Molec. gen. Genet. 160, 59-65 (1978) © by Springer-Verlag 1978
Ribosome Activity and Degradation in Meiotic Cells of Saccharomyces cerevisiae Karen R. Frank and Dallice Mills Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331, USA
Summary. The percentage of active ribosomes in meiotic cells was shown to be influenced by the presporulation carbon source and the age of the culture at the time of inoculation into sporulation medium. Cells harvested at early stationary phase from medium containing glucose (YPG) have a lower percentage (25 to 50%) of active ribosomes at the onset of sporulation than cells harvested from medium containing acetate (YPA). Furthermore, the percentage of active ribosomes in YPD cells typically remains below 50% during the initial 12 h of sporulation. Cells harvested from either YPD or YPA medium during logarithmic growth had 65 to 85% active ribosomes. The YPA cells maintained 65 to 85% active ribosomes during the initial 12 h of sporulation whereas the respiratory inactive YPD cells exhibited a decline to 11% by T4 and, thereafter, never exceeded 20%. The increase in the percentage of active ribosomes in YPA cells between To and T4, and in YPD cells between To and T8 was primarily due to a decrease in the inactive ribosome population resulting from the rapid degradation of ribosomes. Extensive ribosome degradation during meiosis reduced the initial ribosome content in YPD and YPA-grown cells by 50 to 65% at T12. The number of messenger-bound ribosomes remained constant for the first 4 h in YPA cells and for 8 h in YPD cells. Thereafter, the number in both cell types decreased at a rate of 7 to 8% per h.
Introduction Meiosis is a fundamental process of all sexually reproducing eukaryotes. The yeast, Saccharomyces cerevisiae has served as a model organism for characterizing changes in macromolecule synthesis during meiosis For offprints contact." D. Mills
and sporulation. Diploid cells can be induced to carry out meiotic processes when transferred from a growth medium containing nitrogen and glucose or a nonfermentable carbon source, such as acetate, to a nitrogen-free potassium acetate sporulation medium. The active periods of ribonucleic acid (RNA) synthesis accompanying meiosis have been described for several wild-type strains (Croes, 1967; Esposito et al., 1969; Mills, 1972; Hopper etal., 1974), meiotic mutants (Esposito et al., 1970; Roth, 1973) and sporulationincompetent diploid cells homozygous for the a or mating-type alleles (Hopper et al., 1974). Polysomal RNA has been analyzed by polyacrylamide gel electrophoresis (Curiale, Petryna, and Mills, 1976) and shown to contain newly synthesized transfer RNA, ribosomal RNA and poly A-containing RNA. Active periods of protein synthesis, determined by incorporation of radioactive amino acids (Esposito et al., 1969, 1970; Hopper et al., 1974) usually occurs during peak periods of RNA synthesis. Concomitant with protein and RNA synthesis in the meiotic cells is a high degree of protease and ribonuclease activity which ultimately reduces the RNA and protein content of the cell to less than one-half the initial values (Croes, 1967; Hopper et al., 1974). It could be speculated that the large concentration of ribosomes which are present in vegetative yeast serves as the main source of protein and RNA which is degraded. In these experiments, we have measured the percentage of active ribosomes, the number of messenger-bound ribosomes and ribosome degradation in wild-type strains and asporogenous meiotic mutants. The method used for making these determinations has been utilized for similar purposes with mammalian cells (Velez, Farrell, and Freeman, 1971; Storb and Martin, 1972) and bacteria (Zylber and Penman, 1970; Beller and Davis, 1971). It is based on the observation that yeast messenger-bound ribosomes are resistant to dissociation into subunits by high
0026-8925/78/0160/0059/$01.40
60
K.R. Frank and D. Mills: Ribosome Activity and Degradation During Meiosis
ionic strength (Martin and Hartwell, 1970). This procedure is especially suited for characterizing ribosome activity and degradation during meiosis of yeast because it is independent of possible ribonuclease activity associated with the cell lysate and it does not rely upon the in vivo labeling of R N A and protein with precursors which are taken up very poorly by meiotic yeast cells.
