Wong Edith D, Wagner Jennifer A, Scott Sidney V, Okreglak Voytek, Holewinske Timothy J, Cassidy-Stone Ann, Nunnari Jodi
Section of Molecular and Cellular Biology, University of California, Davis, Davis, California, 95616, USA.
J Cell Biol. 2003 Feb 3;160(3):303-11. doi: 10.1083/jcb.200209015.
A balance between fission and fusion events determines the morphology of mitochondria. In yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related GTPase, Dnm1p. Mitochondrial fusion requires two integral outer membrane components, Fzo1p and Ugo1p. Interestingly, mutations in a second mitochondrial-associated dynamin-related GTPase, Mgm1p, produce similar phenotypes to fzo1 and ugo cells. Specifically, mutations in MGM1 cause mitochondrial fragmentation and a loss of mitochondrial DNA that are suppressed by abolishing DNM1-dependent fission. In contrast to fzo1ts mutants, blocking DNM1-dependent fission restores mitochondrial fusion in mgm1ts cells during mating. Here we show that blocking DNM1-dependent fission in Deltamgm1 cells fails to restore mitochondrial fusion during mating. To examine the role of Mgm1p in mitochondrial fusion, we looked for molecular interactions with known fusion components. Immunoprecipitation experiments revealed that Mgm1p is associated with both Ugo1p and Fzo1p in mitochondria, and that Ugo1p and Fzo1p also are associated with each other. In addition, genetic analysis of specific mgm1 alleles indicates that Mgm1p's GTPase and GTPase effector domains are required for its ability to promote mitochondrial fusion and that Mgm1p self-interacts, suggesting that it functions in fusion as a self-assembling GTPase. Mgm1p's localization within mitochondria has been controversial. Using protease protection and immuno-EM, we have shown previously that Mgm1p localizes to the intermembrane space, associated with the inner membrane. To further test our conclusions, we have used a novel method using the tobacco etch virus protease and confirm that Mgm1p is present in the intermembrane space compartment in vivo. Taken together, these data suggest a model where Mgm1p functions in fusion to remodel the inner membrane and to connect the inner membrane to the outer membrane via its interactions with Ugo1p and Fzo1p, thereby helping to coordinate the behavior of the four mitochondrial membranes during fusion.
裂变与融合事件之间的平衡决定了线粒体的形态。在酵母中，线粒体裂变受外膜相关的动力蛋白相关GTP酶Dnm1p调控。线粒体融合需要两个外膜整合成分Fzo1p和Ugo1p。有趣的是，第二个线粒体相关动力蛋白相关GTP酶Mgm1p的突变产生与fzo1和ugo细胞相似的表型。具体而言，MGM1中的突变导致线粒体碎片化和线粒体DNA丢失，通过消除依赖DNM1的裂变可抑制这些现象。与fzo1ts突变体不同，阻断依赖DNM1的裂变可在交配期间恢复mgm1ts细胞中的线粒体融合。在这里我们表明，在Deltamgm1细胞中阻断依赖DNM1的裂变不能在交配期间恢复线粒体融合。为了研究Mgm1p在线粒体融合中的作用我们寻找了与已知融合成分的分子相互作用。免疫沉淀实验表明，Mgm1p在线粒体中与Ugo1p和Fzo1p都有关联，并且Ugo1p和Fzo1p也相互关联。此外，对特定mgm1等位基因的遗传分析表明，Mgm1p的GTP酶和GTP酶效应结构域是其促进线粒体融合能力所必需的，并且Mgm1p自身相互作用，这表明它作为一种自组装GTP酶在融合中发挥作用。Mgm1p在线粒体内的定位一直存在争议。我们之前使用蛋白酶保护和免疫电镜表明，Mgm1p定位于内膜间隙，与内膜相关。为了进一步验证我们的结论，我们使用了一种使用烟草蚀纹病毒蛋白酶的新方法，并证实Mgm1p在体内存在于内膜间隙区室中。综上所述，这些数据提示了一个模型，其中Mgm1p在融合中发挥作用，通过与Ugo1p和Fzo1p的相互作用重塑内膜并将内膜与外膜连接起来，从而有助于在融合过程中协调四个线粒体膜的行为。
