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Decline in synthesis of elongation factor one (EF-1) precedes the decreased synthesis of total protein in aging Drosophila melanogaster
Mechanisms of Ageing and Development, 22 (1983) 121-128 Elsevier ScientificPublishers Ireland Ltd.
121
DECLINE IN SYNTHESIS OF ELONGATION FACTOR ONE (EF-I) PRECEDES THE DECREASED SYNTHESIS OF TOTAL PROTEIN IN AGING DROSOPHILA MELANOGASTER
G.C. W E B S T E R and S.L. W E B S T E R
Depa~'~nent of Biological Sciences, Florida Institute of Technology, Melbourne, Florida32901 (U.S.A.) (Received August 26th, 19821 (Revision received December 14th, 1982) SUMMARY The decrease in the rate of protein synthesis in aging adult Drosophila melanogaster was found previously to be due, to a great extent, to a drop in the rate of peptide chain elongation, and principally to lowered activity of elongation factor one (EF-1). This decrease does not appear to be caused by appearance of an inhibitor of peptide chain elongation. Instead, the synthesis of EF-1 declines markedly early in adult life. This decrease is followed by lowered activity of EF-1 and by a drop in the synthesis of most of the cellular proteins.
Key words: Drosophila; Elongation factor; Protein; Synthesis; Translation
INTRODUCTION Aging in Drosophila melanogaster produces a significant decrease in the rate of protein synthesis [1--4]. This decrease appears to affect most of the cellular proteins rather uniformly [4], and is followed by deterioration of cellular structure and function [5-9]. In an effort to determine which part of the protein synthesis system is responsible for the decrease, the effect of age on the stages of translation was determined previously [10,11,13]. The rates of initiation (measured by the formation of the 40 S and 80 S initiation complexes) and termination (measured as the release of N-formylmethionine from ribosome-bound N-formylmethionyltRNA) did not decrease significantly during the time when total protein synthesis decreased approximately 70% [10,1 1]. The rate or the yield of aminoacylation of t R N A was lowered (up to 50%) for several amino acids [12,13]. However, the rates of aminoacylation far exceeded the rate of translation under similar conditions, so the significance of the reduced aminoacylation for the age-related decrease in translation is not clear [13]. In contrast, the rate of peptide chain elongation declined markedly with age, in parallel with the decrease in overall 1~147-6374/83/$3.110 Printed and Published in Ireland
© 1983 Elsevier ScientificPublishers Ireland Ltd.
122 translation [11]. This decrease was due principally to lowered binding of aminoacyl-tRNA to ribosomes, and specifically to decreased elongation factor 1 (EF-1) activity [ 11]. The decreased EF-1 activity could be due to the age-related formation of an inhibitor of peptide chain elongation, or to a cessation of synthesis of EF-I. As is reported in this paper, we have been unable to find evidence for the age-related appearance of an inhibitor of peptide chain elongation. However, we have found that the synthesis of EF-1 drops abruptly several days prior to the decrease in synthesis of most proteins. MATERIALS AND METHODS
Drosophila melanogaster, strain Oregon R, was grown as described previously [10], and had a mean life-span of 42 days. EF-1 was purified from Drosophila by the method of Pelley and Stafford [14], with a final separation by chromatography on Sephadex G-200 to remove any remaining lower molecular proteins from the m a j o r EF-1 peak. Peptide chain elongation was assayed by measuring the rate of polyuridylate-dependent incorporation of the phenylalanine of phenylalanylt R N A into polypeptide [14] in a reaction system consisting of: 0.05 M T E S (pH 7.6), 0.007 M MgCI2, 0.06M KCI, 0.002M dithiothreitol, 0.0005 M GTP, 0.Ill M phospho(enol)pyruvate, 0.25mg of polyuridylic acid, 400 A2~ units of [3H]phenylalanyl-tRNA, prepared as described previously [11], 0.5 mg of ribosomes, and 0.5 mg of cytosol [14] from 1-day-old adults in a total w)iumc of 0.5 ml. For m e a s u r e m e n t of EF-I synthesis by larvae, 7 5 # C i of a ~H-amino acid mixture was mixed with each 5 ml of culture medium in which the larvae were grown. EF-I synthesis in adults was measured by placing 50-100 adults of known age in a vial containing filter paper soaked with 50/zCi of the 3H-amino acid mixture per ml of 5% sucrose, containing 1 mg/ml yeast. Under these conditions, incorporation of radioactivity into the total protein fraction was linear for at least 24 h. After varying times of up to 24 h, the flies were removed from the vials, washed free of external radioactivity in ice-water, and homogenized as described previously [1l]. An aliquot of the supernatant fluid from the 2 5 0 0 0 g ccntrifugation step was treated as described [2] to measure the rate of overall protein synthesis in the postmitochondrial fraction. EF-I was then purified from the 230 000 g supernatant solution as described above. The purified EF-I from the Sephadex G-200 column was precipitated with 10% trichloroacetic acid (TCA), deposited on a m e m b r a n e filter, washed with 25ml of 1()"/,, T C A , and its raclioactivity was measured in Bray's solution in a Beckman liquid scintillation counter. The results were corrected for variation in the amino acid pool as previously described [2]. In order to correct for variation in feeding by different groups of flies and by flies of different ages, the proteins in 0.5 ml of the 230 000 g supernatant solution were r e m o v e d by precipitation with an equal volume of 10% T C A , and the radioactivity of the non-protein filtrate was determined.