Materials and Methods
homogenate was centrifuged at 12,000 x g for 10 min to sediment the undisrupted cells. A 0.3 ml aliquot of the supernatant was diluted with 0.1 ml of 2.5 M KCI containing 10 gg bovine pancreatic RNase Type 1A and incubated for 5 rain on ice. Routinely, 0.075, 0.1 and 0.125 ml aliquots were layered onto 4 ml 10 30% linear sucrose gradients containing 0.05 M Tris HC1 pH 7.6, 0.88 M KC1 and 0.015 M MgCI2. The gradients were centrifuged at 225,000xg for 90-115 min at 4°C in a Spinco SW56 or SW60 rotor. Sedimentation profiles of active ribosomes were obtained using an Isco Density Gradient Fractionator Model 640 with a UA2 or UA4 absorbance monitor in conjuction with an Omnigraphic Strip Chart Recorder, Series 3000 (Houston Instruments).
Extraction of Polyribosomes from Sporulating and Vegetative Yeast. Yeast Strains. The genotypes of 4579 and the isogenic meiotic mutants D-M10-6A and D-M10-2B have been described by Roth (1973). The genotypes of the homothallic wild-type diploids Z186 and $41 were previously described by Mills (1972) and Esposito et al. (1969). A temperature-sensitive meiotic mutant, C126Y-1A, isogenic with $41 has the following genotype C126Y-1A a/c~ D/D arg 4-1/arg 4-1 cyh 1/cyh 1 spo 2-1/spo 2-1 Symbols are as follows: a, ~, mating type alleles; D, diploidization gene, arg, arginine auxotroph; cyh, cycloheximide resistance.
Cells were harvested, disrupted and the cell lysates centrifuged as described for determining the percentage of active ribosomes. One quarter final volume of 2.5 M KC1 and diethylpyrocarbonate (2% final concentration)were added to the supernatant. Routinely, 0.4 0.8 ml of the cell lysate was layered onto a 12 ml 10-40% sucrose gradient containing 0.05 M Tris HC1 pH 7.6, 0.88 M KC1 and 0.015 M MgCI2 and a 0.65 ml cushion of 50% sucrose. The gradients were centrifuged at 148,000 x g for 90 rain in a Spinco SW41 rotor.
Enumeration of Asci. The method used for counting cells and deterMedia. The composition of the presporulation media which contained yeast extract, peptone and either dextrose (YPD) or acetate (YPA) has been previously described (Fast, 1973). YPD and YPA were supplimented at 50 and 100 mg per liter respectively, with: adenine, arginine, histidine, leucine, lysine, tryptophan, methionine and uracil unless otherwise specified. Sporulation medium consisted of 1% potassium acetate (KAc) pH 6.0.
Growth and Sporulation Conditions. Three to five day old colonies growing on YPD or YPA plates were inoculated into 100 or 200 ml of presporulation growth medium at an initial concentration of 1 × 105 cells/ml. Cells growing in YPA or YPD liquid medium were harvested for sporulation after reaching 2 x 107 and 1 x 108 cells/ml, respectively. The procedure for harvesting cells and the sporulation conditions have been described previously (Mills, 1974).
Determining the Percentage of Disrupted Cells and Active Ribosomes in Sporulating and Vegetative Yeast. Fifteen min before the time of harvest, sporulating cells were collected by centrifugation at 1000xg for 5 rain or filtration on 47 mm, 3 ~t pore or 24mm, 0.45 g pore Millipore filters, washed with ice cold sterile water, and resuspended in fresh KAc medium at 5 x 107 cells per ml to help facilitate uptake of cycloheximide (Mills, 1974). The cells were incubated with shaking for 10 rain and then cycloheximide was added (100gg/ml final concentration) to prevent polysome decay (Hartwell et al., 1970). After an additional 5 min incubation, they were poured over ice and centrifuged at 1000 x g for 5 rain at 4 ° C. The supernatant was poured off and the cell pellet was suspended in 0.1 ml of TKM buffer (0.05 M Tris HC1 pH 7.8, 0.08 M KCI, 0.0125M MgC12, 100 ~xg/ml cycloheximide and 0.001 M dithiothreitol) (Martin, 1973). The cell suspension was then poured into a 17 ml pyrex screwtop tube and 0.45-0.5 mm glass beads were added to the level of the meniscus. The pyrex tube was inserted into a pre-chilled microchamber for the Bronwill Cell Homogenizer Model MSK and the cells were disrupted as previously described (Mills, 1974). The percentage of disrupted cells was determined by hemacytometer counts of intact cells of equivalent aliquots removed before and after homogenization. The glass beads were washed with 0.5 to 1 ml of TKM buffer containing deoxycholate and Triton X-100 (1% final concentration). The cell
mining the percentage of asci in a sporulated culture has been previously reported (Esposito et al., 1970).
