Mechanisms of Ageing and Development, 64 (1992) 235-245
235
Elsevier ScientificPublishers Ireland Ltd.
THE EFFECT OF AGING ON PROTEIN SYNTHESIS IN THE YEAST SACCHAROMYCES CEREVISIAE
MITSUYOSHI MOTIZUKI and KUNIO TSURUGI Department of Biochemistry, Yamanashi Medical College, 1110 Shimokato, Tamaho, Nakakoma, Yamanashi 409-38 (Japan)
(Received September 10th, 1991) (Revision receivedJanuary 22nd, 1992)
SUMMARY The protein synthetic rate in the yeast S. cerevisiae, measured by the incorporation of radioactive amino acids per unit amount of proteins, decreased linearly with age reaching 50% of the rate of 2nd generation cells (young cells) in 20th generation cells (old cells), whereas the RNA content of the old cells was increased three times. Using a cell-free system for poly(U)-directed poly-phenylalanine synthesis, the activity of run-off ribosomes from old cells was shown to be about 40% less than the activity of ribosomes from young cells and the polysome level in old cells was much decreased compared to that in young cells. However, as protein content was increased twice in 20 generations, the cell is considered to maintain a constant level of protein synthesis during the process of aging compensating the decrease in the activity of ribosomes. Thus, it is likely that the decrease in the synthesis of certain proteins whose requirement was raised by the increase in cell volume, which is twice the increase in protein content, causes prolongation of the unbudded phase in old cells.
Key words: Protein synthesis; Yeast; Aging; Cell size; Polysome
INTRODUCTION The budding yeast S. cerevisiae possesses a limited lifespan and the cells increase in cell size and in generation time with age [1]. Recently, Hartwell and Unger reported that these age-related phenomena were observed in yeast cells which were Correspondence to: Kunio Tsurugi, Department of Biochemistry,Yamanashi Medical College, Ill0 Shimokato, Tamaho, Nakakoma, Yamanashi409-38, Japan.
0047-6374/92/$05.00 © 1992ElsevierScientificPublishers Ireland Ltd. Printed and Publishedin Ireland
236
treated with a protein synthesis inhibitor, cycloheximide [2], and suggested that cell growth promoted by protein synthesis and not the events of the DNA division cycle are rate limiting for cellular proliferation. A similar result was obtained by Tyson et al. [3] where the cell growth was controlled by a wide range of different media. These results presented a possibility that the age-related phenomena are caused, in part, by a decline in the activity of protein synthesis. However, the age-related change of protein synthesis in yeast has not been investigated in detail, although a variety of such studies have been reported using higher organisms (for a review, see Ref. 4). In this communication we have measured the activity of protein synthesis in yeast cells of various generations and shown that the yeast cells decrease in protein synthetic activity mainly due to the decrease in the activity of ribosomes. MATERIALS AND METHODS
Materials Yeast Saccharomyces cerevisiae, strain X2180-1A (MATa, suc2, mal, reel, gal2, cupl) [5], was obtained from American Type Culture Collection (ATCC) and used throughout the experiments, tRNA-Phe and poly(U) were purchased from Boehringer Mannheim, Germany and [3H]leucine (120 Ci/mmol) and [3Hlphenylalanine (100 Ci/mmol) were obtained from Amersham, England. Isolation of aged cells Aged yeast cells were prepared according to the method of Egilmetz et al. [1]. Briefly, virgin cells were prepared from a culture grown to a stationary phase by a 10-30% sucrose gradient centrifugation. Then, the growth of virgin cells were synchronized with or-factor and 2nd generation ceils were isolated by centrifugation. The cells of the 2nd generation were grown in YPDG (2% peptone, 1% yeast extract, 0.04% glucose, 1.6% glycerol) and the cells of the 4th generation were isolated. Cells of the desired generations were prepared by repeating these procedures. If small cells were found in the preparation of aged cells, they were removed by centrifugation at 3000 rev./min for 1 min [6]. Determination of generation time and unbudded period Yeast cells were suspended (I × 107 cells/ml) in YPDG medium and the suspension was shaken at 30°C. A 0.l-ml sample was removed at I0 rain intervals and the numbers of total cells and budding cells were counted microscopically. The cell cycle parameters were determined as described in the legends for Fig. 2. Determination of protein synthetic activity of yeast in vivo Yeast cells were suspended in a medium containing yeast nitrogen base (6.7 g/l) and glucose at a final concentration of 4 x 107 cells/ml and cultured at 30°C. At 10-rain intervals, a 0,1-ml sample was removed, added to 1 /zCi [3H]leucine and
237
incubated at 30°C for 10 rain. Then, cells were precipitated, washed and added to TCA at a final concentration of 10%. The radioactivities of the TCA-soluble and insoluble fractions were determined. The rate of protein synthesis was determined by dividing the specific radioactivity per mg protein of the TCA-insoluble fraction by that of the TCA-soluble fraction to rule out the effect of age-related change in the permeability of the amino acid [7]. To determine the extent of the intracellular leucine pool the yeast cells cultured in yeast nitrogen base were harvested and suspended in 5% sulfosalicylic acid. Cells were lysed by a freeze-and-thawing process, performed 4 times after which the supernatant was applied to an amino acid analyzer (Hitachi L8000-500, Japan).
