J. theor. Biol. (1977) 67,287-297
Participation of Chloroplasls and Mitochoudria in Virus Reproduction and the Evolution of the Enkaryotic Cell? S. A. OSTROUMOV
Department of Bioenergetics, Laboratory of Bio-organic Chemistry, Moscow State University and Shemiakin Institute of Bio-organic Chemistry,$ U.S.S.R. (Received 4 September 1975, and in revisedform 27 October 1976) The similarity of prokaryotic cells and the organellesof eukaryotes can be explained not only by the ordinary form of the endosymbiosis hypothesis,but also by another evolutionary model presentedhere. This model postulatesthat portions of DNA of an ancient prokaryote were transferredinto the nucleusof the ancient eukaryote cell. In combination with prokaryotic DNA-encoded proteins and phospholipid membranes prokaryotic DNA may have given rise to a structure resemblinga promitochondrion or a proplastid. The transfer of geneticmaterial into the nucleusor the cytoplasmcould have occurredvia a virus-mediatich.This suggestionis in accordancewith the reported associationof chloroplasts and plant mitochondria with replication of somestrainsof tobacco mosaic virus, cucumbergreenmottle mosaicvirus, barley stripe virus and tobacco rattle virus and with findings of virus-like particles in mammalianand reptilian cells as well as cells of Neurosporacrassa. The terms “mitophages” and “plastophages” (“chlorophages”) are introduced to designate the viruses associatedwith mitochondria or plastids (chloroplasts)during somephaseof development. Participation of virus-mediated DNA transfers can be suggestedfor endosymbiotic organellar origin also.
1. Introduction A much-debated question in the field of evolution of eukaryotic cells is the hypothesis of the endosymbiotic origin of energy-transducing organelles, the chloroplasts and mitochondria (e.g. Sagan, 1967; Skulachev, 1969; t This paperwaspresented in a symposium entitled“The Origin of Eukaryotic Cells” at the XII InternationalBotanical Congress, Leningrad,July 1975. $ Addressfor reprints: Dr S. A. Ostroumov, ShemiakinInstitute of Bio-organic Chemistry,U.S.S.R.Academyof Sciences, Vavilova 32, Moscow117312,U.S.S.R. 287
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Svetailo, 1969; Raven, 1970, 1975; Raff & Mahler, 1972; Takhtajan, 1973; Ostroumov, 1973; Margulis, 1970, 1975; John & Whatley, 1975; Carr & Phillips, 1975; Taylor, 1975). This paper is restricted to a consideration of some aspects of the possibility of the contribution of the genetic determinants of ancient prokaryotes, which apparently dominated in Precambrian Earth’s biota (e.g. Schopf, 1974), to the genetic material of eukaryotic cells. In this connection it seems to be interesting to use some information concerning viruses, which “make ideal mediators of . . . interspecies genetic exchange” (Zhdanov & Tikchonenko, 1974). A significant amount of data have been collected about the participation of mitochondria (Tables 1 and 2) and chloroplasts (Table 1) in the reproduction of several strains of viruses. Because individual stages of multiplication of different strains of the same virus can be localized in different parts of the cell the examples cited below must be interpreted with great caution. 2. Plants (A)
CHLOROPLASTS
Various authors using the electron microscope have reported an association of certain plant viruses with plastids (Bald, 1966; Matthews, 1970). For example, Ralph & Clark (1966) demonstrated that TYMV RNA synthesis and duplex (double-stranded) viral RNA in Chinese cabbage leaf cells systematically infected with TYMV was associated with the chloroplast fraction and did not occur in the cytoplasm or nuclei (for more comprehensive review of data about TYMV see the excellent survey of Matthews, 1973). In 1970 Ushiyama & Matthews obtained new data to support the suggestion that the replicative form of viral RNA is localized in the peripheral vesicles of the chloroplasts of Chinese cabbage (Brassicu pekineprsis) as well as turnip (B. rapa) cells infected with TYMV. The authors suggested that these vesicles could be the site of viral RNA synthesis, and that the viral RNA strands migrated from the vesicles to the cytoplasm where virus protein was synthesized and virus particles were assembled. Barley tissues infected with BSMV were examined with the electron microscope by Carroll (1970). Virions of BSMV were seen in close association with abnormally vesiculated chloroplasts and plastids of immature embryos of barley (Hordeurn vulgare; Carroll, 1970). Some virions surrounded chloroplasts, whereas others were localized in cytoplasmic invaginations into the chloroplasts. Some virions appeared to be attached
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1
Association of replication of somestrains of viruses with chloroplasts and mitochondria of plants Virus ~~ -.___.-~~ Chloroplasts TMV
TYMV BSMV Mitochondria
TMV CGMMV
Tobacco rattle virus Virus-like particles
-__ Tobacco
Reference ____-___ Zaitlin & Boardman, 1958 Gxh, 1967; Esau & Cronshaw, 1967 Shalla, 1968 Singer, 1972 but see Ralph & Clark, 1966 Ralph & Clark, 1966 Ushiyama & Matthews, 1970 Carroll, 1970
Host
Brassica pekinensis B. rapa Hordeum vulgare Tobacco Cucumber, pumpkin, gourd Nicotiana clevelandii Neurospora crassa abnormal-l mutant
TABLE
Hayashi, 1963 Hatta et al., 1971 Harrison, Roberts, 1968 Harrison et al., 1970 Kiintzel et al., 1973
2
Association of mitochondria of animals with production of virus-like particles Virus or virus-like particles ____. Viral nucleocapsids Virus-like particles Virus-like particles Subviral oncogenic ribonucleoprotein particles Virus-like particles
