Naotada Ishihara

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 ΔΨ‐dependent manner.

Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity

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.

Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space

Apoptosis‐inducing factor (AIF) is a mitochondrial intermembrane flavoprotein that is translocated to the nucleus in response to proapoptotic stimuli, where it induces nuclear apoptosis. Here we show that AIF is synthesized as an ∼67‐kDa preprotein with an N‐terminal extension and imported into mitochondria, where it is processed to the ∼62‐kDa mature form. Topology analysis revealed that mature AIF is a type‐I inner membrane protein with the N‐terminus exposed to the matrix and the C‐terminal portion to the intermembrane space. Upon induction of apoptosis, processing of mature AIF to an ∼57‐kDa form occurred caspase‐independently in the intermembrane space, releasing the processed form into the cytoplasm. Bcl‐2 or Bcl‐XL inhibited both these events. These findings indicate that AIF release from mitochondria occurs by a two‐step process: detachment from the inner membrane by apoptosis‐induced processing in the intermembrane space and translocation into the cytoplasm. The results also suggest the presence of a unique protease that is regulated by proapoptotic stimuli in caspase‐independent cell death.

Regulation of mitochondrial morphology by membrane potential, and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells

Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. To analyze the mitochondrial fusion reaction, mitochondria were separately labeled with green or red fluorescent protein (GFP and RFP, respectively) in HeLa cells, and the cells were fused using hemagglutinating virus of Japan (HVJ). The resulting mixing of the fluorescent reporters was then followed using fluorescence microscopy. This system revealed that mitochondria fuse frequently in mammalian cells, and the fusion depends on the membrane potential across the inner membrane. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to fragmentation of the mitochondria and inhibited the fusion reaction. Removal of CCCP recovered the fusion activity to reform filamentous mitochondrial networks. Analysis of the effects of GTP-binding proteins, DRP1 and two FZO1 isoforms, and the GTPase-domain mutants on the CCCP-induced mitochondrial morphologic changes revealed that DRP1 and FZO1 are involved in membrane budding and fusion, respectively. Furthermore, a HVJ-dependent cell fusion assay combined with RNA interference (RNAi) demonstrated that both FZO1 isoforms are essential and must be acting in cis for the mitochondrial fusion reaction to occur.