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Ribosomal subunits in rabbit reticulocytes under different conditions of polyribosome disaggregation
480 M. L. Freedman & M. Rabinowitz REFERENCES 1. Hendler, R W, Dalton, A J & Glenner, G G, J biophys biochem cytol 3 (1957) 325. 2. Makita, T & Nishida, S, Jap j vet sci 17 (1965) 460. 3. - Proc sixth int tong E M II (1966) 777.
4. Richardson, K C, Phil trans b 225 (1935) 149. 5. Sandborn, E B, Makita, T & Lin, K, Anat ret 163 (1969) 255. Received March 3, 1970
Ribosomal subunits in rabbit reticulocytes under different conditions of polyribosome disaggregation M. L. FREEDMAN’
and M. RABINOVITZ,
ratory of Physiology, National Bethesda, Md 20014, USA
Camcer Institute,
L&oNIH,
Summary Disaggregation of reticulocyte polyribosomes caused by tryptophan deficiency was accompanied by an increase in the level of ribosomal subunits, but disaggregation caused by iron deficiency or butanol shock was not. The results are interpreted in terms of the mechanism of action of the various treatments on the translation process.
There has been considerable interest in the role of ribosomal subunits in the initiation step of protein synthesis. Much of the evidence in support of such a mechanism is derived from studies with Escherichia coli [15, 201, where it has been shown that these subunits recycle through ribosomes during both bacterial growth [13] and acellular protein synthesis [12]. Investigations with acellular bacterial systems indicate that the smaller ribosomal subunit associates with initiation factors, messenger RNA (mRNA) and formylmethionine-transfer RNA to form a complex which initiates protein synthesis when the larger subunit attaches to the smaller subunit of the complex [6, 8, 18, 191. The view that the ribosomal subunits rather than intact ribosomes are active in the 1 Present address::Department of Medicine, New York Unersity Medical Center, New York, N.Y. 10016, Exptl
Cell Res 60
initiation of protein synthesis by mammalian cells first came from observations by Bishop [l] on initiation of hemoglobin synthesis by an acellular reticulocyte preparation. However, support for an active role ,of ribosomal subunits in protein synthesis by intact mammalian cells has been varied. Thus, Joklik & Becker [ll] and Hogan [9] found that when HeLa cells are incubated with puromycin, the dissociation of polyribosomes to monomeric ribosomes occurs without a concomitant increase in ribosomal subunits, and similar observations have been made with intact rabbit reticulocytes [2]. This is in contrast to a study with E. coli [22], where the antibiotic was shown to promote the dissociation of monomeric ribosomes into subunits. Fluoride, which appears to be a preferential inhibitor of peptide chain initiation [14], has been reported to promote a decreasein ribosomal subunits concomitant with an increase in monomeric ribosomes [4, 91. Others [2, 161 have reported no significant changes in levels of ribosomal subunits when the level of ribosomal monomers was increased by fluoride. It appears that the abundance of ribosomal subunits relative to monomeric ribosomes of mammalian cells may be determined both by the stage of growth of the cells and the method of isolation of the ribosomes D71. It was of interest to examine the level of ribosomal subunits associated with the increased level of ribosomal monomers caused by three methods of inducing polyribosome disaggregation in reticulocytes, (i) tryptophan deficiency [lo]; (ii) iron (heme) deficiency [21, 24, 251; and (iii) butanol shock [7]. By omitting tryptophan from an otherwise complete incubation medium, translation of mRNA is slowed at the sites of the tryptophan residues [lo], located at positions 14 of the cc-chain[5] and 15and 37 of the,%chain [3]. Sincegrowth of the peptide chains proceeds from the amino-
Ribosomal subunits in rabbit ~et~~~l~cyte~ 481
Fig. I. Abscissa: distance from bottom of tube (cm); ordinate: absorbancy at 260 nm. Polyribosomes, monomeric ribosomes and subunits of intact rabbit reticulocytes. A, Control at 0 time, or after 5 and 20 min incubation in complete medium, B, after disaggregation of polyribosomes by the omission of tryptophan from an otherwise complete medium, incubation time 20 min; C, after disaggregation of poljiribosomes by either 20 min incubation with 2,2’-bipyridine, 2 x IOF M, or 5 min incubation with n-butanoil 0.1 M.