Chemicals. Amino acids, cycloheximide, 7-deoxycholate (sodium salt), ribonuclease-A from bovine pancreas crystallized type 1A, Triton X-100 and Trizma HC1 were obtained from Sigma Chemical Company. Diethylpyrocarbonate was purchased from Nutritional Biochemicals Corporation, and density gradient grade (ribonuclease free) crystalline sucrose from Schwarz-Mann.
Results
Cell Breakage. The optimum conditions for homogenizing cells at intervals during the initial stages of sporulation were determined for strains 4579, Z186 and $41. The percentage of cells disrupted was independent of the presporulation growth conditions. Strain 4579 and Z186 showed essentially similar cell disruption after 5, 10 or 15 s homogenization although the former was harvested for sporulation during exponential growth while Z186 was harvested at early stationary phase (Table 1). The percentage of disrupted cells of both strains increased approximately 20% when homogenization was extended from 5 to 15 s. To determine what effect, if any, increased homogenization had on cell disruption, strain $41 was subjected to homogenization for up to 120 sec at T o and T4. No significant increase in the percentage of disrupted cells was observed when homogenization was extended beyond 15 s. Intervals beyond 12 h were not analyzed because previous results from labeling experiments (Mills, 1972; Hopper et al., 1974) had shown peak periods of transcription and translation to occur before T12.
K.R. Frank and D. Mills: Ribosome Activity and Degradation During Meiosis
61
Table 1. The percentage of meiotic cells disrupted as a function of the duration of homogenization in the Bronwill cell homogenizer
Table 3. Cell disruption and the percentage of active ribosomes following 15 and 30 s homogenization of meiotic cells of $41
Strain
Homogenization Percent cell time ( s e c ) breakage"
Time
Percent cell disruption
Percent active ribosomesa
4579
5 10 15
41.0±4.0 u 54.5_+3.5 b 62.0+_ 3.0 °
(h)
15 s
30 s
15 s
30 s
Z186
5 10 15
40.6_+6.6d 54.4_+4.4d 6h3±5.3 °
0 8 12
62 60 57
61 63 56
67 34 40
19 7 5
$41
15 30 120
63.0_+3.0e 62.9_+3.1 f
a Percentage of active ribosomes from YPD grown cells harvested at late-logarithmic growth phase
a Routinely, 200400 cells were counted to determine the percentage of cells disrupted u Percent breakage was averaged for 0 and 4 h ° Percent breakage was averaged for 0, 4, 6, and 12 h d Percent breakage was averaged for 0, 4 and 8 h e Percent breakage was averaged for 0, 4, 8 and 12 h f Percent breakage was averaged for 0 and 4 h; disruption was for 30, 45, 60, 75, 90, 105 and 120 sec
Table 2. The percentage of active ribosomes and polysomes in 4579 after 6 h in KAc Sample
5g
•
2~
h7
2,
Percentage of ribosomes as: monosomes
RNase treated cell lysates Untreated cell lysates
6G
40S
60S
a c t i v e polyribosomes somes
13.2 10.9
21.6 17.4
65.2 -
71.7
Conversion of Polysomes to Active Ribosomes. After it was d e t e r m i n e d that 15 s h o m o g e n i z a t i o n w o u l d yield the m a x i m u m percentage of disrupted cells, the c o n v e r s i o n o f p o l y s o m a l r i b o s o m e s to active ribosomes was d e t e r m i n e d using mild r i b o n u c l e a s e treatm e n t ( M a r t i n , 1973). The c o n v e r s i o n o f polysomes (72%) to active r i b o s o m e s (65%) in strain 4579 at T 6 was sufficiently c o m p l e t e d within 5 rain (Table 2). A l t h o u g h cell d i s r u p t i o n was n o t significantly increased by h o m o g e n i z i n g in excess of 15 s, our i n t e n t was to d e t e r m i n e whether increased h o m o g e n i z a t i o n affected the percentage of active ribosomes. A significant decrease in the percentage of active ribosomes occurred at all intervals when h o m o g e n i z a t i o n o f $41 was increased f r o m 15 to 30 s even t h o u g h cell disruption r e m a i n e d u n i f o r m (Table 3). Similar results were o b t a i n e d with strain 4579 at selected intervals d u r i n g sporulation. The percentage of active r i b o s o m e s did n o t significantly increase w h e n cells were h o m o genized less t h a n 15 s. Therefore, h o m o g e n i z a t i o n o f all strains for 15 s was a d o p t e d for purposes of determ i n i n g the percentage of active ribosomes.
