The mitochondrial genome. The nucleoid

Mitochondrial DNA (mtDNA) in cells is organized in nucleoids containing DNA and various proteins. This review discusses questions of organization and structural dynamics of nucleoids as well as their protein components. The structures of mt-nucleoid from different organisms are compared. The currently accepted model of nucleoid organization is described and questions needing answers for better understanding of the fine mechanisms of the mitochondrial genetic apparatus functioning are discussed.     

The structure of Tetrahymena pyriformis mitochondrial DNA. II. The complex structure of strain GL mitochondrial DNA.

1. Isolated mtDNA from Tetrahymena pyriformis strain GL is a linear duplex molecule with an average molecular weight of 32.6 - 10(6) and without internal gaps or breaks. Denaturation of this DNA results in single strands with a duplex hairpin at one end. The length of this hairpin varies between 0 and 5 micrometer within one preparation. 2. Uder renaturation conditions the single strands with hairpins are able to circularize in two ways, depending on the length of the hairpin. Circularization is also observed after partial digestion with exonuclease III of native strain GL mtDNA. 3. All these data fit a model (see Fig.2) in which the DNA is heterogeneous in length at both ends. At the left end a 10-micrometer duplication-inversion is present; part of this duplication-inversion is complementary to a region at the right end of the molecule. 4. The analogy between the structural peculiarities of strain GL mtDNA and of some linear viral DNAs is stressed.     

The renaissance of mitochondrial calcium transport.

Although the capacity of mitochondria for accumulating Ca2+ down the electrical gradient generated by the respiratory chain has been known for over three decades, the physiological significance of this phenomenon has been re-evaluated only recently. Indeed, it was long believed that the low affinity of the mitochondrial Ca2+ transporters would allow significant uptake only in conditions of cellular Ca2+ overload. Conversely, the direct measurement of [Ca2+] in the mitochondrial matrix revealed major [Ca2+] increases upon agonist stimulation. In this review, we will summarize: (a) the mechanisms that allow this large response, reconciling the biochemical properties of the transporters and the large amplitude of the mitochondrial [Ca2+] rises, and (b) the biological role of mitochondrial Ca2+ signalling, that encompasses the regulation of mitochondrial function and the modulation of the spatio-temporal pattern of cytosolic [Ca2+] increases.     

The genetic localization of presumptive mitochondrial messenger RNAs on rat-liver mitochondrial DNA.

High molecular weight mitochondrial (mt) RNAs were isolated from rat liver mitochondria and hybridized in the presence of excess competitor mt rRNA and/or mt tRNA to restriction fragments of mtDNA. The data reveals that there are a few areas of the mt-genome on which the complementary of these presumptive messenger RNAs is most pronounced. These areas are away from the parts of the genome which are coding for the mt rRNA or containing the D-loop.     

The effect of age on mitochondrial ultrastructure.

The ultrastructure of perfused livers and of mitochondrial fractions from 6-month-old and 30-month-old C57/BL mice were studied. In old mice the liver cell mitochondria were enlarged and rounded with a light 'foamy', vacuolated matrix, short cristae and a loss of dense granules. Quantitative studies showed a 60% increase in the mean size and an increased proportion of larger mitochondria in intact 30-month-old perfused livers. Endothelial and Kupffer cell mitochondria were smaller than those of the parenchymal cells. Mitochondria in pellets prepared from 6- and 30-month-old livers were rounded and condensed, although there were a few larger and 'foamy' mitochondria in the preparations from old mice. Up to 47% of large mitochondria in the old livers were lost during cell fractionation.     

The mitochondrial localization of coproporphyrinogen III oxidase.

The location of coproporphyrinogen III oxidase in mitochondria was studied in rat liver by using the digitonin method or hypo-osmotic media for fractionation. The enzyme was found in the intermembrane space with a fraction loosely bound to the inner membrane. This fraction was released by washing the inner-membrane-matrix complex with alkaline solutions or solutions of high ionic strength. The enzyme in both fractions had the same Km (0.16 micrometer) for coproporphyrinogen III. When incubation was performed in a medium that avoided destruction of enzyme membrane binding, a dramatic increase in activity was observed after sonication of whole mitochondria or of the inner-membrane-matrix complex.     

The reconstitution of the mitochondrial energy-linked transhydrogenase.

Complex I (NADH-ubiquinone reductase) catalyzes pyridine nucleotide transhydrogenase at rates several fold higher than those found in submitochondrial particles from bovine heart. An ATP-dependent reduction of NADP⁺ by NADH was demonstrated after combination of Complex I with phospholipids, hydrophobic proteins derived from bovine heart mitochondria, and mitochondrial ATPase (F₁)¹. The reaction was inhibited by oligomycin, uncoupling agents and low concentrations of Triton X-100.     

The mitochondrial import machinery for preproteins.

Most mitochondrial proteins are transported from the cytosol into the organelle. Due to the division of mitochondria into an outer and inner membrane, an intermembrane space and a matrix, an elaborated system for recognition and transport of preproteins has evolved. The translocase of the outer mitochondrial membrane (TOM) and the translocases of the inner mitochondrial membrane (TIM) mediate these processes. Receptor proteins on the cytosolic face of mitochondria recognize the cargo proteins and transfer them to the general import pore (GIP) of the outer membrane. Following the passage of preproteins through the outer membrane they are transported with the aid of the TIM23 complex into either the matrix, inner membrane, or intermembrane space. Some preprotein families utilize the TIM22 complex for their insertion into the inner membrane. The identification of protein components, which are involved in these transport processes, as well as significant insights into the molecular function of some of them, has been achieved in recent years. Moreover, we are now approaching a new era in which elaborated techniques have already allowed and will enable us to gather information about the TOM and TIM complexes on an ultrastructural level.     

The effect of tetraphenylborate on mitochondrial energy transduction.

In phosphorylating submitochondrial particles, tetraphenylborate binds to the specific uncoupler binding site and inhibits oxidative phosphorylation, ATP-P_i exchange and ATP-driven reverse electron transport. In contrast, intact mitochondria are unaffected in uncoupler binding and energy transfer at the concentrations used in submitochondrial particles. The proton permeability of submitochondrial particles is only slightly increased (10–20%) at concentrations of tetraphenylborate which cause 50% uncoupling (4–8 μM). These results, and those obtained earlier with picrate, are consistent with a three-step mechanism of uncoupling which involves binding of uncoupler anions, protonation and dissociation of the resulting neutral uncoupler molecule.