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Modern eukaryotic cells were originally believed to have arisen directly from a single prokaryotic ancestor through serial mutation and the process of genetic drift. However, much evidence has lead scientists to believe that eukaryotes are the result of a merger between an aerobic prokaryotic cell, a relative of modern Î±-proteobacteria, that became incorporated by a host archaeon. This is referred to as the endosymbiotic theory. Over the span of millions of years, symbiont DNA was transferred to the nucleus to form the eukaryotic genome and the remains of the symbiont gave rise to mitochondria. As such, the two organisms effectively become a single organism, each unable to survive in the others' absence. The endosymbiotic theory is widely regarded due to the many shared biochemical and morphological characteristics of mitochondria with bacteria, including DNA organization and similarities of the protein synthesising machinery and membrane composition. In reviewing these characteristics, I have concluded that the development of mitochondria within the eukaryotic cell is almost certainly the result of a symbiotic relationship and subsequent gene transfer of symbiont DNA.
The term "endosymbiosis" refers to the event in which one organism takes up permanent residence within another, such that the two develop a mutually beneficial relationship 1.The endosymbiotic theory was developed to explain the evolutionary discontinuity between the appearance of prokaryotes and eukaryotes, and the great many biochemical and structural differences exhibited by the two taxa, some of which are described in Table 1. Included are the overall physiological similarities of mitochondria and prokaryotes, such as the similarity in size compared to eukaryotes, similarities between the protein-synthesising machinery such as the mitochondrial ribosomes, and the presence of a separate mitochondrial genome.3, 8. When the same characteristics of mitochondria, the organelles of eukaryotic cells that carry out aerobic respiration to provide ATP to the cell (Fig.1), are compared, as in Table 1, it is clear that mitochondria are more closely related to the Bacteria than to eukaryotes. These and similar observations led Lynn Margulis (1981) to propose an evolutionary relationship between Bacteria and mitochondria in order to explain the transition from prokaryote to eukaryote. The term endosymbiotic was originally coined by Konstantin MereschkowskiÂ (1905) and was adopted by evolutionary biologists following Margulis' work. 1, 2 Contrary to the traditional view that a series of chance mutations was responsible for the evolution of eukaryotic cells, the endosymbiotic theory states that mitochondria arose by the incorporation of a free living aerobic Î±-proteobacterium, into an anaerobic proto-eukaryote 3. Margulis also proposed, more controversially, that motile prokaryotic species such as SpirochaetaÂ were incorporated and resulted in the evolution of the structures that provide cellular motion. Since this hypothesis is generally not accepted, it is not discussed in further detail here.1
(Above) Figure 1: Structure of an idealised mitochondrion as compared to an electron micrograph. Illustrates the double membrane structure and the infoldings of the inner membrane known as cristae on which oxidative phosphorylation occurs.
Adapted from: Dr Jay Pitocchelli, Saint Anslem College (2001) Lecture Notes for Cell Biology. Available: http://www.anselm.edu/homepage/ jpitocch/genbio/mitochondrion.JPG [accessed 27/04/10]
The first endosymbiotic event is proposed to have occurred approximately 1.5 billion years ago, between the first fossil record of aerobic prokaryotes and eukaryotes (See Table 1), after oxygen had begun to accumulate in Earth's atmosphere as a result of the emergence of photosynthetic organisms 4, 5. Rather than being digested, the prokaryote remained as a symbiont and produced ATP within the host by the process of oxidative phosphorylation, enabling it to survive the increasing oxygen concentrations, thereby giving it a selective advantage over anaerobic cells. Interdependence between the aerobic bacterium and the host cell developed and, the bacterium evolved into the mitochondrion. Photosynthetic eukaryotes originated in a similar manner by a secondary symbiosis between these organisms and photoautotrophic bacteria related to cyanobacteria. 8, 9
Table.1 Summary of the similarities between prokaryotes and eukaryotes, and eukaryotic organelles.