Subviral leukemogenic particles
Source ____. ------~ Hamster cells transformed by RSV Cells line VSW from a tumor bearing Russel’s viper Rous sarcoma cells
Fused hamster tumor, chick embryo cells Hamster tumor induced with the Schmidt-Ruppin strain of Rous sarcoma virus Leukemic spleen cells of mice infected with RML virus
Reference ~--___.-~. - -Gazzalo et al., 1969 Lunger & Clark, 1973 Kleitman et al., 1973 Kara et al., 1971 Kara et al., 1972 Kara & Mach, 1973 see also Bader, 1972 Nass, 1974
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to the outer chloroplast membrane. Carroll suggested that “the plastid vesicles supply structural components or enzymes or both necessary for multiplication of BSMV. These factors for multiplications could pass through the exterior membrane of the envelope and accumulate at sites on the outer surface of this membrane. At these sites the virus may be assembled into whole virions.” The case of TMV is a good example of how complicated the localization problem is (Matthews, 1970). For instance, Ralph & Clark (1966) found that double-stranded TMV RNA in infected tobacco leaves was present in the cytoplasmic fraction of leaf homogenates and not in nuclei or chloroplasts. Singer (1972) obtained evidence that TMV protein synthesis takes place in the chloroplasts. Esau & Cronshaw (1967) published electron micrographs showing clusters of virus-like particles in sections of chloroplasts of plants infected with TMV. The authors argued that the virus did not enter the chloroplasts by the mechanism of invagination but was probably formed there. Impressive data were obtained by Shalla (1968). He showed that sectioned chloroplasts of tobacco plants infected with the U5 strain of TMW contain particles resembling TMV. When chloroplasts were isolated from plants and disrupted, their loss of virions as seen with the electron microscope was correlated with the increase in the infectivity of the ambient medium. This paper showed that no equivalent clusters of virions were seen in chloroplasts of plants infected with the U, strain of TMV. (B)
MITOCHONDRIA
Some indication that the mitochondria of tobacco leaves may be involved in TMV infection comes from the work of Hayashi (1963). He studied incorporation of [14C] amino acids in variou cells fractions of plants infected with TMV and found that soon after infection the mitochondria had the highest incorporation rate per mg of protein. Hatta, Nakamoto, Takagi & Ushiyama (1971) found that crystals of cucumber green mottle mosaic virus (CGMMV) frequently occurred in areas of cytoplasmic invaginations that probably penetrated into mitochondria. Virus particles were not associated with chloroplasts or nuclei. Virus-infected cells contained abnormal mitochondria with small vesicles. Most of them seemed to be located in the space between the inner and outer mitochondrial membranes. Some vesicles seemed to have an opening to the cytoplasm. Another example of association of virus replication with plant mitochondria was provided by Harrison and co-workers. They have shown this for mitochondria of Nicotiana clevelandii infected with the longer type of
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CAM tobacco rattle virus (Harrison & Roberts, 1968) and RNA-producing defective isolate of tobacco rattle virus (Harrison, Stefanae & Roberts, 1970). A very interesting study of virus-like particles isolated from the abnormal- 1 mutant of Neurosporu crussa was made by Ktintzel & co-workers (Ktintzel et al., 1973; see also Schatz & Mason, 1974). Virus-like ribonucleoprotein particles were found both in the cytoplasm and a mitochondrial fraction of abnormal-l cells, but were undetectable in wild-type cells. The particles contained single-stranded 33 S RNA which hybridized with mitochondrial DNA from the abnormal-l mutant or wild type N. crassa. 3. Mitochondria (A)
ELECTRON
of Animals MICROSCOPY
The structures that were interpreted to represent viral nucleocapsids were found in mitochondria of hamster cells transformed by Rous sarcoma virus (Gazzalo, de-The, Vigier & Sarma, 1969). Intramitochondrial viral particles were found in a cell line termed VSW derived from a spleen tumour bearing Russell’s viper (Lunger & Clark, 1973; Kleitmann, Lunger & Clark, 1973). M. M. K. Nass and C. Buck have found numerous virus-like structures in mitochondria of fused hamster tumour-chick embryo cells (Nass, 1974). In this study the cells, derived from a hamster tumour induced with the Schmidt-Ruppin strain of Rous sarcoma virus, were fused with chick embryo fibroblasts using Sendai virus (Nass, 1974). An electron microscope analysis of the isolated Rous sarcoma subviral particles (see next section) was performed (K&a & Mach, 1973) and it supported the assumption that they were located in mitochondria. It is noteworthy that the claims that Rous sarcoma virus production occurs in mitochondria have been challenged by Bader (1972), but his arguments have been subjected to a certain amount of criticism by Nass (1974). (B)
BIOCHEMICAL
DATA
Kara, Cerna & Dvorak (1971) reported the presence and biosynthesis of subviral oncogenic ribonucleoprotein particles (virosomes) in the mitochondria isolated from Rous sarcoma (RS) cells. Virosomes were located in the inner membrane and matrix of RS mitochondria, contained virusspecific proteins (gs-antigens) and RNA-dependent DNA-polymerase (Kara et al., 1972). Recently Kara et al. (1974) presented evidence on biosynthesis of subviral leukemogenic particles in the isolated mitochondria of leukemic spleen cells of mice infected with Rauscher murine leukemic (RML) virus. These
