Distinct types of protease systems are involved in homeostasis regulation of mitochondrial morphology via balanced fusion and fission.

Mitochondrial morphology is dynamically regulated by fusion and fission. Several GTPase proteins control fusion and fission, and posttranslational modifications of these proteins are important for the regulation. However, it has not been clarified how the fusion and fission is balanced. Here, we report the molecular mechanism to regulate mitochondrial morphology in mammalian cells. Ablation of the mitochondrial fission, by repression of Drp1 or Mff, or by over-expression of MiD49 or MiD51, results in a reduction in the fusion GTPase mitofusins (Mfn1 and Mfn2) in outer membrane and long form of OPA1 (L-OPA1) in inner membrane. RNAi- or CRISPR-induced ablation of Drp1 in HeLa cells enhanced the degradation of Mfns via the ubiquitin-proteasome system (UPS). We further found that UPS-related protein BAT3/BAG6, here we identified as Mfn2-interacting protein, was implicated in the turnover of Mfns in the absence of mitochondrial fission. Ablation of the mitochondrial fission also enhanced the proteolytic cleavage of L-OPA1 to soluble S-OPA1, and the OPA1 processing was reversed by inhibition of the inner membrane protease OMA1 independent on the mitochondrial membrane potential. Our findings showed that the distinct degradation systems of the mitochondrial fusion proteins in different locations are enhanced in response to the mitochondrial morphology.

Endogenous Drp1 mediates mitochondrial autophagy and protects the heart against energy stress.

RATIONALE: Both fusion and fission contribute to mitochondrial quality control. How unopposed fusion affects survival of cardiomyocytes and left ventricular function in the heart is poorly understood. OBJECTIVE: We investigated the role of dynamin-related protein 1 (Drp1), a GTPase that mediates mitochondrial fission, in mediating mitochondrial autophagy, ventricular function, and stress resistance in the heart. METHODS AND RESULTS: Drp1 downregulation induced mitochondrial elongation, accumulation of damaged mitochondria, and increased apoptosis in cardiomyocytes at baseline. Drp1 downregulation also suppressed autophagosome formation and autophagic flux at baseline and in response to glucose deprivation in cardiomyocytes. The lack of lysosomal translocation of mitochondrially targeted Keima indicates that Drp1 downregulation suppressed mitochondrial autophagy. Mitochondrial elongation and accumulation of damaged mitochondria were also observed in tamoxifen-inducible cardiac-specific Drp1 knockout mice. After Drp1 downregulation, cardiac-specific Drp1 knockout mice developed left ventricular dysfunction, preceded by mitochondrial dysfunction, and died within 13 weeks. Autophagic flux is significantly suppressed in cardiac-specific Drp1 knockout mice. Although left ventricular function in cardiac-specific Drp1 heterozygous knockout mice was normal at 12 weeks of age, left ventricular function decreased more severely after 48 hours of fasting, and the infarct size/area at risk after ischemia/reperfusion was significantly greater in cardiac-specific Drp1 heterozygous knockout than in control mice. CONCLUSIONS: Disruption of Drp1 induces mitochondrial elongation, inhibits mitochondrial autophagy, and causes mitochondrial dysfunction, thereby promoting cardiac dysfunction and increased susceptibility to ischemia/reperfusion.

Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c.

Mammalian cells typically contain thousands of copies of mitochondrial DNA assembled into hundreds of nucleoids. Here we analyzed the dynamic features of nucleoids in terms of mitochondrial membrane dynamics involving balanced fusion and fission. In mitochondrial fission GTPase dynamin-related protein (Drp1)-deficient cells, nucleoids were enlarged by their clustering within hyperfused mitochondria. In normal cells, mitochondrial fission often occurred adjacent to nucleoids, since localization of Mff and Drp1 is dependent on the nucleoids. Thus, mitochondrial fission adjacent to nucleoids should prevent their clustering by maintaining small and fragmented nucleoids. The enhanced clustering of nucleoids resulted in the formation of highly stacked cristae structures in enlarged bulb-like mitochondria (mito-bulbs). Enclosure of proapoptotic factor cytochrome c, but not of Smac/DIABLO, into the highly stacked cristae suppressed its release from mitochondria under apoptotic stimuli. In the absence of nucleoids, Drp1 deficiency failed to form mito-bulbs and to protect against apoptosis. Thus, mitochondrial dynamics by fission and fusion play a critical role in controlling mitochondrial nucleoid structures, contributing to cristae reformation and the proapoptotic status of mitochondria.

