Volume 2 number 10 October 1975
Nucleic Acids Research
Synthesis of tobacco mosaic virus coat protein following migration of viral RNA into isolated mouse liver mitochondria
G.J.Dimitriadis and J.G.Georgatsos Laboratory of Biochemistry, School of Science, University of Patras, Patras, Greece
Received 8 July 1975 ABSTRACT iNA of Tobacco Mosaic Virus is shown to be able to migrate into isolated mouse liver mitochondria, whence it can be reisolated intact. The migration of RNA is accompanied by enhanced rate of protein synthesis, which is sensitive to chloramphemi&eOl but not to cycloheximide. Evidence is presented showing that, among the products formed is the coat protein of Tobacco Mosaic Virus.
INTRODUCTION
In a recent communication we showed that, under appropriate incubation coditions, mitochondria of Tetrahymena pyriformis are capable of translating RNA of rabbit reticulocytes to form the ca and f chains of rabbit globin (1). Ample evidence that exogenous RNA, either of nuclear origin or synthetic, may enter mitochondria, has been presented in the past by a number of groups (2,3) including ours (4,5). In the present work we investigated the migration of a viral RNA into isolated mitochondria, its fate within the organelles, and finally the ability of the mitochondria to translate the genetic information of the viral genome into
specific products. MATERIALS AND METHODS
Tobacco Mosaic Virus (TMV) was grown on leaves of tobacco plants Nicotiana Tabaccum. The virus was harvested from the leaves and purified as descrived by Fraenkel - Conrat (6). TMV-RNA was isolated by phenol bentonite extraction according to the method of Fraenkel-Conrat et al (7). TMV - coat pro-
1719
Nucleic Acids Research tein was precipitated from the phenol layer by adding ten volumes of methanol and a few crystals of sodium acetate. The precipitate was washed with methanol and ether. Mitochondria were obtained from livers of mice of the pure inbred strain C3Hb, according to the method of Hogeboom (8) modified in that the sucrose was dissolved in buffer composed of 0.1M Tris-HCl, 0.003M EDTA and 0.003M mercaptoethanol at pH 7.4. Subfractionation of mitochondria into outer membranes intermembrane material and outer membranefree mitochondria, was performed according to the method of Sottocasa et al. (9). Mitochondrial RNA was isolated as described by Brawerman et al. (10). Radioactive TMV-RNA was obtained by in vitro labelling, either with 3 H, by means of sodium borotritiide according to the method of De Wachter and Fiers (11), or with 125I, by means of carrier free (125I) sodium iodide according to the method of Shoulder et al. (12). Isotopically labelled reagents were obtained from Amersham, Bucks, England. Electrophoretic analysis on polyacrylamide gels of RNA was performed according to Loening's method (13) and of proteins as described by Weber and Osborn (14). Whenever required, proteins were stained by means of Coomassie blue. Migration of TMV-RNA into isolated mitochondria was tested by a procedure described in detail elsewhere (5). Briefly, mitochondria and exogenous labelled RNA, TMV-RNA in the present case, are incubated in Eagle's medium at 370 for 30 min. The mitochondria are reisolated from the incubation medium by centrifugation at 5000g, and washed 3 times with a buffered sucrose solution in the presence of EDTA and mercaptoethanol. RNA is isolated from the organelles by the method of Brawerman et al (10). Amino acid incorporation into mitochondrial proteins was assayed as described in (1) in a medium suggested by Beattie and Ibrahim (15). Radioactivities were measured in a Packard Tri-Carb Liquid Scintillation Spectrometer. Radioactive polyacrylamide fractions were counted in a Toluene scintillation cocktail that included 3% Protosol of New England Nuclear. Prior to counting, the gel-cocktail mixtures were incubated at 450
1720