Cell-free protein synthesis The activity of protein synthesis in vitro was determined by poly(U)-directed polyphenylalanine synthesis using a system consisting of run-off ribosomes, cytosol (post-ribosomal supernatant) and [3H]phe-tRNA. Ribosomes were prepared as described previously [8] and run off by treatment with high concentrations of KCI and puromycin, according to the method of Blobel and Sabatini [9]. Run-off ribosomes were obtained after a centrifugation at 105 000 x g for 14 h in a discontinuous gradient of 0.7 M and 1.5 M sucrose. The amount of ribosomes suspended in a standard buffer [8] was calculated using absorption at 260 rim, and then stored at -80°C. To examine the purity of the ribosome preparation, RNA was extracted with phenol and applied to 3.5% polyacrylamide gel electrophoresis containing formamide. Gel was stained with pyronin Y and scanned with a densitometer. Charging of tRNA with [3I-I]phenylalanine was carried out using partially purified aminoacyl-tRNA synthetase [10]. Poly(U)-directed poly-phe synthesis was performed at 30°C in a 0.1-ml reaction mixture containing 50 mM Tris-acetate (pH 6.5), 100 mM ammonium acetate, 2 mM GTP, 30 #g of poly(U), 10 mM magnesium acetate, 100#g of yeast extract, 0.5-1 A260 units of run-off ribosomes and 30 pmol of [3H]phe-tRNA (1.4 x 10 4 dpm/6 pmol phe per #g tRNA). The radioactivity incorporated into the labeled TCA-insoluble fraction was measured.
Polysome pattern Post-mitochondrial supernatant (PMS) fraction was prepared according to the method of Gritz et al. [11] with a modification using 200 #g/ml heparin as an RNase inhibitor. A 10-40-#1 quantity of the fraction was applied to a 10-30% sucrose linear gradient and centrifuged at 40 000 rev./min for 1 h using a SW50-1 rotor (Beckman Corp.). The polysome pattern was monitored at 255 nm with a density fractionator (ISCO Co.).
Determination of protein and RNA The amount of proteins was determined by the method of Lowry et al. [12] with bovine serum albumin as standard, and that of RNA was determined by the method of Freck and Monro [13].
238 RESULTS
The age-related changes in protein and RNA contents and in cell cycle parameters in the yeast strain X2180-1A The protein and RNA contents of cells of various generations were determined for the strain used in these experiments (X2180-1A) whose mean life-span was determined to be 24 generations [5] (Fig. 1). Generally, the protein and RNA contents increased linearly with age; during 20 generation times the protein content was doubled and the RNA content increased three and a half times. As most of the RNA is ribosomal (rRNA) in yeast, the ribosome content is considered to increase approximately threefold during aging. Interestingly, the increase in the cell volume (4.3 times during 20 generation times) was much greater than the total increase of protein and RNA contents (about 2.5 times). Then, cell cycle parameters of cells of various generations were determined (Fig. 2) and, as expected, the duration of the unbudded
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AGE (GENERATIONS) Fig. 1. The age-related changes in cell components of S. cerevisiae strain X2180- I A [5]. Cells of various generations were prepared as described in Materials and Methods and the contents of DNA, RNA, protein and cell volume were determined. The increases of RNA (O) and protein (Q) contents were expressed presuming that the DNA contents in cells of all generations are constant and the increase of cell volume (X) was expressed taking the cell size of 2rid generation cells as 1. The contents of the materials in 2nd generation cells were; DNA, 1.0 t~g/108 cells; RNA, 67 #g/108 cells; protein, 134 t~g/108 cells; cell volume, 60/~m 3.
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Fig. 2. The age-related change of cell cycle parameters. (a) The ratio of budding to non-budding cells determined microscopically at a 10-rain interval during incubation at 30°C. Only the first and second buddings from the parent cells were counted eliminating the budding from the first buds which were discriminated from the parent cells by cell size. The generation time was determined to be the interval between the mean time of the appearance of the first bud (T1) and that of the second bud (T3). The budded period was calculated by subtracting T1 from the mean time of the separation of the first bud (T2) and the unbudded period was determined by subtracting the budded periods from the generation time. (b) The changes of the generation time as a function of aging: (0), generation time; (0), budded phase and (X), unbudded phase.
240
phase was shown to be extended with age while the duration of the budded phase was not changed in agreement with the results of other investigators [14,15].
Effect of aging on the rate of protein synthesis in vivo To examine the age-related change of protein synthesis in yeast, the incorporation rate of [3H]leucine into proteins was determined at 30 min of incubation. A 30-min time point was used because the incorporation rate was found to increase during the first 20 min of incubation at different rates among cells of various generations; we speculate that this is due to incomplete equilibration of amino acids between cells and the medium (data not shown). The intracellular leucine pool was determined to be nearly constant during aging showing about 1.5 nmol/mg protein. However, the incorporation rate of [3H]leucine into protein was corrected by the specific radioactivity of the TCA-soluble fraction to eliminate the effect of age-related change in the permeability of amino acids [7]. The incorporation rate, that is, the protein synthetic activity in vivo, decreased linearly with age; the activity in the 20th generation cells (old cells) was 50% less than that of 2nd generation cells (young cells) (Fig. 3). As the cell protein doubles during 20 generation times, it was estimated that the total protein synthesis per whole cell remains nearly constant with age in spite of a t/3 ¢.0 I,I -rI-Z >-
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Fig. 3. The age-related changes in protein synthesis in vivo measured by the incorporation of radioactive amino acid into protein. Cells of various generations were preincubated at 30°C for 30 min, added with 1 t~Ci [3H]leucine and incubated for 10 rain. The radioactivities incorporated into mg protein of the TCA-insoluble fraction were determined and corrected by the specific radioactivity of the TCA-soluble fraction [7]. The relative protein synthetic activity was expressed taking that of 2nd generation cells as 1.