Disruption of mitochondrial fission in the liver protects mice from diet-induced obesity and metabolic deterioration.

AIM/HYPOTHESIS: Mitochondria and the endoplasmic reticulum (ER) physically interact by close structural juxtaposition, via the mitochondria-associated ER membrane. Inter-organelle communication between the ER and mitochondria has been shown to regulate energy metabolism and to be central to the modulation of various key processes such as ER stress. We aimed to clarify the role of mitochondrial fission in this communication. METHODS: We generated mice lacking the mitochondrial fission protein dynamin-related protein 1 (DRP1) in the liver (Drp1LiKO mice). RESULTS: Drp1LiKO mice showed decreased fat mass and were protected from high-fat diet (HFD)-induced obesity. Analysis of liver gene expression profiles demonstrated marked elevation of ER stress markers. In addition, we observed increased expression of the fibroblast growth factor 21 (FGF21) gene through induction of activating transcription factor 4, master regulator of the integrated stress response. CONCLUSIONS/INTERPRETATION: Disruption of mitochondrial fission in the liver provoked ER stress, while inducing the expression of FGF21 to increase energy expenditure and protect against HFD-induced obesity.

Fis1 acts as a mitochondrial recruitment factor for TBC1D15 that is involved in regulation of mitochondrial morphology.

In yeast, C-tail-anchored mitochondrial outer membrane protein Fis1 recruits the mitochondrial-fission-regulating GTPase Dnm1 to mitochondrial fission sites. However, the function of its mammalian homologue remains enigmatic because it has been reported to be dispensable for the mitochondrial recruitment of Drp1, a mammalian homologue of Dnm1. We identified TBC1D15 as a Fis1-binding protein in HeLa cell extracts. Immunoprecipitation revealed that Fis1 efficiently interacts with TBC1D15 but not with Drp1. Bacterially expressed Fis1 and TBC1D15 formed a direct and stable complex. Exogenously expressed TBC1D15 localized mainly in cytoplasm in HeLa cells, but when coexpressed with Fis1 it localized to mitochondria. Knockdown of TBC1D15 induced highly developed mitochondrial network structures similar to the effect of Fis1 knockdown, suggesting that the TBC1D15 and Fis1 are associated with the regulation of mitochondrial morphology independently of Drp1. These data suggest that Fis1 acts as a mitochondrial receptor in the recruitment of mitochondrial morphology protein in mammalian cells.

New insights into the function and regulation of mitochondrial fission.

Mitochondrial morphology changes dynamically by coordinated fusion and fission and cytoskeleton-based transport. Cycles of outer and inner membrane fusion and fission are required for the exchange of damaged mitochondrial genome DNA, proteins, and lipids with those of healthy mitochondria to maintain robust mitochondrial structure and function. These dynamics are crucial for cellular life and death, because they are essential for cellular development and homeostasis, as well as apoptosis. Disruption of these functions leads to cellular dysfunction, resulting in neurologic disorders and metabolic diseases. The cytoplasmic dynamin-related GTPase Drp1 plays a key role in mitochondrial fission, while Mfn1, Mfn2 and Opa1 are involved in fusion reaction. Here, we review current knowledge regarding the regulation and physiologic relevance of Drp1-dependent mitochondrial fission: the initial recruitment and assembly of Drp1 on the mitochondrial fission foci, regulation of Drp1 activity by post-translational modifications, and the role of mitochondrial fission in cell pathophysiology.

