A study was made of theability of cell-free protein synthesis systems from vegeta- tive cells of ... Of the 15 specific aminoacyl-tRNA synthetases assayed, 5 in-.
JOURNAL
OF BACTERIOLOGY, Mar. 1970, p. 755-762 Copyright a 1970 American Society for Microbiology
Vol. 101, No. 3
Prinited ill U.S.A.
In Vitro Protein Synthesis and Aging in Rhizoctonia solani T. G. OBRIG AND DAVID GOTTLIEB Departmentt of Plalit Pathology, Untiversity of Illinois, Urbana, Illintois 61801 Received for publication 25 November 1969
A study was made of the ability of cell-free protein synthesis systems from vegetative cells of different age of the fungus Rhizoctonia solani to produce polyphenylalanine. Polyuridylic acid-directed phenylalanine incorporation into peptides decreased linearly with cell age. The 105,000 x g supernatant fluid and ribosomal fractions were equally responsible for the total loss of synthetic activity of the older cells. Initial rates of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase activity decreased with increasing cell age, which accounted for the defect of the supernatant fraction. An accelerated degradation of soluble phenylalanyl-RNA was associated with the ribosomes of the older cells. In vitro systems from cells of different age transferred phenylalanine from phenylalanyl-tRNA to polyphenylalanine at similar rates. Of the 15 specific aminoacyl-tRNA synthetases assayed, 5 increased and 5 decreased in specific activity with increased age; 3 others did not change during aging and 2 were below acceptable detectable levels.
Previous studies have delineated some of the biochemical changes that occurred in Rhizoctonia solani and Sclerotium baaticola with increasing age of mycelial cells (8, 10, 20, 28). In both species, a general decline in cellular metabolism accompanied the aging process. On a dry-weight basis, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), soluble amino nitrogen, and protein decreased gradually with age in R. solani. Decreases with age in the content of soluble amino nitrogen and protein per unit (dry weight) are quite common in fungi (2, 5, 9, 16, 26). Moreover, the rate of incorporation of '4C-phenylalanine and '4C-leucine into the R. solani cellular protein decreased gradually with increasing age (8). This apparent decrease of synthetic activity was due neither to an amino acid impermeability factor in old cells nor to an isotope dilution from an increase in the amino acid pool size. Reduction in specific activity of protein fractions with age suggests that the decrease in protein synthesis was due to the protein synthesizing mechanism itself. Decreases in the protein synthetic capacity with increasing age has been noted in cells of higher plants (17) and in cell-free preparations of muscle (3), liver (4), and reticulocytes (23). An in vitro system was prepared from the mycelium of the fungus R. solani, with all parameters adjusted for optimal protein synthesis (22). The present investigation was made to determine the location of the defect in protein synthesis as cells of R. solani age. 755
MATERIALS AND METHODS
Six-day-old surface cultures of R. solani Kuhn were grown at 26 C in 30-cm (diameter) culture vessels which contained 4 liters of liquid media. Composition of the chemically defined media is described elsewhere (22). The mycelial pad was divided by two concentric cuts into round segments of cells 0 to 2 (young), 3 to 4 (mature), and 5 to 6 (old) days old with radial widths of 4.3, 3.9, and 1.5 cm, respectively. Preparation of the 105,000 X g supernatant fluid (S-105) and ribosomal pellet (P-105) has been described (22). Protein content of the S-105 fraction was estimated by the Biuret (12) or Folin phenol method (15) by using bovine serum albumin as a standard. RNA content of the P-105 fraction was determined spectrophotometrically by assuming that I mg of ribosomal RNA in 1 ml of water equalled 25 optical density units at 260 nm in a I-cm light
path.