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particles induce splenomegaly and RML 3 weeks after intravenous or interperitoneal administration to white mice. RML virus gs-antigenes were detected by immunoprecipitation in the inner membrane and matrix of the mitochondria of RML spleen cells (K&a et al., 1974). 4. Artificial Systems Study It was shown that mitochondrial RNA-polymerase could recognize transcriptional signals of bacteriophage T3 and T7 DNA (Richter, Herrlich & Schweiger, 1972). Moreover, mitochondrial ribosomes appeared to be capable of synthesizing phage-specific proteins. The ability of mitochondria to transcribe and translate viral RNA was systematically studied by Neifakh et al. (1974) and Zhdanov et al. (1971). In particular, the system of transcription and translation of TMV and Venezuelan equine encephalomyelitis viral RNA was reconstituted using isolated rat liver or yeast mitochondria or submitochondrial preparations (Zhdanov, Tikchonenko, Bocharov & Naroditsky, 1971; Yershov et al., 1971, 1974; Zaitseva et al., 1973). A similar reconstituted system was reported for bacteriophage MS2 genome RNA and rat liver mitochondria (Kisselev, Golubkov, Grabouskaya & Totolian, 1974; Gaitskhoki et al., 1974). 5. Discussion From facts such as those reviewed above it seems likely that some strains of viruses use the enzymes of mitochondria and chloroplasts. The terms “mitophages” and “plastophages” (“chlorophages”) were introduced for the designation of viruses, possessing stages of multiplication localized in mitochondria and chloroplasts (Ostroumov, 1974). It should be emphasized that far-reaching generalizations are premature, and we cannot conclude that all the viruses mentioned above or even a majority of them may be classed as mitophages and plastophages. “The question remains to be answered whether a mitochondrion-associated phase of viral development is indispensable or is merely auxiliary and can easily be carried on extramitochondrially when organelle function is impaired” (Nass, 1974). The same comment could be made about viruses associated with chloroplasts. The data summarized indirectly testify to the possibility of similar associations between viruses and the evolutionary precursors of organelles. We shall discuss this idea in more detail below, following a consideration of evolutionary origin of the organelles. The similarity between prokaryotes and energy-transducing organelles is well-documented and was discussed with reference to the function of energy-
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transforming membranes (e.g. Hind & Olson, 1968; Evans & Whatley, 1970; Henderson, 1971; Skulachev, 1972) and other aspects (e.g. Sagan, 1967; Skulachev, 1969; Svetailo, 1969; Margulis, 1970; Ostroumov, 1973; Taylor, 1974; John & Whatley, 1975). It seems reasonable to suggest a similarity between the nuclear genetic determinants encoding the prokaryotic type proteins of organelles in these eukaryotes and the analogous genetic determinants of prokaryotes. This suggestion might explain the similarity between the nuclear-encoded components of organelles and the analogous components of contemporary prokaryotes. From the evolutionary viewpoint one could propose that this nuclear genetic material originated from the genetic determinants of ancient prokaryotes. However, this still leaves the problem of redistribution of prokaryote genetic material between nuclei and organelles. Two alternatives may be formulated (see Figs 1 and 2). For the sake of clarity and brevity the following designation could be useful : DNA,,-DNA of contemporary energy-transducing organelle or its evolutionary predecessor (a part of DNA of a prokaryote). DNA,,-a part of prokaryotic DNA which was incorporated into nucleus of a protoeukaryotic cell; DNA, encodes organelle prokaryotic type proteins. One model is essentially the well-known endosymbiotic hypothesis with only the qualification that part of the DNA of an endosymbiotic prokaryote has been transferred to the nucleus. In other words, the following sequence of DNA transfers is postulated: (1) Prokaryotic DNA within the prokaryotic cell enters the cytoplasm of the macrocell. (2) DNA, from cytoplasmic endosymbiont enters the nucleus. Another model (Fig. 1) is based on reversed events and does not require a physical symbiosis (see also Zhdanov & Tikchonenko, 1974). First, a fragment of DNA of a prokaryote (without prokaryotic cell) is transferred to the nucleus. Second, DNA, is transported to the cytoplasm, where in combination with DNA,-encoded proteins and phospholipid membrane it gives rise to an organelle-like structure. In this case, the appearance of promitochondria-like or proplastid-like structures containing prokaryotic DNA seems probable. This assumption is in agreement with numerous experiments which demonstrate self-assemblage of phospholipid molecules and formation of T.B. 20
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Profoeukoryoflc
cell
Prokoryote DNA,
Eukoryotic
cell
FIG. I. One of hypothetical schemes of origin of mitochondria and chloroplasts. DNAO-part of ancient prokaryote DNA transferred into the orgunelIe (mitochondrion or chloroplast) predecessor; DNA,-part of ancient prokaryote DNA which after transfer into protoeukaryotic cell encodes organelle proteins and is located in the nucleus. The arrows indicate the transfer of fragments of ancient prokaryote DNA. One of the possibilities is transport by some virus-mediated mechanism.
