ribosomes made during sporulation are not regulated by the r n a mutants. The role of the r n a genes in wild-type cells is not known. All 10 complementation groups appear to have a common mutant phenotype in vegetative cells- a cessation of ribosomal protein synthesis at 34 ° C leading to an inhibition of ribosome synthesis. All of the r n a gene products may interact with a common regulatory mechanism that regulates ribosomal protein synthesis, perhaps by monitoring the availability of nutrients in the cell. Somehow, the regulation associated with each of the 5 r n a mutants we studied is bypassed in sporulating cells. If the r n a mutants somehow signal that the cell is starving, when the temperature is raised to 34 ° C, such information may be " i g n o r e d " by a cell that is induced to differentiate precisely because of nitrogen starvation. The mutations are not completely bypassed, however, as cells are unable to sporulate even though ribosome synthesis is unaffected. If the r n a mutants are bypassed in the regulation of ribosome synthesis, why do the cells not sporulate? One explanation is that the r n a gene products have two roles, one directly involved in metabolism and the other regulatory. For example, such a situation occurs in bacteria. When a single species of tRNA isoacceptor is mostly uncharged within the cell, either due to starvation of a particular amino acid or mutation, there is a specific regulation of transcription of ribosomal protein and ribosomal R N A (Cashel and Gallant, 1974). However, even without its regulatory function, lack of aminoacylation eventually inhibits protein synthesis by directly interfering with translation. An analogous situation may occur in yeast. R N A genes may have two functions, one being the regulation of ribosome synthesis. This regulatory function may be overridden during sporulation, yet cells carrying an r n a mutation could be prevented from completing ascus formation by some direct effect of the mutation on cellular metabolism. Another alternative is that the r n a mutants turn off the transcription of other genes besides ribosomal proteins and that these additional sequences are still regulated during sporulation. Hereford and Rosbash (1977) showed that when a r n a 2 strain was raised to 34 ° C, more than 100 different moderately abundant messages and possibly many less frequent messages were not transcribed. More messages are
affected than can be accounted for by ribosomal proteins, so that other messages necessary for both sporulation and growth may still be regulated. Acknowledgements. We are grateful for the critical reading of this manuscript by Bob Schleif, Jim Hopper and Lynna Hereford. We would also like to thank Peter Wejksnora for many helpful suggestions. N. Pearson was supported by NIH Training Grant GM-1586 in microbiology. This research was supported by USPHS grant GM-20056.
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N.J. Pearson and J.E. Haber: Changes in Regulation of Ribosome Synthesis in Yeast Roth, R., Halvorson, H.O. : Sporulation of yeast harvested during logarithmic growth. J. Bact. 98, 831-832 (1969) Sebastian, J., Mian, F., Halvorson, H.O.: Effect of the growth rate on the level of DNA-dependent RNA polymerase in Saccharomyces cerevisiae. FEBS Lett. 34, 159-162 (1974) Sherman, F., Roman, H. : Evidence for two types of allelic recombination in yeast. Genetics 48, 255 (1963) Slater, M.L.: Rapid nuclear staining method for Saccharomyces cerevisiae. J. Bact. 126, 1339 134I (1970) Warner, J.R., Gorenstein, C.: The synthesis of eucaryotic ribosomal proteins in vitro. Cell 11,201 212 (1977) Warner, J.R., Udem, S.A. : Temperature sensitive mutations affecting ribosome synthesis in Saccharomyces cerevisiae. J. molec. Biol. 65, 243357 (1972)
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Wejksnora, P.J., Haber, J.E.: Methionine dependent synthesis of ribosomal ribonucleic acid during sporulation and vegetative growth of Saccharomyces cerevisiae. J. Bact. 120, 1344-1355 (1974) Zeijst, B.A.M., van der, Kool, A.J., Bloemers, H.P.J.: Isolation of active ribosomal subunits from yeast. Europ. J. Biochem. 30, 15-25 (1972)
Communicated by F. Kaudewitz Received July 18, 1977