Hours
Fig. 1. The percentage of active ribosomes in wild type strain Z186 and a temperature-sensitive meiotic mutant, C126Y-1A, in KAc medium at 30° and 34° C. Symbols: Z186, (i) 30° C, (cz) 34° C; C126Y-1A, (o) 30° C, (©) 34° C
Percentage of Active Ribosomes During Meiosis. The percentage of active ribosomes was d e t e r m i n e d initially for Z186 a n d 4579 after growth in presporulation m e d i u m c o n t a i n i n g glucose a n d acetate, respectively, as the c a r b o n source. The percentage of active r i b o s o m e s in Z186 at To was always significantly lower (Fig. 1) t h a n in 4579 (Fig. 2). The percentage of active ribosomes in Z186 decreased by 50% d u r i n g the first 3 h b u t increased to To levels by T12. A temperature-sensitive meiotic m u t a n t , C126Y-1A, isogenic to Z186, displayed a similar p a t t e r n of active ribosomes at the permissive (30 ° C) a n d n o n p e r m i s sive (34 ° C) temperatures. Since the ts lesion, SPO2-1, in C126Y-1A does n o t affect i n c o r p o r a t i o n of t4Ca d e n i n e or t 4 C - a r g i n i n e into nucleic acids a n d protein, respectively (Esposito et al., 1970), it was expected that the two strains w o u l d have a similar p a t t e r n of active ribosomes. The active ribosomes in 4579 r e m a i n e d at approximately 70 to 75% d u r i n g the initial 12 h of sporulation (Fig. 2). Two isogenic meiotic m u t a n t s , D - M 1 0 6A a n d D - M 1 0 - 2 B which have been previously s h o w n
62
K.R. Frank and D. Mills: Ribosome Activity and Degradation During Meiosis
,00[
Effect of Presporulation Medium and Culture Age on Active Ribosomes During Meiosis. Since the percent-
~i 7 5 I ~ o ~ e - - - - - - m " ' - ' ° -Q
'01
>c
2 0
4
8
12
Hours
Fig. 2. The percentage of active ribosomes during meiosis in 4579 and isogenicmeioticmutants, D-M 10-6Aand D-M 10-2B.Symbols: (o) 4579; (i) D-M10-6A; (,) D-M10-2B
100
8C 6C
o~4C =o 2c ~"
0m
I
I
B
2c
-,-
4
8
-t
12
Hours
Fig. 3A and B. The percentage of active ribosomes in $41 during meiosis after growth in YPA or YPD presporulation medium. A Cells were harvested during exponential growth from YPA medium (o); or at stationary phase from YPD medium (i). B Cells were harvested from YPD during exponental growth (i); and from YPA at early stationary phase (0). The determinations in B were made with 10-30% sucrose gradients which were centrifuged at 35,000rpm for 100rain at 4°C in a Spinco SW60 rotor
to incorporate lower and greater amounts of 14Curacil into nucleic acids than the wild type, respectively (Roth, 1973), were also analyzed (Fig. 2). The active ribosomes in D-M10-6A were approximately two-thirds the level observed in 4579. However, although the percentage of active ribosomes in D-M102B were similar to 4579 at To, they decreased to approximately 28% by T4 and remained at that level through Ts, after which they increased to 50% by T12 (Fig. 2).