Incubation mixtures for the R. solanii peptidesynthesizing system contained, in a final volume of 0.5 ml, tris(hydroxymethyl)aminomethane(Tris)-chloride buffer, 50 ,moles (pH 7.8); magnesium acetate, 10 ,umoles; ammonium chloride, 25 ,umoles; 2-mercaptoethanol, 7.5 sAmoles; reduced glutathione, 0.5 Mmole; adenosine 5'-triphosphate (ATP), 1.5 /Amoles; guanosine 5'-triphosphate (GTP), 0.1 jAmole; phospho(enol) pyruvate (PEP), 2.5 ,moles; pyruvate kinase, 10 ug; yeast-soluble RNA (sRNA), 200 ,g; polyuridylic acid (poly U), 60 Mg; L-phenylalanine-U14C, 0.3 Muc, 800 pmoles; 0.005 ,umoles of each of the other 10 unlabeled amino acids; P-105, 300 Ag of RNA; and S-105, 300 .Ag of protein. When syntheses by different age systems are compared, all mixtures contained the same amount of S-105 protein and
756
OBRIG AND GOTTLIEB
P-105 RNA. The mixtures were incubated for 2.5 to 45 min at 28 C. For measurement of the incorporation of "4C-amino acids into "C-peptides, the reaction was stopped by the addition of 20,moles of unlabeled amino acid in cold (0 to 4 C) trichloroacetic acid so that the final trichloroacetic acid concentration was 8 to 10%. After standing for I hr at 0 to 4 C, the mixtures were heated for 10 min at 90 C and cooled to 21 C. The mixture was filtered through a type HA Millipore filter (0.45 ,um pore diameter), the precipitate was washed with three 5-mi portions of 5% trichloroacetic acid, and the filters were dried in scintillation vials at room temperature for 2 hr and at 80 C for 10 min. The cold trichloroacetic acid assay differed from the hot trichloroacetic acid assay in that all procedures prior to the filter drying were carried out at 0 to 4 C. A counting efficiency of 70% was attained by using liquid scintillation counting conditions previously described (22). Incorporation of "C-amino acids into 14C-aminoacyl-tRNA ("charging reaction") was followed either (i) directly with a cold trichloroacetic acid assay in the absence of peptide synthesis, or (ii) indirectly by subtracting the hot trichloroacetic acid-precipitable radioactivity ("4C-peptide) from the cold trichloroacetic acid-precipitable radioactivity (14C-peptide plus "4C-aminoacyl-tRNA). In the direct method, poly U, GTP, and ribosomes were omitted from the reaction mixture. Where incubations of 2 to 3 min are reported, the reaction was stopped by simultaneously placing all reaction tubes into an acetone-dry ice mixture for 5 to 10 sec and then adding cold trichloroacetic acid as described above. A hot and cold trichloroacetic acid assay was also combined for measuring the incorporation of "4C-phenylalanine from "4C-phenylalanine-tRNA into 14C_
polyphenylalanine. During this "transfer" reaction, the reaction mixture was complete except that labeled and unlabeled free amino acids were omitted. The assay of aminoacyl-tRNA synthetases with hydroxylamine was carried out generally as described by Clark (6), with modifications. The 2.0-ml assay mixture contained: ATP, 20 ,umoles; Trischloride buffer, 50 J,moles (pH 7.2); hydroxylaminehydrochloride, 2 mmoles; magnesium acetate, 20 jAmoles; potassium acetate, 133 jAmoles; L-amino acid, 40 ,umoles; and S-105, 3 to 5 mg of protein. Incubation was at 30 C for 30 min. L-Tyrosine hydroxamate was used as a standard. For the preparation of "4C-phenylalanine-tRNA, amino acid-activating enzymes from Saccharomyces fragilis (ATCC 10022, NRRL-Y665) were used to charge stripped yeast sRNA with "4C-phenylalanine by the methods of So and Davie (25) and Downey et al. (7). Reagent grade chemicals and sources were: Tris, 2-mercaptoethanol, reduced glutathione, pyruvate kinase, ATP, GTP, PEP, sRNA (unstripped), ribonuclease, L-tyrosine hydroxamate from Sigma Chemical Co., St. Louis, Mo.; poly U from Miles Laboratories, Inc., Elkhart, Ind., yeast sRNA (stripped) from General Biochemicals Corp., Chagrin Falls, Ohio; tetracycline from Charles Pfizer & Co., New York,