Nucleic Acids Research overnight. Radioactive precipitates were counted in a dioxane scintillation cocktail after treatment with formic acid in a boiling water bath. RESULTS Table 1 shows that TMV-RNA, like all other RNA species tested thus far, is capable of migrating into isolated mitochondria. The extend of migration is approximately 2% of the RNA offered. In experiment 4 of Table 1 the distribution of radioactivity in the submitochondrial fractions was as follows: outer membranes - 1200 cpmn, inter membrane material 250 cpm and outer membrane - free mitochondria - 580 cpm. An aliquot of the mitochondria of experiment 5 in Table 1 was phenol extracted, according to the method of Brawerman et al (10) in the presence of yeast RNA as carrier, and the
Table 1. Extent of migration of TMV-RNA into isolated mouse liver
mitochondria Experiment No 1 2 3 4 5
Offered RNA (cpm)
16857 19320 42143 88650 37020
Migrated RNA (cpm) 367 382 880 2030 632
Type of label
3H 3H 3H 125I 125I
Percent Migration
2.18 1.98 2.08 2.26 1.70
Incubation conditions as described under Materials and Methods. Each tube contained 3mg mitochondrial protein. Specific radioactivity of tritiated TMV-RNA 5600cpm/A260. SpeciI 39X105cpm/A260. fic radioactivity of RNA isolated total RNA was analyzed by electrophoresis on 2.6% polyacrylamide gels. Fig.1 shows one major peak of radioactivity that coincides exactly with the position to which untreated TMV-RNA- 125I moves under the same experimental conditions. As in the case of nuclear RNA (5), TMV-RNA does not undergo degradation inside the mitochondria. Fig.2 shows the stimulation of amino acid incorporation into TCA precipitable mitochondrial material, as a function of TMV-RNA concentration. The observed stimulation is inhi1721
Nucleic Acids Research
201 15 x
E 10
a
u
5 A
0
24 18 12 Fraction No
6
30
36
Figure 1. Electrophoresis of. mitochondrial RNA on 2.6% polyacrylamide gels following incubation of mitochondria with TMV-RNA-125I. Each fraction corresponds to 2mm of gel.
301
251
0: x
E Q
201
0
30
60
90
TMV-RNA( p9g/ml)
Figure 2. Stimulation of amino acid incorporation into mitochondrial protein by added TMV-RNA. Each tube contained mitochondria (0.7mg protein/ml) preincubated in Eagle's medium with the indicated concentrations of TMV-RNA. Amino acid incorporation into proteins was assayed as described under Materials and Methods in text by including 5UCi 14C-protein hydrolysate (57mCi/mAtom Carbon) per ml of incubation medium. 1722
Nucleic Acids Research bited by chloramphenicol but not by cycloheximide (fig.3). To test whether TMV - coat protein is formed within mitochondria in the presence of TMV-RNA, mitochondria were preincubated in Eagle's medium for 10 min in the presence and absence (control) of TMV-RNA. The preincubated mitochondria were isolated from the medium by centrifugation, washed with buffered sucrose solution in the presence of EDTA and mercaptoethanol to exclude contaminating nonmigrated RNA and were then incubated with radioactive amino acids under conditions described in detail in (1). Mitochondrial proteins were analyzed by means of polyacrylamide gel electrophoresis. Fig.4 is a representative electrophorogram that shows the appearance of a radioactive peak in the exact position that purified TMV - coat protein moves, only when mitochondria were preincubated with TMV-RNA. Fig.5 isa photograph of stained gels following electrophoresis of mitochondrial and viral protein respectively.
0
0.
C'CL Inhibitor (pmobsIsmi) 0
Figure 3. Effect of antibiotics on the TMV-RNA stimullated incorporation of amino acids into mitochondrial proteins. Each tube contained mitochondria (1.2mg protein/ml) preincubated in Eagle's medium with TMV-RNA (300g/ml). Amino acid incorporation into protein was assayed as described under Materials and Methods in text, by including SiCi 14C-protein hydrolysate (57mCi/mAtom Carbon) per ml of incubation medium. 0-S In the presence of cycloheximide, 0-0 in the presence of chloramphenicol.