123 Protein levels were measured with the biuret procedure or by absorbance at 280 nm, using crystalline bovine serum albumin as a standard. All materials and reagents were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A., except for Sephadex G-200 (Pharmacia Fine Chemicals, Piscataway, N J, U.S.A.) and the 3H-amino acid mixture (ICN, Irvine, CA, U.S.A.). RESULTS The marked decrease in peptide chain elongation and particularly in the binding of aminoacyl-tRNA to ribosomes in aging Drosophila [11] could be due to the appearance of an inhibitor of peptide chain elongation. This possibility was examined by the addition of cytosol proteins from old organisms to a peptide chain assay elongation system [11] from 1-day-old adults. As can be seen from Table I, addition of cytosol from 21-day-old adults did not inhibit elongation significantly, although elongation in 21-day-old adults had decreased approximately 70% in comparison with 1-day-old adults [11]. Cytosol from 55-dayold adults, which were considerably beyond the 42-day mean life-span and in which elongation and overall translation had decreased by 80-85%, produced only a 21% inhibition. This small inhibition was apparently not due to a protein, unless it was caused by a heat-resistant protein (such as ribonuclease), because a 16% inhibition could be produced by a cytosol preparation from which most, if not all, proteins had been removed by boiling. Although these observations do not exclude the possibility that the decreased elongation results from the appearance of an inhibitor, they do not provide evidence for the formation of sufficient inhibitor to account for the observed decrease in peptide chain elongation. In agreement with Pelley and Stafford [16], we have found EF-1 from Drosophila melanogaster to be a protein of approximately 250000 molecular weight, based upon its behavior as a single sharp peak upon chromatography on
TABLE 1 EFFECT OF CYTOSOL OF 55-DAY-OLD ADULT DROSOPHILA ON PEPTIDE CHAIN ELONGATION BY I-DAY-OLD ADULTS Each value represents the mean ± S.D. of six measurements. Where indicated, an additional (1.7 mg of cytosol from adults of the ages given above was added to the experimental system.
Addition
Peptide chain elongation (pCi/min per mg ribosomes)
None Cytosol ( l-day-old adults) Cytosol (21-day-old adults) Cytosol (55-day-old adults) Deproteinized cytosol (55-day-old adults)
585 ± 31 573 ± 35 556 ± 28 462 ± 26 491 - 25
(100%) (98%) (95%) (79%) (84%)
124 Sephacryl S-1000 (Pharmacia) in comparison with standards of known molecular weight. As will be reported in detail elsewhere, the EF-1 also migrated as a single component on electrophoresis in a 9% polyacrylamide gel under non-denaturing conditions. The purification procedure described in the Methods section enriched EF-1 activity about 50-fold over that of the supernatant fluid from centrifugation at 230 000 g. That this could produce a highly purified protein is not surprising in view of the finding of Slobin [17] that EF-1 comprises 1-11% of the total cytosol protein in a variety of animal cells, and compares with the 42-fold purification of apparently pure EF-1 from Artemia [18] and 54-fold from calf brain [19]. By sodium dodecyl sulfate polyacrylamide gel electrophoresis, the EF-1 migrated as two polypeptides with molecular weights of approximately 56 000 and 24 500. These findings are comparable with the 53 000 and 30 000 subunits of EF- 1 from rabbit reticulocytes [20] and the 53 000, 51 00(1 and 26 000 molecular weight subunits from the 240 000 molecular weight EF-I from Artemia [18]. In contrast to the lack of evidence for the appearance of an inhibitor of peptide chain elongation, it can be seen from the data of Fig. i that the formation of EF-1, as measured by the rate of incorporation of a mixture of radioactive amino acids into EF-1 protein, decreased about 8 times more rapidly during the first week of adult life than the formation of the bulk of the postmitochondrial proteins. Mitochondrial proteins were omitted from these measurements initially because a small number of mitoehondrial proteins are synthesized by the separate mitochondrial translation system, which we have found to decline differently with age than the ribosomal translation system (P.J. Bailey and G.C. Webster, unpublished observations). Since the decline in EF-I activity was found to parallel approximately the decrease in total protein synthesis [111, the decreased EF-I activity occurred after the drop in EF-I synthesis throughout that period of adult life when protein synthesis decreased maximally. Although the synthesis of EF-I declined to less than 15% of its original value during the first 14 days of adult life, a slow synthesis continued into the later stages of life, and this was paralleled by a continued slow formation of the total postmitochondrial protein fraction. This also is suggestive of a necessity of continued EF-I synthesis for translation to occur. In comparing the decreased synthesis of EF-I with the decreased synthesis of the total postmitochondrial protein fraction, it is important to know whether the amino acids were incorporated into a limited group of postmitochondrial proteins. or whether the decline represented a decreased synthesis of most of the postmitochondrial proteins. In order to obtain information on this question, the postmitochondrial proteins of 1-day-old and 21-day-old organisms that had incorporated a 3H-amino acid mixture, as described in the Methods section, were separated by chromatography on Sephadex G-200 into 9(1 fractions. Although the proteins from each fraction from the 21-day-old organisms exhibited lower specific activities than those from the 1-day-old organisms, the distribution of incorporated radioactivity was essentially the same throughout the fractions from the
125
TOTAL ".'INS 80
i,i Z 0
~
6o
w
EF - I
~. 4o
u.
