The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission

Mitochondrial fusion and structure depend on the dynamin-like GTPase OPA1, whose activity is regulated by proteolytic processing. Constitutive OPA1 cleavage by YME1L and OMA1 at two distinct sites leads to the accumulation of both long and short forms of OPA1 and maintains mitochondrial fusion. Stress-induced OPA1 processing by OMA1 converts OPA1 completely into short isoforms, inhibits fusion, and triggers mitochondrial fragmentation. Here, we have analyzed the function of different OPA1 forms in cells lacking YME1L, OMA1, or both. Unexpectedly, deletion of Oma1 restored mitochondrial tubulation, cristae morphogenesis, and apoptotic resistance in cells lacking YME1L. Long OPA1 forms were sufficient to mediate mitochondrial fusion in these cells. Expression of short OPA1 forms promoted mitochondrial fragmentation, which indicates that they are associated with fission. Consistently, GTPase-inactive, short OPA1 forms partially colocalize with ER–mitochondria contact sites and the mitochondrial fission machinery. Thus, OPA1 processing is dispensable for fusion but coordinates the dynamic behavior of mitochondria and is crucial for mitochondrial integrity and quality control.     

Loss of Drp1 function alters OPA1 processing and changes mitochondrial membrane organization

RNAi mediated loss of Drp1 function changes mitochondrial morphology in cultured HeLa and HUVEC cells by shifting the balance of mitochondrial fission and fusion towards unopposed fusion. Over time, inhibition of Drp1 expression results in the formation of a highly branched mitochondrial network along with “bulge”-like structures. These changes in mitochondrial morphology are accompanied by a reduction in levels of Mitofusin 1 (Mfn1) and 2 (Mfn2) and a modified proteolytic processing of OPA1 isoforms, resulting in the inhibition of cell proliferation. In addition, our data imply that bulge formation is driven by Mfn1 action along with particular proteolytic short-OPA1 (s-OPA1) variants: Loss of Mfn2 in the absence of Drp1 results in an increase of Mfn1 levels along with processed s-OPA1-isoforms, thereby enhancing continuous “fusion” and bulge formation. Moreover, bulge formation might reflect s-OPA1 mitochondrial membrane remodeling activity, resulting in the compartmentalization of cytochrome c deposits. The proteins Yme1L and PHB2 appeared not associated with the observed enhanced OPA1 proteolysis upon RNAi of Drp1, suggesting the existence of other OPA1 processing controlling proteins. Taken together, Drp1 appears to affect the activity of the mitochondrial fusion machinery by unbalancing the protein levels of mitofusins and OPA1.     

Regulation of mitochondrial morphology through proteolytic cleavage of OPA1

The dynamin-like GTPase OPA1, a causal gene product of human dominant optic atrophy, functions in mitochondrial fusion and inner membrane remodeling. It has several splice variants and even a single variant is found as several processed forms, although their functional significance is unknown. In yeast, mitochondrial rhomboid protease regulates mitochondrial function and morphology through proteolytic cleavage of Mgm1, the yeast homolog of OPA1. We demonstrate that OPA1 variants are synthesized with a bipartite-type mitochondrial targeting sequence. During import, the matrix-targeting signal is removed and processed forms (L-isoforms) are anchored to the inner membrane in type I topology. L-isoforms undergo further processing in the matrix to produce S-isoforms. Knockdown of OPA1 induced mitochondrial fragmentation, whose network morphology was recovered by expression of L-isoform but not S-isoform, indicating that only L-isoform is fusion-competent. Dissipation of membrane potential, expression of m-AAA protease paraplegin, or induction of apoptosis stimulated this processing along with the mitochondrial fragmentation. Thus, mammalian mitochondrial function and morphology is regulated through processing of OPA1 in a DeltaPsi-dependent manner.     

Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells

The mammalian mitochondrial inner membrane fusion protein OPA1 is controlled by complex patterns of alternative splicing and proteolysis. A subset of OPA1 isoforms is constitutively cleaved by YME1L. Other isoforms are not cleaved by YME1L, but they are cleaved when mitochondria lose membrane potential or adenosine triphosphate. In this study, we show that this inducible cleavage is mediated by a zinc metalloprotease called OMA1. We find that OMA1 small interfering RNA inhibits inducible cleavage, helps retain fusion competence, and slows the onset of apoptosis, showing that OMA1 controls OPA1 cleavage and function. We also find that OMA1 is normally cleaved from 60 to 40 kD by another as of yet unidentified protease. Loss of membrane potential causes 60-kD protein to accumulate, suggesting that OMA1 is attenuated by proteolytic degradation. We conclude that a proteolytic cascade controls OPA1. Inducible cleavage provides a mechanism for quality control because proteolytic inactivation of OPA1 promotes selective removal of defective mitochondrial fragments by preventing their fusion with the mitochondrial network.     

OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L

OPA1, a dynamin-related guanosine triphosphatase mutated in dominant optic atrophy, is required for the fusion of mitochondria. Proteolytic cleavage by the mitochondrial processing peptidase generates long isoforms from eight messenger RNA (mRNA) splice forms, whereas further cleavages at protease sites S1 and S2 generate short forms. Using OPA1-null cells, we developed a cellular system to study how individual OPA1 splice forms function in mitochondrial fusion. Only mRNA splice forms that generate a long isoform in addition to one or more short isoforms support substantial mitochondrial fusion activity. On their own, long and short OPA1 isoforms have little activity, but, when coexpressed, they functionally complement each other. Loss of mitochondrial membrane potential destabilizes the long isoforms and enhances the cleavage of OPA1 at S1 but not S2. Cleavage at S2 is regulated by the i-AAA protease Yme1L. Our results suggest that mammalian cells have multiple pathways to control mitochondrial fusion through regulation of the spectrum of OPA1 isoforms.     

Membrane depolarization activates the mitochondrial protease OMA1 by stimulating self‐cleavage

Mitochondrial inner membrane fusion depends on the dynamin‐related GTPase OPA1 and the function of OPA1 is regulated by proteolytic cleavage. The mitochondrial proteases Yme1L and OMA1 cleave OPA1 at S2 and S1 sites, respectively. Here, we show that OMA1 is cleaved to a short form (S‐OMA1) by itself upon mitochondrial membrane depolarization; S‐OMA1 is degraded quickly but could be stabilized by CCCP treatment or Prohibitin knockdown in cells. In addition, OMA1 processing is positively correlated with OPA1 cleavage at the S1 site and the regulation of mitochondrial morphology. Thus, our results reveal the molecular mechanism for OMA1 activation toward OPA1 processing.     

Opa1 Is Required for Proper Mitochondrial Metabolism in Early Development

Opa1 catalyzes fusion of inner mitochondrial membranes and formation of the cristae. OPA1 mutations in humans lead to autosomal dominant optic atrophy. OPA1 knockout mice lose viability around embryonic day 9 from unknown reasons, indicating that OPA1 is essential for embryonic development. Zebrafish are an attractive model for studying vertebrate development and have been used for many years to describe developmental events that are difficult or impractical to view in mammalian models. In this study, Opa1 was successfully depleted in zebrafish embryos using antisense morpholinos, which resulted in disrupted mitochondrial morphology. Phenotypically, these embryos exhibited abnormal blood circulation and heart defects, as well as small eyes and small pectoral fin buds. Additionally, startle response was reduced and locomotor activity was impaired. Furthermore, Opa1 depletion caused bioenergetic defects, without impairing mitochondrial efficiency. In response to mitochondrial dysfunction, a transient upregulation of the master regulator of mitochondrial biogenesis, pgc1a, was observed. These results not only reveal a new Opa1-associated phenotype in a vertebrate model system, but also further elucidates the absolute requirement of Opa1 for successful vertebrate development.     

Loss of OMA1 delays neurodegeneration by preventing stress-induced OPA1 processing in mitochondria

Proteolytic cleavage of the dynamin-like guanosine triphosphatase OPA1 in mitochondria is emerging as a central regulatory hub that determines mitochondrial morphology under stress and in disease. Stress-induced OPA1 processing by OMA1 triggersmitochondrial fragmentation, which is associated with mitophagy and apoptosis in vitro. Here, we identify OMA1 as a critical regulator of neuronal survival in vivo and demonstrate that stress-induced OPA1 processing by OMA1 promotes neuronal death and neuroinflammatory responses. Using mice lacking prohibitin membrane scaffolds as a model of neurodegeneration, we demonstrate that additional ablation of Oma1 delays neuronal loss and prolongs lifespan. This is accompanied by the accumulation of fusion-active, long OPA1 forms, which stabilize the mitochondrial genome but do not preserve mitochondrial cristae or respiratory chain supercomplex assembly in prohibitin-depleted neurons. Thus, long OPA1 forms can promote neuronal survival independently of cristae shape, whereas stress-induced OMA1 activation and OPA1 cleavage limit mitochondrial fusion and promote neuronal death.     