241
3.5-fold increase of RNA. This result suggested that the activity and/or recruitment rather than the content of ribosomes had declined in older cells.
The age-related change of protein synthetic activity of ribosomes in vitro The activity of ribosomes was determined in vitro using a cell-free system for poly(U)-directed poly-phenylalanine synthesis consisting of run-off ribosomes, cytosol and [3H]phe-tRNA (Fig. 4). The translational activity of the system from the old cells was 40% less than that from the young cells and this decrease was considered to be caused by run-off ribosomes as the exchange of cytosols from either young or old cells did not affect the translational activity. It should be added that RNAs extracted from run-off ribosomes from the young and old cells showed the same patterns with equivalent ratios of 17S and 26S rRNAs on polyacrylamide gel electrophoresis indicating that there are no significant differences in the purity of ribosomes between them (data not shown). Thus, it is suggested that the activity of
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TIME ( MIN ) Fig. 4. The age-related change in poly(U)-directed poly-phenylalanine synthesis. The protein synthesis activity was determined in vitro using a cell-free system consisting of run-off ribosomes and cell extract from the young and old cells. Poly-phe synthesis was performed at 30°C with [3H]phe-tRNA and the radioactivity incorporated into the hot TCA-insoluble fraction was measured at 5-min intervals. Time courses of cell-free systems consisting of: (O), young cell ribosomes and young cell extract; (0), young cell ribosomes and old cell extract; (El), old cell ribosomes and old cell extract; (ll), old cell ribosomes and young cell extract.
242 ribosomes is decreased in old yeast cells at least at the elongation level of translation, as the poly(U)-directed poly-phe synthesis is carried out only by an elongation reaction.
The change of polysome pattern with age To examine whether the recruitment of ribosomes in the cytoplasm is affected in vivo, the polysome pattern was compared between the young and old cells (Fig. 5). Polysome was prepared using heparin as an RNase inhibitor and polysome patterns from both cells were obtained reproducibly. The polysome patterns show apparently that the proportion of polysomes, especially that of large polysomes, was significantly decreased; the ratios of polysome to monosome levels were calculated to be 4 and I in young cells and old cells, respectively. The absolute amount of polysomes was not calculated because the cell's sensitivity to mechanical disruption was markedly different between the young and old cells, probably due to the difference in cell size. However, this result indicates that the recruitment rate of ribosomes was decreased in old cells resulting in substantial decrease of the polysome level although it does not necessarily mean that the resting ribosomes are dysfunctional.
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"X. Fig. 5. The age-related changes in polysome patterns. Polysome patterns of the young (solid line) and old (broken line) cells were obtained by centrifugation of the post-mitochondrial supernatant fractions on 10-30% sucrose linear gradient at 40 000 × g, for I h. Polysomepattern was monitored with a density fractionator (ISCO Co.) and the proportion of monosome and polysome fractions were estimated from their peak area. The proportions of monomers of age 4 and age 20 cells were determined to be 21% and 50%, respectively, from three separate experiments.
243
Changes in protein synthesis during the cell cycle Previously, Elliot and McLaughlin reported that protein synthesis of yeast increases exponentially as the cells grow exponentially [16]. It is possible, however, that the protein synthesis in old cells does not increase exponentially as ribosome activity decreases and a large proportion of ribosomes are kept at rest. However, the incorporation rate of [3H]leucine did not change during the cell cycle in either the young or old cells (Fig. 6), an indication that the protein synthesis is continued exponentially in either old or young cells. The result also indicated that the prolongation of the unbudded phase is not caused by a phase-specific decrease of protein synthesis in old cells.
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TIME ( MIN ) Fig. 6. The rate of protein synthesis during cell cycle in the young and old cells. Cells were grown as described in the legend for Fig. 2 and the rate of protein synthesis was determined as described in those for Fig. 3. (O) and (0), relative rate of protein synthesis of young and old cells, respectively, taking the maximal rate as 1. (X), percentage of budding cells.