KLP6: a newly identified kinesin that regulates the morphology and transport of mitochondria in neuronal cells.

Mitochondria utilize diverse cytoskeleton-based mechanisms to control their functions and morphology. Here, we report a role for kinesin-like protein KLP6, a newly identified member of the kinesin family, in mitochondrial morphology and dynamics. An RNA interference screen using Caenorhabditis elegans led us to identify a C. elegans KLP-6 involved in maintaining mitochondrial morphology. We cloned a cDNA coding for a rat homolog of C. elegans KLP-6, which is an uncharacterized kinesin in vertebrates. A rat KLP6 mutant protein lacking the motor domain induced changes in mitochondrial morphology and significantly decreased mitochondrial motility in HeLa cells, but did not affect the morphology of other organelles. In addition, the KLP6 mutant inhibited transport of mitochondria during anterograde movement in differentiated neuro 2a cells. To date, two kinesins, KIF1Bα and kinesin heavy chain (KHC; also known as KIF5) have been shown to be involved in the distribution of mitochondria in neurons. Expression of the kinesin heavy chain/KIF5 mutant prevented mitochondria from entering into neurites, whereas both the KLP6 and KIF1Bα mutants decreased mitochondrial transport in axonal neurites. Furthermore, both KLP6 and KIF1Bα bind to KBP, a KIF1-binding protein required for axonal outgrowth and mitochondrial distribution. Thus, KLP6 is a newly identified kinesin family member that regulates mitochondrial morphology and transport.

Mammalian Oxa1 protein is useful for assessment of submitochondrial protein localization and mitochondrial membrane integrity.

Taking advantage of the unique topology of oxidase assembly 1 (Oxa1) protein, a mitochondrial inner membrane protein with N (intermembrane space)-C (matrix) orientation, we explored the usefulness of the protein as a marker for submitochondrial protein localization. Mammalian Oxa1 protein exhibited different proteolytic patterns depending on mitochondrial membrane integrity, and in mitochondria with a disrupted outer membrane and outer and inner membranes, the proteolytic patterns of Oxa1 protein were consistent with those of mitochondrial intermembrane space and matrix marker proteins, respectively, suggesting that Oxa1 protein, a single molecule, can serve as a versatile submitochondrial localization marker that doubles as a membrane integrity marker.

MAVS is energized by Mff which senses mitochondrial metabolism via AMPK for acute antiviral immunity.

Mitochondria are multifunctional organelles that produce energy and are critical for various signaling pathways. Mitochondrial antiviral signaling (MAVS) is a mitochondrial outer membrane protein essential for the anti-RNA viral immune response, which is regulated by mitochondrial dynamics and energetics; however, the molecular link between mitochondrial metabolism and immunity is unclear. Here we show in cultured mammalian cells that MAVS is activated by mitochondrial fission factor (Mff), which senses mitochondrial energy status. Mff mediates the formation of active MAVS clusters on mitochondria, independent of mitochondrial fission and dynamin-related protein 1. Under mitochondrial dysfunction, Mff is phosphorylated by the cellular energy sensor AMP-activated protein kinase (AMPK), leading to the disorganization of MAVS clusters and repression of the acute antiviral response. Mff also contributes to immune tolerance during chronic infection by disrupting the mitochondrial MAVS clusters. Taken together, Mff has a critical function in MAVS-mediated innate immunity, by sensing mitochondrial energy metabolism via AMPK signaling.

Identification of Tom5 and Tom6 in the preprotein translocase complex of human mitochondrial outer membrane.