I
I0
15
ADULT AGE (DAYS)
Fig. 1. Effect of age of adult Drosophila on the rate of EF-I synthesis and on the synthesis of the total postmitochondrial protein fraction. Each value represents the mean 4- S.D. of four m e a s u r e m e n t s . Specific activities of EF-I from l-day-old organisms and from larvae were 8 2 6 d p m / m g and 1192dpm/mg, respectively. Specific activities of total postmitochondrial proteins from l-day-old organisms and from larvae were 1737dpm/mg and 1996dpm/mg. respectively. Essentially identical results were obtained when the total protein fraction was substituted for the postmitochondrial fraction.
two ages, indicating that the measured synthesis of total postmitochondrial proteins in Fig. 1 was not into a limited subset of these proteins. Further evidence for this was obtained by examining the effect of age on the synthesis of several purified proteins. Alcohol dehydrogenase has been characterized extensively from Drosophila, and was obtained in pure form by the procedure of Thatcher [21]. As can be seen in Table II, the age-related decrease in the synthesis of alcohol dehydrogenase essentially paralleled the age-related decrease in synthesis of the total postmitochondrial protein fraction and was distinctly different from the decrease in EF-I synthesis. The possibility that proteins that are present in amounts roughly equal to that of EF-1 may exhibit a decreased synthesis with age which parallels that of EF-1, rather than that of the total postmitochondrial protein fraction, was examined also. The effect of age was determined on the synthesis of the proteins of three chromatographic fractions, designated UF1, UF2 and UF3, whose peak
126
T A B L E II D E C R E A S E IN S Y N T H E S I S OF F O U R D E C R E A S E D S Y N T H E S I S O F EF-I
CYTOPLASMIC PROTEINS
COMPARED
WITH
UF1, UF2 and UF3 were eluted from the 10 × 0.5 cm Sephadex G-200 column in fractions 15, 42 and 57, respectively. Specific activities were approximately those in Fig. 1. Each value represents the mean ±S.D. of four measurements.
Age (days)
Specific activity (per cent of I-day-old) EF- 1 ADH a UF 1b
UF2
UF3
1 7 14 21
1 (}(I 71} ± 9 12+4 7±5
1 (Ill 89 _+ 5 82_+6 45-+7
I l)(I 96 ± 3 75_+9 53_+8
1 (lO 100 72+8 37+-7
l{}l) ll}(I 77+-t~ 52+5
aADH = alcohol dehydrogenase. bUF = unidentified fraction.
heights indicated an abundance comparable to that of EF-1. Although the identity of the proteins of these fractions is not known, Table II shows that the age-related decrease in their synthesis parallels the decrease in synthesis of the total protein fraction, rather than the decrease in EF-I synthesis. These observations are in agreement with the autoradiograms prepared by Parker et al. [4] of the twodimensional gel electrophoresis separations of the proteins of young and old Drosophila. The autoradiograms showed little difference in the labeling pattern, although a marked, age-related decline in synthesis was found. Thus, the decrease in synthesis of the total protein fraction appears to be representative of the decline in synthesis of most postmitochondrial proteins. A decreased synthesis of EF-I would cause a drop in the rate of replacement of EF-I molecules. This could result in a decrease in EF-1 levels, if the spontaneously unfolding polypeptide chains of EF-I were hydrolyzed by proteolytic enzymes, or it could result in an accumulation of inactive EF-I which had unfolded or been otherwise subjected to posttranslational modification [22]. Table III presents the amounts of EF-1 which it was possible to isolate from organisms of different ages by the procedure described in the Methods section. It can be seen that there was a sharply decreased amount of EF-1 which it was possible to isolate after the decline in synthesis had occurred. Although this failure to isolate EF-1 from older organisms could be due to a posttranslational modification of the EF-1 that caused it to behave differently in the isolation procedure, it also could be due to an actual decline in EF-1 levels resulting from a shut-down of synthesis and concurrent proteolytic activity. In either event, an important factor in the lowered EF-I activity appears to be the drop in synthesis which would allow posttranslational modification or proteolytic activity (or a combination of the two) to decrease total EF-1 activity. Further information on
127
T A B L E II1 L E V E L S O F EF-1 I S O L A T E D F R O M D R O S O P H I L A O F D I F F E R E N T A G E S Values given are the means ± S.D. of five preparations.