244 DISCUSSION
In this communication we show that protein synthesis decreases with age in the yeast S. cerevisiae by in vivo and in vitro experiments. In old cells (20th generation cells) the incorporation rate of labeled amino acid into proteins was half the rate of young cells (2nd generation cells) while the ribosome content was estimated to be much increased in the old cells. The age-related decline in protein synthesis despite the increase in ribosome content was shown to be caused partly by the decline in the activity of ribosomes as shown by poly(U)-directed poly-phenylalanine synthesis. The result showing the age-related decrease in ribosome activity is consistent with the results using a cell-free system from Drosophila melanogaster [17], nematode [18] and rat [19] although a contradictory result was reported using rat liver ribosomes [20]. Furthermore, the lack of an effect due to an exchange of cell extracts between young and old yeast in a cell-free protein synthesis system, is consistent with the results from similar experiments using rat [19] and D. melanogaster [21]. However, as run-off ribosomes are known to bind some translation factors, it is still possible that some translational factors are limiting in old cells. It is important to point out that, although protein synthesis measured by leucine incorporation into unit amounts of protein was decreased by half during 20 generation times, protein synthesis per cell remains constant with age as protein content was doubled during 20 generation times. Thus, the cell appears to try and succeed to maintain a constant level of protein synthesis in the face of some process that is damaging its ribosomes and/or translational factors during aging. We have also shown that the unbudded phase was extended with age, consistent with the result of Egilmez et al. [1]. As the G1 phase has been shown to be the most variable phase in the cell cycle [14], it is probably the G1 phase that is extended with age in yeast cells. Considered together with the result by Hartwell and Unger [2] showing that the treatment with a protein synthesis inhibitor, cycloheximide, resulted in the elongation of the unbudded phase, our results suggested that the elongation of the generation time found in old cells is caused, at least in part, by the decrease in protein synthetic activity. As it has been known that some specific proteins such as cyclins are required for the GI to S-phase transmission [22], the decline in synthesis of these proteins in old cells might cause the age-related extension of generation time. However, as protein synthesis per cell is constant during aging it is more likely that the decrease in the synthesis of certain proteins whose requirement was raised by the increase in cell volume causes prolongation of the unbudded phase in old cells. REFERENCES 1 N.K. Egilmez, J.B. Chen and S.M. Jazwinski, Preparation and partial characterization of old yeast cells. J. Gerontol., 45 (1990) B9-17. 2 L.H. Hartwell and M.W. Unger, Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division. J. Cell Biol., 75 (1977) 422-435.
245 3 4 5 6 7 8 9 10 1!
12 13 14 15 16 17 18 19 20 21
22
C.B. Tyson, P.G. Lord and A.E. Wheals, Dependency of size of Saccharomyces cerevisiae cells on growth rate. J. Bacteriol., 138 (1979) 92-98. S.C. Makrides, Protein synthesis and degradation during aging and senescence. Biol. Rev., 58 (1983) 343 -422. N.K. Egilmez and S.M. Jazwinski, Evidence for the involvement of a cytoplasmic factor in the aging of the yeast Saccharomyces cerevisiae. J. Bacteriol., 171 (1989) 37-42. M. Hayashibe and N. Sando, Characterization of different sized cells of baker's yeast. J. Gen. Appl. Microbiol., 16 (1970) 15-27. D. Gottlieb, P. Molitoris and J.L. van Etten, Changes in fungi with age III. Incorporation of amino acids into cells of Rhizoctoria solani and Scleritiun bataticola. Arch. Mikrobiol., 61 (1968) 394-398. C.F. Heredia and H.O. Halverson, Transfer of amino acids from amino acyl soluble ribonucleic acid to protein by cell-free extracts from yeast. Biochemistry, 5 (1966) 946-951. G. Blobel and D. Sabatini, Dissociation of mammalian polysomes into subunits by puromycin. Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 390-394. P. Berg, F.H. Bergrnann, E.J. Ofengang and M. Dieckmann, The enzymic synthesis of amino acyl derivatives of ribonucleic acid. J. Biol. Chem., 236 (1961) 1726-1734. L. Gritz, N. Abovich, J.L. Teem and M, Rosbash, Posttranscriptional regulation and assembly into ribosomes of a Saccharomyces cerevisiae ribosomal protein-B-galactosidase fusion. Mol. Cell. Biol., 5 (1985) 3436-3442. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent. J. Biol. Chem., 193 (1951) 265-275. A. Freck and H.N. Munro, The precision of ultraviolet absorption measurements in the SchmidtThannhauser procedure for nucleic acid estimation. Biochim. Biophys. Acta, 55 (1962) 571-583. M.L. Slater, S.O. Sharrow and J.J. Gart, Cell cycle of Saccharomyces cerevisiae in populations growing at different rates. Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3850-3854. G.L. Grove and V.J. Cristofolo, Characterization of the cell cycle of cultured human diploid cells: effects of aging and hydrocortisone. J. Cell. Physiol., 90 (1976) 415-422. S.G. Elliot and C.S. McLaughlin, Rate of macromolecular synthesis through the cell cycle of the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 4384-4388. G.C. Webster and S.L. Webster, Effects of age on the post-initiation stages of protein synthesis. Mech. Ageing Dev., 18 (1982) 369-378. N.K. Egilmez and M. Rothstein, The effect of aging on cell-free protein synthesis in the free-living nematode Turbatrix aceti. Biochim. Biophys. Acta, 840 (1985) 355-363. H.T. Sojor and M. Rothstein, Protein synthesis by liver ribosomes from aged rats. Mech. Ageing Dev., 35 (1986) 47-57. M. Laughrea and J. Latulippe, The poly(U) translational capacity of Fischer 344 rat liver does not deteriorate with age and is not affected by dietary regime. Mech. Ageing Dev., 45 (1988) 137-143. G.C. Webster and S.L. Webster, Decline in synthesis of elongation factor one (EF-I) precedes the decreased synthesis of total protein in aging Drosophila melanogaster. Mech. Ageing Dev., 22 (1983) 121-128. L. Dirick and K. Nasmyth, Positive feedback in the activation of GI cyclins in yeast. Nature, 351 (1991) 754-757.
