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First published online 30 November 2004
doi: 10.1242/jcs.01565

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

Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan

^* Author for correspondence (e-mail: mihara{at}cell.med.kyushu-u.ac.jp)

Accepted 24 September 2004

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.

Key words: Mitochondria, Membrane fusion, GTPase, Mitofusins, Fzo1

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