Age (days)
mg EF-1 isolated per g organisms
Larvae 1 7 13 16 21
1.60 ±_0.25 1.55 ± 0.19 2.(15 ± 0.26 1.35 ± 0.21 0.60±0.11 0.60 ± 0.10
this question will require measurements of EF-1 protein levels by reaction with specific antibodies. DISCUSSION
The experiments reported here have not provided evidence for the appearance of an inhibitor of elongation in aging adults, but they have produced evidence for a marked drop in the synthesis of EF-1, which precedes temporally the great decline in translation. It is not clear whether the observed cessation of EF-1 synthesis is the principal cause of decreased protein synthesis in aging Drosophila cells, but it may be an important contributor. If so, there is approximately a 5-day period between the time when EF-1 synthesis stops and its effect appears as decreased EF-1 activity, with the subsequent impact on the synthesis of the other postmitochondrial proteins. The most likely possibility is that the 5-day period represents the time required for posttranslationai modification of the EF-1 to an inactive form in the environment of the cell. Probably the most important observation of this investigation is that the synthesis of a single protein, EF-I, decreases prior to the decrease in synthesis of most other postmitochondrial proteins. It is not clear as yet whether the synthesis of every postmitochondrial protein decreases only after the drop in EF-1 synthesis and its consequent loss of activity, or whether the synthesis of one or more other proteins stops at the same time as the synthesis Of EF-1. In either case, it seems likely that the synthesis of most of the postmitochondrial proteins drops after the decline in EF-1 synthesis. A final answer to this question will require the separation of each postmitochondrial protein by two-dimensional polyacrylamide gel electrophoresis and measurement of the effect of age on the specific activity of each separated protein. The cause of this seemingly selective early decline in EF-1 synthesis is not known, but experiments are in progress on the possibility of a specific loss of the m R N A for EF-1 in aging cells.
128 ACKNOWLEDGEMENTS
We wish to thank Laurie Kuestner for technical assistance. This work was reported at the 1982 meeting of the Federation of American Societies for Experimental Biology [15]. REFERENCES 1 P.A. Baumann and P.S. Chen, Alterung und Proteinsynthese bei Drosophila melanogaster. Rev. Suisse Zool., 75 (1968) 1051-1058. 2 G.C. Webster and S.L. Webster, Decreased protein synthesis by microsomes from aging Drosophila melanogaster. Exp. Gerontol., 14 (19791 343-348. 3 G.C. Webster, V.T. Beachell and S.L. Webster, Differential decrease in protein synthesis by microsomes from aging Drosophila melanogaster. Exp. Gerontol., 15 (19801 495-497. 4 J. Parker, J. Flanagan, J. Murphy and J. Gallant, On the accuracy of protein synthesis in Drosophila melanogaster. Mech. Ageing Dev., 16 (19811 127-139. 5 J. Miquel, Aging of male Drosophila melanogaster: histological, histochemical and ultrastructural observations. Adv. Gerontol. Res., 3 (19711 39-71. 6 A.C. Vann and G.C. Webster, Age-related changes in mitochondrial function in Drosophila rnelanogaster. Exp. Gerontol., 12 (19771 1-5. 7 J. Miquel and J.E. Johnson, Jr., Senescent changes in the ribosomes of animal cells in vivo. Mech. Ageing Dev., 9 (1979) 247-266. 8 G.C. Webster and S.L. Webster, Lysosomal activity during aging in Drosophila melanogaster. Exp. Gerontol., 13 (1978) 343-,347. 9 J. Miquel, A.C. Economos, K.G. Bensch, H. Atlan and J.E. Johnson, Jr., Review of cell aging in Drosophila and mouse. Age, 2 (1979) 78-88. l0 G.C. Webster, S.L. Webster and W.A. Landis, The effect of age on the initiation of protein synthesis in Drosophila melanogaster. Mech. Ageing Dev., 16 (1981) 71-79. 11 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. 12 H.A. Hosbach and E. Kubli, Transfer RNA in aging Drosophila: i. Extent of aminoacylation. Mech. Ageing Dev., 10 (1979) 131-140. 13 G.C. Webster and S.L. Webster, Aminoacylation of tRNA by cell-free preparations from aging Drosophila melanogaster. Exp. Gerontol., 16 (1981)487-494. 14 J.W. Pelley and D.W. Stafford, Studies on the enzymatic binding of aminoacyl transfer ribonucleic acid to ribosomes in a Drosophila in vitro system. Biochemistry, 9 (19701 34118-3414. 15 G.C. Webster and S.L. Webster, Decreased synthesis of elongation factor I (EF-1): an early event in the age-related reduction in protein synthesis. Fed. Proc., 41 (1982) 383. 16 J.W. Pelley and D.W. Stafford, Partial purification of the aminoacyl-tRNA binding enzyme from Drosophila larvae. Biochim. Biophys. Acta, 204 (19701 40~405. 17 L.I. Slobin, The role of eucaryotic elongation factor Tu in protein synthesis. Eur. J. Biochem., llO (19801 555-563. 18 L.I. Slobin and W. Moiler, Characterization of developmentally regulated forms of elongation factor 1 in Artemia salina. Eur. J. Biochem., 69 (1976) 351-366. 19 H. Moon, B. Redfield, S. Millard, F. Vane and H. Weissbach, Multiple forms of elongation factor I from calf brain. Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 3282-3286. 20 L.I. Slobin, Eucaryotic elongation factor Ts is an integral component of rabbit reticulocytc elongation factor 1. Eur. J. Biochem., 96 (1979) 287-293. 21 D.R. Thatcher, Enzyme instability and proteolysis during the purification of an alcohol dehydrogenase from Drosophila melanogaster. Biochem. J., 163 (19771 317-323. 22 M. Rothstein, The formation of altered enzymes in aging animals. Mech. Ageing De~., 9 (19791 197-2O2.
