Introduction

Cells must generate new cytoplasmic organelles if they are to grow and divide. They must also replenish organelles that are degraded as part of the continual process of organelle turnover in nonproliferating cells. Organelle biosynthesis requires the ordered synthesis of the requisite proteins and lipids and the delivery of each component to the correct organelle subcompartment. In Chapter 12we discussed how selected proteins and lipids are imported into mitochondria and chloroplasts from elsewhere in the cell. Here we describe the contributions that these energy-converting organelles make to their own biogenesis.

The Biosynthesis of Mitochondria and Chloroplasts Involves the Contribution of Two Separate Genetic Systems ⁴³

While most of the proteins in mitochondria and chloroplasts are encoded by nuclear DNA and imported into the organelle from the cytosol after they are synthesized on cytosolic ribosomes, some are encoded by organelle DNA and synthesized on ribosomes within the organelle. The protein traffic between the cytosol and these organelles seems to be unidirectional, as no protein is known to be exported from mitochondria or chloroplasts to the cytosol.

The contributions from the two genetic systems to the construction of mitochondria and chloroplasts are closely coordinated in the cell. Isolated organelles in a test tube continue to make organelle DNA, RNA, and proteins for brief periods, however, thereby providing one means of determining which proteins are encoded in organelle DNA and which in nuclear DNA. Another approach uses specific inhibitors on intact cells. The drug cycloheximide, for example, inhibits cytosolic protein synthesis but does not inhibit organelle protein synthesis. Conversely, various antibiotics (such as chloramphenicol, tetracycline, and erythromycin) inhibit protein synthesis in mitochondria and chloroplasts but have little effect on cytosolic protein synthesis (Figure 14-62). These inhibitors are widely used in studies of the functions of these organelles.

Figure 14-62

An overview of the biosynthesis of mitochondrial and chloroplast proteins. Each red arrow indicates the site of action of an inhibitor that is specific for either organelle or cytosolic protein synthesis.

Organelle Growth and Division Maintain the Number of Mitochondria and Chloroplasts in a Cell ⁴⁴

Mitochondria and chloroplasts are never made de novo. They always arise by the growth and division of existing mitochondria and chloroplasts. Observations of living cells indicate that mitochondria not only divide but also fuse with one another. On average, however, each organelle must double in mass and then divide in half once in each cell generation. Electron microscopic studies suggest that organelle division begins by an inward furrowing of the inner membrane, as occurs in cell division in many bacteria (Figures14-63 and 14-64), implying that it is a controlled process rather than a chance pinching in two.

Figure 14-63

Diagram of a dividing mitochondrion. The pathway shown has been postulated from static views of dividing mitochondria like that in Figure 14-64.

Figure 14-64

Electron micrograph of a dividing mitochondrion in a liver cell. (Courtesy of Daniel S. Friend.)

In most cells individual energy-converting organelles divide throughout interphase, out of phase with one another and with the division of the cell. Similarly, the replication of organelle DNA is not limited to the S phase, when nuclear DNA replicates, but occurs throughout the cell cycle. Individual organelle DNA molecules seem to be selected at random for replication, so that in a given cell cycle some may replicate more than once and others not at all. Nonetheless, under constant conditions the process is regulated to ensure that the total number of organelle DNA molecules doubles in every cell cycle, so that each cell type maintains a constant amount of organelle DNA.

The number of organelles per cell can be regulated according to need; a large increase in mitochondria (as much as five- to tenfold), for example, is observed if a resting skeletal muscle is repeatedly stimulated to contract for a prolonged period. Moreover, in special circumstances, organelle division is precisely controlled by the cell: thus, in some algae that contain only one or a few chloroplasts, the organelle divides just prior to cytokinesis in a plane that is identical to the future plane of cell division.

The Genomes of Chloroplasts and Mitochondria Are Usually Circular DNA Molecules ⁴⁵

Organelle DNA molecules are relatively small and simple, and, except for the mitochondrial genomes of some algae and protozoans, they are circular. The chloroplast genome (which is identical to the genomes of the other plastids in a plant) has a similar size in all organisms examined, but the mitochondrial genome is very much larger in plants than in animals (Table 14-2).