235
Elsevier ScientificPublishers Ireland Ltd.
THE EFFECT OF AGING ON PROTEIN SYNTHESIS IN THE YEAST SACCHAROMYCES CEREVISIAE
MITSUYOSHI MOTIZUKI and KUNIO TSURUGI Department of Biochemistry, Yamanashi Medical College, 1110 Shimokato, Tamaho, Nakakoma, Yamanashi 409-38 (Japan)
(Received September 10th, 1991) (Revision receivedJanuary 22nd, 1992)
SUMMARY The protein synthetic rate in the yeast S. cerevisiae, measured by the incorporation of radioactive amino acids per unit amount of proteins, decreased linearly with age reaching 50% of the rate of 2nd generation cells (young cells) in 20th generation cells (old cells), whereas the RNA content of the old cells was increased three times. Using a cell-free system for poly(U)-directed poly-phenylalanine synthesis, the activity of run-off ribosomes from old cells was shown to be about 40% less than the activity of ribosomes from young cells and the polysome level in old cells was much decreased compared to that in young cells. However, as protein content was increased twice in 20 generations, the cell is considered to maintain a constant level of protein synthesis during the process of aging compensating the decrease in the activity of ribosomes. Thus, it is likely that the decrease in the synthesis of certain proteins whose requirement was raised by the increase in cell volume, which is twice the increase in protein content, causes prolongation of the unbudded phase in old cells.
Key words: Protein synthesis; Yeast; Aging; Cell size; Polysome
INTRODUCTION The budding yeast S. cerevisiae possesses a limited lifespan and the cells increase in cell size and in generation time with age [1]. Recently, Hartwell and Unger reported that these age-related phenomena were observed in yeast cells which were Correspondence to: Kunio Tsurugi, Department of Biochemistry,Yamanashi Medical College, Ill0 Shimokato, Tamaho, Nakakoma, Yamanashi409-38, Japan.
0047-6374/92/$05.00 © 1992ElsevierScientificPublishers Ireland Ltd. Printed and Publishedin Ireland
236
treated with a protein synthesis inhibitor, cycloheximide [2], and suggested that cell growth promoted by protein synthesis and not the events of the DNA division cycle are rate limiting for cellular proliferation. A similar result was obtained by Tyson et al. [3] where the cell growth was controlled by a wide range of different media. These results presented a possibility that the age-related phenomena are caused, in part, by a decline in the activity of protein synthesis. However, the age-related change of protein synthesis in yeast has not been investigated in detail, although a variety of such studies have been reported using higher organisms (for a review, see Ref. 4). In this communication we have measured the activity of protein synthesis in yeast cells of various generations and shown that the yeast cells decrease in protein synthetic activity mainly due to the decrease in the activity of ribosomes. MATERIALS AND METHODS
Materials Yeast Saccharomyces cerevisiae, strain X2180-1A (MATa, suc2, mal, reel, gal2, cupl) [5], was obtained from American Type Culture Collection (ATCC) and used throughout the experiments, tRNA-Phe and poly(U) were purchased from Boehringer Mannheim, Germany and [3H]leucine (120 Ci/mmol) and [3Hlphenylalanine (100 Ci/mmol) were obtained from Amersham, England. Isolation of aged cells Aged yeast cells were prepared according to the method of Egilmetz et al. [1]. Briefly, virgin cells were prepared from a culture grown to a stationary phase by a 10-30% sucrose gradient centrifugation. Then, the growth of virgin cells were synchronized with or-factor and 2nd generation ceils were isolated by centrifugation. The cells of the 2nd generation were grown in YPDG (2% peptone, 1% yeast extract, 0.04% glucose, 1.6% glycerol) and the cells of the 4th generation were isolated. Cells of the desired generations were prepared by repeating these procedures. If small cells were found in the preparation of aged cells, they were removed by centrifugation at 3000 rev./min for 1 min [6]. Determination of generation time and unbudded period Yeast cells were suspended (I × 107 cells/ml) in YPDG medium and the suspension was shaken at 30°C. A 0.l-ml sample was removed at I0 rain intervals and the numbers of total cells and budding cells were counted microscopically. The cell cycle parameters were determined as described in the legends for Fig. 2. Determination of protein synthetic activity of yeast in vivo Yeast cells were suspended in a medium containing yeast nitrogen base (6.7 g/l) and glucose at a final concentration of 4 x 107 cells/ml and cultured at 30°C. At 10-rain intervals, a 0,1-ml sample was removed, added to 1 /zCi [3H]leucine and
237
incubated at 30°C for 10 rain. Then, cells were precipitated, washed and added to TCA at a final concentration of 10%. The radioactivities of the TCA-soluble and insoluble fractions were determined. The rate of protein synthesis was determined by dividing the specific radioactivity per mg protein of the TCA-insoluble fraction by that of the TCA-soluble fraction to rule out the effect of age-related change in the permeability of the amino acid [7]. To determine the extent of the intracellular leucine pool the yeast cells cultured in yeast nitrogen base were harvested and suspended in 5% sulfosalicylic acid. Cells were lysed by a freeze-and-thawing process, performed 4 times after which the supernatant was applied to an amino acid analyzer (Hitachi L8000-500, Japan).