1723
Nucleic Acids Research
6-)
0
E 0. u
Frac_ion
No
f ra ct i on
No
Figure 4. Following incubation of mitochondria, under amino acid incorporation conditions, the mitochondria were removed from the medium, washed and dissolved in 10% SDS in electrophoresis buffer in the presence of 10-1M mercaptoethanol. Gel size 0.45X10cm. Electrophoresis for 5hr in 10% polyacrylamide according to the method of Weber and Osborn (14). Each fraction corresponds to 2mm of gel. The figure shows radioactive fractior-3when mitochondria were preincubated in the presence (0-@) and in the absence (O---0) of TMV-RNA. The bar shows the position of Coomassie blue staining when TMVcoat protein is subjected to electrophoresis -nder identical conditions (cf. Fig.5).
Figure 5.
Photograph of 10% polycrylamide gels stained with Coomassie blue, following electrophoresis of mitochondrial (left) and TMV-coat protein (right), by the method of Weber and Osborn (1969).
1724
Nucleic Acids Research DISCUSSION
The results of the present investigation show that TMVRNA can migrate into mouse liver mitochondria whence it can be reisolated intact. The presence of TMV-RNA in mitochondria induces a stimulation to the protein synthetic activity of the organelles, that is sensitive to chloramphenicol, but not to cycloheximide. Strong evidence has been obtained, indicating that among the products formed within the mitochondrion, in the presence of TMV-RNA, is TMV - coat protein. Until evidence is obtained that the mechanism of translation of exogenous informational RNA by mitochondria is also operative in vivo, we may not speculate on the biological significance of this process. The discovery by KAra and his group (16,17) of the presence and biosynthesis of subviral oncogenic ribonucleoprotein particles in mitochondria from Rous sarcoma tissue, constitutes indirect evidence that such a mechanism may indeed be operative in vivo. Irrespective of this aspect, however, we wish to point out that the present, as well as our previous work (1), has shown that the mitochondrion is a "packaged" cell-free protein synthesizing system that may translate a diversity of informational RNAs.
REFERENCES
1 2 3
4 5 6 7 8 9 10
11
Dimitriadis, G.J. and Georgatsos, J.G. (1974) FEBS Letters 46, 96. Swanson, V.F. (1971) Nature 231, 31. Gaitskhoki, V.S., Kisselev, O.I. and Neifakh, S.A.(1973) FEBS Letters 31, 93. Georgatsos, J.G. (1972), Proc. Soc. Exp. Biol. and Med. 139, 667. Kyriakidis, D. and Georgatsos, J.G., (1973) Biochem. Biophys. Res. Commun. 53, 1167. Fraenkel-Conrat, H. (1967) in Procedures in Nucleic Acids Research (Cantoni, G.L. and Davies, D.R. Ed.), Academic Press, N.Y. pp. 481-484. Fraenkel-Conrat, H., Singer, B., Tsucita, A., (1961). Virology, 14, 54. Hogeboom, G.H. (1955) Methods in Enzymol. 1, 16. Sottocasa, G.L., Kuylenstierma, B., Ernster, L. and Bergstrand, A. (1967), J. Cell Biol. 32, 415. Brawerman, G., Mendeki, J., and Lee, S.Y. (1972) Biochemistry 11, 637. De Wachter, R. and Fiers, W. (1967), J. Mol. Biol. 30, 507. 1725
Nucleic Acids Research 12 13 14
Shoulder, A., Darby, G., and Minson, T. (1974), Nature, 251, 733. Loening, V.E. (1969), Biochem. J. 113, 131. Weber, K. and Osborn, M. (1969), J. Biol. Chem. 244, 4406.
15
Beattie, D.C. and Ibrahim, N.G. (1973) Biochemistry 12,
16
K&ra, J., Mach, 0. and Cerna, H. (1971) Biochem. Biophys Res. Commun. 44, 162. Klra, J. and Mach, 0. (1973) Folia Biol. (Praha) 19, 78.
