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The human adenine nucleotide translocase-2 (ANT2) promoter contains a silencer region that confers partial repression on the heterologous herpes simplex virus thymidine kinase (HSVtk) promoter [Barath, P., Albert-Fournier, B., Luciakova, K., Nelson, B.D. (1999) 1 Biol. Chem. 274, 3378-33841. Two sequences in the silencer (Site-2 and Site-3) are protected in the DNase I assay in vitro, and one of these is a repeated GTCCTG element previously shown to act as the active repressor element. We have now purified the DNA binding protein, and identified it using MALDI-TOF MS as a 33-kDa member of the nuclear factor 1 (NF1) family of transcription factors. NF1 purified from rat liver and HeLa cell nuclei bind to both silencer Site-2 and Site-3, resulting in a DNase I footprint identical to that obtained with purified recombinant NFl. Furthermore, transient transfection experiments with reporter constructs containing mutated silencer Site-2 and/or Site-3 show that both sites contribute to repression of the HSVtk promoter. Finally, chromatin immunoprecipitation analysis reveals that NF1 is bound to both elements on the endogenous HeLa cell ANT2 promoter. Our data support the belief that NF1 acts as a repressor when bound to silencing Site-2 and Site-3 of the ANT2 gene. [ABSTRACT FROM AUTHOR]

The arrangement of the peptides of free complex III and complex III incorporated into phospholipid vesicles was investigated by labeling with 125I + lactoperoxidase and with p-diazoniumbenzene [35S]sulfonate (N=N-PhSO3H). The peptides were separated by sodium dodecylsulfate acrylamide gel electrophoresis and scanned for radioactivity. In free complex III, the most extensive iodination occurred in peptides II and IV, whereas all polypeptides (I -VI), except peptide VII, could be coupled to N=N-PhSO3H. Peptide VII was, however, labeled in the sodium-dodecylsulfate- dissociated enzyme. These results show that, with the possible exception of peptide VII, all peptides are at least partially exposed on the surface of the isolated complex. Complex III incorporated into phospholipid vesicles showed a labeling pattern which differed from the free enzyme, both with respect to the total incorporation and to the relative incorporation between the peptides. Total incorporation of N=N-Ph35SO3H and 125I was decreased by 60 - 70% in complex III=vesicles, suggesting that a large number of the binding sites on the free enzyme become buried within the membrane. In addition, peptides I, II, IV and V were relatively less labeled in complex III-vesicles than were peptides III and VI, suggesting a deeper location of the former peptides within the membrane. This is supported by the finding that labeling of deoxycholate-treated complex III-vesicles with N = N - Ph35SO3H results in an incorporation pattern similar to that in free complex III. Binding of N = N -PhSO3H and its effects on enzymatic activity was also studied in free and membrane-bound complex III. Both the binding of N = N -Ph35SO3H and inactivation of duroquinol -cytochrome c reductase were significantly slower in complex Ill-vesicles. These results indicate that many of the N = N -PhCO3H-binding sites on the free complex are inaccessible when the enzyme is incorporated into a vesicle. This approach was also used to ascertain intactness of the vesicles, and to show that N = N -PhSO3H labeled only those peptides exposed on the outer surface. [ABSTRACT FROM AUTHOR]

The peptides of purified complex III from beef heart mitochondria have been studied by electrophoresis on dodecylsulfate gels. Of the 12 peptides consistently observed, only eight appear to be integral peptides of the functional complex. Attempts to identify these peptides have been made through co-electrophoresis of complex [I and fractions in which the individual peptides were either purified or greatly enriched. Electrophoresis of complex III preparations which were not reduced by mercaptoethanol indicates that intermolecular disulfide bonds play no significant role in stabilizing the complex. [ABSTRACT FROM AUTHOR]

Thyroid hormone is one of the few known physiological regulators of mammalian mitochondrial biogenesis. Although it exerts a global effect on biogenesis, it does so by regulating the expression of a limited number of unidentified mitochondrial proteins. We have investigated these hormoneregulated proteins in rat liver. Hormone injection induced a 30-fold increase in the levels of cytochrome-c1 mRNA after 3 d. In addition, the mRNA for the growth-activated adenine-nucleotide translocator, ANT2, was increased 13-fold and that for the ATPase N,N'-dicyclohexylcarbodiimidebinding protein increased 4-5-fold. Mitochondrial transcripts of cytochrome-oxidase subunit I also increased. No changes were found in the mRNA levels for the FI-ATPase β-subunit or cytochrome oxidase IV. A single low dose of triiodothyronine induces rapid increases in cytochrome-c1 and ANT2 mRNA species which parallel changes in the activity of the hormone-responsive malic enzyme, but are earlier than other mitochondrial biogenetic events. These data strengthen the view that thyroid hormone regulates synthesis of specific components within each respiratory-chain complex and that these products apparently play key roles in inner-membrane biogenesis and assembly. The significance of ANT2 induction is also discussed with respect to the rapid respiratory response induced by thyroid hormone. [ABSTRACT FROM AUTHOR]