Cell-free protein synthesis The activity of protein synthesis in vitro was determined by poly(U)-directed polyphenylalanine synthesis using a system consisting of run-off ribosomes, cytosol (post-ribosomal supernatant) and [3H]phe-tRNA. Ribosomes were prepared as described previously [8] and run off by treatment with high concentrations of KCI and puromycin, according to the method of Blobel and Sabatini [9]. Run-off ribosomes were obtained after a centrifugation at 105 000 x g for 14 h in a discontinuous gradient of 0.7 M and 1.5 M sucrose. The amount of ribosomes suspended in a standard buffer [8] was calculated using absorption at 260 rim, and then stored at -80°C. To examine the purity of the ribosome preparation, RNA was extracted with phenol and applied to 3.5% polyacrylamide gel electrophoresis containing formamide. Gel was stained with pyronin Y and scanned with a densitometer. Charging of tRNA with [3I-I]phenylalanine was carried out using partially purified aminoacyl-tRNA synthetase [10]. Poly(U)-directed poly-phe synthesis was performed at 30°C in a 0.1-ml reaction mixture containing 50 mM Tris-acetate (pH 6.5), 100 mM ammonium acetate, 2 mM GTP, 30 #g of poly(U), 10 mM magnesium acetate, 100#g of yeast extract, 0.5-1 A260 units of run-off ribosomes and 30 pmol of [3H]phe-tRNA (1.4 x 10 4 dpm/6 pmol phe per #g tRNA). The radioactivity incorporated into the labeled TCA-insoluble fraction was measured.
Polysome pattern Post-mitochondrial supernatant (PMS) fraction was prepared according to the method of Gritz et al. [11] with a modification using 200 #g/ml heparin as an RNase inhibitor. A 10-40-#1 quantity of the fraction was applied to a 10-30% sucrose linear gradient and centrifuged at 40 000 rev./min for 1 h using a SW50-1 rotor (Beckman Corp.). The polysome pattern was monitored at 255 nm with a density fractionator (ISCO Co.).
Determination of protein and RNA The amount of proteins was determined by the method of Lowry et al. [12] with bovine serum albumin as standard, and that of RNA was determined by the method of Freck and Monro [13].
238 RESULTS
The age-related changes in protein and RNA contents and in cell cycle parameters in the yeast strain X2180-1A The protein and RNA contents of cells of various generations were determined for the strain used in these experiments (X2180-1A) whose mean life-span was determined to be 24 generations [5] (Fig. 1). Generally, the protein and RNA contents increased linearly with age; during 20 generation times the protein content was doubled and the RNA content increased three and a half times. As most of the RNA is ribosomal (rRNA) in yeast, the ribosome content is considered to increase approximately threefold during aging. Interestingly, the increase in the cell volume (4.3 times during 20 generation times) was much greater than the total increase of protein and RNA contents (about 2.5 times). Then, cell cycle parameters of cells of various generations were determined (Fig. 2) and, as expected, the duration of the unbudded
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Fig. 2. The age-related change of cell cycle parameters. (a) The ratio of budding to non-budding cells determined microscopically at a 10-rain interval during incubation at 30°C. Only the first and second buddings from the parent cells were counted eliminating the budding from the first buds which were discriminated from the parent cells by cell size. The generation time was determined to be the interval between the mean time of the appearance of the first bud (T1) and that of the second bud (T3). The budded period was calculated by subtracting T1 from the mean time of the separation of the first bud (T2) and the unbudded period was determined by subtracting the budded periods from the generation time. (b) The changes of the generation time as a function of aging: (0), generation time; (0), budded phase and (X), unbudded phase.
240
phase was shown to be extended with age while the duration of the budded phase was not changed in agreement with the results of other investigators [14,15].
Effect of aging on the rate of protein synthesis in vivo To examine the age-related change of protein synthesis in yeast, the incorporation rate of [3H]leucine into proteins was determined at 30 min of incubation. A 30-min time point was used because the incorporation rate was found to increase during the first 20 min of incubation at different rates among cells of various generations; we speculate that this is due to incomplete equilibration of amino acids between cells and the medium (data not shown). The intracellular leucine pool was determined to be nearly constant during aging showing about 1.5 nmol/mg protein. However, the incorporation rate of [3H]leucine into protein was corrected by the specific radioactivity of the TCA-soluble fraction to eliminate the effect of age-related change in the permeability of amino acids [7]. The incorporation rate, that is, the protein synthetic activity in vivo, decreased linearly with age; the activity in the 20th generation cells (old cells) was 50% less than that of 2nd generation cells (young cells) (Fig. 3). As the cell protein doubles during 20 generation times, it was estimated that the total protein synthesis per whole cell remains nearly constant with age in spite of a t/3 ¢.0 I,I -rI-Z >-
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Fig. 3. The age-related changes in protein synthesis in vivo measured by the incorporation of radioactive amino acid into protein. Cells of various generations were preincubated at 30°C for 30 min, added with 1 t~Ci [3H]leucine and incubated for 10 rain. The radioactivities incorporated into mg protein of the TCA-insoluble fraction were determined and corrected by the specific radioactivity of the TCA-soluble fraction [7]. The relative protein synthetic activity was expressed taking that of 2nd generation cells as 1.