Adapted from: Indiana University-Purdue University Department of Biology (2004) Class Notes: The Endosymbiotic Theory Available: http://www.biology.iupui.edu/biocourses/n100/2k4endosymb.html [Accessed 16/04/10]
1 single, circular chromosome
Multiple linear chromosomes
compartmentalized in a nucleus
1 single, circularÂ
Binary FissionÂ involving Fts proteins
Process akin to binary Fission involving dynamin proteins
30S and 50S Subunits
40S and 60S Subunits
30S and 50S Subunits
Electron Transport Chain
Found in the plasma membrane around cell
Found only in the cell's mitochondria
Found in the plasma membrane around mitochondrion
~50 - 500 Î¼m
First appearance in fossil record
~3.8 Billion years ago
~2.5 Billion years ago
~1.5 billion years ago
~1.5 billion years ago
What the endosymbiotic hypothesis does not make clear is the order of events regarding the formation of the nucleus and the acquisition of the prokaryotic cell containing the precursor mitochondrialÂ genome. Two hypotheses have been put forward for the formation of the eukaryotic cell, illustrated in Fig.2.
Figure 2: Two models for the origin of the eukaryotic cell.
Nucleus formation preceded symbiosis of mitochondrion and chloroplast
Nucleus formed after symbiosis of mitochondrion and before acquisition of the chloroplast
From Michael Madigan et al. Brock Biology of Microorganisms
(2008) 12th edition, Pearson Education Inc
The most widely regarded, summarised in Fig.3, proposes that an ancestral prokaryote first developed a membrane around its DNA from infolding of the plasma, similar to the way in which the endomembranous system of the endoplasmic rectilium and Golgi apparatus is thought to have arisen 3. This organism, dubbed the "protoeukaryote" engulfed a small heterotrophic prokaryote, shown on Fig.2 as the 'ancestor of mitochondrion' 8.
Figure 3: A proposed view of the endosymbiotic theory and origin of mitochondria and chloroplasts.
From Niel A Campbell, Jane B Reece (2009) Biology
Pearson Education Inc.
The second hypothesis, in contrast, considers that theÂ nucleus was formed after the acquisition of the protomitochondrion through endocytosis by a member of the Archaea 9. This is known as the hydrogen hypothesis, proposed by Martin and Muller in 1998, who claimed that the symbiotic relationship between the two cells was initially based on the host's dependence on the hydrogen evolved by the symbiont as a by-product of anaerobic respiration as a source of energy. The nucleus was formed from the mitochondrial DNA from the symbiont and the free DNA residing in the nucleus 10, 11. Both models thus involve the transfer of a large portion of mitochondrial DNA to the host nucleus, resulting in the dependence of the symbiont upon the host.
An alternative hypothesis has recently been forwarded by Davidov and Jurkevitch, who propose thatÂ the ancestors of mitochondria were not endocytosed by ArchaeaÂ but were predators that penetrated the host andÂ devoured the host. The prey managed to survive and established a mutualistic relationship as in the previous hypotheses. This appears to be supported by the finding that certain species of Rickettsia, obligate intracellular parasites, have more similar genomes to the than mitochondrion 12, 13.
However the eukaryotic cell arose, abundant evidence has accumulated that supports the endosymbiotic theory, and the evidence of similarities relating to different functions of bacteria and mitochondria are reviewed in this essay. There is also an increasing body of experimental evidence that suggests that endosymbiotic events occur in modern cells, and two such experiments and their implications in the endosymbiotic theory are reviewed here. Aside from these experiments, the evidence presented in this essay relates entirely to the emergence of mitochondria.
The Mitochondrial Genome
Mitochondria possess their own genomes that replicate independently from the nucleus, using DNA polymerases specific to the mitochondria.8 The presence of a separate mitochondrial genome in itself is evidence that it was formed from a separate organism, taken in by the eukaryotic cell in which the organelle resides. The processes of DNA replication, as well as the subsequent DNA transcription and protein synthesis take place in the matrix of mitochondria and occur throughout the cell cycle, which parallels the situation in bacteria but is not true of nuclear DNA2. The DNA of mitochondria is a single circular molecule of a much smaller size than the nuclear genome, for example the genome of yeast, the example shown in Table 2. The mean GC content of mitochondrial DNA ranges from 20-50%, which is close to the content of bacterial species but is greater than the content of eukaryotic DNA, reflecting the phylogenetic relationships of mitochondria and Bacteria. Furthermore, like bacterial DNA, the DNA of mitochondria lacks both intervening sequences and the organisation into histones present in bacteria 2, 3. Comparative molecular sequencing of mitochondrial genes has revealed that the mitochondrial genome is more closely related to that of organisms such as the Î±-proteobacterium Rickettsia prowazekii than to the rest of the eukaryotic cell, indicating an extracellular origin 14, 15. Similar sequencing by Ito and Braithewaite has revealed that yeast mitochondrial DNA polymerase I is homologous in amino acid sequence to the DNA polymerases of E. coli and Streptococcus pneumoniae. The similarity of the mitochondrial DNA replication machinery when compared to that of the nucleus further implies that mitochondria evolved from prokaryotes 16.