Table 14-2

The Size of Organelle Genomes*.

Many organelle DNA molecules are about the same size as typical viral DNAs. In mammals, for example, the mitochondrial genome is a DNA circle of about 16,500 base pairs (less than 10^-5 times the size of the nuclear genome). It is nearly the same size in animals as diverse as Drosophila and sea urchins (Figure 14-65). Plants, however, contain a circular mitochondrial genome that is 10 to 150 times larger, depending on the plant. The largest of these are about half the size of typical bacterial genomes, which are also circular DNA molecules.

Figure 14-65

Electron micrograph of an animal mitochondrial DNA molecule caught during the process of DNA replication. The circular DNA genome has replicated only between the two points marked by arrows (yellow strands). (Courtesy of David Clayton.)

All mitochondria and chloroplasts contain multiple copies of the organelle DNA molecule (Table 14-3). The molecules are usually distributed in several clusters in the matrix of the mitochondrion and in the stroma of the chloroplast, where they are thought to be attached to the inner membrane. Although it is not known how the DNA is packaged, the genome structure is likely to resemble that in bacteria rather than eucaryotic chromatin. As in bacteria, for example, there are no histones.

In mammalian cells mitochondrial DNA makes up less than 1% of the total cellular DNA. In other cells, however, such as the leaves of higher plants or the very large egg cells of amphibia, a much larger fraction of the cellular DNA may be present in the energy-converting organelles (see Table 14-3), and a larger fraction of RNA and protein synthesis takes place there.

Mitochondria and Chloroplasts Contain Complete Genetic Systems ⁴⁶

Despite the small number of proteins encoded in their genomes, mitochondria and plastids carry out their own DNA replication, DNA transcription, and protein synthesis. These processes take place in the matrix in mitochondria and in the stroma in chloroplasts. Although the proteins that mediate these genetic processes are unique to the organelle, most of them are encoded in the nuclear genome. This is all the more surprising because the protein-synthesis machinery of the organelles resembles that of bacteria rather than that of eucaryotes. The resemblance is particularly close in the case of chloroplasts:

1.

Chloroplast ribosomes are very similar to E. coli ribosomes, both in their sensitivity to various antibiotics (such as chloramphenicol, streptomycin, erythromycin, and tetracycline) and in their structure. Not only are the nucleotide sequences of the ribosomal RNAs of chloroplasts and E. coli strikingly similar, but chloroplast ribosomes are able to use bacterial tRNAs in protein synthesis. In all these respects, chloroplast ribosomes differ from those found in the cytosol of the same plant cell.

2.

Protein synthesis in chloroplasts starts with N-formylmethionine, as in bacteria, and not with methionine, as in the cytosol of eucaryotic cells.

3.

Unlike nuclear DNA, chloroplast DNA can be transcribed by the RNA polymerase enzyme from E. coli to produce chloroplast mRNAs, and these mRNAs are efficiently translated by an E. coli protein-synthesizing system.

Although mitochondrial genetic systems are much less similar to those of present-day bacteria than are the genetic systems of chloroplasts, their ribosomes are also sensitive to antibacterial antibiotics, and protein synthesis in mitochondria also starts with N-formylmethionine.

The Chloroplast Genome of Higher Plants Contains About 120 Genes ⁴⁷

The best-studied chloroplast genomes are those of green algae and higher plants, whose chloroplasts are very similar circular DNA molecules. The complete nucleotide sequences have been determined for the chloroplasts of tobacco and liver-wort. The results indicate that these two distantly related higher plants contain nearly identical chloroplast genes. In addition to four ribosomal RNAs, these genomes encode about 20 chloroplast ribosomal proteins, selected subunits of the chloroplast RNA polymerase, several proteins that are part of photosystems I and II, subunits of the ATP synthase, portions of enzyme complexes in the electron-transport chain, one of the two subunits of ribulose bisphosphate carbox-ylase, and 30 tRNAs (Figure 14-66). In addition, the DNA sequences present seem to encode at least 40 proteins whose functions are unknown. Paradoxically, all of the known proteins encoded in the chloroplast are part of larger protein complexes that also contain one or more subunits encoded in the nucleus. Possible reasons will be discussed later.