terminal end, during a deficiency of this four times that used. Transferrin and Fez’ amino acid the rate of translation of mRNA were omitted when the cells were incubated is retarded near the proximal end. If the other with the 2,2’-bipyridine. The cells were iso-, essential amino acids are present, a normal lated by centrifugation at 4”C, lysed and the rate of translation occurs beyond these sites, coarse particulate fraction removed [21, 2% thus causing polyribosome disaggregation be- 251. The total ribosomal component was cause of a failure to maintain the steady state isolated from the lysate by centrifugalion at number of ribosomes on the mRNA. In addi- 100 000 g for 3 h. Similar results were obtion, polyribosomes are disaggregated by in- tained if the centrifugation was contirmed for ducing a heme deficiency with the iron chelat- 9 h. The ribosomal pellet was resuspende ing agent, 2,2’-bipyridine [21, 24, 251, or by gently in 1.2 ml of standard buffer, cleared of interfering with cell membrane function with any coarse material by centrifugation at 600 g n-butanol [7]. With these treatments the ribo- for 10 min and 1 ml of this was layered on a somes already on mRNA complete transla- 15-30 y0 linear sucrose gradient. This was tion and are inhibited in their ability to reini- centrifuged for 5 h on a Spinco SW25.1 rotor tiate protein synthesis. and analyzed as previously described. As may be seen in fig. 1, of the ~~~~it~~~s Preparation of reticulocytes and incubation of the washed cells with glucose, amino acids, which increase the level of monomeric ribobuffered salt solution, Fez+ and transferrin somes (80 S), only a tryptophan deficiency were identical to that previously described also increases the amount of subunits. The [21, 24, 251, except that the volumes were changes in subunit levels, although small,
482
M. L. Freedman & M. Rabinowitz
were consistent and could be estimated from the peak heights by comparison with a calibration curve obtained with portions of a preparation of monomeric ribosomes and subunits treated identically. By this method it was found that under conditions of tryptophan deficiency there was approximately a twofold increase in monomeric ribosomes and a 50 y0 increase in the 40 S subunits. An increase in Ihe level of 60 S subunits was also noted; however, since they were not completely resolved from the 80 S ribosomes, no quantitation was attempted. In seven experiments, the percentage increase in monomeric ribosomes ranged from 55 to 120 while the increase in 40 S subunits ranged from 30 to 57. The significance of this small increase is reinforced by the failure to find any increase in subunit levels under the other conditions of polyribosome disaggregation (fig. 1 C). These observations support the view that there is equilibration between the polyribosomes, monomeric ribosomes and subunits in the intact mammalian cell. Under conditions of tryptophan deficiency, a retardation of ribosomal movement occurs at the sites of the tryptophan codons, resulting in a decreased rate of initiation. Under these conditions the initiation step itself is not affected, but it cannot be expressed because of the mechanical block caused by a retarded ribosome occupying its site. It would therefore appear that conditions which promote polyribosome disaggregation without causing a corresponding increase in ribosomal subunits, such as iron deficiency or butanol treatment, interfere with chain initiation at the stage of ribosome dissociation. REFERENCES 1. Bishop, J 0, Biochim biophys acta 119 (1966) 130. 2. - Arch biochem biophys 125 (1968) 449. 3. Braunitzer, G, Best, J S, Flamm, U & Schrank, B, Z physiol Chem 347 (1966) 207. Exptl Cell Res 60