The import and processing of cytochrome c1 and the iron sulfur protein of the cytochrome b-c1 complex were studied in Zajdela hepatoma ascites cells. Both peptides were synthesized as larger percursor molecules which were approximately 2–3 kDa and 5–6kDa larger than the mature forms of apocytochrome c1 and apo-iron sulfur protein, respectively. Comparison of these precursors to those reported for functionally homologous peptides in yeast and Neurospora indicate significant size changes have occurred in mammals. Rhodamine 6G, a specific vital stain for mitochondria, is a potent inhibitor of precursor processing in isolated hepatoma cells. Both precursor to cytochrome c1 and precursor to FeS accumulate in the soluble and particulate fractions obtained by digitonin treatment of tumor cells treated with Rhodamine 6G. Appearance of the mature peptides was abolished. The precursors are unstable, however, and disappear from the cytosolic and membrane fractions during a 10min chase. Comparison of the effects of Rhodamine 6G and carbonylcyanide m-chloro- phenylhydrazone on precursor processing shows that: (a) Rhodamine 6G is a more effective inhibitor of processing. (b) it has less of an inhibitory effect on cellular protein synthesis, and (c) it inhibits processing trader conditions in which it appears to have little influence on coupled respiration in whole cells. The data suggest that the most likely mode of action of Rhodamine 6G is on the matrix processing step. [ABSTRACT FROM AUTHOR]

Purified complex III (ubiquinol-cytochrome c reductase) from beef heart mitochondria was alkylated with iodo[1-14C]acetamide. After 6–8 h of incubation with iodo[1-14C]acetamide, duroquinol and ubiquinol-2-cytochrome c reductase activities were inhibited approximately 50%. During this time 4.5 ± 1.6 nmol of iodo[1-14C]acetamide reacted per mg of complex III protein. Experiments carried out over 24 h indicated that enzyme activity could be inhibited to 70% and that alkylation of complex III was proportional to inhibition. The rates of cytochrome b and c1 reduction by duroquinol are also decreased upon treatment of complex III with iodoacetamide. Separation of the peptides of complex III by electrophoresis in sodium dodecylsulfate shows that all of the radioactivity is located in a single peptide of 50000 molecular weight, which has been identified as one of the two core proteins. The possible functions of core protein are discussed. [ABSTRACT FROM AUTHOR]

1. The incorporation of small amounts of ubiquinone (Q) into pentane-extracted submitochondrial particles has been studied. A procedure is described which allows nearly quantitative incorporation of as little as 0.2 to 0.3 nmol Q/mg protein, i.e: less than 10 % of the Q present in the lyophilized particles prior to extraction. 2. It is shown that both NADH oxidase and succinate oxidase activities can be restored to 100 % of that in lyophilized particles by incorporation of 6-8 nmol Q/mg protein. The relative activities of both oxidases show a parallel response to an increase of the Q content of the particles between 0.2 and 22 nmol Q/mg protein, 50% reactivation being obtained at about 2 nmol Q/mg protein. 3. 50 to 60% of the incorporated Q can be reduced by NADH or succinate, independent of the total concentration of Q in the membrane. Similar reduction values were obtained for lyophilized particles prior to extraction, suggesting that incorporated Q functions similarly to that originally present in the particles. 4. Succinate dehydrogenase activity, which is decreased % by extraction of Q. can be completely restored upon incorporation of only 1.5 nmol Q/mg protein. 5. Extraction of lyophilized particles with pentane + 10% acetone results in a more effective removal of Q, but also in a differential Q requirement for NADH oxidase and succinate oxidase. Thin-layer chromatography shows that extraction with pentane + acetone removes an additional, unidentified, nonpolar lipid. 6. The present data do not support the belief that succinate oxidase and NADH oxidase communicate with separate pools of Q. [ABSTRACT FROM AUTHOR]