Figure 14-66

The organization of the liverwort chloroplast genome. The complete nucleotide sequence of this genome has been determined. The organization of the chloroplast genome is very similar in all higher plants, although the size varies from species to species (more...)

The similarities between the genomes of chloroplasts and bacteria are striking. The basic regulatory sequences, such as transcription promoters and terminators, are virtually identical in the two cases. Protein sequences encoded in chloroplasts are clearly recognizable as bacterial, and several clusters of genes with related functions (for example, those encoding ribosomal proteins) are organized in the same way in the genomes of chloroplasts, E. coli, and cyanobacteria.

Detailed comparisons of large numbers of homologous nucleotide sequences should help to clarify the exact evolutionary pathway from bacteria to chloroplasts, but several conclusions can already be drawn. (1) Chloroplasts in higher plants arose from photosynthetic bacteria. (2) The chloroplast genome has been stably maintained for at least several hundred million years, the estimated time of divergence of liverwort and tobacco. (3) Many of the genes of the original bacterium are now present in the nuclear genome, where they have been transferred and stably maintained. In higher plants, for example, two-thirds of the 60 or so chloroplast ribosomal proteins are encoded in the cell nucleus, although the genes have a clear bacterial ancestry and the chloroplast ribosomes retain their original bacterial properties.

Mitochondrial Genomes Have Several Surprising Features ⁴⁸

The chloroplast genome was not the first organelle genome to be sequenced completely. The relatively small size of the human mitochondrial genome made it a particularly attractive target for molecular geneticists equipped with newly devised DNA-sequencing techniques, and in 1981 the complete sequence of its 16,569 nucleotides was published. By comparing this sequence with known mitochondrial tRNA sequences and with the partial amino acid sequences available for proteins encoded by the mitochondrial DNA, it has been possible to locate all of the human mitochondrial genes on the circular DNA molecule (Figure 14-67).

Figure 14-67

The organization of the human mitochondrial genome. The genome contains 2 rRNA genes, 22 tRNA genes, and 13 protein-coding sequences. The DNAs of several other animal mitochondrial genomes have also been completely sequenced and have the same genes and (more...)

Compared to nuclear, chloroplast, and bacterial genomes, the human mitochondrial genome has several surprising features. (1) Unlike other genomes, nearly every nucleotide appears to be part of a coding sequence, either for a protein or for one of the rRNAs or tRNAs. Since these coding sequences run directly into each other, there is very little room left for regulatory DNA sequences. (2) Whereas 30 or more tRNAs specify amino acids in the cytosol and in chloroplasts, only 22 tRNAs are required for mitochondrial protein synthesis. The normal codon-anticodon pairing rules are relaxed in mitochondria, so that many tRNA molecules recognize any one of the four nucleotides in the third (wobble) position. Such "2 out of 3" pairing allows one tRNA to pair with any one of four codons and permits protein synthesis with fewer tRNA molecules. (3) Perhaps most surprising, comparison of mitochondrial gene sequences and the amino acid sequences of the corresponding proteins indicates that the genetic code is different, so that 4 of the 64 codons have different "meanings" from those of the same codons in other genomes (Table 14-4).

Table 14-4

Some Differences Between the "Universal" Code and Mitochondrial Genetic Codes^*.

