Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity
Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity
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DOI：
10.1242/jcs.01565
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发表时间：
2004-12-15
影响因子：
4
通讯作者：
Mihara, K
中科院分区：
文献类型：
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作者：
Ishihara, N;Eura, Y;Mihara, K
酵母和果蝇Fzo的哺乳动物同源物，线粒体融合蛋白（Mfn）1和2，都是线粒体融合和线粒体形态维持所必需的。虽然GTP酶结构域是Mfn蛋白功能所必需的，但GTP酶依赖性反应的分子机制以及两种Mfn蛋白的功能划分尚不清楚。为了检查Mfn蛋白的功能，通过荧光显微镜使用绿色荧光蛋白-或红色荧光蛋白-标记的和Mfn 1-表达的线粒体，或通过免疫沉淀使用携带HA-或FLAG-标记的Mfn蛋白的线粒体，在体外测量线粒体膜的束缚。这些实验表明，携带Mfn 1的线粒体以GTP依赖性方式有效地拴系，而携带Mfn 2的线粒体仅以低效率拴系。蔗糖密度梯度离心后免疫共沉淀显示Mfn 1以GTP依赖的方式产生类似于250 kDa和类似于450 kDa的寡聚化复合物。类似于450 kDa的复合物含有来自不同并置膜的寡聚化Mfn 1（对接复合物），而类似于250 kDa的复合物由存在于相同膜上或膜溶解状态的Mfn 1组成（顺式复合物）。这些结果也使用蓝色非变性PAGE证实。Mfn 1对该反应表现出比Mfn 2更高的活性。纯化的重组Mfn 1比Mfn 2表现出相似的高至8倍的GT酶活性。这些发现表明，这两个Mfn蛋白具有不同的活动，并建议Mfn 1是主要负责GTP依赖的膜拴系。
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 similar to250 kDa and similar to450 kDa complexes in a GTP-dependent manner. The similar to450 kDa complex contained oligomerized Mfn1 from distinct apposing membranes (docking complex), whereas the similar to250 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 similar toeightfold 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.