4. Colombo, B, Vesco, C & Baglioni, C, Proc natl acad sci US 61 (1968) 651. 5. von Ehrenstein, G, Cold Spring Harbor symp quant biol 31 (1966) 705. 6. Eisenstadt, J M & Brawerman, G, Proc natl acad sci US 58 (1967) 1560. 7. Freedman, M L, Hori, M & Rabinovitz, M, Science 157 (1967) 323. 8. Hille, M B, Miller, M J, Iwasaki, K & Wahba, A J, Proc natl acad sci US 58 (1967) 1652. 9. Hogan, B L M, Biochim biophys acta 182 (1969) 264. 10. Hori, M, Fisher, J M & Rabinovitz, M, Science 155 (1967) 83. 11. Joklik, W K &Becker, Y, J mol biol13 (1965) 496. 12. Kaempfer, R, Proc natl acad sci US 61 (1968) 106. 13. Kaempfer, R 0 R, Meselson, M & Raskas, H J, J mol biol 31 (1968) 277. 14. Lin, S, Mosteller, R D & Hardesty, B, J mol biol 21 (1966) 51. 15. Mangiarotti, G & Schlessinger, D, J mol biol 29 (1967) 395. 16. Marks, PA & Kovach, J S, Current topics in developmental biology (ed A A Moscona &A Monroy) vol. I, p. 230. Academic Press, New York (1966). 17. McConkey, C N & Hopkins, J W, J mol biol 14 (1965) 257. 18. Nomura, M & Lowry, C V, Proc natl acad sci US 58 (1967) 946. 19. Nomura, M, Lowry, C V & Guthrie, C, Proc natl acad sci US 58 (1967) 1487. 20. Pestka, S & Nirenberg, M, J mol biol 21 (1966) 145. 21. Rabinovitz, I____. ^^_ M & Waxman, H S, Nature 206 (IYb5)
X9-l.
22. Schlessinger, D, Mangiarotti, G & Apirion, D, Proc natl acad sci US 58 (1967) 1782. 23. Warner, J R, Knopf, P hi & Rich, A, Proc natl acad sci US 49 (1963) 122. 24. Waxman, H S & Rabinovitz, M, Biochem biophys res commun 19 (1965) 538. 25. - Biochim biophys acta 129 (1966) 369. Received March 9, 1970
A serum macromolecule-supplemented medium for frog cell lines1 LOUISE E. SOOY and LISELOTTE MEZGERFREED, The Institute for Cancer Research, Philadelphia, Pa 19111, USA
A basal medium for cultured frog (Rana pipiens) cells has been developed for the isolation of genetic variants with nutritional deficiencies. Until now frog cell lines have been maintained exclusively in media with whole serum [6, 7, 141.Since whole serum contains significant concentrations of compounds of
4. Richardson, K C, Phil trans b 225 (1935) 149. 5. Sandborn, E B, Makita, T & Lin, K, Anat ret 163 (1969) 255. Received March 3, 1970
Ribosomal subunits in rabbit reticulocytes under different conditions of polyribosome disaggregation M. L. FREEDMAN’
and M. RABINOVITZ,
ratory of Physiology, National Bethesda, Md 20014, USA
Camcer Institute,
L&oNIH,
Summary Disaggregation of reticulocyte polyribosomes caused by tryptophan deficiency was accompanied by an increase in the level of ribosomal subunits, but disaggregation caused by iron deficiency or butanol shock was not. The results are interpreted in terms of the mechanism of action of the various treatments on the translation process.