The observation that the genetic code is nearly the same in all organisms provides strong evidence that all cells have evolved from a common ancestor. How, then, does one explain the few differences in the genetic code in mitochondria? A hint comes from the recent finding that the mitochondrial genetic code is different in different organisms. Thus UGA, which is a stop codon elsewhere, is read as tryptophan in mitochondria of mammals, fungi, and protozoans but as stop in plant mitochondria. Similarly, the codon AGG normally codes for arginine, but it codes for stop in the mitochondria of mammals and for serine in Drosophila (see Table 14-4). Such variation suggests that a random drift can occur in the genetic code in mitochondria. Presumably, the unusually small number of proteins encoded by the mitochondrial genome makes an occasional change in the meaning of a rare codon tolerable, whereas such a change in a large genome would alter the function of many proteins and thereby destroy the cell.

Animal Mitochondria Contain the Simplest Genetic Systems Known ⁴⁹

Comparisons of DNA sequences in different organisms reveal that the rate of nucleotide substitution during evolution has been 10 times greater in mitochondrial genomes than in nuclear genomes, which presumably is due to a reduced fidelity of mitochondrial DNA replication, DNA repair, or both. Because only about 16,500 DNA nucleotides need to be replicated and expressed as RNAs and proteins in animal cell mitochondria, the error rate per nucleotide copied by DNA replication, maintained by DNA repair, transcribed by RNA polymerase, or translated into protein by mitochondrial ribosomes can be relatively high without damaging one of the relatively few gene products. This could explain why the mechanisms that carry out these processes are relatively simple compared to those used for the same purpose elsewhere in cells. The presence of only 22 tRNAs and the unusually small size of the rRNAs (less than two-thirds the size of the E. coli rRNAs), for example, would be expected to reduce the fidelity of protein synthesis in mitochondria, although this has not yet been tested adequately.

The relatively high rate of evolution of mitochondrial genes makes mitochondrial DNA sequence comparisons especially useful for estimating the dates of relatively recent evolutionary events, such as the steps in primate development.

Why Are Plant Mitochondrial Genomes So Large? ⁵⁰

Mitochondrial genomes are much larger in plant than in animal cells, and they vary remarkably in their DNA content, ranging from about 150,000 to about 2.5 x 10⁶ nucleotide pairs. Yet these genomes seem to encode only a few more proteins than do animal mitochondrial genomes. The paradox is compounded by the observation that in one family of plants, the cucurbits, mitochondrial genomes vary in size by as much as sevenfold. The green alga Chlamydomonas has a linear mitochondrial genome of only 16,000 nucleotide pairs, the same size as in animals.

Although very little sequence information is available for higher plant mitochondrial DNA molecules, almost all of the 70,000 nucleotide pairs in the large mitochondrial genome of the yeast Saccharomyces cerevisiae have been sequenced, and only about one-third of them code for protein. This finding raises the possibility that much of the extra DNA in yeast mitochondria, and possibly in plant mitochondria as well, is "junk DNA" of little consequence to the organism.

Some Organelle Genes Contain Introns ⁵¹

The processing of precursor RNAs plays an important role in the two mitochondrial systems studied in most detail - human and yeast. In human cells both strands of the mitochondrial DNA are transcribed at the same rate from a single promoter region on each strand, producing two different giant RNA molecules, each containing a full-length copy of one DNA strand. Transcription is, therefore, completely symmetric. The transcripts made on one strand - called the heavy strand (H strand) because of its density in CsCl - are extensively processed by nuclease cleavage to yield the two rRNAs, most of the tRNAs, and about 10 poly-A-containing RNAs. In contrast, the light strand (L strand) transcript is processed to produce only eight tRNAs and one small poly-A-containing RNA; the remaining 90% of this transcript apparently contains no useful information (being complementary to coding sequences synthesized on the other strand) and is degraded. The poly-A-containing RNAs are the mitochondrial mRNAs: although they lack a cap structure at their 5' end, they carry a poly-A tail at their 3' end that is added posttranscriptionally by a mitochondrial poly-A polymerase.

Unlike human mitochondrial genes, some plant and fungal (including yeast) mitochondrial genes contain introns, which must be removed by RNA splicing. Introns have also been found in about 20 plant chloroplast genes. Many of the introns in organelle genes consist of related nucleotide sequences that are capable of splicing themselves out of the RNA transcripts by RNA-mediated catalysis (see p. 109), although these self-splicing reactions are generally aided by proteins. The presence of introns in organelle genes is surprising, as introns are not common in the genes of the bacteria whose ancestors are thought to have given rise to mitochondria and plant chloroplasts.