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membraneous vesicles (liposomes). Liposomes readily incorporate proteins and can transform energy (e.g. Skulachev, 1972; Drachev et al., 1974) and produce ATP (e.g. Racker & Stoeckenius, 1974). The appearance of ATP producing membraneous vesicles in cytosol of protoeukaryotic cell could have given cell evolutionary advantage, because membrane-dependent phosphorylation of ADP is more efficient than phosphorylation during glycolysis. Could the transfer of DNA, into the membrane structures in the cytosol give the cell some evolutionary advantage? Yes, because the incorporation of hydrophobic proteins (encoded in DNA,) into the inner surface of membrane could improve the functioning of energy-transducing membraneous structures. The important link in both schemes is transfer of parts of prokaryotic DNA from the endosymbiont to the nucleus or to the cytoplasm. The aboveconsidered data prompt, as one of possibilities, transfer by some virusmediated mechanism. The study of association of some viruses with contemporary energytransducing organelles will provide an important additional source of information for understanding evolutionary and individual (Neifakh, 1974) development of mitochondria and chloroplasts. The author gratefully acknowledges Academician A. L. Takhtajan for his interest and support; Corresponding Member of Academy of Sciences of U.S.S.R. V. P. Skulachev, Professor Peter H. Raven and Dr A. S. Antonov for valuable advice and reading the manuscript; Professor V. I. Ago], Professor I. G. Atabekov Professor S. E. Bresler, Professor L. Margulis, Dr H. Mikelsaar, Professor M. M. K. Nass, Professor S. A. Neifakh, Professor W. Schopf, Dr E. N. Svetailo and Professor T. I. Tikchonenko, for discussion on some problems considered in the paper; and Miss T. I. Kheifets for correcting the English version of the paper. A part of this work was carried out in a group of Dr I. I. Tovarova, Institute of Bio-organic Chemistry.
APPENDIX
Abbreviations Used BSMV CGMMV RML RSV TMV TYMV
barley stripe mosaic virus. cucumber
green mottle mosaic virus.
Rausher murine leukemia. Rous sarcoma virus. tobacco mosaic virus. turnip yellow mosaic virus.
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REFERENCES BADER, A. V. (1972). J. Cell Biol. 55, lla. BALD, J. G. (1966). Adv. Virus Res. 12, 103. CARR, N. G. & PHILLIPS, C. 0. (1975). XII International Botanical Congress, Leningrad. CARROLL, T. W. (1970). ViroZogy 42, 1015. CECH, M. (1967). Phytopathol. Z. 59, 72. DRACHEV, L. A., JASAITIS, A. A., KAULEN, A. D., KONDRASHINA, A. A., LIBERMAN, E. A., NEMECEK, I. B., OSTROUMOV, S., SEMENOV, A. & Yu, S. (1974). Nature, Lond. 249, 321. &AU, K., CRONSHAW, J. (1967). J. Cell Biol. 33, 665. EVANS, M. C. W. & WHATLEY F. R. (1970). In Organization and Control in Procaryotic and Eucaryotic Ceils, pp. 203-220. Cambridge: Society of General Microbiology GAITSKHOKI, V. S., GOLUBKOV, V. I., GRABOVSKAJA, K. B., KISSELEV, 0. I., TOTOLAN, A. A. & NEIFAKH, S. A. (1974). Dokl. Acad. Nauk, U.S.S.R. 215, 1487. GAZZALQ, L., DE-THE, G., VIGIER, P. & SARMA, P. S. (1969). C. R. Acad. Sci. 268, 1668. HARRISON, D. D., STEFANAC, Z. & ROBERTS, I. M. (1970). J. gen. Viral. 6, 127. HARRISON, B. D. & ROBERTS, I. M. (1968). J. gen. Viral. 3, 121. HATTA, T., NAKAMOTO, T., TAKAGI, Y. & USHIYAMA, R. (1971). Virology 45,292. HAYASHI, Y. (1963). Virology 21, 226. HENDERSON, P. J. F. (1971). A. Rev. Microbial. 25, 393. HIND, G. & OLSON, J. M. (1968). A. Rev. PI. Physiol. 19, 249. JOHN, P. & WHATLEY, F. R. (1975). Nature, Land. 254,495. K.&RA, J., MACH, 0. & CERNA, H. (1971). Biochem. Biophys. Commun. 44, 162. KARA, J., DVORAK, M. & CERNA, H. (1972). FEBS Lett. 25, 33. KP;RA, J. & MACH, 0. (1973). Foliu Biol 19, 78. K&A, J., CERNA, H. & DVORAK, M. (1974). Absfruct of the Reports at the Symposium, Leningrad, November 27-29, pp. 63-65. KISSELEV, 0. I., GOLUBKOV, V. I., GRABOVSKAYA, K. B. & TOTOLIAN, A. A. (1974). Abstract of the Reports at the Symposium, Leningrad, November 27-29, pp. 67-69. KLEITMANN, W., LUNGER, P. D. & CLARK, H. F. (1973). Molecular Biology of Viruses, pp. 172-182. KUNTZEL, H., BARATH, Z., ALI, J., KIND, J. & ALTHAUS, H.