There has been considerable interest in the role of ribosomal subunits in the initiation step of protein synthesis. Much of the evidence in support of such a mechanism is derived from studies with Escherichia coli [15, 201, where it has been shown that these subunits recycle through ribosomes during both bacterial growth [13] and acellular protein synthesis [12]. Investigations with acellular bacterial systems indicate that the smaller ribosomal subunit associates with initiation factors, messenger RNA (mRNA) and formylmethionine-transfer RNA to form a complex which initiates protein synthesis when the larger subunit attaches to the smaller subunit of the complex [6, 8, 18, 191. The view that the ribosomal subunits rather than intact ribosomes are active in the 1 Present address::Department of Medicine, New York Unersity Medical Center, New York, N.Y. 10016, Exptl
Cell Res 60
initiation of protein synthesis by mammalian cells first came from observations by Bishop [l] on initiation of hemoglobin synthesis by an acellular reticulocyte preparation. However, support for an active role ,of ribosomal subunits in protein synthesis by intact mammalian cells has been varied. Thus, Joklik & Becker [ll] and Hogan [9] found that when HeLa cells are incubated with puromycin, the dissociation of polyribosomes to monomeric ribosomes occurs without a concomitant increase in ribosomal subunits, and similar observations have been made with intact rabbit reticulocytes [2]. This is in contrast to a study with E. coli [22], where the antibiotic was shown to promote the dissociation of monomeric ribosomes into subunits. Fluoride, which appears to be a preferential inhibitor of peptide chain initiation [14], has been reported to promote a decreasein ribosomal subunits concomitant with an increase in monomeric ribosomes [4, 91. Others [2, 161 have reported no significant changes in levels of ribosomal subunits when the level of ribosomal monomers was increased by fluoride. It appears that the abundance of ribosomal subunits relative to monomeric ribosomes of mammalian cells may be determined both by the stage of growth of the cells and the method of isolation of the ribosomes D71. It was of interest to examine the level of ribosomal subunits associated with the increased level of ribosomal monomers caused by three methods of inducing polyribosome disaggregation in reticulocytes, (i) tryptophan deficiency [lo]; (ii) iron (heme) deficiency [21, 24, 251; and (iii) butanol shock [7]. By omitting tryptophan from an otherwise complete incubation medium, translation of mRNA is slowed at the sites of the tryptophan residues [lo], located at positions 14 of the cc-chain[5] and 15and 37 of the,%chain [3]. Sincegrowth of the peptide chains proceeds from the amino-
Ribosomal subunits in rabbit ~et~~~l~cyte~ 481
Fig. I. Abscissa: distance from bottom of tube (cm); ordinate: absorbancy at 260 nm. Polyribosomes, monomeric ribosomes and subunits of intact rabbit reticulocytes. A, Control at 0 time, or after 5 and 20 min incubation in complete medium, B, after disaggregation of polyribosomes by the omission of tryptophan from an otherwise complete medium, incubation time 20 min; C, after disaggregation of poljiribosomes by either 20 min incubation with 2,2’-bipyridine, 2 x IOF M, or 5 min incubation with n-butanoil 0.1 M.
terminal end, during a deficiency of this four times that used. Transferrin and Fez’ amino acid the rate of translation of mRNA were omitted when the cells were incubated is retarded near the proximal end. If the other with the 2,2’-bipyridine. The cells were iso-, essential amino acids are present, a normal lated by centrifugation at 4”C, lysed and the rate of translation occurs beyond these sites, coarse particulate fraction removed [21, 2% thus causing polyribosome disaggregation be- 251. The total ribosomal component was cause of a failure to maintain the steady state isolated from the lysate by centrifugalion at number of ribosomes on the mRNA. In addi- 100 000 g for 3 h. Similar results were obtion, polyribosomes are disaggregated by in- tained if the centrifugation was contirmed for ducing a heme deficiency with the iron chelat- 9 h. The ribosomal pellet was resuspende ing agent, 2,2’-bipyridine [21, 24, 251, or by gently in 1.2 ml of standard buffer, cleared of interfering with cell membrane function with any coarse material by centrifugation at 600 g n-butanol [7]. With these treatments the ribo- for 10 min and 1 ml of this was layered on a somes already on mRNA complete transla- 15-30 y0 linear sucrose gradient. This was tion and are inhibited in their ability to reini- centrifuged for 5 h on a Spinco SW25.1 rotor tiate protein synthesis. and analyzed as previously described. As may be seen in fig. 1, of the ~~~~it~~~s Preparation of reticulocytes and incubation of the washed cells with glucose, amino acids, which increase the level of monomeric ribobuffered salt solution, Fez+ and transferrin somes (80 S), only a tryptophan deficiency were identical to that previously described also increases the amount of subunits. The [21, 24, 251, except that the volumes were changes in subunit levels, although small,
