Organization and Function of Mitochondria

Mitochondria are surrounded by a double-membrane system, consisting of inner and outer mitochondrial membranes separated by an intermembrane space (Figure 10.1). The inner membrane forms numerous folds (cristae), which extend into the interior (or matrix) of the organelle. Each of these components plays distinct functional roles, with the matrix and inner membrane representing the major working compartments of mitochondria.

Figure 10.1

Structure of a mitochondrion. Mitochondria are bounded by a double-membrane system, consisting of inner and outer membranes. Folds of the inner membrane (cristae) extend into the matrix. (Micrograph by K. R. Porter/Photo Researchers, Inc.)

The matrix contains the mitochondrial genetic system as well as the enzymes responsible for the central reactions of oxidative metabolism (Figure 10.2). As discussed in Chapter 2, the oxidative breakdown of glucose and fatty acids is the principal source of metabolic energy in animal cells. The initial stages of glucose metabolism (glycolysis) occur in the cytosol, where glucose is converted to pyruvate (see Figure 2.32). Pyruvate is then transported into mitochondria, where its complete oxidation to CO₂ yields the bulk of usable energy (ATP) obtained from glucose metabolism. This involves the initial oxidation of pyruvate to acetyl CoA, which is then broken down to CO₂ via the citric acid cycle (see Figures 2.33 and 2.34). The oxidation of fatty acids also yields acetyl CoA (see Figure 2.36), which is similarly metabolized by the citric acid cycle in mitochondria. The enzymes of the citric acid cycle (located in the matrix of mitochondria) thus are central players in the oxidative breakdown of both carbohydrates and fatty acids.

Figure 10.2

Metabolism in the matrix of mitochondria. Pyruvate and fatty acids are imported from the cytosol and converted to acetyl CoA in the mitochondrial matrix. Acetyl CoA is then oxidized to CO₂ via the citric acid cycle, the central pathway of oxidative metabolism. (more...)

The oxidation of acetyl CoA to CO₂ is coupled to the reduction of NAD⁺ and FAD to NADH and FADH₂, respectively. Most of the energy derived from oxidative metabolism is then produced by the process of oxidative phosphorylation (discussed in detail in the next section), which takes place in the inner mitochondrial membrane. The high-energy electrons from NADH and FADH₂ are transferred through a series of carriers in the membrane to molecular oxygen. The energy derived from these electron transfer reactions is converted to potential energy stored in a proton gradient across the membrane, which is then used to drive ATP synthesis. The inner mitochondrial membrane thus represents the principal site of ATP generation, and this critical role is reflected in its structure. First, its surface area is substantially increased by its folding into cristae. In addition, the inner mitochondrial membrane contains an unusually high percentage (greater than 70%) of proteins, which are involved in oxidative phosphorylation as well as in the transport of metabolites (e.g., pyruvate and fatty acids) between the cytosol and mitochondria. Otherwise, the inner membrane is impermeable to most ions and small molecules—a property critical to maintaining the proton gradient that drives oxidative phosphorylation.

In contrast to the inner membrane, the outer mitochondrial membrane is freely permeable to small molecules. This is because it contains proteins called porins, which form channels that allow the free diffusion of molecules smaller than about 6000 daltons. The composition of the intermembrane space is therefore similar to the cytosol with respect to ions and small molecules. Consequently, the inner mitochondrial membrane is the functional barrier to the passage of small molecules between the cytosol and the matrix and maintains the proton gradient that drives oxidative phosphorylation.

The Genetic System of Mitochondria

Mitochondria contain their own genetic system, which is separate and distinct from the nuclear genome of the cell. As reviewed in Chapter 1, mitochondria are thought to have evolved from bacteria that developed a symbiotic relationship in which they lived within larger cells (endosymbiosis). This hypothesis has recently been substantiated by the results of DNA sequence analysis, which revealed striking similarities between the genomes of mitochondria and of the bacterium Rickettsia prowazekii. Rickettsia are intracellular parasites which, like mitochondria, are only able to reproduce within eukaryotic cells. Consistent with their similar symbiotic lifestyles, the genomic DNA sequences of Rickettsia and mitochondria suggest that they share a common ancestor, from which the genetic system of present-day mitochondria evolved.