-H. (1973). Proc. Natn. Acad. Sci. U.S.A. 70, 1574. LUNGER, P. D. & CLARK, H. F. (1973). J. Nutn. Cancer Inst. 50, 111. MARGULIS, L. (1970). Origin of Eucoryotic Cells. New Haven: Yale University Press. MARGULIS, L. (1975). MATHEWS, R. E. F. (1970). Plant Virology. New York and London: Academic Press. MATHEWS, R. E. F. (1973). Ann. Rev. Phytopathology 11, 147. NASS, M. M. K. (1974). In Hormones and Cancer, pp. 261-305. New York, San Francisco and London: Academic Press. NEIFAKH, S. A. (1974). Abstracts of Reports at the Symposium, Leningrad, November 27-29, pp. 55-58. OSTROUMOV, S. A. (1973). Priroda, U.S.S.R. 3, 21. OSTROUMOV, S. A. (1974). Priroda, U.S.S.R. 10, 108. RACKER, E. & STOECKENIUS, W. (1974). J. biol. Chem. 249,662. RAFF, R. A. & MAHLER, H. R. (1972). Science, N. Y. 177, 575. RALPH, R. K. & CL-ARK, M. F. (1966). Biochem. biophys. Actu 119, 29. RAVEN, P. H. (1970). Science, N. Y. 169, 641. RAVEN, P. H. (1975). RICHTER, D., HERRLICH, P. SCHWEIGER, M. (1972). Nature New Biol. 238, 74. SAGAN, L. (1967). J. theor. Biof. 14,225. SCHATZ, G. & MASON, T. L. (1974). A. Rev. Biochem. 43, 51. SCHOPP, J. W. (1974). Orig. Life 5, 119. SHALLA, T. A. (1968). Virology 35, 194.
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SINGER, B. (1972). Virology 47, 397. SKULACHEV, V. P. (1969). Energy Accumulation in the Cell. Moscow: Nauka. SHULACHEV, V. P. (1972). Energy Transformation in Biomembranes. Moscow: Nauka. SVETAILO, E. N. (1969). Thesis, Moscow. TAKHTAJAN, A. J. (1973). Priroda, U.S.S.R. 2, 22. TAYUIR, F. P. R. (1974). Taxon 23, 229. TAYLDR, F. J. R. (1975). USHIYAMA, R. MA~WS, R. E. F. (1970). Virology 42, 293. Y~RSHOV, F. I., GAITSKHOKI, V. S., KHSELEV, 0. I.. ZAITSEVA, 0. V., MENSHIKH, L. K., URWAEV, L. B., NEIFAKH, S. A. & ZHDANOV, V. M. (1971). Voprosy virusologii, U.S.S.R. 3, 274. ZAITLIN, M. & BOARDMAN, N. K. (1958). Virology 6, 743. ZAITSEVA, 0. V., MENSHIKH, L. K., KISSELEV, 0. I., GAITSKHOKI, V. S., YERSHOV, F. I., UR~AEV, L. B., ZHDANOV, V. M. & NEIFAKH, S. A. (1973). Dokl. Adad. Nauk, U.S.S.R.
208,985. ZHDANOV,
V. M.,
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Adv. virus res. 19, 361.
B. A. (1971).
Participation of Chloroplasls and Mitochoudria in Virus Reproduction and the Evolution of the Enkaryotic Cell? S. A. OSTROUMOV
Department of Bioenergetics, Laboratory of Bio-organic Chemistry, Moscow State University and Shemiakin Institute of Bio-organic Chemistry,$ U.S.S.R. (Received 4 September 1975, and in revisedform 27 October 1976) The similarity of prokaryotic cells and the organellesof eukaryotes can be explained not only by the ordinary form of the endosymbiosis hypothesis,but also by another evolutionary model presentedhere. This model postulatesthat portions of DNA of an ancient prokaryote were transferredinto the nucleusof the ancient eukaryote cell. In combination with prokaryotic DNA-encoded proteins and phospholipid membranes prokaryotic DNA may have given rise to a structure resemblinga promitochondrion or a proplastid. The transfer of geneticmaterial into the nucleusor the cytoplasmcould have occurredvia a virus-mediatich.This suggestionis in accordancewith the reported associationof chloroplasts and plant mitochondria with replication of somestrainsof tobacco mosaic virus, cucumbergreenmottle mosaicvirus, barley stripe virus and tobacco rattle virus and with findings of virus-like particles in mammalianand reptilian cells as well as cells of Neurosporacrassa. The terms “mitophages” and “plastophages” (“chlorophages”) are introduced to designate the viruses associatedwith mitochondria or plastids (chloroplasts)during somephaseof development. Participation of virus-mediated DNA transfers can be suggestedfor endosymbiotic organellar origin also.