In yeasts the same mitochondrial gene may have an intron in one strain but not in another. Such "optional introns" seem to be able to move in and out of genomes like transposable elements. On the other hand, introns in other yeast mitochondrial genes have been found in a corresponding position in the mitochondria of Aspergillus and Neurospora, implying that they were inherited from a common ancestor of these three fungi. It seems likely that the intron sequences themselves are of ancient origin and that, while they have been lost from many bacteria, they have been preferentially retained in those organelle genomes where RNA splicing is regulated to help control gene expression.

Mitochondrial Genes Can Be Distinguished from Nuclear Genes by Their Non-Mendelian (Cytoplasmic) Inheritance ⁵²

Most experiments on the mechanisms of mitochondrial biogenesis have been performed with Saccharomyces cerevisiae (baker's yeast). There are several reasons for this. First, when grown on glucose, this yeast has an ability to live by glycolysis alone and can therefore survive without functional mitochondria, which are required for oxidative phosphorylation. This makes it possible to grow cells with mutations in mitochondrial or nuclear DNA that drastically interfere with mitochondrial biogenesis; such mutations are lethal in many other eucaryotes. Second, yeasts are simple unicellular eucaryotes that are easy to grow and characterize biochemically. Finally, these yeast cells normally reproduce asexually by budding (asymmetrical mitosis), but they can also reproduce sexually. During sexual reproduction two haploid cells mate and fuse to form a diploid zygote, which can either grow mitotically or divide by meiosis to produce new haploid cells. The ability to control the alternation between asexual and sexual reproduction in the laboratory greatly facilitates genetic analyses. Because mutations in mitochondrial genes are not inherited according to the Mendelian rules that govern the inheritance of nuclear genes, genetic studies reveal which of the genes involved in mitochondrial function are located in the nucleus and which in the mitochondria.

An example of non-Mendelian (cytoplasmic) inheritance of mitochondrial genes in a haploid yeast cell is illustrated in Figure 14-68. In this example we follow the inheritance of a mutant gene that makes mitochondrial protein synthesis resistant to chloramphenicol. When a chloramphenicol-resistant haploid cell mates with a chloramphenicol-sensitive wild-type haploid cell, the resulting diploid zygote will contain a mixture of mutant and wild-type mitochondria. But when the zygote undergoes mitosis to produce a diploid daughter, the mutant and wild-type mitochondria will be distributed at random between the mother and the daughter cell, so that each daughter is likely to inherit more mutant or more wild-type mitochondria. With successive mitotic divisions, either the mutant or the wild-type mitochondria will gradually be diluted out of some daughters by the same random process, leaving mitochondria of only one type. Thereafter, all of the progeny from that daughter will have mitochondria that are genetically identical. Thus this random process, called mitotic segregation, will eventually produce diploid yeast cells with only a single type of mitochondrial DNA. When such diploid cells undergo meiosis to form four haploid daughter cells, each of the four daughters receives the same mitochondrial genes. This type of inheritance is called non-Mendelian, or cytoplasmic, to contrast it with the Mendelian inheritance of nuclear genes (see Figure 14-68). When it occurs, it demonstrates that the gene in question is located outside the nuclear chromosomes and therefore probably in the yeast mitochondria.

Figure 14-68

The difference in the pattern of inheritance between mitochondrial and nuclear genes of yeast. For each nuclear gene two of the four cells that result from meiosis inherit the gene from one of the original haploid parent cells and the remaining two cells (more...)