J. BACTERIOL.
N.Y.; L-phenylalanine-U-'4C (368 /Ac/jumole), L-"Cphenylalanine-tRNA from E. coli (UL, 0.19 ,uc/mg), and 15 individual L-14C-amino acids (10 mc/mmole each) from New England Nuclear Corp., Boston, Mass.; and liquid scintillation reagents from Packard Instrument Co., Downers Grove, Ill. RESULTS R. solani did not produce spores under the described culture conditions. Surface cultures grew in a typical sigmoidal-growth-curve fashion when measured by diameter of the fungal pad. The growth rates (Fig. 1) increased until the 5th day and then decreased sharply. This inherent growth characteristic was not due to the depletion of nutrients in the medium or to the synthesis of an exoenzyme (unpublished data). Viability of individual cells was previously shown to be almost identical for the three different age groups of R. solani. There was decreased peptide synthesis in cellfree systems with increasing age (Fig. 2). After 30 min, the young system had incorporated twice as much "4C-phenylalanine into peptides than did the old system. An attempt was made to locate the rate-limiting step of polyphenylalanine synthesis in the older cells. The ribosomal (P-105) and supernatant (S-105) fractions from young, mature, and old cells were used in all possible combinations, and the relative ability of such homologous and heterologous systems to incorporate phenylalanine into peptides was measured. The results indicate that the activities of both the P-105 and S-105 fractions decreased with increasing age of the cells (Table 1). In addition, 50% of the total loss of activity in the homologous mature or old cell systems was due to the P-105 fraction; the other half was due to the old S-105 fraction. A comparison of the different age preparations was then made by investigating the individual E 32E 2824a -C
20
-
16
0
a° 0..
8 1 2 3 4 5 6 7 "Ic Incubation (days) FIG. 1. Growth rate of R. solanti surface culture.
PROTEIN SYNTHESIS IN R. SOLANI
VOL. 101, 1970
nr 0
.I I
5
15
45
30
Incubation (minutes) FiG. 2. Effect of cell age on in vitro polyphenylalanine synthesis by R. solani. The assay was carried out as described in Materials and Methods. Each assay contained 360,g of S-105 protein and 290.ug of P-105 RNA; 570 counts/min per pmole of phenylalanine. TABLE 1. Incorporation of phenylalanine into polyphenylalanine by homologous- and heterologousage cell-free systems from R. solania Riobosomesg Supernatant fraction
Young .......... Mature ......... Old ...........
Young
Mature
Old
23,600
21,400
18,100 15,600 12,000
19,800 17,600
17,600 13,500
757
alanyl-tRNA formation or an increased rate of degradation of the phenylalanyl-tRNA, or both. These experiments were conducted in the presence of saturating quantities of tRNA and '4C-phenylalanine. If the amount of phenylalanyl-tRNA formed determined the rate of peptide synthesis, then young, mature, or old systems, when supplied with "4C-phenylalanyl-tRNA, should transfer the amino acid into peptides at equal rates in the "transfer" reaction. When this reaction was compared for the three systems with a limiting amount of phenylalanyl-tRNA, after 30 min of incubation there was less than a 15% difference among any of the systems, either in production of "4C-polyphenylalanine or in decrease of the '4C-phenylalanyl-tRNA substrate (Fig. 4). In all cases, between 60 and 66% of the radioactivity which disappeared as substrate-"IC was recovered as peptide-14C. Similar results were obtained with charged tRNA from yeast or bacteria. Amino acid activation with ATP and tRNA charging by phenylalanyl-tRNA synthetases in the absence of peptide synthesis was studied in young, mature, and old systems. First, when peptide synthesis was inhibited 50 to 60% with a high concentration of tetracycline by suppressing binding of phenylalanyl-tRNA to polyribosomes, "4C-phenylalanine was incorporated into
a Values represent counts/min per milligram of ribosomal RNA. Each figure is the average of three separate experiments (30-min incubation). Contents of the reaction mixtures were as stated in Materials and Methods, with S-105 protein and P-105 RNA ranging from 0.400 to 0.420 mg and 310 to 335 ug per assay, respectively; 570 counts/ min equals 1 pmole of phenylalanine.