age of active ribosomes in Z186 and 4579 differed significantly at To and throughout the first 12 h of sporulation, it was of interest to determine whether the differences were due to the presporulation growth media, the stage at which the cells were harvested for sporulation, or both factors. Strain $41 sporulates efficiently when harvested from YPD medium at early stationary phase or from YPA medium during exponential growth (Fast, 1973). Consequently, the percentage of active ribosomes were determined for a single strain during sporulation when cells were harvested from YPD and YPA during exponential growth and at early stationary phase. Under normal sporulation conditions, cells harvested during exponential growth from YPA presporulation medium had approximately 80% active ribosomes at To, whereas cells harvested from YPD medium at early stationary phase had less than 30% (Fig. 3A). Over the initial 12 h of sporulation, there was a net increase in active ribosomes in cells harvested from YPD but a decrease in cells harvested from YPA. It was of interest, therefore, to determine whether the high level of active ribosomes observed during sporulation after cells were harvested during exponential growth (Figs. 2 and 3A) was a consequence of t h e age of the culture or reflective of the carbon source. Cells of $41 were harvested from YPD medium during exponential growth rather than at early stationary phase. The percentage of active ribosomes in these cells was high (80%) at To but fell to about 10% at T¢ and at all subsequent intervals was less than 20%. Sporulation was also greatly reduced (Table 4). However, cells harvested at early stationary phase from YPA medium exhibited a pattern of active ribosomes which was similar to that observed when cells were harvested during exponential growth. The percentage of sporulation of $41 following growth in YPD and YPA is presented in Table 4.
Ribosome Degradation and Relative Number of Message-bound Ribosomes During Meiosis. The design of the experiment presented in Figure 3A also provided a method for estimating the amount of ribosome degradation and the relative number of ribosomes that were bound to messenger R N A . Since cell breakage was uniform and the volumes of buffers and lysates layered onto each gradient were constant throughout, a change in the total area under the ribosome peaks was taken as an indication of a change in the ribosome content (Table 5). Cells grown in either presporulation medium showed a steady decrease in the ribosome content during the first 12 h of sporulation (Fig. 4). Only about 35% of the initial ribosome con-
K.R. Frank and D. Mills: Ribosome Activity and Degradation During Meiosis Table 4. Percentage of sporulation of $41 after removal of cells from YPD or YPA presporulation medium during exponential growth or at early stationary phase Pre-
Percentage of Sporulation
Growth phase
YPA YPD
"6 1.0 I-"
o
0.8
r~ co
sporulation medium
T2~
T30
T48
exponential early stationary
49 70
72
79 -
exponential early stationary
48 1.5
10
60 -
63
0.6
O3 O3
0.4 ©
E
0.2
O ¢~-
J.
Table 5. Measurements of ribosome content in $41 harvested from YPA during exponential growth and YPD at early stationary phase Growth medium
Time in sporulation medium (h)
Area under ribosome peaks"
YPA
0 4 8 12
4.47_+0.95 4.09_+0.70 3.23_+0.10 1.62+0.08
YPD
0 4 8 12
10.40 + 1.60 8.95_+0.70 5.35_+0.53 5.15_+0.48
]2
Hours
Fig. 4. Ribosome degradation in strain $41 during the initial 12 h of sporulation. Cells were grown in either YPA (e) or YPD ( i ) . T(ol is the ribosome content of cells at the onset of sporulation; T(x) is the ribosome content at subsequent h. The area under the ribosome peaks at To and at each interval was normalized to 60% cell breakage
1.2
,g E o co
" Area determined and normalized to 60% cell breakage and 4.5 x 107 cells/ml. The relative units of area were determined by planimetric analysis of the A 2~4 nm tracings
8
>.