Table 2: Sizes and the number of proteins encoded by some organelle and prokaryote genomes, compared to the yeast nuclear genome.
Adapted from Jeremy N. Timmis, Michael A. Ayliffe, Chun Y. HuangÂ & William MartinÂ (2004) Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes Nature Reviews GeneticsÂ 5,Â 123-135Â
DNA sequence analysis has also demonstrated the presence of mitochondrial DNA in the nucleus 17. Biologists originally believed that the mitochondrial proteins were coded for purely by the mitochondrion. However, Margulis reasoned that, if the endosymbiotic hypothesis represents the true course of events in the evolution of mitochondria, then upon entering a symbiotic partnership, the symbionts would lose all synthetic capabilities except the ability to replicate their own DNA. Human mitochondria, contain approximately 3000 proteins, of which only 13 are products of mitochondrial DNA 3, 9. Sequenced mitochondrial genomes encode anything from 3 to 67 proteins (Table 2). However, due to the presence of the separate mitochondrial genome, nuclear genes direct the synthesis of only some of the mitochondrial proteins, thus cells which lack mitochondria cannot generate them 1. Many of the proteins that mediate function of the mitochondrion are encoded must be imported to the mitochondrion, and the process by which mitochondrial protein is formed from nuclear precursor protein is shown in shown in Fig.4.
The size of the mitochondrial genome appears to be the result of extensive gene transfer that took place over the 1.5 billion years since the ancestral cells merged. The transfer of essential genes to the host nucleus resulted in the progressive loss of independence after the host acquired the symbiont, resulting in an organelle unable to survive in oxic conditions outside the host 3. O Daley proposes that the large transfer of mitochondrial DNA may have been the result of evolutionary pressure to accumulate DNA inside the nucleus, due to increased genetic variation 17, 18.The High rate transfer of DNA between the nucleus and mitochondria demonstrates that the incorporation of the mitochondrial genome into the eukaryotic cell was vital in defining the eukaryotic genome18. The loss of synthetic ability in mitochondria therefore supports Margulis' hypothesis and explains why the mitochondrial genome is so small when compared to nuclear genomes in Table 2.
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Fig 4: Transfer of nuclear precursor proteins from the nucleus to the mitochondrion and subsequent production of mitochondrial protein. Red arrows indicate the site of action of a specific inhibitor of either nuclear or mitochondrial protein synthesis, discussed in more detail later in of this essay.
From Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson (1994) Molecular Biology of the Cell. 3rdÂ edition, Garland Publishing Inc., New York.
The mitochondrial genome provides further evidence for the symbiotic concept due to the differences in inheritance of DNA between the mitochondria and the nucleus. While nuclear DNA is contributed by both parents in sexual organisms, mitochondrial DNA is derived purely from the mitochondria present in the oocyte, and therefore the mtRNA is inherited through the maternal line 15. This DNA, as previously mentioned, is inherited independently of fertilization and meiosis.Â The symbiotic concept accounts for this non-Mendelian mode of inheritance, as the symbiont continued to replicate within the host and thus DNA was inherited only from the mitochondria from which it arose 7.