1. Introduction A much-debated question in the field of evolution of eukaryotic cells is the hypothesis of the endosymbiotic origin of energy-transducing organelles, the chloroplasts and mitochondria (e.g. Sagan, 1967; Skulachev, 1969; t This paperwaspresented in a symposium entitled“The Origin of Eukaryotic Cells” at the XII InternationalBotanical Congress, Leningrad,July 1975. $ Addressfor reprints: Dr S. A. Ostroumov, ShemiakinInstitute of Bio-organic Chemistry,U.S.S.R.Academyof Sciences, Vavilova 32, Moscow117312,U.S.S.R. 287
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Svetailo, 1969; Raven, 1970, 1975; Raff & Mahler, 1972; Takhtajan, 1973; Ostroumov, 1973; Margulis, 1970, 1975; John & Whatley, 1975; Carr & Phillips, 1975; Taylor, 1975). This paper is restricted to a consideration of some aspects of the possibility of the contribution of the genetic determinants of ancient prokaryotes, which apparently dominated in Precambrian Earth’s biota (e.g. Schopf, 1974), to the genetic material of eukaryotic cells. In this connection it seems to be interesting to use some information concerning viruses, which “make ideal mediators of . . . interspecies genetic exchange” (Zhdanov & Tikchonenko, 1974). A significant amount of data have been collected about the participation of mitochondria (Tables 1 and 2) and chloroplasts (Table 1) in the reproduction of several strains of viruses. Because individual stages of multiplication of different strains of the same virus can be localized in different parts of the cell the examples cited below must be interpreted with great caution. 2. Plants (A)
CHLOROPLASTS
Various authors using the electron microscope have reported an association of certain plant viruses with plastids (Bald, 1966; Matthews, 1970). For example, Ralph & Clark (1966) demonstrated that TYMV RNA synthesis and duplex (double-stranded) viral RNA in Chinese cabbage leaf cells systematically infected with TYMV was associated with the chloroplast fraction and did not occur in the cytoplasm or nuclei (for more comprehensive review of data about TYMV see the excellent survey of Matthews, 1973). In 1970 Ushiyama & Matthews obtained new data to support the suggestion that the replicative form of viral RNA is localized in the peripheral vesicles of the chloroplasts of Chinese cabbage (Brassicu pekineprsis) as well as turnip (B. rapa) cells infected with TYMV. The authors suggested that these vesicles could be the site of viral RNA synthesis, and that the viral RNA strands migrated from the vesicles to the cytoplasm where virus protein was synthesized and virus particles were assembled. Barley tissues infected with BSMV were examined with the electron microscope by Carroll (1970). Virions of BSMV were seen in close association with abnormally vesiculated chloroplasts and plastids of immature embryos of barley (Hordeurn vulgare; Carroll, 1970). Some virions surrounded chloroplasts, whereas others were localized in cytoplasmic invaginations into the chloroplasts. Some virions appeared to be attached
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Association of replication of somestrains of viruses with chloroplasts and mitochondria of plants Virus ~~ -.___.-~~ Chloroplasts TMV
TYMV BSMV Mitochondria
TMV CGMMV
Tobacco rattle virus Virus-like particles
-__ Tobacco
Reference ____-___ Zaitlin & Boardman, 1958 Gxh, 1967; Esau & Cronshaw, 1967 Shalla, 1968 Singer, 1972 but see Ralph & Clark, 1966 Ralph & Clark, 1966 Ushiyama & Matthews, 1970 Carroll, 1970
Host
Brassica pekinensis B. rapa Hordeum vulgare Tobacco Cucumber, pumpkin, gourd Nicotiana clevelandii Neurospora crassa abnormal-l mutant
TABLE
Hayashi, 1963 Hatta et al., 1971 Harrison, Roberts, 1968 Harrison et al., 1970 Kiintzel et al., 1973
2
Association of mitochondria of animals with production of virus-like particles Virus or virus-like particles ____. Viral nucleocapsids Virus-like particles Virus-like particles Subviral oncogenic ribonucleoprotein particles Virus-like particles
Subviral leukemogenic particles
Source ____. ------~ Hamster cells transformed by RSV Cells line VSW from a tumor bearing Russel’s viper Rous sarcoma cells
Fused hamster tumor, chick embryo cells Hamster tumor induced with the Schmidt-Ruppin strain of Rous sarcoma virus Leukemic spleen cells of mice infected with RML virus
Reference ~--___.-~. - -Gazzalo et al., 1969 Lunger & Clark, 1973 Kleitman et al., 1973 Kara et al., 1971 Kara et al., 1972 Kara & Mach, 1973 see also Bader, 1972 Nass, 1974
Kara et al., 1974
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to the outer chloroplast membrane. Carroll suggested that “the plastid vesicles supply structural components or enzymes or both necessary for multiplication of BSMV. These factors for multiplications could pass through the exterior membrane of the envelope and accumulate at sites on the outer surface of this membrane. At these sites the virus may be assembled into whole virions.” The case of TMV is a good example of how complicated the localization problem is (Matthews, 1970). For instance, Ralph & Clark (1966) found that double-stranded TMV RNA in infected tobacco leaves was present in the cytoplasmic fraction of leaf homogenates and not in nuclei or chloroplasts. Singer (1972) obtained evidence that TMV protein synthesis takes place in the chloroplasts. Esau & Cronshaw (1967) published electron micrographs showing clusters of virus-like particles in sections of chloroplasts of plants infected with TMV. The authors argued that the virus did not enter the chloroplasts by the mechanism of invagination but was probably formed there. Impressive data were obtained by Shalla (1968). He showed that sectioned chloroplasts of tobacco plants infected with the U5 strain of TMW contain particles resembling TMV. When chloroplasts were isolated from plants and disrupted, their loss of virions as seen with the electron microscope was correlated with the increase in the infectivity of the ambient medium. This paper showed that no equivalent clusters of virions were seen in chloroplasts of plants infected with the U, strain of TMV. (B)
