MITOL Regulates Endoplasmic Reticulum-Mitochondria Contacts via Mitofusin2
MITOL Regulates Endoplasmic Reticulum-Mitochondria Contacts via Mitofusin2
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DOI：
10.1016/j.molcel.2013.04.023
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发表时间：
2013-07-11
期刊：
影响因子：
16
通讯作者：
Yanagi, Shigeru
中科院分区：
文献类型：
--
作者：
Sugiura, Ayumu;Nagashima, Shun;Yanagi, Shigeru
线粒体泛素连接酶MITOL调节线粒体！动力学我们在这里报告，MITOL调节通过nnitofusin 2（Mfn 2）与内质网（ER）膜（MAM）结构域的形成。MITOL与线粒体Mfn 2相互作用并使其泛素化，但不与ER相关的Mfn 2相互作用并使其泛素化。突变分析鉴定了MITOL C-末端结构域与Mfn 2 HR 1结构域之间的特异性相互作用。MITOL介导的赖氨酸-63连接的多聚泛素链添加到Mfn 2，但不是其蛋白酶体降解。MITOL敲低抑制Mfn 2复合物的形成并引起Mfn 2错误定位和MAM功能障碍。蔗糖密度梯度离心和蓝色非变性PAGE阻滞试验证明Mfn 2寡聚化需要MITOL。MITOL敲低减少Mfn 2 GTP结合，导致GTP水解减少。我们确定Mfn 2的GTdR结构域中的K192为MITOL的主要泛素化位点。即使在GTP存在下，K192 R突变也能阻断寡聚化。总之，这些结果表明，MITOL通过K192泛素化激活Mfn 2来调节ER与线粒体的连接。
The mitochondrial ubiquitin ligase MITOL regulates mitochondria! dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through nnitofusin2 (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.