Possibly the most compelling evidence of the extracellular origin of mitochondria is the similarity between the inner mitochondrial membrane and the membrane possessed by bacteria. Mitochondria are surrounded by two phospholipid membranes, and while the outer resembles eukaryotic membranes, including the membranes of other cellular organelles such as that of the nucleus and endoplasmic rectilium, the innermost membrane is chemically distinct to those found elsewhere in the eukaryotic cell 3, 6. Furthermore, microscopic observation has enabled the comparison of cristae, invaginations of the inner mitochondrial membrane, shown in Fig.1, to the mesosomes possessed by many species of Bacteria, shown in Fig.4. Both structures increase the surface area of their respective membranes and provide a site for the process of oxidative phosphorylation. Margulis has suggested that the similarity between these structures also alludes to the extracellular origin of mitochondria 3.
Figre 5 : Electron micrograph of thin section of T.dentrificans showing 'inverted Y' form of simple mesosome-like body.
From: J W Greenawalt and T L Whiteside (Dec 1975). Mesosomes: membranous bacterial organelles. Bacteriological Review.Â Â 39(4): 405-463.
The inner mitochondria and bacterial membranes also share many biochemical features. Table 3 illustrates the observations made by Parsons, that the outer mitochondrial membrane is more similar in density and lipid composition to that of the endoplasmic reticulum of Serratia than of the inner mitochondrial membrane 3 19. It has also been noted that Î²-barrel transmembrane proteins are exclusively found in the bacterial membranesÂ and in theÂ outer membraneÂ of mitochondria, and that the amino acid sequences of these proteins show high similarity 20. Additionally, LACTB, a protein that derives from bacterial penicillin-binding protein of peptidoglycan, has been found in the intermembrane space of eukaryotic mitochondria 21. While mitochondria lack peptidoglycan, the presence of a vestigial peptidoglycan-forming protein provided further evidence that mitochondria are descended from bacteria.
Table 3: Comparison of structural features and lipid composition of inner and outer Mitochondrial membranes and ER of Serratia.
Adapted from D.F. Parsons (1867) Ultrastructural and molecular aspects of cell membranes. Proceeings of the Seventh Canadian Cancer Research ConferenceÂ 7:193-246.
Mitochondrial Inner Membrane
Mitochondrial Outer Membrane
Smooth Endoplasmic Rectilium
"Globules" 9.0 nm membrane subunits
"Globules" 6.0 nm membrane subunits
"Globules" polysaccharide fringe
Cardiolipin (% of total phospholipids)
Cytochrome a and a3 (Î¼g/mol)
Cytochrome b5 (Î¼g/mol)
Effect of ATP
The nature of the mitochondrial respiratory system raises another significant line of evidence supporting the endosymbiotic theory. The production of energy via the electron transport chain by mitochondria is associated only with the inner membrane, as in prokaryotes, and does not occur in the outer membrane, as evidenced by the difference in electron transport protein content in the mitochondrial membranes, shown in Table 3 9. Additionally, the membrane potential across the inner membrane that is necessary for the production of ATP is not found in the outer membrane of the mitochondrion or in eukaryotic membranes. Table 4 demonstrates that that bacteria such as P. denitrificans and mitochondria share many respiratory features, such as the sensitivity of the oxidative chain to antimycin, which disrupts proton gradient formation across the membrane. Table 4 also shows that the electron-transport chains of bacteria and mitochondria both contain a membrane-bound enzyme complex that accepts electrons solely from ubiquinone-10 quinine carrier 3. Taken together, this evidence is consistent with the theory that the inner mitochondrial membrane once belonged to the bacterial symbiont, and that the outer membrane was a remnant of the phagocytic vacuole in which the symbiont was engulfed by the host cell, the process of which is shown in Fig. 6. Eventually, this resulted in the development of cristae from mesosomes 2 9.
Table 4: Mitochondrial features of Paracoccus denitrificans
Illustrates the similarities of the respiratory system of mitochondria to the systems of Paracoccus , that are also found in many other bacteria. These strikingly similar respiratory features indicate that microbes such as P. denitrificans may be the ancestors of mitochondria. Many of these features are found in other bacteria. After Whateley (1977) Adapted from Lynn Margulis. Symbiosis in Cell EvolutionÂ (1981) W.H.Freeman & Co Ltd.
