Proapoptotic Bcl-2 family members have been proposed to play a central role in regulating apoptosis. However, mice lacking bax display limited phenotypic abnormalities. As presented here, bak(-/-) mice were found to be developmentally normal and reproductively fit and failed to develop any age-related disorders. However, when Bak-deficient mice were mated to Bax-deficient mice to create mice lacking both genes, the majority of bax(-/-)bak(-/-) animals died perinatally with fewer than 10% surviving into adulthood. bax(-/-)bak(-/-) mice displayed multiple developmental defects, including persistence of interdigital webs, an imperforate vaginal canal, and accumulation of excess cells within both the central nervous and hematopoietic systems. Thus, Bax and Bak have overlapping roles in the regulation of apoptosis during mammalian development and tissue homeostasis.

A sudden increase in permeability of the inner mitochondrial membrane, the so-called mitochondrial permeability transition, is a common feature of apoptosis and is mediated by the mitochondrial permeability transition pore (mtPTP). It is thought that the mtPTP is a protein complex formed by the voltage-dependent anion channel, members of the pro-and anti-apoptotic BAX-BCL2 protein family, cyclophilin D, and the adenine nucleotide (ADP/ATP) translocators (ANTs) 1,2 . The latter exchange mitochondrial ATP for cytosolic ADP and have been implicated in cell death. To investigate the role of the ANTs in the mtPTP, we genetically inactivated the two isoforms of ANT [3][4][5] in mouse liver and analysed mtPTP activation in isolated mitochondria and the induction of cell death in hepatocytes. Mitochondria lacking ANT could still be induced to undergo permeability transition, resulting in release of cytochrome c. However, more Ca 2+ than usual was required to activate the mtPTP, and the pore could no longer be regulated by ANT ligands. Moreover, hepatocytes without ANT remained competent to respond to various initiators of cell death. Therefore, ANTs are non-essential structural components of the mtPTP, although they do contribute to its regulation.To investigate the role of ANTs in the mtPTP, we inactivated both the heart-muscle (Ant1) and the systemic (Ant2) ANT isoform genes in the mouse liver. Humans have three ANT genes 6,7 , whereas results of complementary DNA library screening 5 and northern and western analyses 3 have suggested that mouse has only two Ant genes. To verify this, we screened the Celera and Ensembl mouse genome assemblies, as well as the respective EST 1a). Targeted embryonic stem cells (Fig. 1b) were used to introduce the conditional floxed allele into the mouse germ line. Liver-specific inactivation of the Ant2 fl allele was achieved by breeding Ant2 fl mice with an Alb-Cre line of transgenic mice that expresses CRE under control of the liver-specific albumin promoter, which results in the excision of exons 3 and 4 of Ant2 (Fig. 1a) Endogenous respiration rates of ANT1/ANT2-deficient mitochondria were almost twice that of control mitochondria (34.58 ± 1.6 versus 18.12 ± 1.1 nmol O min -1 per mg protein) (Fig. 2b) and the mitochondrial membrane potential (ΔP = ΔΨ + ΔpH) of the ANT-deficient mitochondria was higher than that of controls (191.7 ± 4.9 versus 172.9 ± 3.5 mV). Analysis of the specific activity of OXPHOS enzyme complexes in ANT-deficient mitochondria revealed that complex IV (cytochrome c oxidase, COX) was increased more than twofold compared with controls (P < 0.01) (Fig. 2c). This was confirmed by western blot analysis, which revealed that the mitochondrial COX subunit I (COI) and cytochrome c proteins were more abundant in the ANT-deficient mitochondria (Fig. 2d). Hence, the increased respiration rate is likely to be the result of the specific upregulation in COX activity, suggesting that COX activity may modulate respiration rate. Because ΔP is the product of proton pumpin...

In an attempt to create an animal model of tissue-specific mitochondrial disease, we generated 'knockout' mice deficient in the heart/muscle isoform of the adenine nucleotide translocator (Ant1). Histological and ultrastructural examination of skeletal muscle from Ant1 null mutants revealed ragged-red muscle fibers and a dramatic proliferation of mitochondria, while examination of the heart revealed cardiac hypertrophy with mitochondrial proliferation. Mitochondria isolated from mutant skeletal muscle exhibited a severe defect in coupled respiration. Ant1 mutant adults also had a resting serum lactate level fourfold higher than that of controls, indicative of metabolic acidosis. Significantly, mutant adults manifested severe exercise intolerance. Therefore, Ant1 mutant mice have the biochemical, histological, metabolic and physiological characteristics of mitochondrial myopathy and cardiomyopathy.

The majority of mitochondrial DNA (mtDNA) mutations that cause human disease are mild to moderately deleterious, yet many random mtDNA mutations would be expected to be severe. To determine the fate of the more severe mtDNA mutations, we introduced mtDNAs containing two mutations that affect oxidative phosphorylation into the female mouse germ line. The severe ND6 mutation was selectively eliminated during oogenesis within four generations, whereas the milder COI mutation was retained throughout multiple generations even though the offspring consistently developed mitochondrial myopathy and cardiomyopathy. Thus, severe mtDNA mutations appear to be selectively eliminated from the female germ line, thereby minimizing their impact on population fitness.