121
DECLINE IN SYNTHESIS OF ELONGATION FACTOR ONE (EF-I) PRECEDES THE DECREASED SYNTHESIS OF TOTAL PROTEIN IN AGING DROSOPHILA MELANOGASTER
G.C. W E B S T E R and S.L. W E B S T E R
Depa~'~nent of Biological Sciences, Florida Institute of Technology, Melbourne, Florida32901 (U.S.A.) (Received August 26th, 19821 (Revision received December 14th, 1982) SUMMARY The decrease in the rate of protein synthesis in aging adult Drosophila melanogaster was found previously to be due, to a great extent, to a drop in the rate of peptide chain elongation, and principally to lowered activity of elongation factor one (EF-1). This decrease does not appear to be caused by appearance of an inhibitor of peptide chain elongation. Instead, the synthesis of EF-1 declines markedly early in adult life. This decrease is followed by lowered activity of EF-1 and by a drop in the synthesis of most of the cellular proteins.
Key words: Drosophila; Elongation factor; Protein; Synthesis; Translation
INTRODUCTION Aging in Drosophila melanogaster produces a significant decrease in the rate of protein synthesis [1--4]. This decrease appears to affect most of the cellular proteins rather uniformly [4], and is followed by deterioration of cellular structure and function [5-9]. In an effort to determine which part of the protein synthesis system is responsible for the decrease, the effect of age on the stages of translation was determined previously [10,11,13]. The rates of initiation (measured by the formation of the 40 S and 80 S initiation complexes) and termination (measured as the release of N-formylmethionine from ribosome-bound N-formylmethionyltRNA) did not decrease significantly during the time when total protein synthesis decreased approximately 70% [10,1 1]. The rate or the yield of aminoacylation of t R N A was lowered (up to 50%) for several amino acids [12,13]. However, the rates of aminoacylation far exceeded the rate of translation under similar conditions, so the significance of the reduced aminoacylation for the age-related decrease in translation is not clear [13]. In contrast, the rate of peptide chain elongation declined markedly with age, in parallel with the decrease in overall 1~147-6374/83/$3.110 Printed and Published in Ireland
© 1983 Elsevier ScientificPublishers Ireland Ltd.
122 translation [11]. This decrease was due principally to lowered binding of aminoacyl-tRNA to ribosomes, and specifically to decreased elongation factor 1 (EF-1) activity [ 11]. The decreased EF-1 activity could be due to the age-related formation of an inhibitor of peptide chain elongation, or to a cessation of synthesis of EF-I. As is reported in this paper, we have been unable to find evidence for the age-related appearance of an inhibitor of peptide chain elongation. However, we have found that the synthesis of EF-1 drops abruptly several days prior to the decrease in synthesis of most proteins. MATERIALS AND METHODS
Drosophila melanogaster, strain Oregon R, was grown as described previously [10], and had a mean life-span of 42 days. EF-1 was purified from Drosophila by the method of Pelley and Stafford [14], with a final separation by chromatography on Sephadex G-200 to remove any remaining lower molecular proteins from the m a j o r EF-1 peak. Peptide chain elongation was assayed by measuring the rate of polyuridylate-dependent incorporation of the phenylalanine of phenylalanylt R N A into polypeptide [14] in a reaction system consisting of: 0.05 M T E S (pH 7.6), 0.007 M MgCI2, 0.06M KCI, 0.002M dithiothreitol, 0.0005 M GTP, 0.Ill M phospho(enol)pyruvate, 0.25mg of polyuridylic acid, 400 A2~ units of [3H]phenylalanyl-tRNA, prepared as described previously [11], 0.5 mg of ribosomes, and 0.5 mg of cytosol [14] from 1-day-old adults in a total w)iumc of 0.5 ml. For m e a s u r e m e n t of EF-I synthesis by larvae, 7 5 # C i of a ~H-amino acid mixture was mixed with each 5 ml of culture medium in which the larvae were grown. EF-I synthesis in adults was measured by placing 50-100 adults of known age in a vial containing filter paper soaked with 50/zCi of the 3H-amino acid mixture per ml of 5% sucrose, containing 1 mg/ml yeast. Under these conditions, incorporation of radioactivity into the total protein fraction was linear for at least 24 h. After varying times of up to 24 h, the flies were removed from the vials, washed free of external radioactivity in ice-water, and homogenized as described previously [1l]. An aliquot of the supernatant fluid from the 2 5 0 0 0 g ccntrifugation step was treated as described [2] to measure the rate of overall protein synthesis in the postmitochondrial fraction. EF-I was then purified from the 230 000 g supernatant solution as described above. The purified EF-I from the Sephadex G-200 column was precipitated with 10% trichloroacetic acid (TCA), deposited on a m e m b r a n e filter, washed with 25ml of 1()"/,, T C A , and its raclioactivity was measured in Bray's solution in a Beckman liquid scintillation counter. The results were corrected for variation in the amino acid pool as previously described [2]. In order to correct for variation in feeding by different groups of flies and by flies of different ages, the proteins in 0.5 ml of the 230 000 g supernatant solution were r e m o v e d by precipitation with an equal volume of 10% T C A , and the radioactivity of the non-protein filtrate was determined.
