title: MITOL regulates endoplasmic reticulum-mitochondria contacts via Mitofusin2.
abstract: The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.
authors.affiliation: Laboratory of Molecular Biochemistry, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Ayumu
authors.initials: A
authors.lastname: Sugiura
authors.forename: Shun
authors.initials: S
authors.lastname: Nagashima
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tokuyama
authors.forename: Taku
authors.initials: T
authors.lastname: Amo
authors.forename: Yohei
authors.initials: Y
authors.lastname: Matsuki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ishido
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Kudo
authors.forename: Heidi M
authors.initials: HM
authors.lastname: McBride
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Fukuda
authors.forename: Nobuko
authors.initials: N
authors.lastname: Matsushita
authors.forename: Ryoko
authors.initials: R
authors.lastname: Inatome
authors.forename: Shigeru
authors.initials: S
authors.lastname: Yanagi
mesh_terms: D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000032:analysis / Q000378:metabolism* / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000032:analysis / Q000378:metabolism* / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism / Q000502:physiology*; D054875:Ubiquitination
chemical_list: D008565:Membrane Proteins; D024101:Mitochondrial Proteins; C515578:MARCHF5 protein, human; D044767:Ubiquitin-Protein Ligases; D020558:GTP Phosphohydrolases; C470018:MFN2 protein, human