411
1.0
--II
t-,
0.8 E 0
A
~ I.-20. 6
..Q~
tent was present at T12 in cells previously grown in YPA and about 50% in cells harvested from YPD medium. The increased percentage of active ribosomes in YPA cells at r 4 and in YPD cells at T4 and T 8 (Fig. 3 A) could have resulted from a diminishing free pool of inactive ribosomes or increased messenger R N A synthesis and its subsequent translation. An estimate of the number of messenger-bound ribosomes was determined, therefore, using the following equation : MBR(Tx) =
An-x)' (C(Tx)/C(To)) A(To)
where MBR(T~) is the number of messenger-bound ribosomes at intervals(T~) during meiosis relative to the number at To; A(Tx) and A(ro) represent the fraction of active ribosomes at Tx and To, respectively; C(To) and C(T~) represent the ribosome content at To and Tx, respectively (Table 5). C(To) was normalized to 1.0 and C(T~) was expressed as a fraction of the To content. The number of messenger-bound ribosomes in YPA-grown cells decreased by 4% at T4, and thereaf-
th
0.4
E 0.2
~:
0
0
I
I
4
8
12
Hours
Fig. 5. Relative number of ribosomes engaged in protein synthesis during the initial 12 h of sporulation in strain $41. The ribosome content engaged in protein synthesis at the onset of sporulation (To) was normalized to I. T(×), the amount of messenger-bound ribosomes during sporulation was determined to be the product of the ribosome content and the percent active ribosomes at each interval. Symbols: (o) YPA-grown cells; (-,) YPD-grown cells
ter, the number decreased by about 8% per h (Fig. 5). Cells l~arvested from YPD medium maintained a constant number of messenger-bound ribosomes during the initial 8 h. Thereafter, the number also decreased at a rate of 7 to 8% per h. However, unlike YPA cells which had only 28% of the To number of messenger-bound ribosomes at T12, YPD cells had 73%.
64
K.R. Frank and D. Mills: RibosomeActivityand DegradationDuring Meiosis
Discussion
The percentage of ribosomes active in protein synthesis has been one technique used for measuring changes in macromolecule synthesis in mammalian cells (Valdez, Farrell, and Freedman, 1971; Zylber and Penman, 1970) and in microorganisms (Martin and Hartwell, 1970; Beller and Davis, 1971; Martin, 1973). This method measures the percentage of the population of ribosomes in a lysate which is bound to messenger RNA and, therefore, the results are not dependent on uniform cellular disruption. If the number of ribosomes per cell were decreasing as the result of increased protease and ribonuclease activity, fewer ribosomes would be available to the cell and an increase in the percentage of active ribosomes would be required to maintain a constant level of translation. Under those conditions, the increase in percentage of active ribosomes would not be signaling a period of increased protein synthesis or an increase in the number of messenger-bound ribosomes. The number of message-bound ribosomes and relative number of ribosomes per cell can be determined, however, if cellular disruption is uniform and buffer volumes of cellular lysates are kept constant at all sampling periods. Three strains of S. cerevisiae which were subjected to 15 s homogenization at intervals throughout the first 12 h of sporulation were uniformly disrupted without loss of message-bound ribosomes; an essential requirement for measuring ribosome activity and content. The percentage of active ribosomes of Z186 and 4579 during the initial 12 h of meiosis were very different (Figs. 1 and 2). The generally low percentage of active ribosomes in Z186 may have been the result of the lower metabolic activity associated with stationary phase cells, as these cells were inoculated into sporulation medium at early stationary phase. An isogenic temperature-sensitive meiotic mutant had similar profiles of active ribosomes at the permissive and nonpermissive temperatures. Since Esposito et al. (1970) had concluded that the temperature-sensitive lesion did not result in decreased RNA or protein synthesis, similar profiles were expected. However, when transcription or translation in isogenic meiotic mutants appear different from the wild type, a corresponding change may be expected in the percentage of active ribosomes. Strain 4579 and isogenic mutants D-M10-6A and D-M10-2B have different percentages of active ribosomes during the initial 12 h of sporulation. The lower level of incorporation of 14C-uracil and 14C-leucine by D-M10-6A (Roth, 1973), is also accompanied by a lower percentage of active ribosomes (Fig. 2). D-M10-2B which has rates of incorporation of uracil and leucine comparable to 4579 at