123 Protein levels were measured with the biuret procedure or by absorbance at 280 nm, using crystalline bovine serum albumin as a standard. All materials and reagents were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A., except for Sephadex G-200 (Pharmacia Fine Chemicals, Piscataway, N J, U.S.A.) and the 3H-amino acid mixture (ICN, Irvine, CA, U.S.A.). RESULTS The marked decrease in peptide chain elongation and particularly in the binding of aminoacyl-tRNA to ribosomes in aging Drosophila [11] could be due to the appearance of an inhibitor of peptide chain elongation. This possibility was examined by the addition of cytosol proteins from old organisms to a peptide chain assay elongation system [11] from 1-day-old adults. As can be seen from Table I, addition of cytosol from 21-day-old adults did not inhibit elongation significantly, although elongation in 21-day-old adults had decreased approximately 70% in comparison with 1-day-old adults [11]. Cytosol from 55-dayold adults, which were considerably beyond the 42-day mean life-span and in which elongation and overall translation had decreased by 80-85%, produced only a 21% inhibition. This small inhibition was apparently not due to a protein, unless it was caused by a heat-resistant protein (such as ribonuclease), because a 16% inhibition could be produced by a cytosol preparation from which most, if not all, proteins had been removed by boiling. Although these observations do not exclude the possibility that the decreased elongation results from the appearance of an inhibitor, they do not provide evidence for the formation of sufficient inhibitor to account for the observed decrease in peptide chain elongation. In agreement with Pelley and Stafford [16], we have found EF-1 from Drosophila melanogaster to be a protein of approximately 250000 molecular weight, based upon its behavior as a single sharp peak upon chromatography on
TABLE 1 EFFECT OF CYTOSOL OF 55-DAY-OLD ADULT DROSOPHILA ON PEPTIDE CHAIN ELONGATION BY I-DAY-OLD ADULTS Each value represents the mean ± S.D. of six measurements. Where indicated, an additional (1.7 mg of cytosol from adults of the ages given above was added to the experimental system.
Addition
Peptide chain elongation (pCi/min per mg ribosomes)
None Cytosol ( l-day-old adults) Cytosol (21-day-old adults) Cytosol (55-day-old adults) Deproteinized cytosol (55-day-old adults)
585 ± 31 573 ± 35 556 ± 28 462 ± 26 491 - 25
(100%) (98%) (95%) (79%) (84%)
124 Sephacryl S-1000 (Pharmacia) in comparison with standards of known molecular weight. As will be reported in detail elsewhere, the EF-1 also migrated as a single component on electrophoresis in a 9% polyacrylamide gel under non-denaturing conditions. The purification procedure described in the Methods section enriched EF-1 activity about 50-fold over that of the supernatant fluid from centrifugation at 230 000 g. That this could produce a highly purified protein is not surprising in view of the finding of Slobin [17] that EF-1 comprises 1-11% of the total cytosol protein in a variety of animal cells, and compares with the 42-fold purification of apparently pure EF-1 from Artemia [18] and 54-fold from calf brain [19]. By sodium dodecyl sulfate polyacrylamide gel electrophoresis, the EF-1 migrated as two polypeptides with molecular weights of approximately 56 000 and 24 500. These findings are comparable with the 53 000 and 30 000 subunits of EF- 1 from rabbit reticulocytes [20] and the 53 000, 51 00(1 and 26 000 molecular weight subunits from the 240 000 molecular weight EF-I from Artemia [18]. In contrast to the lack of evidence for the appearance of an inhibitor of peptide chain elongation, it can be seen from the data of Fig. i that the formation of EF-1, as measured by the rate of incorporation of a mixture of radioactive amino acids into EF-1 protein, decreased about 8 times more rapidly during the first week of adult life than the formation of the bulk of the postmitochondrial proteins. Mitochondrial proteins were omitted from these measurements initially because a small number of mitoehondrial proteins are synthesized by the separate mitochondrial translation system, which we have found to decline differently with age than the ribosomal translation system (P.J. Bailey and G.C. Webster, unpublished observations). Since the decline in EF-I activity was found to parallel approximately the decrease in total protein synthesis [111, the decreased EF-I activity occurred after the drop in EF-I synthesis throughout that period of adult life when protein synthesis decreased maximally. Although the synthesis of EF-I declined to less than 15% of its original value during the first 14 days of adult life, a slow synthesis continued into the later stages of life, and this was paralleled by a continued slow formation of the total postmitochondrial protein fraction. This also is suggestive of a necessity of continued EF-I synthesis for translation to occur. In comparing the decreased synthesis of EF-I with the decreased synthesis of the total postmitochondrial protein fraction, it is important to know whether the amino acids were incorporated into a limited group of postmitochondrial proteins. or whether the decline represented a decreased synthesis of most of the postmitochondrial proteins. In order to obtain information on this question, the postmitochondrial proteins of 1-day-old and 21-day-old organisms that had incorporated a 3H-amino acid mixture, as described in the Methods section, were separated by chromatography on Sephadex G-200 into 9(1 fractions. Although the proteins from each fraction from the 21-day-old organisms exhibited lower specific activities than those from the 1-day-old organisms, the distribution of incorporated radioactivity was essentially the same throughout the fractions from the
125
TOTAL ".'INS 80
i,i Z 0
~
6o
w
EF - I
~. 4o
u.