Regulation of the mitochondrial dynamin-like protein Opa1 by proteolytic cleavage

The dynamin-related protein Opa1 is localized to the mitochondrial intermembrane space, where it facilitates fusion between mitochondria. Apoptosis causes Opa1 release into the cytosol and causes mitochondria to fragment. Loss of mitochondrial membrane potential also causes mitochondrial fragmentation but not Opa1 release into the cytosol. Both conditions induce the proteolytic cleavage of Opa1, suggesting that mitochondrial fragmentation is triggered by Opa1 inactivation. The opposite effect was observed with knockdown of the mitochondrial intermembrane space protease Yme1. Knockdown of Yme1 prevents the constitutive cleavage of a subset of Opa1 splice variants but does not affect carbonyl cyanide m-chlorophenyl hydrazone or apoptosis-induced cleavage. Knockdown of Yme1 also increases mitochondrial connectivity, but this effect is independent of Opa1 because it also occurs in Opa1 knockdown cells. We conclude that Yme1 constitutively regulates a subset of Opa1 isoforms and an unknown mitochondrial morphology protein, whereas the loss of membrane potential induces the further proteolysis of Opa1.     

L‐OPA1 regulates mitoflash biogenesis independently from membrane fusion

Mitochondrial flashes mediated by optic atrophy 1 (OPA1) fusion protein are bioenergetic responses to stochastic drops in mitochondrial membrane potential (Δψ_m) whose origin is unclear. Using structurally distinct genetically encoded pH‐sensitive probes, we confirm that flashes are matrix alkalinization transients, thereby establishing the pH nature of these events, which we renamed “mitopHlashes”. Probes located in cristae or intermembrane space as verified by electron microscopy do not report pH changes during Δψ_m drops or respiratory chain inhibition. Opa1 ablation does not alter Δψ_m fluctuations but drastically decreases the efficiency of mitopHlash/Δψ_m coupling, which is restored by re‐expressing fusion‐deficient OPA1^K301A and preserved in cells lacking the outer‐membrane fusion proteins MFN1/2 or the OPA1 proteases OMA1 and YME1L, indicating that mitochondrial membrane fusion and OPA1 proteolytic processing are dispensable. pH/Δψ_m uncoupling occurs early during staurosporine‐induced apoptosis and is mitigated by OPA1 overexpression, suggesting that OPA1 maintains mitopHlash competence during stress conditions. We propose that OPA1 stabilizes respiratory chain supercomplexes in a conformation that enables respiring mitochondria to compensate a drop in Δψ_m by an explosive matrix pH flash.     

A proteomic screen with Drosophila Opa1-like identifies Hsc70-5/Mortalin as a regulator of mitochondrial morphology and cellular homeostasis

Mitochondrial morphology is regulated by conserved proteins involved in fusion and fission processes. The mammalian Optic atrophy 1 (OPA1) that functions in mitochondrial fusion is associated with Optic Atrophy and has been implicated in inner membrane cristae remodeling during cell death. Here, we show Drosophila Optic atrophy 1-like (Opa1-like) influences mitochondrial morphology through interaction with ‘mitochondria-shaping’ proteins like Mitochondrial assembly regulatory factor (Marf) and Drosophila Mitofilin (dMitofilin). To gain an insight into Opa1-like's network, we delineated bonafide interactors like dMitofilin, Marf, Serine protease High temperature requirement protein A2 (HTRA2), Rhomboid-7 (Rho-7) along with novel interactors such as Mortalin ortholog (Hsc70-5) from Drosophila mitochondrial extract. Interestingly, RNAi mediated down-regulation of hsc70-5 in Drosophila wing imaginal disc's peripodial cells resulted in fragmented mitochondria with reduced membrane potential leading to proteolysis of Opa1-like. Increased ecdysone activity induced dysfunctional fragmented mitochondria for clearance through lysosomes, an effect enhanced in hsc70-5 RNAi leading to increased cell death. Over-expression of Opa1-like rescues mitochondrial morphology and cell death in prepupal tissues expressing hsc70-5 RNAi. Taken together, we have identified a novel interaction between Hsc70-5/Mortalin and Opa1-like that influences cellular homeostasis through mitochondrial fusion.     