steps involved in the synthesis of polyphenylalanine. The relative ability of the three homologousage S-105 and P-105 systems to incorporate 14C-phenylalanine into "4C-phenylalanyl-tRNA and "4C-polyphenylalanine was followed over a 30-min period. Rates of peptide synthesis and net phe-tRNA formation decreased with increasing age of the preparations (Fig. 3). Between 20 15 10 5 and 15 min, phenylalanyl-tRNA formation Incubation (minutes) in the young system was still increasing, but it was FiG. 3. Effect of cell age on in vitro incorporation of decreasing in the mature and in the old systems. phenylalanine into phenylalanyl-tRNA and polyThe data suggest that the ability to carry out a phenylalanine by R. solani. Assay conditions were as net charging of tRNA might be limiting peptide described in Materials and Methods. Each assay consynthesis. The data suggest that with increasing tained 400 ,g of S-105 protein and 300 ,g of P-105 age, there is either a decrease in the rate of phenyl- RNA; 570 countslmin per pmole of phenylalanine.
758
OBRIG AND GOTTLIEB
J. B A(C I L' RI ()L.
TABLE 2. lIicorporacili0/ ot/ pheli / lly/ l(ciiiic, ilito phenylalanyl-tRNA: eJfct of S-105 age oli /Ic chairgilig reactioir, Incorporation at minutes of incubation
z
Age
cr -
25
-
2.5
i.0
1(.(
11,500
14,900 11,500
15,'950 13,100
15.
E
Young. Old ..
0 In
° 20 .0
13,350 12,400
Values represenlt counts/min per milligram tof S-105 protein. The younlg and old systemiis contained 306 and 320 ,g of protein per assay, respectively. The incubation mixture was complete as listed in Materials and Methods, except P-1)5 and poly U were omitted; 570 counlts/iimm per pmole of phenylalanine.
E 15 I0 -
9,450
10
x E u
W 0
0
5
10
15
20
25
30
Incubation (minutes) FIG. 4. Effect of'cell age Oli ill vitro inicorpor-ation of phentylalaniine Jrom phenylalanyl-tRNA (Escherichia
coli) iitto polyphenylalaitinie by R. solaini. Assay contditionis were as described inl Materials anid Methods except that '4C-pheniylalaninle was omitted aind tRNA was replaced with 0.15 mg (6 nimoles) of 14C-phentylalaniniietRNA from Escherichia coli; 7,300 coaints/mini per lmole o/'phenylalanine-tRNA.
TABLE 3. Ilicorporatioii of plheny/alanine ilto aminloacyl-iRNA in/ the presence or in tile abscnce of tetracycline"' Incubation (min)
7 12 17 12
Tetracycline (400 A.g/ml)
Present', Present'
Presen1t' Absenrv
tiounts/minn le a-assy a per
Young
Mature
Old
3,826 4,483 3,724 4,325
4,427 3,638 3 053
2,618 2,429
4,825
2,087 3,208
'4C-phenylalanyl-tRNA differently by the three Cold trichloroacetic acid-precipitable radiodifferent-age systems; the phenylalanyl-tRNA synthetase activity usually decreased with in- activity; 570 counts/min per pmole of' phenylal-tcreasing age of the cells (Table 3). After 17 min, nine. b Assay mixtures were complete as listed in Mathe mature and old systems incorporated only 82 terials and Methods, with 415 jAg of S-l05 protein and 56c/;, respectively, as much amino acid into and 310,ug of P-105 RNA per assay. aminoacyl-tRNA as did the young system. c Assay mixtures were exceplt that Second, when peptide synthesis was eliminated by ribosomes and poly U werecomplete omitted. omitting P-105 and poly U from an otherwise complete reaction mixture (more than 95r of synthetase activity was located in the S-105 fraction), '4C-phenylalanyl-tRNA formation by TABLE 4. Specific activ,it)y o p,e,nylalanvl1-i RNA, the young, mature, and old systems was 100, 110, syiltletalses from cdiflierent-age cells of R. soloiti: and 74%7,, respectively, after 12 min of incubation hydroxylanmine aissay, (Table 3). Third, an aminoacyl-tRNA synthetase of product ~~~Amtforme(l' assay based on the reaction of hydroxylamine Assay" Assay" with aminoacyl adenylates indicated that the phenylalanyl-tRNA synthetase activity de- Complete, young S-105. 0.86 creased in the mature (48%/6) and old (67 %) Complete, mature S-105 0. 45 systems when compared to activity of the young Complete, old S-105 0.28