The fungal preprotein translocase of the mitochondrial outer membrane (TOM complex) comprises import receptors Tom70, Tom20, and Tom22, import channel Tom40, and small Tom proteins Tom5, Tom6, and Tom7, which regulate TOM complex assembly. These components are conserved in mammals; unlike the other components, however, Tom5 and Tom6 remain unidentified in mammals. We immuno-isolated the TOM complex from HeLa cells expressing hTom22-FLAG and identified the human counterparts of Tom5 and Tom6, together with the other components including Tom7. These small Tom proteins are associated with Tom40 in the TOM complex. Knockdown of Tom7, but not Tom5 and Tom6, strongly compromised stability of the TOM complex. Conversely, knockdown of hTom40 decreased the level of all small Tom proteins. Matrix import of preprotein was affected by double knockdown of any combination of small Tom proteins. These results indicate that human small Tom proteins maintain the structural integrity of the TOM complex.

An RNAi screen for mitochondrial proteins required to maintain the morphology of the organelle in Caenorhabditis elegans.

Mitochondria are dynamic organelles that frequently divide and fuse together, resulting in the formation of intracellular tubular networks. In yeast and mammals, several factors including Drp1/Dnm1 and Mfn/Fzo1 are known to regulate mitochondrial morphology by controlling membrane fission or fusion. Here, we report the systematic screening of Caenorhabditis elegans mitochondrial proteins required to maintain the morphology of the organelle using an RNA interference feeding library. In C. elegans body wall muscle cells, mitochondria usually formed tubular structures and were severely fragmented by the mutation in fzo-1 gene, indicating that the body wall muscle cells are suitable for monitoring changes in mitochondrial morphology due to gene silencing. Of 719 genes predicted to code for most of mitochondrial proteins, knockdown of >80% of them caused abnormal mitochondrial morphology, including fragmentation and elongation. These findings indicate that most fundamental mitochondrial functions, including metabolism and oxidative phosphorylation, are necessary for maintenance of the tubular networks as well as membrane fission and fusion. This is the first evidence that known mitochondrial activities are prerequisite for regulating the morphology of the organelle. Furthermore, 88 uncharacterized or poorly characterized genes were found in the screening to be implicated in mitochondrial morphology.

Switching the sorting mode of membrane proteins from cotranslational endoplasmic reticulum targeting to posttranslational mitochondrial import.

Hydrophobic membrane proteins are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, mediated by hydrophobic signal sequence. Mitochondrial membrane proteins escape this mechanism despite their hydrophobic character. We examined sorting of membrane proteins into the mitochondria, by using mitochondrial ATP-binding cassette (ABC) transporter isoform (ABC-me). In the absence of 135-residue N-terminal hydrophilic segment (N135), the membrane domain was integrated into the ER membrane in COS7 cells. Other sequences that were sufficient to import soluble protein into mitochondria could not import the membrane domain. N135 imports other membrane proteins into mitochondria. N135 prevents cotranslational targeting of the membrane domain to ER and in turn achieves posttranslational import into mitochondria. In a cell-free system, N135 suppresses targeting to the ER membranes, although it does not affect recognition of hydrophobic segments by signal recognition particle. We conclude that the N135 segment blocks the ER targeting of membrane proteins even in the absence of mitochondria and switches the sorting mode from cotranslational ER integration to posttranslational mitochondrial import.

Circadian Control of DRP1 Activity Regulates Mitochondrial Dynamics and Bioenergetics.

Mitochondrial fission-fusion dynamics and mitochondrial bioenergetics, including oxidative phosphorylation and generation of ATP, are strongly clock controlled. Here we show that these circadian oscillations depend on circadian modification of dynamin-related protein 1 (DRP1), a key mediator of mitochondrial fission. We used a combination of in vitro and in vivo models, including human skin fibroblasts and DRP1-deficient or clock-deficient mice, to show that these dynamics are clock controlled via circadian regulation of DRP1. Genetic or pharmacological abrogation of DRP1 activity abolished circadian network dynamics and mitochondrial respiratory activity and eliminated circadian ATP production. Pharmacological silencing of pathways regulating circadian metabolism and mitochondrial function (e.g., sirtuins, AMPK) also altered DRP1 phosphorylation, and abrogation of DRP1 activity impaired circadian function. Our findings provide new insight into the crosstalk between the mitochondrial network and circadian cycles.