Organelle Genes Are Maternally Inherited in Many Organisms ⁵³

The consequences of cytoplasmic inheritance are more profound for some organisms, including ourselves, than they are for yeasts. In yeasts, when two haploid cells mate, they are equal in size and contribute equal amounts of mitochondrial DNA to the zygote (see Figure 14-68). Mitochondrial inheritance in yeasts is therefore biparental: both parents contribute equally to the mitochondrial gene pool of the progeny (although, as we have just seen, after several generations of vegetative growth the individual progeny often contain mitochondria from only one parent). In higher animals, by contrast, the egg cell always contributes much more cytoplasm to the zygote than does the sperm. One would expect mitochondrial inheritance in higher animals, therefore, to be nearly uniparental (or more precisely, maternal). Such maternal inheritance has been demonstrated in laboratory animals. When animals carrying type A mitochondrial DNA are crossed with animals carrying type B, the progeny contain only the maternal type of mitochondrial DNA. Similarly, by following the distribution of variant mitochondrial DNA sequences in large families, human mitochondrial DNA has been shown to be maternally inherited.

In about two-thirds of higher plants the chloroplasts from the male parent (contained in pollen grains) do not enter the zygote, so that chloroplast as well as mitochondrial DNA is maternally inherited. In other plants the pollen chloroplasts enter the zygote, making chloroplast inheritance biparental. In such plants defective chloroplasts are a cause of variegation: a mixture of normal and defective chloroplasts in a zygote may sort out by mitotic segregation during plant growth and development, thereby producing alternating green and white patches in leaves. The green patches contain normal chloroplasts, while the white patches contain defective chloroplasts.

Petite Mutants in Yeasts Demonstrate the Overwhelming Importance of the Cell Nucleus for Mitochondrial Biogenesis ⁵⁴

Genetic studies of yeasts have played a crucial part in the analysis of mitochondrial biogenesis. A striking example is provided by studies of yeast mutants that contain large deletions in their mitochondrial DNA, so that all mitochondrial protein synthesis is abolished. Not surprisingly, these mutants cannot make respiring mitochondria. Some of these mutants lack mitochondrial DNA altogether. Because they form unusually small colonies when grown in media with low glucose, all mutants with such defective mitochondria are called cytoplasmic petite mutants.

Although petite mutants cannot synthesize proteins in their mitochondria and therefore cannot make mitochondria that produce ATP, they nevertheless contain mitochondria. These mitochondria have a normal outer membrane and an inner membrane with poorly developed cristae (Figure 14-69), and they contain virtually all of the mitochondrial proteins that are specified by nuclear genes and imported from the cytosol - including DNA and RNA polymerases, all of the citric acid cycle enzymes, and most inner membrane proteins - demonstrating the overwhelming importance of the nucleus in mitochondrial biogenesis. The petite mutants also show that an organelle that divides by fission can replicate indefinitely in the cytoplasm of proliferating eucaryotic cells even in the complete absence of its own genome. Many biologists believe that peroxisomes normally replicate in this way (see Figure 12-29).

Figure 14-69

Electron micrographs of yeast cells showing the structure of normal mitochondria (A) and mitochondria in a petite mutant (B). In petite mutants all of the mitochondrion-encoded gene products are missing, and so the organelle is constructed entirely from (more...)

For chloroplasts the nearest equivalent to yeast mitochondrial petite mutants are mutants of unicellular algae such as Euglena. Cells in which no chloroplast protein synthesis occurs still contain chloroplasts and are perfectly viable if oxidizable substrates are provided. If the development of mature chloroplasts is blocked in higher plants, however, either by raising the plants in the dark or because chloroplast DNA is defective or absent, the plants die as soon as their food stores run out.

Mitochondria and Chloroplasts Contain Tissue-specific Proteins ⁵⁵

Mitochondria can have specialized functions in particular types of cells. The urea cycle, for example, is the central metabolic pathway in mammals for disposing of cellular breakdown products that contain nitrogen. These products are excreted in the urine as urea. Nuclear-encoded enzymes in the mitochondrial matrix carry out several steps in the cycle. Urea synthesis occurs in only a few tissues, such as the liver, and the required enzymes are synthesized and imported into mitochondria only in these tissues. In addition, the respiratory enzyme complexes in the mitochondrial inner membrane of mammals contain several tissue-specific, nuclear-encoded subunits that are thought to act as regulators of electron transport. Thus some humans with a genetic muscle disease have a defective subunit of cytochrome oxidase; since the subunit is specific to skeletal muscle cells, their other cells, including their heart muscle cells, function normally, allowing the individuals to survive. As would be expected, tissue-specific differences are also found among the nuclear-encoded proteins in chloroplasts.