482
M. L. Freedman & M. Rabinowitz
were consistent and could be estimated from the peak heights by comparison with a calibration curve obtained with portions of a preparation of monomeric ribosomes and subunits treated identically. By this method it was found that under conditions of tryptophan deficiency there was approximately a twofold increase in monomeric ribosomes and a 50 y0 increase in the 40 S subunits. An increase in Ihe level of 60 S subunits was also noted; however, since they were not completely resolved from the 80 S ribosomes, no quantitation was attempted. In seven experiments, the percentage increase in monomeric ribosomes ranged from 55 to 120 while the increase in 40 S subunits ranged from 30 to 57. The significance of this small increase is reinforced by the failure to find any increase in subunit levels under the other conditions of polyribosome disaggregation (fig. 1 C). These observations support the view that there is equilibration between the polyribosomes, monomeric ribosomes and subunits in the intact mammalian cell. Under conditions of tryptophan deficiency, a retardation of ribosomal movement occurs at the sites of the tryptophan codons, resulting in a decreased rate of initiation. Under these conditions the initiation step itself is not affected, but it cannot be expressed because of the mechanical block caused by a retarded ribosome occupying its site. It would therefore appear that conditions which promote polyribosome disaggregation without causing a corresponding increase in ribosomal subunits, such as iron deficiency or butanol treatment, interfere with chain initiation at the stage of ribosome dissociation. REFERENCES 1. Bishop, J 0, Biochim biophys acta 119 (1966) 130. 2. - Arch biochem biophys 125 (1968) 449. 3. Braunitzer, G, Best, J S, Flamm, U & Schrank, B, Z physiol Chem 347 (1966) 207. Exptl Cell Res 60
4. Colombo, B, Vesco, C & Baglioni, C, Proc natl acad sci US 61 (1968) 651. 5. von Ehrenstein, G, Cold Spring Harbor symp quant biol 31 (1966) 705. 6. Eisenstadt, J M & Brawerman, G, Proc natl acad sci US 58 (1967) 1560. 7. Freedman, M L, Hori, M & Rabinovitz, M, Science 157 (1967) 323. 8. Hille, M B, Miller, M J, Iwasaki, K & Wahba, A J, Proc natl acad sci US 58 (1967) 1652. 9. Hogan, B L M, Biochim biophys acta 182 (1969) 264. 10. Hori, M, Fisher, J M & Rabinovitz, M, Science 155 (1967) 83. 11. Joklik, W K &Becker, Y, J mol biol13 (1965) 496. 12. Kaempfer, R, Proc natl acad sci US 61 (1968) 106. 13. Kaempfer, R 0 R, Meselson, M & Raskas, H J, J mol biol 31 (1968) 277. 14. Lin, S, Mosteller, R D & Hardesty, B, J mol biol 21 (1966) 51. 15. Mangiarotti, G & Schlessinger, D, J mol biol 29 (1967) 395. 16. Marks, PA & Kovach, J S, Current topics in developmental biology (ed A A Moscona &A Monroy) vol. I, p. 230. Academic Press, New York (1966). 17. McConkey, C N & Hopkins, J W, J mol biol 14 (1965) 257. 18. Nomura, M & Lowry, C V, Proc natl acad sci US 58 (1967) 946. 19. Nomura, M, Lowry, C V & Guthrie, C, Proc natl acad sci US 58 (1967) 1487. 20. Pestka, S & Nirenberg, M, J mol biol 21 (1966) 145. 21. Rabinovitz, I____. ^^_ M & Waxman, H S, Nature 206 (IYb5)
X9-l.
22. Schlessinger, D, Mangiarotti, G & Apirion, D, Proc natl acad sci US 58 (1967) 1782. 23. Warner, J R, Knopf, P hi & Rich, A, Proc natl acad sci US 49 (1963) 122. 24. Waxman, H S & Rabinovitz, M, Biochem biophys res commun 19 (1965) 538. 25. - Biochim biophys acta 129 (1966) 369. Received March 9, 1970
A serum macromolecule-supplemented medium for frog cell lines1 LOUISE E. SOOY and LISELOTTE MEZGERFREED, The Institute for Cancer Research, Philadelphia, Pa 19111, USA
A basal medium for cultured frog (Rana pipiens) cells has been developed for the isolation of genetic variants with nutritional deficiencies. Until now frog cell lines have been maintained exclusively in media with whole serum [6, 7, 141.Since whole serum contains significant concentrations of compounds of