Identification and characterization of a new tom40 isoform, a central component of mitochondrial outer membrane translocase.

Newly synthesized precursors are transported into mitochondria through an outer membrane translocase, TOM. Tom40, a central pore-forming component, interacts directly with precursors to help them translocate across the outer membrane. We identified a new isoform of rat Tom40, Tom40B, which is conserved among mammals and exhibits significant similarities to Tom40 in other eukaryotes. Tom40B is an integral protein localized on the mitochondrial outer membrane, and expressed widely in all tissues examined except testis. Deletion mutant analysis revealed that the 28 amino acid residues at the carboxyl terminus were crucial for the mitochondrial targeting of Tom40B. Tom40B co-precipitated with other Tom components and formed a large protein complex. Furthermore, Tom40B directly bound to precursors of the matrix-targeted proteins with high affinities, comparable to those of Tom40A, a previously identified isoform. These findings indicate that Tom40B is a functional component of mitochondrial outer membrane translocase.

Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane.

We analyzed the signal that directs the outer membrane protein with the C-terminal transmembrane segment (TMS) to mammalian mitochondria by using yeast Tom5 as a model and green fluorescent protein as a reporter. Deletions or mutations were systematically introduced into the TMS or the flanking regions and their intracellular localization in COS-7 cells was examined using confocal microscopy and cell fractionation. 1) Three basic amino acid residues within the C-terminal five-residue segment (C-segment) contained the information required for mitochondrial-targeting. Reduction of the net positive charge in this segment decreased mitochondrial specificity, and the mutants were distributed throughout the intracellular membranes. 2) Elongation of the TMS interfered with the function of the C-segment and the mutants were delivered to the intracellular membranes. 3) Separation of the TMS and C-segment by linker insertion severely impaired mitochondrial targeting function, leading to mislocalization to the cytoplasm. 4) Mutations or small deletions in the region of the TMS flanking the C-segment also impaired the mitochondrial targeting. Therefore, the moderate length of the TMS, the positive charges in the C-segment, and the distance between or context of the TMS and C-segment are critical for the targeting signal. The structural characteristics of the signal thus defined were also confirmed with mammalian C-tail-anchored protein OMP25.

PARL paves the way to apoptosis.

Although the mitochondrial inner membrane rhomboid peptidase PARL is known to participate in critical signalling cascades, its role in apoptosis has remained unclear. PARL is now shown to process the mitochondrial pro-apoptotic protein Smac (also known as DIABLO) for its subsequent release into the cytosol where it antagonizes XIAP-mediated caspase inhibition to promote apoptosis.

Cell-free mitochondrial fusion assay detected by specific protease reaction revealed Ca2+ as regulator of mitofusin-dependent mitochondrial fusion.

Mitochondrial dynamic by frequent fusion and fission have important roles in various cellular signalling processes and pathophysiology in vivo. However, the molecular mechanisms that regulate mitochondrial fusion, especially in mammalian cells, are not well understood. Accordingly, we developed a novel biochemical cell-free mitochondrial fusion assay system using isolated human mitochondria. We used a protease and its specific substrate that are essential for yeast autophagy; Atg4 protease is required for maturation and the de-conjugation of the ubiquitin-like modifier Atg8. Atg4-FLAG and Atg8-GFP were separately expressed in the mitochondrial matrix of HeLa cells. Isolated mitochondria were then mixed and packed in the presence of energy regeneration mix. Immunoblotting with an anti-GFP antibody revealed Atg8 processing, suggesting that the double membranes of isolated mitochondria were indeed fused. The mitochondrial fusion reaction required GTP hydrolysis, mitochondrial membrane potential and intact outer membrane proteins containing two mitofusin isoforms. Using this assay, we searched for stimulators of mitochondrial fusion and found that rabbit reticulocyte lysate and Ca2+ chelator EGTA stimulate mitochondrial fusion. This novel cell-free assay system using isolated human mitochondria is simple, sensitive and reproducible; thus, it is useful for screening proteins and molecules that modulate mitochondrial fusion.

Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin.

Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. Optic atrophy 1 (OPA1) is an essential GTPase protein for both mitochondrial inner membrane (IM) fusion and cristae morphology. Under mitochondria-stress conditions, membrane-anchored L-OPA1 is proteolytically cleaved to form peripheral S-OPA1, leading to the selection of damaged mitochondria for mitophagy. However, molecular details of the selective mitochondrial fusion are less well understood. Here, we showed that L-OPA1 and cardiolipin (CL) cooperate in heterotypic mitochondrial IM fusion. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 protein expressed in silkworm, and found that L-OPA1 on one side of the membrane and CL on the other side are sufficient for fusion. GTP-independent membrane tethering through L-OPA1 and CL primes the subsequent GTP-hydrolysis-dependent fusion, which can be modulated by the presence of S-OPA1. These results unveil the most minimal intracellular membrane fusion machinery. In contrast, independent of CL, a homotypic trans-OPA1 interaction mediates membrane tethering, thereby supporting the cristae structure. Thus, multiple OPA1 functions are modulated by local CL conditions for regulation of mitochondrial morphology and quality control.

Three-dimensional analysis of somatic mitochondrial dynamics in fission-deficient injured motor neurons using FIB/SEM.

Mitochondria undergo morphological changes through fusion and fission for their quality control, which are vital for neuronal function. In this study, we examined three-dimensional morphologies of mitochondria in motor neurons under normal, nerve injured, and nerve injured plus fission-impaired conditions using the focused ion beam/scanning electron microscopy (FIB/SEM), because the FIB/SEM technology is a powerful tool to demonstrate both 3D images of whole organelle and the intra-organellar structure simultaneously. Crossing of dynamin-related protein 1 (Drp1) gene-floxed mice with neuronal injury-specific Cre driver mice, Atf3:BAC Tg mice, allowed for Drp1 ablation specifically in injured neurons. FIB/SEM analysis demonstrated that somatic mitochondrial morphologies in motor neurons were not altered before or after nerve injury. However, the fission impairment resulted in prominent somatic mitochondrial enlargement, which initially induced complex morphologies with round regions and long tubular processes, subsequently causing a decrease in the number of processes and further enlargement of the round regions, which eventually resulted in big spheroidal mitochondria without processes. The abnormal mitochondria exhibited several degradative morphologies: local or total cristae collapse, vacuolization, and mitophagy. These suggest that mitochondrial fission is crucial for maintaining mitochondrial integrity in injured motor neurons, and multiple forms of mitochondria degradation may accelerate neuronal degradation.

Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling.

Mitochondrial fission facilitates cytochrome c release from the intracristae space into the cytoplasm during intrinsic apoptosis, although how the mitochondrial fission factor Drp1 and its mitochondrial receptors Mff, MiD49, and MiD51 are involved in this reaction remains elusive. Here, we analyzed the functional division of these receptors with their knockout (KO) cell lines. In marked contrast to Mff-KO cells, MiD49/MiD51-KO and Drp1-KO cells completely resisted cristae remodeling and cytochrome c release during apoptosis. This phenotype in MiD49/51-KO cells, but not Drp1-KO cells, was completely abolished by treatments disrupting cristae structure such as OPA1 depletion. Unexpectedly, OPA1 oligomers generally thought to resist cytochrome c release by stabilizing the cristae structure were similarly disassembled in Drp1-KO and MiD49/51-KO cells, indicating that disassembly of OPA1 oligomers is not directly linked to cristae remodeling for cytochrome c release. Together, these results indicate that Drp1-dependent mitochondrial fission through MiD49/MiD51 regulates cristae remodeling during intrinsic apoptosis.