To (Roth, 1973), also has similar levels of active ribosomes. However, at subsequent intervals when transcription is 2 to 3 fold greater in D-M10-2B, the percentage of active ribosomes is 2 to 3 fold lower than was observed in 4579 (Fig. 2). These results suggest that the increased transcription may be due to increased synthesis of ribosomal RNA which is processed into functional ribosomes. If protein synthesis were not increased during this interval, while the ribosome content per cell increased, a smaller percentage of ribosomes would be active in protein synthesis. That Z186 and 4579 exhibited greatly different profiles of active ribosomes, suggested either significant differences between nonisogenic strains, or differences in the physiology of the strains as the result of the presporulation carbon source or the age of the culture at the time of transfer to sporulation medium. It was possible to determine how much influence the presporulation conditions and the physiological state of the cells have on ribosome activity and degradation because $41 sporulates efficiently after growth in either YPA or YPD presporulation medium. The results were in general aggreement with those observed for 4579 and Z186. Cells harvested at early stationary phase have lower percentages of active ribosomes at the onset of sporulation reflecting the lower metabolic activity of stationary phase cells. However, a net increase in the percentage of active ribosomes was observed at T12 (Fig. 3A, B). Cells harvested during exponential growth from YPA or YPD have 65 to 80% active ribosomes at To and maintain a high level only if they are capable of utilizing the acetate of the sporulation medium (Fig. 3 A, B). The respiratory inactive YPD cells which were harvested for sporulation during exponential growth underwent an immediate decline in the percentage of active ribosomes, from 66% at To to 11% at T4 (Fig. 3 B) and sporulation was greatly inhibited (Table 4). Roth and Halvorson (1969) have previously shown that sporulation was greatly inhibited when cells were harvested from glucose medium during exponential growth. A presporulation medium containing glucose represses the tricarboxylic acid cycle enzymes and maturation of mitochondria resulting in an inactive respiratory system (Eaton and Klein, 1954). Respiratory adaptation occurs when the glucose of the YPD medium is depleted as early stationary growth phase is approached and it continues during the initial hours of sporulation (Esposito et al., 1969). Once cells are respiratory active, they can be induced to sporulate even in the absence of a functional mitochondrial genome (Kuenzi, Tingle, and Halvorson, 1974). The increase in percentage of active ribosomes
K.R. Frank and D. Mills: Ribosome Activity and Degradation During Meiosis
in sporulating yeast appesrs to be entirely due to a decrease in ribosomes available for translation rather than an increase in the number of messagebound ribosomes. The percentage of active ribosomes increased about 50% between To and T8 in cells harvested from YPD (Fig. 3A) while over the same interval, nearly 50% of the ribosomes were degraded (Fig. 4 and Table 5). Consequently, the number of ribosomes active in protein synthesis remained essentially constant (Fig. 5). Ribosome degradation during the initial 12 h was more pronounced (64%) in YPA cells and the relative number of message-bound ribosomes fell to 28%. The degradation of 50 to 65% of the ribosome population in sporulating cells is consistent with previous observations of total RNA and protein degradation during sporulation, Croes (1967) first observed a 50% reduction in total cellular RNA content by T12. Hopper et al. (1974) measured the breakdown of labeled vegetative protein and RNA in sporulating cells by the loss of radioactivity from the cells and its appearance in the sporulation medium. They concluded that 50 to 70% of the RNA and up to 30% of the protein was degraded by T2~. Electron microscopy has revealed that the large concentration of ribosomes which is present in vegetative cells decreases during sporulation (Munkur, 196l). The extent to which amino acids and ribonucleotides of degraded ribosomes contribute to the cellular pools is not known. However, it could be an essential requirement of sporulation since : i) extensive ribosome degradation occurs during active periods of transcription, translation and DNA replication and ii) the rate of RNA and protein degradation in asporogenous diploid strains is at least 2-fold lower than in sporulating diploids (Hopper et al., 1974). Acknowledgements. We thank Lynn Peterson for excellent technical assistance during part of this study. This investigation was supported by NIH g r a n t / / G M 24040.
References Beller, R.J., Davis, B.D. : Selective dissociation of free ribosomes of Escherichia coli by sodium ions. J. molec. Biol. 55, 477 485 (1971) Croes, A.F.: Induction of meiosis in baker's yeast. I. Timing of cytological and biochemical events. Planta (Berl.) 76, 209226 (1967)
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Communicated by F. Kaudewitz Received August 16, 1977