Localization of the dynamin-related GTPase Drp1 to mitochondria relies on the mitochondrial fission factor Mff.

Organelles are inherited to daughter cells beyond dynamic changes of the membrane structure during mitosis. Mitochondria are dynamic entities, frequently dividing and fusing with each other, during which dynamin-related GTPase Drp1 is required for the fission reaction. In this study, we analyzed mitochondrial dynamics in mitotic mammalian cells. Although mitochondria in interphase HeLa cells are long tubular network structures, they are fragmented in early mitotic phase, and the filamentous network structures are subsequently reformed in the daughter cells. In marked contrast, tubular mitochondrial structures are maintained during mitosis in Drp1 knockdown cells, indicating that the mitochondrial fragmentation in mitosis requires mitochondrial fission by Drp1. Drp1 was specifically phosphorylated in mitosis by Cdk1/cyclin B on Ser-585. Exogenous expression of unphosphorylated mutant Drp1 S585A led to reduced mitotic mitochondrial fragmentation. These results suggest that phosphorylation of Drp1 on Ser-585 promotes mitochondrial fission in mitotic cells.

Mitochondrial morphology is dynamically controlled by a balance between fusion and fission. The physiological importance of mitochondrial fission in vertebrates is less clearly defined than that of mitochondrial fusion. Here we show that mice lacking the mitochondrial fission GTPase Drp1 have developmental abnormalities, particularly in the forebrain, and die after embryonic day 12.5. Neural cell-specific (NS) Drp1(-/-) mice die shortly after birth as a result of brain hypoplasia with apoptosis. Primary culture of NS-Drp1(-/-) mouse forebrain showed a decreased number of neurites and defective synapse formation, thought to be due to aggregated mitochondria that failed to distribute properly within the cell processes. These defects were reflected by abnormal forebrain development and highlight the importance of Drp1-dependent mitochondrial fission within highly polarized cells such as neurons. Moreover, Drp1(-/-) murine embryonic fibroblasts and embryonic stem cells revealed that Drp1 is required for a normal rate of cytochrome c release and caspase activation during apoptosis, although mitochondrial outer membrane permeabilization, as examined by the release of Smac/Diablo and Tim8a, may occur independently of Drp1 activity.

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 DW-dependent manner.

The mammalian homologues of yeast and Drosophila Fzo, mitofusin (Mfn) 1 and 2, are both essential for mitochondrial fusion and maintenance of mitochondrial morphology. Though the GTPase domain is required for Mfn protein function, the molecular mechanisms of the GTPase-dependent reaction as well as the functional division of the two Mfn proteins are unknown. To examine the function of Mfn proteins, tethering of mitochondrial membranes was measured in vitro by fluorescence microscopy using green fluorescence protein- or red fluorescent protein-tagged and Mfn1-expressing mitochondria, or by immunoprecipitation using mitochondria harboring HA- or FLAG-tagged Mfn proteins. These experiments revealed that Mfn1-harboring mitochondria were efficiently tethered in a GTP-dependent manner, whereas Mfn2-harboring mitochondria were tethered with only low efficiency. Sucrose density gradient centrifugation followed by co-immunoprecipitation revealed that Mfn1 produced oligomerized ∼250 kDa and ∼450 kDa complexes in a GTP-dependent manner. The ∼450 kDa complex contained oligomerized Mfn1 from distinct apposing membranes (docking complex), whereas the ∼250 kDa complex was composed of Mfn1 present on the same membrane or in the membrane-solubilized state (cis complex). These results were also confirmed using blue-native PAGE. Mfn1 exhibited higher activity for this reaction than Mfn2. Purified recombinant Mfn1 exhibited ∼eightfold higher GTPase activity than Mfn2. These findings indicate that the two Mfn proteins have distinct activities, and suggest that Mfn1 is mainly responsible for GTP-dependent membrane tethering.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with a cleavable N-terminal presequence and are imported into mitochondria. We report here the NMR structure of a general import receptor, rat Tom20, in a complex with a presequence peptide derived from rat aldehyde dehydrogenase. The cytosolic domain of Tom20 forms an all alpha-helical structure with a groove to accommodate the presequence peptide. The bound presequence forms an amphiphilic helical structure with hydrophobic leucines aligned on one side to interact with a hydrophobic patch in the Tom20 groove. Although the positive charges of the presequence are essential for import ability, presequence binding to Tom20 is mediated mainly by hydrophobic rather than ionic interactions.

Editorial, see p 225Mitochondria are dynamic organelles that constantly undergo fusion and fission 5 to adapt to changes in the cellular environment. Although mitochondrial fusion allows mitochondria to maintain membrane potential by fusing depolarized mitochondria to intact ones, fission allows the segregation of unrecoverable mitochondria so that they can be eliminated by autophagy or mitophagy, a specialized form of autophagy.6 Mitochondrial fusion is critically

Summary How mitochondrial dynamism (fission and fusion) affects mitochondrial quality control is unclear. We uncovered distinct effects on mitophagy of inhibiting Drp1-mediated mitochondrial fission versus mitofusin-mediated mitochondrial fusion. Conditional cardiomyocyte-specific Drp1 ablation evoked mitochondrial enlargement, lethal dilated cardiomyopathy, and cardiomyocyte necrosis. Conditionally ablating cardiomyocyte mitofusins (Mfn) caused mitochondrial fragmentation with eccentric remodeling and no cardiomyocyte dropout. Parallel studies in cultured murine embryonic fibroblasts (MEFs) and in vivo mouse hearts revealed that Mfn1/Mfn2 deletion provoked accumulation of defective mitochondria exhibiting an unfolded protein response, without appropriately increasing mitophagy. Conversely, interrupting mitochondrial fission by Drp1 ablation increased mitophagy and caused a generalized loss of mitochondria. Mitochondrial permeability transition pore (MPTP) opening in Drp1 null mitochondria was associated with mitophagy in MEFs and contributed to cardiomyocyte necrosis and dilated cardiomyopathy in mice. Drp1, MPTP, and cardiomyocyte mitophagy are functionally integrated. Mitochondrial fission and fusion have opposing roles during in vivo cardiac mitochondrial quality control.