The PINK1-Parkin pathway is involved in the regulation of mitochondrial remodeling process

The two Parkinson’s disease (PD) genes, PTEN-induced kinase 1 (PINK1) and parkin, are linked in a common pathway which affects mitochondrial integrity and function. However, it is still not known what this pathway does in the mitochondria. Therefore, we investigated its physiological function in Drosophila. Because Drosophila PINK1 and parkin mutants show changes in mitochondrial morphology in both indirect flight muscles and dopaminergic neurons, we here investigated whether the PINK1-Parkin pathway genetically interacts with the regulators of mitochondrial fusion and fission such as Drp1, which promotes mitochondrial fission, and Opa1 or Marf, which induces mitochondrial fusion. Surprisingly, DrosophilaPINK1 and parkin mutant phenotypes were markedly suppressed by overexpression of Drp1 or downregulation of Opa1 or Marf, indicating that the PINK1-Parkin pathway regulates mitochondrial remodeling process in the direction of promoting mitochondrial fission. Therefore, we strongly suggest that mitochondrial fusion and fission process could be a prominent therapeutic target for the treatment of PD.     

A new vicious cycle involving glutamate excitotoxicity, oxidative stress and mitochondrial dynamics

Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1^enu/+ mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1^enu/+ mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.     

Mitochondrial retention of Opa1 is required for mouse embryogenesis

Mitochondria are dynamic cellular organelles that balance fission and fusion to regulate organelle morphology, distribution, and activity, and Opa1 is one of three GTPases known to regulate mitochondrial fusion. In humans, loss of a single Opa1 allele causes dominant optic atrophy, a degenerative condition that leads to loss of vision. Here we demonstrate that the lilR3 mutant mouse phenotype is due to a point mutation in the Opa1 gene resulting in mislocalized Opa1 protein from the mitochondria to the cytosol. Importantly, the mutation is in the middle domain of the Opa1 protein, for which no function had been described. Lack of mitochondrial retention of Opa1 is sufficient to cause the cellular Opa1 loss-of-function phenotype as the mitochondria are fragmented, indicating an inability to fuse. Despite the normally ubiquitous expression of Opa1 and the essential nature of mitochondria, embryos with aberrant Opa1 survived through midgestation and died at E11.5. These mutants displayed growth retardation, exencephaly, and abnormal patterning along the anterior-posterior axis, although the A–P axis itself was intact. The complex relationship between mitochondrial dynamics and cell death is emphasized by apoptosis in specific cell populations of lilR3 embryos. Our results define, for the first time, a function of the middle domain of the Opa1 protein and demonstrate that mitochondrial retention of Opa1 protein is essential for normal embryogenesis.     

Mitochondrial fusion regulates proliferation and differentiation in the type II neuroblast lineage in Drosophila

Optimal mitochondrial function determined by mitochondrial dynamics, morphology and activity is coupled to stem cell differentiation and organism development. However, the mechanisms of interaction of signaling pathways with mitochondrial morphology and activity are not completely understood. We assessed the role of mitochondrial fusion and fission in the differentiation of neural stem cells called neuroblasts (NB) in the Drosophila brain. Depleting mitochondrial inner membrane fusion protein Opa1 and mitochondrial outer membrane fusion protein Marf in the Drosophila type II NB lineage led to mitochondrial fragmentation and loss of activity. Opa1 and Marf depletion did not affect the numbers of type II NBs but led to a decrease in differentiated progeny. Opa1 depletion decreased the mature intermediate precursor cells (INPs), ganglion mother cells (GMCs) and neurons by the decreased proliferation of the type II NBs and mature INPs. Marf depletion led to a decrease in neurons by a depletion of proliferation of GMCs. On the contrary, loss of mitochondrial fission protein Drp1 led to mitochondrial clustering but did not show defects in differentiation. Depletion of Drp1 along with Opa1 or Marf also led to mitochondrial clustering and suppressed the loss of mitochondrial activity and defects in proliferation and differentiation in the type II NB lineage. Opa1 depletion led to decreased Notch signaling in the type II NB lineage. Further, Notch signaling depletion via the canonical pathway showed mitochondrial fragmentation and loss of differentiation similar to Opa1 depletion. An increase in Notch signaling showed mitochondrial clustering similar to Drp1 mutants. Further, Drp1 mutant overexpression combined with Notch depletion showed mitochondrial fusion and drove differentiation in the lineage, suggesting that fused mitochondria can influence differentiation in the type II NB lineage. Our results implicate crosstalk between proliferation, Notch signaling, mitochondrial activity and fusion as an essential step in differentiation in the type II NB lineage.     

Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies caused by a dysfunction of mitochondria and raise the possibility that tau pathologies are associated with other neurodegenerative disorders caused by deficiencies in mitochondrial dynamics.     

Proteolytic processing of OPA1 links mitochondrial dysfunction to alterations in mitochondrial morphology

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 gamma. 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.     