Succinate and NADH dehydrogenases
Ubiquinone-10 is the sole quinine
Cytochromes a and a3 act as oxidase
Sensitive to low concentrations of antimycin
Respiratory control is released by ADP or by uncouplers of oxidative phosphorylation
ATPase has tightly bound nucleotise exchangeable on energization
Phosphotidylcholine is the main constituent
All fatty acids are straight-chain and monounsaturated
Fig 6: Theorised formation of the mitochondrial double membrane from a phagocytic membrane.
From: Indiana University-Purdue University Department of Biology (2004) Class Notes: The Endosymbiotic Theory Available: http://www.biology.iupui.edu/biocourses/n100/2k4endosymb.html [Accessed 16/04/10]
The way in which mitochondria divide also provides evidence for their extracellular origin. These organelles multiply semi-autonomously, not through mitosis as eukaryotic cells are, but rather by a process similar toÂ the binary fission of Bacteria, such that mitochondria only arise through pre-existing organelles 22. Bacterial division is aided by the FtsZ, filamentous temperature sensitive proteins that interact to form a divisome ring complex. FtsZ1 proteins constrict the membrane form a division furrow on the inside of the membrane, while FtsZ2 proteins have been found to form a protein ring that constricts the membrane from the outside. Mitochondria have been found to possess dynamin proteins, mechanochemicalÂ GTPases that are related to the FtsZ2 proteins 23. Mitochondrial Dnm1 and Drp1 proteins act by a similar mechanism to FtsZ2 protein, illustrated in Fig.7. However, FtsZ proteins themselves have been found in the mitochondria of the alga Mallomonas splendens that are closely related in DNA sequence to those of Î±-proteobacteria.Â Fig.8 shows the presence and location of several types of division proteins present in mitochondria and Bacteria, 33 and shows that some more primitive eukaryotes have mitochondria with FtsZ1 proteins. The similarities in the molecules involved in initiating bacterial and organelle division further emphasise the evolutionary ties that these organelles have to bacteria9, 23, 24, 25
Find diagram of FtsZ
Figure 7: A model of the mechanism of mitochondrial division: Dynamins are targeted to their site of action by other proteins. After modification by GTP, Dynamins from rings that tighten around the site of division and constrict to cut the mitochondrion into two. This mecahism parrales that of FtsZ, the tubulin protein of bacteria.
Adapted from: Suzanne Hoppins,Â Laura Lackner, and Jodi Nunnari (2007) The Machines that Divide and Fuse Mitochondria. Annual Review of Biochemistry Vol. 76: 751-780
Figure 8: The use of FtsZ or alternative proteins for the diviosion of mitochondrial compared to prokaryotic organisms.
A green ring represents FtsZ1 protein, found in most euryarchaea and many bacteria, and forms the Z ring around the inner membrane. FtsZ2 protein is not shown.
A yellow ring represents the other division protein possessed by some Crenarchaea that also performs division in a similar manner.
A red ring represents dynamin or similar proteins, localized on the outer membrane. Dynamin is possessed by most mitochondria, including those of fungi and animals, from on the cytoplasmic face.
A Blue rings also represents the dividing ring possessed by the mitochondria of some primitive eukaryotes.