MITOCHONDRIA
Some indication that the mitochondria of tobacco leaves may be involved in TMV infection comes from the work of Hayashi (1963). He studied incorporation of [14C] amino acids in variou cells fractions of plants infected with TMV and found that soon after infection the mitochondria had the highest incorporation rate per mg of protein. Hatta, Nakamoto, Takagi & Ushiyama (1971) found that crystals of cucumber green mottle mosaic virus (CGMMV) frequently occurred in areas of cytoplasmic invaginations that probably penetrated into mitochondria. Virus particles were not associated with chloroplasts or nuclei. Virus-infected cells contained abnormal mitochondria with small vesicles. Most of them seemed to be located in the space between the inner and outer mitochondrial membranes. Some vesicles seemed to have an opening to the cytoplasm. Another example of association of virus replication with plant mitochondria was provided by Harrison and co-workers. They have shown this for mitochondria of Nicotiana clevelandii infected with the longer type of
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CAM tobacco rattle virus (Harrison & Roberts, 1968) and RNA-producing defective isolate of tobacco rattle virus (Harrison, Stefanae & Roberts, 1970). A very interesting study of virus-like particles isolated from the abnormal- 1 mutant of Neurosporu crussa was made by Ktintzel & co-workers (Ktintzel et al., 1973; see also Schatz & Mason, 1974). Virus-like ribonucleoprotein particles were found both in the cytoplasm and a mitochondrial fraction of abnormal-l cells, but were undetectable in wild-type cells. The particles contained single-stranded 33 S RNA which hybridized with mitochondrial DNA from the abnormal-l mutant or wild type N. crassa. 3. Mitochondria (A)
ELECTRON
of Animals MICROSCOPY
The structures that were interpreted to represent viral nucleocapsids were found in mitochondria of hamster cells transformed by Rous sarcoma virus (Gazzalo, de-The, Vigier & Sarma, 1969). Intramitochondrial viral particles were found in a cell line termed VSW derived from a spleen tumour bearing Russell’s viper (Lunger & Clark, 1973; Kleitmann, Lunger & Clark, 1973). M. M. K. Nass and C. Buck have found numerous virus-like structures in mitochondria of fused hamster tumour-chick embryo cells (Nass, 1974). In this study the cells, derived from a hamster tumour induced with the Schmidt-Ruppin strain of Rous sarcoma virus, were fused with chick embryo fibroblasts using Sendai virus (Nass, 1974). An electron microscope analysis of the isolated Rous sarcoma subviral particles (see next section) was performed (K&a & Mach, 1973) and it supported the assumption that they were located in mitochondria. It is noteworthy that the claims that Rous sarcoma virus production occurs in mitochondria have been challenged by Bader (1972), but his arguments have been subjected to a certain amount of criticism by Nass (1974). (B)
BIOCHEMICAL
DATA
Kara, Cerna & Dvorak (1971) reported the presence and biosynthesis of subviral oncogenic ribonucleoprotein particles (virosomes) in the mitochondria isolated from Rous sarcoma (RS) cells. Virosomes were located in the inner membrane and matrix of RS mitochondria, contained virusspecific proteins (gs-antigens) and RNA-dependent DNA-polymerase (Kara et al., 1972). Recently Kara et al. (1974) presented evidence on biosynthesis of subviral leukemogenic particles in the isolated mitochondria of leukemic spleen cells of mice infected with Rauscher murine leukemic (RML) virus. These
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particles induce splenomegaly and RML 3 weeks after intravenous or interperitoneal administration to white mice. RML virus gs-antigenes were detected by immunoprecipitation in the inner membrane and matrix of the mitochondria of RML spleen cells (K&a et al., 1974). 4. Artificial Systems Study It was shown that mitochondrial RNA-polymerase could recognize transcriptional signals of bacteriophage T3 and T7 DNA (Richter, Herrlich & Schweiger, 1972). Moreover, mitochondrial ribosomes appeared to be capable of synthesizing phage-specific proteins. The ability of mitochondria to transcribe and translate viral RNA was systematically studied by Neifakh et al. (1974) and Zhdanov et al. (1971). In particular, the system of transcription and translation of TMV and Venezuelan equine encephalomyelitis viral RNA was reconstituted using isolated rat liver or yeast mitochondria or submitochondrial preparations (Zhdanov, Tikchonenko, Bocharov & Naroditsky, 1971; Yershov et al., 1971, 1974; Zaitseva et al., 1973). A similar reconstituted system was reported for bacteriophage MS2 genome RNA and rat liver mitochondria (Kisselev, Golubkov, Grabouskaya & Totolian, 1974; Gaitskhoki et al., 1974). 5. Discussion From facts such as those reviewed above it seems likely that some strains of viruses use the enzymes of mitochondria and chloroplasts. The terms “mitophages” and “plastophages” (“chlorophages”) were introduced for the designation of viruses, possessing stages of multiplication localized in mitochondria and chloroplasts (Ostroumov, 1974). It should be emphasized that far-reaching generalizations are premature, and we cannot conclude that all the viruses mentioned above or even a majority of them may be classed as mitophages and plastophages. “The question remains to be answered whether a mitochondrion-associated phase of viral development is indispensable or is merely auxiliary and can easily be carried on extramitochondrially when organelle function is impaired” (Nass, 1974). The same comment could be made about viruses associated with chloroplasts. The data summarized indirectly testify to the possibility of similar associations between viruses and the evolutionary precursors of organelles. We shall discuss this idea in more detail below, following a consideration of evolutionary origin of the organelles. The similarity between prokaryotes and energy-transducing organelles is well-documented and was discussed with reference to the function of energy-
