[Abstract unavailable

Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase ␥. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network.

Mitochondrial dysfunction is linked to apoptosis, aging, cancer, and a number of neurodegenerative and muscular disorders. The interplay between mitophagy and mitochondrial dynamics has been linked to the removal of dysfunctional mitochondria ensuring mitochondrial quality control. An open question is what role mitochondrial fission plays in the removal of mitochondria after mild and transient oxidative stress; conditions reported to result in moderately elevated reactive oxygen species (ROS) levels comparable to physical activity. Here we show that applying such conditions led to fragmentation of mitochondria and induction of mitophagy in mouse and human cells. These conditions increased ROS levels only slightly and neither triggered cell death nor led to a detectable induction of non-selective autophagy. Starvation led to hyperfusion of mitochondria, to high ROS levels, and to the induction of both non-selective autophagy and to a lesser extent to mitophagy. We conclude that moderate levels of ROS specifically trigger mitophagy but are insufficient to trigger non-selective autophagy. Expression of a dominant-negative variant of the fission factor DRP1 blocked mitophagy induction by mild oxidative stress as well as by starvation. Taken together, we demonstrate that in mammalian cells under mild oxidative stress a DRP1-dependent type of mitophagy is triggered while a concomitant induction of non-selective autophagy was not observed. We propose that these mild oxidative conditions resembling well physiological situations are thus very helpful for studying the molecular pathways governing the selective removal of dysfunctional mitochondria.

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Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death. Progressive degeneration and death of neurons are the major features of several acute and chronic neurodegenerative diseases such as ischemic stroke, Alzheimer's disease, or Parkinson's disease. 1 The main mechanisms of neuronal cell death are, for example, disturbed calcium homeostasis, oxidative stress, breakdown of the mitochondrial membrane potential, and release of mitochondrial factors that initiate downstream apoptotic cell death programs.2 In particular, mitochondrial membrane permeabilization is considered as a critical step for the release of pro-apoptotic proteins such as cytochrome c, Smac/Diablo (second mitochondria-derived activator of caspase/direct IAP binding protein with low pI), HtrA2/Omi, apoptosis-inducing factor (AIF), or endonuclease G, which trigger caspase-dependent or caspase-independent mechanisms of DNA degradation and cell death. 3,4 An increasing number of recent studies provide evidence that AIF is a major factor for an alternative post-mitochondrial cell death pathway, for example, following hypoxia, 5,6 ischemia, 7,8 or excitotoxic lesions. 9,10 AIF is a 63 kDa flavoprotein located at the inner mitochondrial membrane that is released early after oxygen-glucose deprivation in vitro or cerebral ischemia in vivo.7 Using harlequin (Hq) mutant mice expressing low AIF levels and small interfering RNA (siRNA) approaches, we recently demonstrated a causal role of AIF in neuronal cell death in models of i...

Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8 m . Due to the presence of ARG8 m , accumulation of ؊ / 0 petites issued from large deletions in mtDNA could be restricted to 20 -30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steadystate level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion.In the mitochondrial inner membrane, the F 1 F 0 -type ATP synthase produces ATP from ADP and inorganic phosphate by using the energy of the transmembrane electrochemical proton gradient generated by the respiratory chain in the course of electron transfer to oxygen. The ATP synthase harbors two major structural domains, a transmembrane component (F 0 ) containing a proton-permeable pore and a peripheral, matrixlocalized, catalytic component (F 1 ) where the ATP is synthesized (1-4). In the F 0 , the core of the proton channel consists of a ring of c subunits (ten in yeast (4)) and one a subunit (Atp6p). Proton movement through this channel coincides with rotation of the subunit c ring (5-9), which results in conformational changes favoring ATP synthesis in the F 1 (1).Due to its good fermenting capacity the yeast Saccharomyces cerevisiae has been extensively used as a genetic system for the study of the mitochondrial ATP synthase (for reviews see Refs. 10 and 11). As in most eukaryotes, the yeast ATP synthase has a dual genetic origin, nuclear and mitochondrial. The yeast mitochondrial ATP synthase genes (ATP6, ATP9, and ATP8) encode the proton channel subunits a and c (usually referred to in yeast as Atp6p and Atp9p), respectively, and a third F 0 subunit (Atp8p) of unknown function. Dozens of mutations in the nuclear ATP synthase genes have provided much information on their protein products (10, 11). In contrast, only a very few mutants of the mitochondrial ATP synthase genes have been reported. Random generation of respiratory growth-deficient yeast strains issued fro...

