Reactive oxygen species (ROS), continuously generated as byproducts of respiration, inflict more damage on the mitochondrial (mt) than on the nuclear genome because of the nonchromatinized nature and proximity to the ROS source of the mitochondrial genome. Such damage, particularly single-strand breaks (SSBs) with 5-blocking deoxyribose products generated directly or as repair intermediates for oxidized bases, is repaired via the base excision/SSB repair pathway in both nuclear and mt genomes. Here, we show that EXOG, a 5-exo/endonuclease and unique to the mitochondria unlike FEN1 or DNA2, which, like EXOG, has been implicated in the removal of the 5-blocking residue, is required for repairing endogenous SSBs in the mt genome. EXOG depletion induces persistent SSBs in the mtDNA, enhances ROS levels, and causes apoptosis in normal cells but not in mt genome-deficient rho0 cells. Thus, these data show for the first time that persistent SSBs in the mt genome alone could provide the initial trigger for apoptotic signaling in mammalian cells.A mammalian cell contains up to several thousands copies of duplex, circular 16.5-kb mt 2 genome within 80 -700 mitochondria depending on the cell type (1, 2). The mtDNA encodes essential subunits of the respiratory chain, tRNA and rRNAs, all of which are critical for maintaining oxidative phosphorylation (OXPHOS) (3). OXPHOS accounts for about 85% of oxygen consumed by the cell, and early reports estimated that under physiological conditions ϳ5% of consumed oxygen is partially reduced to ROS (4). Although recent reports indicate a much lower level of ROS production in normal cells, even a low and persistent ROS level has long term detrimental effects (5, 6). Thus, the mitochondria, the major cellular site for ROS generation, are under continuous oxidative stress that results in oxidative damage to DNA, as well as proteins and lipids (7).ROS-induced DNA damage includes multitude of mutagenic oxidized bases and single-strand breaks (SSBs) containing 3Ј-and 5Ј-blocking groups in DNA, which are generated both directly or as intermediates during BER (8, 9). Because of close proximity of the site of ROS generation and nonchromatinized state of the mt genome, the mutation rate in human mtDNA is 20 -100-fold higher relative to the nDNA (10). As summarized in recent reviews (11-13), repair of oxidized base lesions or abnormal bases is initiated with their excision by a DNA glycosylase. A monofunctional glycosylase, such as uracil-DNA glycosylase, excises U from the DNA to generate an abasic (AP) site, which is then cleaved by AP endonuclease (APE1) in mammalian cells to generate 3Ј-OH and nonligatable 5Ј-deoxyribose phosphate (dRP) residues. In the nucleus, the 5Ј-dRP could be removed by DNA polymerase ␤ (pol ␤) via its intrinsic dRP lyase activity. In the mitochondria, the DNA polymerase ␥ (pol ␥) with similar dRP lyase activity is also able to remove the dRP moiety (14). In the case of oxidized base repair by DNA glycosylases with intrinsic AP lyase activity, such as 8-oxoguanine-DNA g...