I
I0
15
ADULT AGE (DAYS)
Fig. 1. Effect of age of adult Drosophila on the rate of EF-I synthesis and on the synthesis of the total postmitochondrial protein fraction. Each value represents the mean 4- S.D. of four m e a s u r e m e n t s . Specific activities of EF-I from l-day-old organisms and from larvae were 8 2 6 d p m / m g and 1192dpm/mg, respectively. Specific activities of total postmitochondrial proteins from l-day-old organisms and from larvae were 1737dpm/mg and 1996dpm/mg. respectively. Essentially identical results were obtained when the total protein fraction was substituted for the postmitochondrial fraction.
two ages, indicating that the measured synthesis of total postmitochondrial proteins in Fig. 1 was not into a limited subset of these proteins. Further evidence for this was obtained by examining the effect of age on the synthesis of several purified proteins. Alcohol dehydrogenase has been characterized extensively from Drosophila, and was obtained in pure form by the procedure of Thatcher [21]. As can be seen in Table II, the age-related decrease in the synthesis of alcohol dehydrogenase essentially paralleled the age-related decrease in synthesis of the total postmitochondrial protein fraction and was distinctly different from the decrease in EF-I synthesis. The possibility that proteins that are present in amounts roughly equal to that of EF-1 may exhibit a decreased synthesis with age which parallels that of EF-1, rather than that of the total postmitochondrial protein fraction, was examined also. The effect of age was determined on the synthesis of the proteins of three chromatographic fractions, designated UF1, UF2 and UF3, whose peak
126
T A B L E II D E C R E A S E IN S Y N T H E S I S OF F O U R D E C R E A S E D S Y N T H E S I S O F EF-I
CYTOPLASMIC PROTEINS
COMPARED
WITH
UF1, UF2 and UF3 were eluted from the 10 × 0.5 cm Sephadex G-200 column in fractions 15, 42 and 57, respectively. Specific activities were approximately those in Fig. 1. Each value represents the mean ±S.D. of four measurements.
Age (days)
Specific activity (per cent of I-day-old) EF- 1 ADH a UF 1b
UF2
UF3
1 7 14 21
1 (}(I 71} ± 9 12+4 7±5
1 (Ill 89 _+ 5 82_+6 45-+7
I l)(I 96 ± 3 75_+9 53_+8
1 (lO 100 72+8 37+-7
l{}l) ll}(I 77+-t~ 52+5
aADH = alcohol dehydrogenase. bUF = unidentified fraction.
heights indicated an abundance comparable to that of EF-1. Although the identity of the proteins of these fractions is not known, Table II shows that the age-related decrease in their synthesis parallels the decrease in synthesis of the total protein fraction, rather than the decrease in EF-I synthesis. These observations are in agreement with the autoradiograms prepared by Parker et al. [4] of the twodimensional gel electrophoresis separations of the proteins of young and old Drosophila. The autoradiograms showed little difference in the labeling pattern, although a marked, age-related decline in synthesis was found. Thus, the decrease in synthesis of the total protein fraction appears to be representative of the decline in synthesis of most postmitochondrial proteins. A decreased synthesis of EF-I would cause a drop in the rate of replacement of EF-I molecules. This could result in a decrease in EF-1 levels, if the spontaneously unfolding polypeptide chains of EF-I were hydrolyzed by proteolytic enzymes, or it could result in an accumulation of inactive EF-I which had unfolded or been otherwise subjected to posttranslational modification [22]. Table III presents the amounts of EF-1 which it was possible to isolate from organisms of different ages by the procedure described in the Methods section. It can be seen that there was a sharply decreased amount of EF-1 which it was possible to isolate after the decline in synthesis had occurred. Although this failure to isolate EF-1 from older organisms could be due to a posttranslational modification of the EF-1 that caused it to behave differently in the isolation procedure, it also could be due to an actual decline in EF-1 levels resulting from a shut-down of synthesis and concurrent proteolytic activity. In either event, an important factor in the lowered EF-I activity appears to be the drop in synthesis which would allow posttranslational modification or proteolytic activity (or a combination of the two) to decrease total EF-1 activity. Further information on
127
T A B L E II1 L E V E L S O F EF-1 I S O L A T E D F R O M D R O S O P H I L A O F D I F F E R E N T A G E S Values given are the means ± S.D. of five preparations.