Mitochondrial genomes are usually circular DNA molecules, like those of bacteria, which are present in multiple copies per organelle. They vary considerably in size between different species. The genomes of human and most other animal mitochondria are only about 16 kb, but substantially larger mitochondrial genomes are found in yeasts (approximately 80 kb) and plants (more than 200 kb). However, these larger mitochondrial genomes are composed predominantly of noncoding sequences and do not appear to contain significantly more genetic information. For example, the largest sequenced mitochondrial genome is that of the plant Arabidopsis thaliana. Although Arabidopsis mitochondrial DNA is approximately 367 kb, it encodes only 32 proteins: just more than twice the number encoded by human mitochondrial DNA. The largest number of mitochondrial genes has been found in mitochondrial DNA of the protozoan Reclinomonas americana, which is 69 kb and contains 97 genes. The mitochondrial genome of Reclinomonas appears to more closely resemble the bacterial genome from which mitochondria evolved than most present-day mitochondrial genomes, which encode only a small number of proteins that are essential components of the oxidative phosphorylation system. In addition, mitochondrial genomes encode all of the ribosomal RNAs and most of the transfer RNAs needed for translation of these protein-coding sequences within mitochondria. Other mitochondrial proteins are encoded by nuclear genes, which are thought to have been transferred to the nucleus from the ancestral mitochondrial genome.

The human mitochondrial genome encodes 13 proteins involved in electron transport and oxidative phosphorylation (Figure 10.3). In addition, human mitochondrial DNA encodes 16S and 12S rRNAs and 22 tRNAs, which are required for translation of the proteins encoded by the organelle genome. The two rRNAs are the only RNA components of animal and yeast mitochondrial ribosomes, in contrast to the three rRNAs of bacterial ribosomes (23S, 16S, and 5S). Plant mitochondrial DNAs, however, also encode a third rRNA of 5S. The mitochondria of plants and protozoans also differ in importing and utilizing tRNAs encoded by the nuclear as well as the mitochondrial genome, whereas in animal mitochondria, all the tRNAs are encoded by the organelle.

Figure 10.3

The human mitochondrial genome. The genome contains 13 proteincoding sequences, which are designated as components of respiratory complexes I, III, IV, or V. In addition, the genome contains genes for 12S and 16S rRNAs and for 22 tRNAs, which are designated (more...)

The small number of tRNAs encoded by the mitochondrial genome highlights an important feature of the mitochondrial genetic system—the use of a slightly different genetic code, which is distinct from the “universal” genetic code used by both prokaryotic and eukaryotic cells (Table 10.1). As discussed in Chapter 3, there are 64 possible triplet codons, of which 61 encode the 20 different amino acids incorporated into proteins (see Table 3.1). Many tRNAs in both prokaryotic and eukaryotic cells are able to recognize more than a single codon in mRNA because of “wobble,” which allows some mispairing between the tRNA anticodon and the third position of certain complementary codons (see Figure 7.3). However, at least 30 different tRNAs are required to translate the universal code according to the wobble rules. Yet human mitochondrial DNA encodes only 22 tRNA species, and these are the only tRNAs used for translation of mitochondrial mRNAs. This is accomplished by an extreme form of wobble in which U in the anticodon of the tRNA can pair with any of the four bases in the third codon position of mRNA, allowing four codons to be recognized by a single tRNA. In addition, some codons specify different amino acids in mitochondria than in the universal code.

Table 10.1

Differences between the Universal and Mitochondrial Genetic Codes.

Like the DNA of nuclear genomes, mitochondrial DNA can be altered by mutations, which are frequently deleterious to the organelle. Since almost all the mitochondria of fertilized eggs are contributed by the oocyte rather than by the sperm, germ-line mutations in mitochondrial DNA are transmitted to the next generation by the mother. Such mutations have been associated with a number of diseases. For example, Leber's hereditary optic neuropathy, a disease that leads to blindness, can be caused by mutations in mitochondrial genes that encode components of the electron transport chain. In addition, the progressive accumulation of mutations in mitochondrial DNA during the lifetime of individuals has been suggested to contribute to the process of aging.

Protein Import and Mitochondrial Assembly

In contrast to the RNA components of the mitochondrial translation apparatus (rRNAs and tRNAs), most mitochondrial genomes do not encode the proteins required for DNA replication, transcription, or translation. Instead, the genes that encode proteins required for the replication and expression of mitochondrial DNA are contained in the nucleus. In addition, the nucleus contains the genes that encode most of the mitochondrial proteins required for oxidative phosphorylation and all of the enzymes involved in mitochondrial metabolism (e.g., enzymes of the citric acid cycle). The proteins encoded by these genes (more than 95% of mitochondrial proteins) are synthesized on free cytosolic ribosomes and imported into mitochondria as completed polypeptide chains. Because of the double-membrane structure of mitochondria, the import of proteins is considerably more complicated than the transfer of a polypeptide across a single phospholipid bilayer. Proteins targeted to the matrix have to cross both the inner and outer mitochondrial membranes, while other proteins need to be sorted to distinct compartments within the organelle (e.g., the intermembrane space).