Eight human OPA1 isoforms,long and short: What are they for?

OPA1 is a dynamin-related GTPase that controls mitochondrial dynamics, cristae integrity, energetics and mtDNA maintenance. The exceptional complexity of this protein is determined by the presence, in humans, of eight different isoforms that, in turn, are proteolytically cleaved into combinations of membrane-anchored long forms and soluble short forms. Recent advances highlight how each OPA1 isoform is able to fulfill “essential” mitochondrial functions, whereas only some variants carry out “specialized” features. Long forms determine fusion, long or short forms alone build cristae, whereas long and short forms together tune mitochondrial morphology. These findings offer novel challenging therapeutic potential to gene therapy.     

Metalloprotease-mediated OPA1 processing is modulated by the mitochondrial membrane potential

Background information. Human OPA1 (optic atrophy type 1) is a dynamin‐related protein of the mitochondrial IMS (intermembrane space) involved in membrane fusion and remodelling. Similarly to its yeast orthologue Mgm1p that exists in two isoforms generated by the serine protease Pcp1p/Rbd1p, OPA1 exists in various isoforms generated by alternative splicing and processing. In the present paper, we focus on protease processing of OPA1. Results. We find that various mammalian cell types display a similar pattern of OPA1 isoforms [two L‐OPA1 (long isoforms of OPA1) and three S‐OPA1 (short isoforms of OPA1)] and that loss of the inner membrane potential, but not inhibition of oxidative phosphorylation or glycolysis, induces rapid and complete processing of L‐OPA1 to S‐OPA1. In isolated mitochondria, OPA1 processing was inhibited by heavy‐metal chelators, pointing to processing by a mitochondrial metalloprotease. The pattern of OPA1 isoforms and its processing kinetics were normal in mitochondria devoid of the serine protease PARL (presenilins‐associated rhomboid‐like protein) – the human orthologue of Pcp1/Rbd1 – and in cells from patients carrying homozygous mutations in SPG7 (spastic paraplegia type 7), a gene encoding the matrix‐oriented metalloprotease paraplegin. In contrast, OPA1 processing kinetics were delayed upon knock‐down of YME1L (human yme1‐like protein), an IMS‐oriented metalloprotease. OPA1 processing was also stimulated during apoptosis, but inhibition of this processing did not affect apoptotic release of OPA1 and cytochrome c. Finally, we show that all OPA1 isoforms interact with Mfn1 (mitofusin 1) and Mfn2 and that these interactions are not affected by dissipation of ΔΨm (inner mitochondrial membrane potential) or OPA1 processing. Conclusions. Metalloprotease‐mediated processing of OPA1 is modulated by the inner membrane potential and is likely to be mediated by the YME1L protease.     

Loss of Yme1L perturbates mitochondrial dynamics

Yme1L is an AAA protease that is embedded in the mitochondrial inner membrane with its catalytic domain facing the mitochondrial inner-membrane space. However, how Yme1L regulates mammalian mitochondrial function is still obscure. We find that endogenous Yme1L locates at punctate structures of mitochondria, and that loss of Yme1L in mouse embryonic fibroblast (MEF) cells results in mitochondrial fragmentation and leads to significant increased ‘kiss-and-run'' type of mitochondrial fusion; however, Yme1L knockdown (shYme1L (short hairpin-mediated RNA interference of Yme1L)) cells still remain normal mitochondrial fusion although shYme1L mitochondria have a little bit less fusion and fission rates, and the shYme1L-induced fragmentation is due to a little bit more mitochondrial fission than fusion in cells. Furthermore, shYme1L-induced mitochondrial fragmentation is independent on optic atrophy 1 (OPA1) S1 or S2 processing, and shYme1L results in the stabilization of OPA1 long form (L-OPA1); in addition, the exogenous expression of OPA1 or L-OPA1 facilitates the shYme1L-induced mitochondrial fragmentation, thus this fragmentation induced by shYme1L appears to be associated with L-OPA1''s stability. ShYme1L also causes a slight increase of mitochondrial dynamics proteins of 49 kDa and mitochondrial fission factor (Mff), which recruit mitochondrial key fission factor dynamin-related protein 1 (Drp1) into mitochondria in MEF cells, and loss of Drp1 or Mff inhibits the shYme1L-induced mitochondrial fragmentation. In addition, there is interaction between SLP-2 with Yme1L and shYme1L cells retain stress-induced mitochondrial hyperfusion. Taken together, our results clarify how Yme1L regulates mitochondrial morphology.