176. 17
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Nucleic Acids Research
Synthesis of tobacco mosaic virus coat protein following migration of viral RNA into isolated mouse liver mitochondria
G.J.Dimitriadis and J.G.Georgatsos Laboratory of Biochemistry, School of Science, University of Patras, Patras, Greece
Received 8 July 1975 ABSTRACT iNA of Tobacco Mosaic Virus is shown to be able to migrate into isolated mouse liver mitochondria, whence it can be reisolated intact. The migration of RNA is accompanied by enhanced rate of protein synthesis, which is sensitive to chloramphemi&eOl but not to cycloheximide. Evidence is presented showing that, among the products formed is the coat protein of Tobacco Mosaic Virus.
INTRODUCTION
In a recent communication we showed that, under appropriate incubation coditions, mitochondria of Tetrahymena pyriformis are capable of translating RNA of rabbit reticulocytes to form the ca and f chains of rabbit globin (1). Ample evidence that exogenous RNA, either of nuclear origin or synthetic, may enter mitochondria, has been presented in the past by a number of groups (2,3) including ours (4,5). In the present work we investigated the migration of a viral RNA into isolated mitochondria, its fate within the organelles, and finally the ability of the mitochondria to translate the genetic information of the viral genome into
specific products. MATERIALS AND METHODS
Tobacco Mosaic Virus (TMV) was grown on leaves of tobacco plants Nicotiana Tabaccum. The virus was harvested from the leaves and purified as descrived by Fraenkel - Conrat (6). TMV-RNA was isolated by phenol bentonite extraction according to the method of Fraenkel-Conrat et al (7). TMV - coat pro-
1719
Nucleic Acids Research tein was precipitated from the phenol layer by adding ten volumes of methanol and a few crystals of sodium acetate. The precipitate was washed with methanol and ether. Mitochondria were obtained from livers of mice of the pure inbred strain C3Hb, according to the method of Hogeboom (8) modified in that the sucrose was dissolved in buffer composed of 0.1M Tris-HCl, 0.003M EDTA and 0.003M mercaptoethanol at pH 7.4. Subfractionation of mitochondria into outer membranes intermembrane material and outer membranefree mitochondria, was performed according to the method of Sottocasa et al. (9). Mitochondrial RNA was isolated as described by Brawerman et al. (10). Radioactive TMV-RNA was obtained by in vitro labelling, either with 3 H, by means of sodium borotritiide according to the method of De Wachter and Fiers (11), or with 125I, by means of carrier free (125I) sodium iodide according to the method of Shoulder et al. (12). Isotopically labelled reagents were obtained from Amersham, Bucks, England. Electrophoretic analysis on polyacrylamide gels of RNA was performed according to Loening's method (13) and of proteins as described by Weber and Osborn (14). Whenever required, proteins were stained by means of Coomassie blue. Migration of TMV-RNA into isolated mitochondria was tested by a procedure described in detail elsewhere (5). Briefly, mitochondria and exogenous labelled RNA, TMV-RNA in the present case, are incubated in Eagle's medium at 370 for 30 min. The mitochondria are reisolated from the incubation medium by centrifugation at 5000g, and washed 3 times with a buffered sucrose solution in the presence of EDTA and mercaptoethanol. RNA is isolated from the organelles by the method of Brawerman et al (10). Amino acid incorporation into mitochondrial proteins was assayed as described in (1) in a medium suggested by Beattie and Ibrahim (15). Radioactivities were measured in a Packard Tri-Carb Liquid Scintillation Spectrometer. Radioactive polyacrylamide fractions were counted in a Toluene scintillation cocktail that included 3% Protosol of New England Nuclear. Prior to counting, the gel-cocktail mixtures were incubated at 450
1720