Mitochondria Import Most of Their Lipids; Chloroplasts Make Most of Theirs ⁵⁶

The biosynthesis of new mitochondria and chloroplasts requires lipids in addition to nucleic acids and proteins. Chloroplasts tend to make the lipids they require. In spinach leaves, for example, all cellular fatty acid synthesis takes place in the chloroplast, although desaturation of the fatty acids occurs elsewhere. The major glycolipids of the chloroplast are also synthesized locally.

Mitochondria, on the other hand, import most of their lipids. In animal cells the phospholipids phosphatidylcholine and phosphatidylserine are synthesized in the endoplasmic reticulum and then transferred to the outer membrane of mitochondria. In addition to decarboxylating imported phosphatidylserine to phosphatidylethanolamine, the main reaction of lipid biosynthesis catalyzed by the mitochondria themselves is the conversion of imported lipids to cardiolipin (bisphosphatidylglycerol). Cardiolipin is a "double" phospholipid that contains four fatty-acid tails; it is found mainly in the mitochondrial inner membrane, where it constitutes about 20% of the total lipid.

We have discussed the important question of how specific cytosolic proteins are imported into mitochondria and chloroplasts in detail in Chapter 12.

Both Mitochondria and Chloroplasts Probably Evolved from Endosymbiotic Bacteria ⁵⁷

As discussed in Chapter 1, the procaryotic character of the organelle genetic systems, especially striking in chloroplasts, suggests that mitochondria and chloroplasts evolved from bacteria that were endocytosed more than a billion years ago. According to this endosymbiont hypothesis, eucaryotic cells started out as anaerobic organisms without mitochondria or chloroplasts and then established a stable endosymbiotic relation with a bacterium, whose oxidative phosphorylation system they subverted for their own use (Figure 14-70). The endocytic event that led to the development of mitochondria is presumed to have occurred when oxygen entered the atmosphere in substantial amounts, about 1.5 x 10⁹ years ago, before animals and plants separated (see Figure 14-59). Plant and algal chloroplasts seem to have been derived later from an endocytic event involving an oxygen-evolving photosynthetic bacterium. In order to explain the different pigments and properties of the chloroplasts found in present-day higher plants and algae, it is usually assumed that at least three separate events of this kind occurred.

Figure 14-70

A suggested evolutionary pathway for the origin of mitochondria. Microsporidia and Giardia are two present-day anaerobic single-celled eucaryotes (protozoa) without mitochondria. Because they have an rRNA sequence that suggests a great deal of evolutionary (more...)

Since most of the genes encoding present-day mitochondrial and chloroplast proteins are in the cell nucleus, it seems that an extensive transfer of genes from organelle to nuclear DNA has occurred during eucaryote evolution. This would explain why some of the nuclear genes encoding mitochondrial proteins resemble bacterial genes: the amino acid sequence of the chicken mitochondrial enzyme superoxide dismutase, for example, resembles the corresponding bacterial enzyme much more than it resembles the superoxide dismutase found in the cytosol of the same eucaryotic cells. Further evidence that such DNA transfers have occurred during evolution comes from the discovery of some noncoding DNA sequences in nuclear DNA that seem to be of recent mitochondrial origin; they have apparently integrated into the nuclear genome as "junk DNA."

What type of bacterium gave rise to the mitochondrion? Protein and nucleotide sequence analyses have provided evidence for the evolutionary tree shown previously in Figure 14-60. It appears that mitochondria are descendants of a particular type of purple photosynthetic bacterium that had previously lost its ability to carry out photosynthesis and was left with only a respiratory chain. It is not clear that all mitochondria have originated from a single endosymbiotic event, however. While the mitochondria from protozoans have distinctly procaryotic features, for example, some of them are sufficiently different from plant and animal mitochondria to suggest a separate origin.