241
3.5-fold increase of RNA. This result suggested that the activity and/or recruitment rather than the content of ribosomes had declined in older cells.
The age-related change of protein synthetic activity of ribosomes in vitro The activity of ribosomes was determined in vitro using a cell-free system for poly(U)-directed poly-phenylalanine synthesis consisting of run-off ribosomes, cytosol and [3H]phe-tRNA (Fig. 4). The translational activity of the system from the old cells was 40% less than that from the young cells and this decrease was considered to be caused by run-off ribosomes as the exchange of cytosols from either young or old cells did not affect the translational activity. It should be added that RNAs extracted from run-off ribosomes from the young and old cells showed the same patterns with equivalent ratios of 17S and 26S rRNAs on polyacrylamide gel electrophoresis indicating that there are no significant differences in the purity of ribosomes between them (data not shown). Thus, it is suggested that the activity of
2
o
5
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15
TIME ( MIN ) Fig. 4. The age-related change in poly(U)-directed poly-phenylalanine synthesis. The protein synthesis activity was determined in vitro using a cell-free system consisting of run-off ribosomes and cell extract from the young and old cells. Poly-phe synthesis was performed at 30°C with [3H]phe-tRNA and the radioactivity incorporated into the hot TCA-insoluble fraction was measured at 5-min intervals. Time courses of cell-free systems consisting of: (O), young cell ribosomes and young cell extract; (0), young cell ribosomes and old cell extract; (El), old cell ribosomes and old cell extract; (ll), old cell ribosomes and young cell extract.
242 ribosomes is decreased in old yeast cells at least at the elongation level of translation, as the poly(U)-directed poly-phe synthesis is carried out only by an elongation reaction.
The change of polysome pattern with age To examine whether the recruitment of ribosomes in the cytoplasm is affected in vivo, the polysome pattern was compared between the young and old cells (Fig. 5). Polysome was prepared using heparin as an RNase inhibitor and polysome patterns from both cells were obtained reproducibly. The polysome patterns show apparently that the proportion of polysomes, especially that of large polysomes, was significantly decreased; the ratios of polysome to monosome levels were calculated to be 4 and I in young cells and old cells, respectively. The absolute amount of polysomes was not calculated because the cell's sensitivity to mechanical disruption was markedly different between the young and old cells, probably due to the difference in cell size. However, this result indicates that the recruitment rate of ribosomes was decreased in old cells resulting in substantial decrease of the polysome level although it does not necessarily mean that the resting ribosomes are dysfunctional.
80S
$
"X. Fig. 5. The age-related changes in polysome patterns. Polysome patterns of the young (solid line) and old (broken line) cells were obtained by centrifugation of the post-mitochondrial supernatant fractions on 10-30% sucrose linear gradient at 40 000 × g, for I h. Polysomepattern was monitored with a density fractionator (ISCO Co.) and the proportion of monosome and polysome fractions were estimated from their peak area. The proportions of monomers of age 4 and age 20 cells were determined to be 21% and 50%, respectively, from three separate experiments.
243
Changes in protein synthesis during the cell cycle Previously, Elliot and McLaughlin reported that protein synthesis of yeast increases exponentially as the cells grow exponentially [16]. It is possible, however, that the protein synthesis in old cells does not increase exponentially as ribosome activity decreases and a large proportion of ribosomes are kept at rest. However, the incorporation rate of [3H]leucine did not change during the cell cycle in either the young or old cells (Fig. 6), an indication that the protein synthesis is continued exponentially in either old or young cells. The result also indicated that the prolongation of the unbudded phase is not caused by a phase-specific decrease of protein synthesis in old cells.
1.0 w -r' I-,Z >.-
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0.5
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TIME ( MIN ) Fig. 6. The rate of protein synthesis during cell cycle in the young and old cells. Cells were grown as described in the legend for Fig. 2 and the rate of protein synthesis was determined as described in those for Fig. 3. (O) and (0), relative rate of protein synthesis of young and old cells, respectively, taking the maximal rate as 1. (X), percentage of budding cells.