We report here that blocking the activity of the 26 S proteasome results in drastic changes in the morphology of the mitochondria and accumulation of intermembrane space (IMS) proteins. Using endonuclease G (endoG) as a model IMS protein, we found that accumulation of wild-type but to a greater extent mutant endoG leads to changes in the morphology of the mitochondria similar to those observed following proteasomal inhibition. Further, we show that wildtype but to a greater extent mutant endoG is a substrate for ubiquitination, suggesting the presence of a protein quality control. Conversely, we also report that wild-type but not mutant endoG is a substrate for the mitochondrial protease Omi but only upon inhibition of the proteasome. These findings suggest that although elimination of mutant IMS proteins is strictly dependent on ubiquitination, elimination of excess or spontaneously misfolded wild-type IMS proteins is monitored by ubiquitination and as a second checkpoint by Omi cleavage when the proteasome function is deficient. One implication of our finding is that in the context of attenuated proteasomal function, accumulation of IMS proteins would contribute to the collapse of the mitochondrial network such as that observed in neurodegenerative diseases. Another implication is that such collapse could be accelerated either by mutations in IMS proteins or by mutations in Omi itself.The elimination of misfolded proteins represents an important mechanism for the maintenance of cellular viability. Such protein quality controls (PQC) 4 involve the binding of a chaperone to the misfolded protein and its presentation to the ubiquitin-dependent proteasome degradation pathway (1, 2). Linkage of ubiquitin to a protein is a highly organized process involving the sequential action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligase (E3) (3). Most of the regulation of the ubiquitination pathway occurs at the level of the ubiquitin ligase. This enzymatic cascade results in the attachment of a polyubiquitin chain onto specific lysine residues of a substrate. Although several types of polyubiquitin chains have now been reported, chains that are built on lysine 48 of ubiquitin serve as a signal for degradation by the 26 S proteasome. The 20 S catalytic core of the proteasome consists of three distinct proteolytic activities within a cylindrical barrel that is flanked by two 19 S regulatory caps. The proteasomes are localized both in the nucleus and in the cytoplasm, where they exist free or in association with organelles including the endoplasmic reticulum (4, 5).PQC in the cytoplasm involves chaperones of the heat shock protein (hsp) family that act in concert with ubiquitin ligases (2). A similar PQC acting in the nucleus has recently been reported (6). Ubiquitin-dependent PQC was also found to monitor the folding of proteins present in the lumen of the endoplasmic reticulum, a result that was at first surprising since proteins that are translocated across the ER membrane ...

No abstract

P II proteins are widespread and highly conserved signal transduction proteins occurring in bacteria, Archaea, and plants and play pivotal roles in controlling nitrogen assimilatory metabolism. This study reports on biochemical properties of the P IIhomologue GlnK (originally termed NrgB) in Bacillus subtilis (BsGlnK). Like other P II proteins, the native BsGlnK protein has a trimeric structure and readily binds ATP in the absence of divalent cations, whereas 2-oxoglutarate is only weakly bound. In contrast to other P II -like proteins, Mg 2؉ severely affects its ATP-binding properties. BsGlnK forms a tight complex with the membrane-bound ammonium transporter AmtB (NrgA), from which it can be relieved by millimolar concentrations of ATP. Immunoprecipitation and co-localization experiments identified a novel interaction between the BsGlnK-AmtB complex and the major transcription factor of nitrogen metabolism, TnrA. In vitro in the absence of ATP, TnrA is completely tethered to membrane (AmtB)-bound GlnK, whereas in extracts from BsGlnK-or AmtB-deficient cells, TnrA is entirely soluble. The presence of 4 mM ATP leads to concomitant solubilization of BsGlnK and TnrA. This ATP-dependent membrane re-localization of TnrA by BsGlnK/AmtB may present a novel mechanism to control the global nitrogen-responsive transcription regulator TnrA in B. subtilis under certain physiological conditions.

Evolutionary conserved mitochondrial nucleases are involved in programmed cell death and normal cell proliferation in lower and higher eukaryotes. The endo/exonuclease Nuc1p, also termed ‘yeast Endonuclease G (EndoG)’, is a member of this class of enzymes that differs from mammalian homologs by the presence of a 5′–3′ exonuclease activity in addition to its broad spectrum endonuclease activity. However, this exonuclease activity is thought to be essential for a function of the yeast enzyme in DNA recombination and repair. Here we show that higher eukaryotes in addition to EndoG contain its paralog ‘EXOG’, a novel EndoG-like mitochondrial endo/exonuclease. We find that during metazoan evolution duplication of an ancestral nuclease gene obviously generated the paralogous EndoG- and EXOG-protein subfamilies in higher eukaryotes, thereby maintaining the full endo/exonuclease activity found in mitochondria of lower eukaryotes. We demonstrate that human EXOG is a dimeric mitochondrial enzyme that displays 5′–3′ exonuclease activity and further differs from EndoG in substrate specificity. We hypothesize that in higher eukaryotes the complementary enzymatic activities of EndoG and EXOG probably together account for both, the lethal and vital functions of conserved mitochondrial endo/exonucleases.