Why Do Mitochondria and Chloroplasts Have Their Own Genetic Systems? ⁵⁸

Why do mitochondria and chloroplasts require their own separate genetic systems when other organelles that share the same cytoplasm, such as peroxisomes and lysosomes, do not? The question is not trivial because maintaining a separate genetic system is costly: more than 90 proteins - including many ribosomal proteins, aminoacyl-tRNA synthases, DNA and RNA polymerases, and RNA-processing and -modifying enzymes - must be encoded by nuclear genes specifically for this purpose (Figure 14-71). The amino acid sequences of most of these proteins in mitochondria and chloroplasts differ from those of their counterparts in the nucleus and cytosol, and there is reason to think that these organelles have relatively few proteins in common with the rest of the cell. This means that the nucleus must provide at least 90 genes just to maintain each organelle genetic system. The reason for such a costly arrangement is not clear, and the hope that the nucleotide sequences of mitochondrial and chloroplast genomes would provide the answer has proved unfounded. We cannot think of compelling reasons why the proteins made in mitochondria and chloroplasts should be made there rather than in the cytosol.

Figure 14-71

The origins of mitochondrial RNAs and proteins. The proteins imported from the cytosol play a major part in creating the genetic system of the mitochondrion in addition to contributing most of the organelle protein. The mitochondrion itself contributes (more...)

At one time it was suggested that some proteins have to be made in the organelle because they are too hydrophobic to get to their site in the membrane from the cytosol. More recent studies, however, make this explanation implausible. In many cases even highly hydrophobic subunits are synthesized in the cytosol. Moreover, although the individual protein subunits in the various mitochondrial enzyme complexes are highly conserved in evolution, their site of synthesis is not. The diversity in the location of the genes coding for the subunits of functionally equivalent proteins in different organisms is difficult to explain by any hypothesis that postulates a specific evolutionary advantage of present-day mitochondrial or chloroplast genetic systems.

Perhaps the organelle genetic systems are an evolutionary dead end. In terms of the endosymbiont hypothesis, this would mean that the process whereby the endosymbionts transferred most of their genes to the nucleus stopped before it was complete. Further transfers may have been ruled out, in the case of mitochondria, by recent alterations in the mitochondrial genetic code that made the remaining mitochondrial genes nonfunctional if they were transferred to the nucleus.

Summary

Mitochondria and chloroplasts grow and divide in two in a coordinated process that requires the contribution of two separate genetic systems - that of the organelle and that of the cell nucleus. Most of the proteins in these organelles are encoded by nuclear DNA, synthesized in the cytosol, and then imported individually into the organelle. Some organelle proteins and RNAs are encoded by the organelle DNA and are synthesized in the organelle itself. The human mitochondrial genome contains about 16,500 nucleotides and encodes 2 ribosomal RNAs, 22 transfer RNAs, and 13 different polypeptide chains. Chloroplast genomes are about 10 times larger and contain about 120 genes. But partially functional organelles will form in normal numbers even in mutants that lack a functional organelle genome, demonstrating the overwhelming importance of the nucleus for the biogenesis of both organelles.

The ribosomes of chloroplasts closely resemble bacterial ribosomes, while mitochondrial ribosomes show both similarities and differences that make their origin more difficult to trace. Protein similarities, however, suggest that both organelles originated when a primitive eucaryotic cell entered into a stable endosymbiotic relationship with a bacterium: a purple bacterium is thought to have given rise to the mitochondrion, and (later) a relative of a cyanobacterium is thought to have given rise to the plant chloroplast. Although many of the genes of these ancient bacteria still function to make organelle proteins, most of them have become integrated into the nuclear genome, where they encode bacterial-like enzymes that are synthesized on cytosolic ribosomes and then imported into the organelle.