Age (days)
mg EF-1 isolated per g organisms
Larvae 1 7 13 16 21
1.60 ±_0.25 1.55 ± 0.19 2.(15 ± 0.26 1.35 ± 0.21 0.60±0.11 0.60 ± 0.10
this question will require measurements of EF-1 protein levels by reaction with specific antibodies. DISCUSSION
The experiments reported here have not provided evidence for the appearance of an inhibitor of elongation in aging adults, but they have produced evidence for a marked drop in the synthesis of EF-1, which precedes temporally the great decline in translation. It is not clear whether the observed cessation of EF-1 synthesis is the principal cause of decreased protein synthesis in aging Drosophila cells, but it may be an important contributor. If so, there is approximately a 5-day period between the time when EF-1 synthesis stops and its effect appears as decreased EF-1 activity, with the subsequent impact on the synthesis of the other postmitochondrial proteins. The most likely possibility is that the 5-day period represents the time required for posttranslationai modification of the EF-1 to an inactive form in the environment of the cell. Probably the most important observation of this investigation is that the synthesis of a single protein, EF-I, decreases prior to the decrease in synthesis of most other postmitochondrial proteins. It is not clear as yet whether the synthesis of every postmitochondrial protein decreases only after the drop in EF-1 synthesis and its consequent loss of activity, or whether the synthesis of one or more other proteins stops at the same time as the synthesis Of EF-1. In either case, it seems likely that the synthesis of most of the postmitochondrial proteins drops after the decline in EF-1 synthesis. A final answer to this question will require the separation of each postmitochondrial protein by two-dimensional polyacrylamide gel electrophoresis and measurement of the effect of age on the specific activity of each separated protein. The cause of this seemingly selective early decline in EF-1 synthesis is not known, but experiments are in progress on the possibility of a specific loss of the m R N A for EF-1 in aging cells.
128 ACKNOWLEDGEMENTS
We wish to thank Laurie Kuestner for technical assistance. This work was reported at the 1982 meeting of the Federation of American Societies for Experimental Biology [15]. REFERENCES 1 P.A. Baumann and P.S. Chen, Alterung und Proteinsynthese bei Drosophila melanogaster. Rev. Suisse Zool., 75 (1968) 1051-1058. 2 G.C. Webster and S.L. Webster, Decreased protein synthesis by microsomes from aging Drosophila melanogaster. Exp. Gerontol., 14 (19791 343-348. 3 G.C. Webster, V.T. Beachell and S.L. Webster, Differential decrease in protein synthesis by microsomes from aging Drosophila melanogaster. Exp. Gerontol., 15 (19801 495-497. 4 J. Parker, J. Flanagan, J. Murphy and J. Gallant, On the accuracy of protein synthesis in Drosophila melanogaster. Mech. Ageing Dev., 16 (19811 127-139. 5 J. Miquel, Aging of male Drosophila melanogaster: histological, histochemical and ultrastructural observations. Adv. Gerontol. Res., 3 (19711 39-71. 6 A.C. Vann and G.C. Webster, Age-related changes in mitochondrial function in Drosophila rnelanogaster. Exp. Gerontol., 12 (19771 1-5. 7 J. Miquel and J.E. Johnson, Jr., Senescent changes in the ribosomes of animal cells in vivo. Mech. Ageing Dev., 9 (1979) 247-266. 8 G.C. Webster and S.L. Webster, Lysosomal activity during aging in Drosophila melanogaster. Exp. Gerontol., 13 (1978) 343-,347. 9 J. Miquel, A.C. Economos, K.G. Bensch, H. Atlan and J.E. Johnson, Jr., Review of cell aging in Drosophila and mouse. Age, 2 (1979) 78-88. l0 G.C. Webster, S.L. Webster and W.A. Landis, The effect of age on the initiation of protein synthesis in Drosophila melanogaster. Mech. Ageing Dev., 16 (1981) 71-79. 11 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. 12 H.A. Hosbach and E. Kubli, Transfer RNA in aging Drosophila: i. Extent of aminoacylation. Mech. Ageing Dev., 10 (1979) 131-140. 13 G.C. Webster and S.L. Webster, Aminoacylation of tRNA by cell-free preparations from aging Drosophila melanogaster. Exp. Gerontol., 16 (1981)487-494. 14 J.W. Pelley and D.W. Stafford, Studies on the enzymatic binding of aminoacyl transfer ribonucleic acid to ribosomes in a Drosophila in vitro system. Biochemistry, 9 (19701 34118-3414. 15 G.C. Webster and S.L. Webster, Decreased synthesis of elongation factor I (EF-1): an early event in the age-related reduction in protein synthesis. Fed. Proc., 41 (1982) 383. 16 J.W. Pelley and D.W. Stafford, Partial purification of the aminoacyl-tRNA binding enzyme from Drosophila larvae. Biochim. Biophys. Acta, 204 (19701 40~405. 17 L.I. Slobin, The role of eucaryotic elongation factor Tu in protein synthesis. Eur. J. Biochem., llO (19801 555-563. 18 L.I. Slobin and W. Moiler, Characterization of developmentally regulated forms of elongation factor 1 in Artemia salina. Eur. J. Biochem., 69 (1976) 351-366. 19 H. Moon, B. Redfield, S. Millard, F. Vane and H. Weissbach, Multiple forms of elongation factor I from calf brain. Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 3282-3286. 20 L.I. Slobin, Eucaryotic elongation factor Ts is an integral component of rabbit reticulocytc elongation factor 1. Eur. J. Biochem., 96 (1979) 287-293. 21 D.R. Thatcher, Enzyme instability and proteolysis during the purification of an alcohol dehydrogenase from Drosophila melanogaster. Biochem. J., 163 (19771 317-323. 22 M. Rothstein, The formation of altered enzymes in aging animals. Mech. Ageing De~., 9 (19791 197-2O2.