244 DISCUSSION
In this communication we show that protein synthesis decreases with age in the yeast S. cerevisiae by in vivo and in vitro experiments. In old cells (20th generation cells) the incorporation rate of labeled amino acid into proteins was half the rate of young cells (2nd generation cells) while the ribosome content was estimated to be much increased in the old cells. The age-related decline in protein synthesis despite the increase in ribosome content was shown to be caused partly by the decline in the activity of ribosomes as shown by poly(U)-directed poly-phenylalanine synthesis. The result showing the age-related decrease in ribosome activity is consistent with the results using a cell-free system from Drosophila melanogaster [17], nematode [18] and rat [19] although a contradictory result was reported using rat liver ribosomes [20]. Furthermore, the lack of an effect due to an exchange of cell extracts between young and old yeast in a cell-free protein synthesis system, is consistent with the results from similar experiments using rat [19] and D. melanogaster [21]. However, as run-off ribosomes are known to bind some translation factors, it is still possible that some translational factors are limiting in old cells. It is important to point out that, although protein synthesis measured by leucine incorporation into unit amounts of protein was decreased by half during 20 generation times, protein synthesis per cell remains constant with age as protein content was doubled during 20 generation times. Thus, the cell appears to try and succeed to maintain a constant level of protein synthesis in the face of some process that is damaging its ribosomes and/or translational factors during aging. We have also shown that the unbudded phase was extended with age, consistent with the result of Egilmez et al. [1]. As the G1 phase has been shown to be the most variable phase in the cell cycle [14], it is probably the G1 phase that is extended with age in yeast cells. Considered together with the result by Hartwell and Unger [2] showing that the treatment with a protein synthesis inhibitor, cycloheximide, resulted in the elongation of the unbudded phase, our results suggested that the elongation of the generation time found in old cells is caused, at least in part, by the decrease in protein synthetic activity. As it has been known that some specific proteins such as cyclins are required for the GI to S-phase transmission [22], the decline in synthesis of these proteins in old cells might cause the age-related extension of generation time. However, as protein synthesis per cell is constant during aging it is more likely that the decrease in the synthesis of certain proteins whose requirement was raised by the increase in cell volume causes prolongation of the unbudded phase in old cells. REFERENCES 1 N.K. Egilmez, J.B. Chen and S.M. Jazwinski, Preparation and partial characterization of old yeast cells. J. Gerontol., 45 (1990) B9-17. 2 L.H. Hartwell and M.W. Unger, Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division. J. Cell Biol., 75 (1977) 422-435.
245 3 4 5 6 7 8 9 10 1!
12 13 14 15 16 17 18 19 20 21
22
C.B. Tyson, P.G. Lord and A.E. Wheals, Dependency of size of Saccharomyces cerevisiae cells on growth rate. J. Bacteriol., 138 (1979) 92-98. S.C. Makrides, Protein synthesis and degradation during aging and senescence. Biol. Rev., 58 (1983) 343 -422. N.K. Egilmez and S.M. Jazwinski, Evidence for the involvement of a cytoplasmic factor in the aging of the yeast Saccharomyces cerevisiae. J. Bacteriol., 171 (1989) 37-42. M. Hayashibe and N. Sando, Characterization of different sized cells of baker's yeast. J. Gen. Appl. Microbiol., 16 (1970) 15-27. D. Gottlieb, P. Molitoris and J.L. van Etten, Changes in fungi with age III. Incorporation of amino acids into cells of Rhizoctoria solani and Scleritiun bataticola. Arch. Mikrobiol., 61 (1968) 394-398. C.F. Heredia and H.O. Halverson, Transfer of amino acids from amino acyl soluble ribonucleic acid to protein by cell-free extracts from yeast. Biochemistry, 5 (1966) 946-951. G. Blobel and D. Sabatini, Dissociation of mammalian polysomes into subunits by puromycin. Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 390-394. P. Berg, F.H. Bergrnann, E.J. Ofengang and M. Dieckmann, The enzymic synthesis of amino acyl derivatives of ribonucleic acid. J. Biol. Chem., 236 (1961) 1726-1734. L. Gritz, N. Abovich, J.L. Teem and M, Rosbash, Posttranscriptional regulation and assembly into ribosomes of a Saccharomyces cerevisiae ribosomal protein-B-galactosidase fusion. Mol. Cell. Biol., 5 (1985) 3436-3442. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent. J. Biol. Chem., 193 (1951) 265-275. A. Freck and H.N. Munro, The precision of ultraviolet absorption measurements in the SchmidtThannhauser procedure for nucleic acid estimation. Biochim. Biophys. Acta, 55 (1962) 571-583. M.L. Slater, S.O. Sharrow and J.J. Gart, Cell cycle of Saccharomyces cerevisiae in populations growing at different rates. Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3850-3854. G.L. Grove and V.J. Cristofolo, Characterization of the cell cycle of cultured human diploid cells: effects of aging and hydrocortisone. J. Cell. Physiol., 90 (1976) 415-422. S.G. Elliot and C.S. McLaughlin, Rate of macromolecular synthesis through the cell cycle of the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 4384-4388. G.C. Webster and S.L. Webster, Effects of age on the post-initiation stages of protein synthesis. Mech. Ageing Dev., 18 (1982) 369-378. N.K. Egilmez and M. Rothstein, The effect of aging on cell-free protein synthesis in the free-living nematode Turbatrix aceti. Biochim. Biophys. Acta, 840 (1985) 355-363. H.T. Sojor and M. Rothstein, Protein synthesis by liver ribosomes from aged rats. Mech. Ageing Dev., 35 (1986) 47-57. M. Laughrea and J. Latulippe, The poly(U) translational capacity of Fischer 344 rat liver does not deteriorate with age and is not affected by dietary regime. Mech. Ageing Dev., 45 (1988) 137-143. G.C. Webster and S.L. Webster, Decline in synthesis of elongation factor one (EF-I) precedes the decreased synthesis of total protein in aging Drosophila melanogaster. Mech. Ageing Dev., 22 (1983) 121-128. L. Dirick and K. Nasmyth, Positive feedback in the activation of GI cyclins in yeast. Nature, 351 (1991) 754-757.