Adapted from William Margolin (2005) FtsZ and the division of prokaryotic cells and organelles. Nature Reviews Molecular Cell BiologyÂ 6,Â 862-871Â
The protein-synthesising machinery of mitochondria shares more similarities with bacteria than that of the eukaryote cytoplasm. For example, the initiating amino acid in the transcripts of bacteria and mitochondria is N-formylmethionine, whereas protein synthesis in the cytosol of eukaryotic cells begins with methionine 8. The structure of the mitochondrial ribosomes also differs from those found in the eukaryotic cytoplasm, in that they are more similar in size and share the same subunit structure, (See Table 1) 3, 9. The sequences of 16S ribosomal RNAs are closer to certain aerobic Eubacteria than many other bacteria are, for example, Wolters and Erdmann have confirmed, by phylogenetic analyses, that the primary and secondary structure of 5S and 16S rRNA of angiosperm mitochondria share specific signatures with a particular type of purple bacteria, the rhodobacteria 26. Conversely, mitochondria show no homologies in these traits with the eukaryotic cell cytoplasm. These similarities appear to confirm the phylogenetic relationship of these organisms and the organelles. 3
Similarities between the ribosomes of bacteria and mitochondria are further evidenced by the action of a number of antibiotics that affect only bacterial and mitochondrial ribosomal protein-synthesising machinery. Fig.3, above, demonstrates several specific inhibitors of protein synthesis. The similarities in the action of antibiotics between the three types of ribosomes are illustrated in Table 5 3. Neomycin and streptomycin act by binding the 30S subunit of mitochondria and bacteria and inhibiting protein chain initiation, while chloramphenicol blocks the attachment of amino acid to tRNA9. None of these chemicals interfere with protein synthesis in the cytoplasm of the eukaryotes. Conversely, cyclohexamide and anisomycin affect only the protein synthetic machinery of eukaryotic cells, and have no inhibitory effects on mitochondria or on bacteria. Another example, rifampicin, inhibits the RNA polymerase of bacteria and mitochondria, but has no such effect on eukaryotic nuclear RNA polymerase. It is notable that each antibiotic, except for Puromycin, affects both mitochondrial and bacterial or cytoplasmic ribosomes, and this would appear to suggest a relationship between the protein synthesising machinery of mitochondria and that of prokaryotic bacteria from which they were thought to have originated 3.
Table 5: Antibiotic sensitivities of Mitochondria, Bacteria and Cytoplasmic ribosomes. A Cross indicates that the structure is sensitive to that particular antibiotic.
Adapted from: Lynn Margulis. Symbiosis in Cell EvolutionÂ (1981) W.H.Freeman & Co LtdÂ
Laboratory experiments have been conducted to confirm the establishment of endosymbioses in several organisms. Kwang Jeon of the University of Tennessee has demonstrated that, under laboratory conditions, it is possible to observe the establishment of a stable amoeba-bacteria symbiosis. After over 20 years of culture, a strain of Amoeba proteus became infected with a large number of bacteria. These became integrated as necessary cell components after initially being pathogenic to the host cells 29. The amoeba's dependence on the endosymbiotic bacteria was demonstrated by removing the nucleus of an infected cell and placing it into another cell that had previously had its nucleus removed. Only those cells with symbionts could support a transplanted nucleus. Treatment with chloramphenicol also killed the majority of the endosymbiotic bacteria, which rendered the amoebae unable to survive. Thus, Jeon had proven that and extensive gene transfer had occurred and the host amoeba had become dependent on the symbionts to carry out aerobic respiration 3, 28, 29.
Finally, Okamoto and Inouye have shown that some organisms can take opportunistic advantage of a similar process to endosymbiosis, by observation of a heterotrophicÂ protist that engulfed a unicellularÂ green alga and used the products of its photosynthesis. Inside the host cell, the alga underwent morphological changes, including the loss of flagella and cytoskeleton. The heterotrophic host switched its source of nutrition and became an autotroph, capable of phototaxis 27. The acquisition of the alga by the protist and subsequent changes in both cells are believed to represent the early stages of a secondary endosymbiosis in process. Although neither experiment proves that endosymbiosis did occur, the conclusions of both experiments illustrate the possibility of endosymbiosis occurring in modern cells in a similar way in which the symbionts from which mitochondria are descended were thought to have been acquired 30.
Based upon the large body of available evidence contributed by scientists in the years since the endosymbiotic hypothesis was first proposed, including the conclusions of various experiments and the sequence data of mitochondrial nucleic acids and proteins, I have concluded that modern eukaryotic cells arose by a stable incorporation of prokaryotic endosymbionts. This dramatic change was then the driving force behind the evolution of new species and eventually more complex organisms 4. However, the question of which order eukaryotes came to possess nuclei and respiratory organelles is still the subject of much debate, and the fact that some genes remain encoded in the mitochondria rather than being completely transferred to the nucleus has not been accounted for16. Despite these uncertainties, the endosymbiotic theory remains the most probable explanation for the similarities between mitochondria and Bacteria, and the large disparity between Bacteria and eukaryotes. The next steps in the development of this theory may require new methods of reconstructing events that occurred billions of years ago, in order to answer one of the greatest uncertainties in evolutionary biology, regarding the origin of the eukaryotes.