Nucleic Acids Research overnight. Radioactive precipitates were counted in a dioxane scintillation cocktail after treatment with formic acid in a boiling water bath. RESULTS Table 1 shows that TMV-RNA, like all other RNA species tested thus far, is capable of migrating into isolated mitochondria. The extend of migration is approximately 2% of the RNA offered. In experiment 4 of Table 1 the distribution of radioactivity in the submitochondrial fractions was as follows: outer membranes - 1200 cpmn, inter membrane material 250 cpm and outer membrane - free mitochondria - 580 cpm. An aliquot of the mitochondria of experiment 5 in Table 1 was phenol extracted, according to the method of Brawerman et al (10) in the presence of yeast RNA as carrier, and the
Table 1. Extent of migration of TMV-RNA into isolated mouse liver
mitochondria Experiment No 1 2 3 4 5
Offered RNA (cpm)
16857 19320 42143 88650 37020
Migrated RNA (cpm) 367 382 880 2030 632
Type of label
3H 3H 3H 125I 125I
Percent Migration
2.18 1.98 2.08 2.26 1.70
Incubation conditions as described under Materials and Methods. Each tube contained 3mg mitochondrial protein. Specific radioactivity of tritiated TMV-RNA 5600cpm/A260. SpeciI 39X105cpm/A260. fic radioactivity of RNA isolated total RNA was analyzed by electrophoresis on 2.6% polyacrylamide gels. Fig.1 shows one major peak of radioactivity that coincides exactly with the position to which untreated TMV-RNA- 125I moves under the same experimental conditions. As in the case of nuclear RNA (5), TMV-RNA does not undergo degradation inside the mitochondria. Fig.2 shows the stimulation of amino acid incorporation into TCA precipitable mitochondrial material, as a function of TMV-RNA concentration. The observed stimulation is inhi1721
Nucleic Acids Research
201 15 x
E 10
a
u
5 A
0
24 18 12 Fraction No
6
30
36
Figure 1. Electrophoresis of. mitochondrial RNA on 2.6% polyacrylamide gels following incubation of mitochondria with TMV-RNA-125I. Each fraction corresponds to 2mm of gel.
301
251
0: x
E Q
201
0
30
60
90
TMV-RNA( p9g/ml)
Figure 2. Stimulation of amino acid incorporation into mitochondrial protein by added TMV-RNA. Each tube contained mitochondria (0.7mg protein/ml) preincubated in Eagle's medium with the indicated concentrations of TMV-RNA. Amino acid incorporation into proteins was assayed as described under Materials and Methods in text by including 5UCi 14C-protein hydrolysate (57mCi/mAtom Carbon) per ml of incubation medium. 1722
Nucleic Acids Research bited by chloramphenicol but not by cycloheximide (fig.3). To test whether TMV - coat protein is formed within mitochondria in the presence of TMV-RNA, mitochondria were preincubated in Eagle's medium for 10 min in the presence and absence (control) of TMV-RNA. The preincubated mitochondria were isolated from the medium by centrifugation, washed with buffered sucrose solution in the presence of EDTA and mercaptoethanol to exclude contaminating nonmigrated RNA and were then incubated with radioactive amino acids under conditions described in detail in (1). Mitochondrial proteins were analyzed by means of polyacrylamide gel electrophoresis. Fig.4 is a representative electrophorogram that shows the appearance of a radioactive peak in the exact position that purified TMV - coat protein moves, only when mitochondria were preincubated with TMV-RNA. Fig.5 isa photograph of stained gels following electrophoresis of mitochondrial and viral protein respectively.
0
0.
C'CL Inhibitor (pmobsIsmi) 0
Figure 3. Effect of antibiotics on the TMV-RNA stimullated incorporation of amino acids into mitochondrial proteins. Each tube contained mitochondria (1.2mg protein/ml) preincubated in Eagle's medium with TMV-RNA (300g/ml). Amino acid incorporation into protein was assayed as described under Materials and Methods in text, by including SiCi 14C-protein hydrolysate (57mCi/mAtom Carbon) per ml of incubation medium. 0-S In the presence of cycloheximide, 0-0 in the presence of chloramphenicol.
