title: Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity.
abstract: 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 approximately 250 kDa and approximately 450 kDa complexes in a GTP-dependent manner. The approximately 450 kDa complex contained oligomerized Mfn1 from distinct apposing membranes (docking complex), whereas the approximately 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 approximately 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.
authors.affiliation: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Naotada
authors.initials: N
authors.lastname: Ishihara
authors.forename: Yuka
authors.initials: Y
authors.lastname: Eura
authors.forename: Katsuyoshi
authors.initials: K
authors.lastname: Mihara
mesh_terms: D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology*; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000502:physiology
chemical_list: D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases; C470018:MFN2 protein, human; C470017:Mfn1 protein, human