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transforming membranes (e.g. Hind & Olson, 1968; Evans & Whatley, 1970; Henderson, 1971; Skulachev, 1972) and other aspects (e.g. Sagan, 1967; Skulachev, 1969; Svetailo, 1969; Margulis, 1970; Ostroumov, 1973; Taylor, 1974; John & Whatley, 1975). It seems reasonable to suggest a similarity between the nuclear genetic determinants encoding the prokaryotic type proteins of organelles in these eukaryotes and the analogous genetic determinants of prokaryotes. This suggestion might explain the similarity between the nuclear-encoded components of organelles and the analogous components of contemporary prokaryotes. From the evolutionary viewpoint one could propose that this nuclear genetic material originated from the genetic determinants of ancient prokaryotes. However, this still leaves the problem of redistribution of prokaryote genetic material between nuclei and organelles. Two alternatives may be formulated (see Figs 1 and 2). For the sake of clarity and brevity the following designation could be useful : DNA,,-DNA of contemporary energy-transducing organelle or its evolutionary predecessor (a part of DNA of a prokaryote). DNA,,-a part of prokaryotic DNA which was incorporated into nucleus of a protoeukaryotic cell; DNA, encodes organelle prokaryotic type proteins. One model is essentially the well-known endosymbiotic hypothesis with only the qualification that part of the DNA of an endosymbiotic prokaryote has been transferred to the nucleus. In other words, the following sequence of DNA transfers is postulated: (1) Prokaryotic DNA within the prokaryotic cell enters the cytoplasm of the macrocell. (2) DNA, from cytoplasmic endosymbiont enters the nucleus. Another model (Fig. 1) is based on reversed events and does not require a physical symbiosis (see also Zhdanov & Tikchonenko, 1974). First, a fragment of DNA of a prokaryote (without prokaryotic cell) is transferred to the nucleus. Second, DNA, is transported to the cytoplasm, where in combination with DNA,-encoded proteins and phospholipid membrane it gives rise to an organelle-like structure. In this case, the appearance of promitochondria-like or proplastid-like structures containing prokaryotic DNA seems probable. This assumption is in agreement with numerous experiments which demonstrate self-assemblage of phospholipid molecules and formation of T.B. 20
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Profoeukoryoflc
cell
Prokoryote DNA,
Eukoryotic
cell
FIG. I. One of hypothetical schemes of origin of mitochondria and chloroplasts. DNAO-part of ancient prokaryote DNA transferred into the orgunelIe (mitochondrion or chloroplast) predecessor; DNA,-part of ancient prokaryote DNA which after transfer into protoeukaryotic cell encodes organelle proteins and is located in the nucleus. The arrows indicate the transfer of fragments of ancient prokaryote DNA. One of the possibilities is transport by some virus-mediated mechanism.
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membraneous vesicles (liposomes). Liposomes readily incorporate proteins and can transform energy (e.g. Skulachev, 1972; Drachev et al., 1974) and produce ATP (e.g. Racker & Stoeckenius, 1974). The appearance of ATP producing membraneous vesicles in cytosol of protoeukaryotic cell could have given cell evolutionary advantage, because membrane-dependent phosphorylation of ADP is more efficient than phosphorylation during glycolysis. Could the transfer of DNA, into the membrane structures in the cytosol give the cell some evolutionary advantage? Yes, because the incorporation of hydrophobic proteins (encoded in DNA,) into the inner surface of membrane could improve the functioning of energy-transducing membraneous structures. The important link in both schemes is transfer of parts of prokaryotic DNA from the endosymbiont to the nucleus or to the cytoplasm. The aboveconsidered data prompt, as one of possibilities, transfer by some virusmediated mechanism. The study of association of some viruses with contemporary energytransducing organelles will provide an important additional source of information for understanding evolutionary and individual (Neifakh, 1974) development of mitochondria and chloroplasts. The author gratefully acknowledges Academician A. L. Takhtajan for his interest and support; Corresponding Member of Academy of Sciences of U.S.S.R. V. P. Skulachev, Professor Peter H. Raven and Dr A. S. Antonov for valuable advice and reading the manuscript; Professor V. I. Ago], Professor I. G. Atabekov Professor S. E. Bresler, Professor L. Margulis, Dr H. Mikelsaar, Professor M. M. K. Nass, Professor S. A. Neifakh, Professor W. Schopf, Dr E. N. Svetailo and Professor T. I. Tikchonenko, for discussion on some problems considered in the paper; and Miss T. I. Kheifets for correcting the English version of the paper. A part of this work was carried out in a group of Dr I. I. Tovarova, Institute of Bio-organic Chemistry.
APPENDIX
Abbreviations Used BSMV CGMMV RML RSV TMV TYMV
barley stripe mosaic virus. cucumber
green mottle mosaic virus.
Rausher murine leukemia. Rous sarcoma virus. tobacco mosaic virus. turnip yellow mosaic virus.
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