1723
Nucleic Acids Research
6-)
0
E 0. u
Frac_ion
No
f ra ct i on
No
Figure 4. Following incubation of mitochondria, under amino acid incorporation conditions, the mitochondria were removed from the medium, washed and dissolved in 10% SDS in electrophoresis buffer in the presence of 10-1M mercaptoethanol. Gel size 0.45X10cm. Electrophoresis for 5hr in 10% polyacrylamide according to the method of Weber and Osborn (14). Each fraction corresponds to 2mm of gel. The figure shows radioactive fractior-3when mitochondria were preincubated in the presence (0-@) and in the absence (O---0) of TMV-RNA. The bar shows the position of Coomassie blue staining when TMVcoat protein is subjected to electrophoresis -nder identical conditions (cf. Fig.5).
Figure 5.
Photograph of 10% polycrylamide gels stained with Coomassie blue, following electrophoresis of mitochondrial (left) and TMV-coat protein (right), by the method of Weber and Osborn (1969).
1724
Nucleic Acids Research DISCUSSION
The results of the present investigation show that TMVRNA can migrate into mouse liver mitochondria whence it can be reisolated intact. The presence of TMV-RNA in mitochondria induces a stimulation to the protein synthetic activity of the organelles, that is sensitive to chloramphenicol, but not to cycloheximide. Strong evidence has been obtained, indicating that among the products formed within the mitochondrion, in the presence of TMV-RNA, is TMV - coat protein. Until evidence is obtained that the mechanism of translation of exogenous informational RNA by mitochondria is also operative in vivo, we may not speculate on the biological significance of this process. The discovery by KAra and his group (16,17) of the presence and biosynthesis of subviral oncogenic ribonucleoprotein particles in mitochondria from Rous sarcoma tissue, constitutes indirect evidence that such a mechanism may indeed be operative in vivo. Irrespective of this aspect, however, we wish to point out that the present, as well as our previous work (1), has shown that the mitochondrion is a "packaged" cell-free protein synthesizing system that may translate a diversity of informational RNAs.
REFERENCES
1 2 3
4 5 6 7 8 9 10
11
Dimitriadis, G.J. and Georgatsos, J.G. (1974) FEBS Letters 46, 96. Swanson, V.F. (1971) Nature 231, 31. Gaitskhoki, V.S., Kisselev, O.I. and Neifakh, S.A.(1973) FEBS Letters 31, 93. Georgatsos, J.G. (1972), Proc. Soc. Exp. Biol. and Med. 139, 667. Kyriakidis, D. and Georgatsos, J.G., (1973) Biochem. Biophys. Res. Commun. 53, 1167. Fraenkel-Conrat, H. (1967) in Procedures in Nucleic Acids Research (Cantoni, G.L. and Davies, D.R. Ed.), Academic Press, N.Y. pp. 481-484. Fraenkel-Conrat, H., Singer, B., Tsucita, A., (1961). Virology, 14, 54. Hogeboom, G.H. (1955) Methods in Enzymol. 1, 16. Sottocasa, G.L., Kuylenstierma, B., Ernster, L. and Bergstrand, A. (1967), J. Cell Biol. 32, 415. Brawerman, G., Mendeki, J., and Lee, S.Y. (1972) Biochemistry 11, 637. De Wachter, R. and Fiers, W. (1967), J. Mol. Biol. 30, 507. 1725
Nucleic Acids Research 12 13 14
Shoulder, A., Darby, G., and Minson, T. (1974), Nature, 251, 733. Loening, V.E. (1969), Biochem. J. 113, 131. Weber, K. and Osborn, M. (1969), J. Biol. Chem. 244, 4406.
15
Beattie, D.C. and Ibrahim, N.G. (1973) Biochemistry 12,
16
K&ra, J., Mach, 0. and Cerna, H. (1971) Biochem. Biophys Res. Commun. 44, 162. Klra, J. and Mach, 0. (1973) Folia Biol. (Praha) 19, 78.
176. 17
1726