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria. INTRODUCTIONMitochondria form large networks of interconnected tubules that are maintained by balanced fission and fusion events (Nunnari et al., 1997;Okamoto and Shaw, 2005). The morphology and ultrastructure of mitochondria depend on the tissue, on the physiological condition of the cell, and, in particular, on the functional status of mitochondria. Dynamic processes associated with mitochondria are apparently crucial for the cell, e.g., in apoptosis (Frank et al., 2001;Karbowski et al., 2002;Lee et al., 2004;Jagasia et al., 2005). Likewise, formation of dendritic spines and synapses (Li et al., 2004;Verstreken et al., 2005) and functional complementation of mitochondrial DNA (mtDNA) mutations by content mixing Ono et al., 2001) depend on dynamics of mitochondria. In contrast, vast morphological alterations of mitochondria have been reported to occur in human disorders. Impairment of mitochondrial fusion is causative of neurodegenerative diseases such as CharcotMarie-Tooth disease type 2A and 4A, and optic atrophy type 1 (Alexander et al., 2000;Delettre et al., 2000;Zuchner et al., 2004;Niemann et al., 2005).A key player in regulating mitochondrial fusion is the dynamin-like GTPase OPA1 (Olichon et al., 2003;Cipolat et al., 2004). Mutations in the OPA1 gene cause autosomal dominant optic atrophy type I, a prevalent hereditary neuropathy of the optic nerve (Alexander et al., 2000;Delettre et al., 2000). Down-regulation of OPA1 leads to fragmentation of mitochondria, mitochondrial dysfunction, altered maintenance of mtDNA, altered mitochondrial inner membrane morphology, and increased propensity for apoptosis (Olichon et al., 2003;Griparic ...

Mitochondrial genomes generally encode a minimal set of tRNAs necessary for protein synthesis. However, a number of eukaryotes import tRNAs from the cytoplasm into their mitochondria. For instance, Saccharomyces cerevisiae imports cytoplasmic tRNA Gln into the mitochondrion without any added protein factors. Here, we examine the existence of a similar active tRNA import system in mammalian mitochondria. We have used subcellular RNA fractions from rat liver and human cells to perform RT-PCR with oligonucleotide primers specific for nucleus-encoded tRNA CUG Gln and tRNA UUG Gln species, and we show that these tRNAs are present in rat and human mitochondria in vivo. Import of in vitro transcribed tRNAs, but not of heterologous RNAs, into isolated mitochondria also demonstrates that this process is tRNA-specific and does not require the addition of cytosolic factors. Although this in vitro system requires ATP, it is resistant to inhibitors of the mitochondrial electrochemical gradient, a key component of protein import. tRNA Gln import into mammalian mitochondria proceeds by a mechanism distinct from protein import. We also show that both tRNA Gln species and a bacterial pre-tRNA Asp can be imported in vitro into mitochondria isolated from myoclonic epilepsy with ragged-red fiber cells if provided with sufficient ATP (2 mM). This work suggests that tRNA import is more widespread than previously thought and may be a universal trait of mitochondria. Mutations in mitochondrial tRNA genes have been associated with human disease; the tRNA import system described here could possibly be exploited for the manipulation of defective mitochondria.human ͉ MERFF D ifferent protein-synthesizing systems exist in the cytoplasm and organelles (mitochondria and chloroplasts) of eukaryotic cells. Reflecting the evolutionary origin of the organelle, the mitochondrial system is bacteria-like (1, 2). For instance, the tRNAs and aminoacyl-tRNA synthetases present in the mitochondria are closely related to their bacterial counterparts. The mitochondrial genome of higher eukaryotes encodes all of the tRNA species necessary for protein synthesis (3), whereas the aminoacyl-tRNA synthetases are nucleus-encoded and imported into the organelle. The mitochondrial protein-synthesizing system is important for the synthesis of a number of proteins for multienzyme complexes involved in oxidative phosphorylation (1). Since the original suggestion 40 years ago (4), tRNA import into mitochondria has been demonstrated in Tetrahymena, trypanosomatids, yeast, plants, and marsupials (reviewed in refs. 5 and 6). The detailed nature of the different tRNA import mechanisms is not yet known; however, ATP hydrolysis is always required. In the yeast system, the importance of some mitochondrial membrane proteins as well as of specific cytosolic proteins (enolase and lysyl-tRNA synthetase) has been established (reviewed in refs. 5 and 6). After the early observation of the presence of a cytoplasmic tRNA CUU Lys in Saccharomyces cerevisiae mitochondria (7), import o...