Various human being diseases are connected with mitochondrial DNA (mtDNA) mutations, but heteroplasmythe coexistence of mutant and wild-type mtDNAcomplicates their study. diseases. INTRODUCTION Despite its diminutive size (17 kb in mammals), mitochondrial DNA (mtDNA) encodes 13 essential subunits of the electron transport complexes (Wallace, 2005 ) and is vital for life. Various human diseases stem from mutations in mtDNA (Taylor and Turnbull, 2005 ; Wallace, 2005 ). mtDNA diseases often affect tissues buy Palosuran with high-energy demand, such as muscles and the nervous system (DiMauro and Schon, 2003 ), which may reflect mitochondria’s primary buy Palosuran role in energy homeostasis. However, mtDNA diseases also feature great complexity along with a broad spectrum of symptoms that can be manifested in various tissues, suggesting the disruption of pathways other than energy homeostasis. These pathways include reactive oxygen species (ROS) generation and signaling, apoptosis, and calcium homeostasis (Chan, 2006 ; McBride mtDNAs (Oliveira as a model to understand Rabbit polyclonal to AK3L1 the function and regulation of mtDNA. There is a single locus on mtDNA. Expression of a mitochondrially targeted oxidase subunit I (CoI) protein (Hill, Chen, flies developed normally at 18C but fail to eclose at 29C (Hill, Chen, flies only survive up to 5 d (Hill, Chen, level in heteroplasmic flies remains constant over many generations at 18C, it is dramatically reduced during oogenesis and eventually purged from the population at 29C (Hill, Chen, flies to inquire questions about this mtDNA mutation that would be difficult to address in other systems. The homoplasmic flies provided material for a detailed biochemical characterization of the phenotype. The heteroplasmic flies allowed us to model the age-dependent and tissue-specific phenotypes typically observed in human mtDNA diseases. In particular, heteroplasmic flies provided a healthy background in which we were able to induce tissue-specific homoplasmy, which in turn allowed us to study some tissue-specific phenotypes of the mutation. RESULTS disrupts cytochrome oxidase activity To understand the biochemical basis of the temperature sensitivity of and wt flies (Physique 1A), cytochrome oxidase (COX) activity in the mutant buy Palosuran was decreased to 30% of wt activity at 25C (Supplemental Physique S1A). The mutant COX appears unstable at restrictive condition, as the COX activity of extract quickly diminished to <5% of wild type after incubating at 29C for 40 min (Hill, Chen, disrupts cytochrome oxidase activity. (A) Western blot analysis of total buy Palosuran tissue extracts of and wt flies cultured at 25 or 29C after eclosion at 25C, with antibodies against CoI, CoIV, ATP synthase -subunit ... COX activity depends on the association of CoI with two heme a cofactors (Babcock and Wikstrom, 1992 ). Spectral analyses showed that cytochrome amounts were markedly decreased in flies, whereas the amount of cytochrome was normal (Physique 1, B and C). In addition, the heme a cofactors further dissociated from COX in mitochondrial extracts after a brief incubation at 29C (Physique 1C), rendering them spectrally invisible due to the low solubility and high reactivity of free hemes (Severance and Hamza, 2009 ). These results suggest that the mutation reduces COX activity by weakening the conversation between the a hemes and CoI. Consistent with this hypothesis, the residue mutated in is located in transmembrane helix VIII of the CoI protein, which interacts with the a hemes (Tsukihara mitochondria compared with wild type based on the blue native PAGE analysis (Supplemental Physique S1B). This suggests that the mutation might affect the assembly or the stability of the whole complex. We also found that the level of ATP synthase -subunit, a routinely used mitochondrial marker, was comparable in mutant and wild type (Physique 1A). The amounts of complexes I, III, and V were all comparable between wt and mutant on.