The import of proteins to the matrix is the best-understood aspect of mitochondrial protein sorting (Figure 10.4). Most proteins are targeted to mitochondria by amino-terminal sequences of 20 to 35 amino acids (called presequences) that are removed by proteolytic cleavage following their import into the organelle. The presequences of mitochondrial proteins, first characterized by Gottfried Schatz, contain multiple positively charged amino acid residues, usually in an amphipathic α helix. The first step in protein import is the binding of these presequences to receptors on the surface of mitochondria. The polypeptide chains are then inserted into a protein complex that directs translocation across the outer membrane (the translocase of the outer membrane or Tom complex). The proteins are then transferred to a second protein complex in the inner membrane (the translocase of the inner membrane or Tim complex). Continuing protein translocation requires the electrochemical potential established across the inner mitochondrial membrane during electron transport. As discussed in the next section of this chapter, the transfer of high-energy electrons from NADH and FADH₂ to molecular oxygen is coupled to the transfer of protons from the mitochondrial matrix to the intermembrane space. Since protons are charged particles, this transfer establishes an electric potential across the inner membrane, with the matrix being negative. During protein import, this electric potential drives translocation of the positively charged presequence.

Figure 10.4

Import of proteins into mitochondria. Proteins are targeted for mitochondria by an amino-terminal presequence containing positively charged amino acids. Proteins are maintained in a partially unfolded state by association with a cytosolic Hsp70 and are (more...)

To be translocated across the mitochondrial membrane, proteins must be at least partially unfolded. Consequently, protein import into mitochondria requires molecular chaperones in addition to the membrane proteins involved in translocation (see Figure 10.4). On the cytosolic side, members of the Hsp70 family of chaperones maintain proteins in a partially unfolded state so that they can be inserted into the mitochondrial membrane. As they cross the inner membrane, the unfolded polypeptide chains bind to another member of the Hsp70 family, which is associated with the Tim complex and acts as a motor that drives protein import. The polypeptide is then transferred to a chaperone of the Hsp60 family (a chaperonin), within which protein folding takes place. Since these interactions of polypeptide chains with molecular chaperones depend on ATP, protein import requires ATP both outside and inside the mitochondria, in addition to the electric potential across the inner membrane.

As noted above, some mitochondrial proteins are targeted to the outer membrane, inner membrane, or intermembrane space rather than to the matrix, so additional mechanisms are needed to direct these proteins to the correct submitochondrial compartment. These proteins are targeted to their destinations by a second sorting signal following the positively charged presequence that directs mitochondrial import. The targeting of proteins to the mitochondrial membranes appears to be mediated by hydrophobic stop-transfer sequences that halt translocation of the polypeptide chains through the Tim or Tom complexes, leading to their insertion into the inner or outer mitochondrial membranes, respectively (Figure 10.5). Proteins may be targeted to the intermembrane space by several different mechanisms (Figure 10.6). Some proteins are transferred across the outer membrane through the Tom complex but are then released within the intermembrane space instead of being transferred to the Tim complex. Other proteins are transferred to the Tim complex but are then released into the intermembrane space as a result of cleavage of hydrophobic stop-transfer sequences. Still other proteins may be completely imported into the mitochondrial matrix and then exported back across the inner membrane to the intermembrane space.

Figure 10.5

Insertion of mitochondrial membrane proteins. Proteins targeted for the mitochondrial membranes contain hydrophobic stop-transfer sequences that halt their translocation through the Tom or Tim complexes and lead to their incorporation into the outer or (more...)

Figure 10.6

Sorting proteins to the intermembrane space. Proteins can be targeted to the intermembrane space by several mechanisms. Some proteins (I) are translocated through the Tom complex and released into the intermembrane space. Other proteins (II) are transferred (more...)

Not only the proteins, but also the phospholipids of mitochondrial membranes are imported from the cytosol. In animal cells, phosphatidylcholine and phosphatidylethanolamine are synthesized in the ER and carried to mitochondria by phospholipid transfer proteins, which extract single phospholipid molecules from the membrane of the ER. The lipid can then be transported through the aqueous environment of the cytosol, buried in a hydrophobic binding site of the protein, and released when the complex reaches a new membrane, such as that of mitochondria. The mitochondria then synthesize phosphatidylserine from phosphatidylethanolamine, in addition to catalyzing the synthesis of the unusual phospholipid cardiolipin, which contains four fatty acid chains (Figure 10.7).

Figure 10.7

Structure of cardiolipin. Cardiolipin is an unusual “double” phospholipid, containing four fatty acid chains, that is found primarily in the inner mitochondrial membrane.

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Molecular Medicine: Diseases of Mitochondria: Leber's Hereditary Optic Neuropathy.