The dynamin-related protein Mgm1p assembles into oligomers and hydrolyzes GTP to function in mitochondrial membrane fusion.
The dynamin-related protein Mgm1p assembles into oligomers and hydrolyzes GTP to function in mitochondrial membrane fusion.
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DOI：
10.1021/bi801723d
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发表时间：
2009-02
期刊：
影响因子：
2.9
通讯作者：
Gabriela Meglei;G. A. McQuibban
中科院分区：
文献类型：
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作者：
Gabriela Meglei;G. A. McQuibban
真核生物正常的细胞功能需要由竞争性膜融合和裂变反应引起的线粒体动力学。Mgm1p是一种动力蛋白相关蛋白，是酵母线粒体融合的关键成分，在进化上是保守的。先前的研究表明，Mgm1p介导线粒体内膜融合的方式与其他动力蛋白类似，这些动力蛋白利用GTP水解和寡聚诱导脂质双层的结构变化；但是，还没有提出这些活动的直接证明。在这里，我们发现纯化的Mgm1p形成依赖于蛋白质浓度的低阶低聚物，表明动态和可逆的相互作用。我们进一步证明Mgm1p具有GTPase活性和与机械酶一致的动力学性质，并在内膜线粒体融合中发挥作用。GTPase结构域保守基序的关键残基突变显示GTPase活性显著降低或减弱。GTPase效应域的突变，涉及组装和组装刺激的GTP水解，具有与野生型Mgm1p相似的基础GTPase活性，但形成低聚物的倾向较弱。最后，我们的数据表明，Mgm1p与线粒体膜中发现的带负电荷的磷脂特异性相互作用，而预测的脂质结合域的点突变取消了这些相互作用。这些发现表明存在一个假定的脂质结合结构域，为这种蛋白质如何介导内膜融合提供了深入的了解。综上所述，这些数据表明Mgm1p通过寡聚化、GTP水解和脂质结合介导融合，其方式与其他动力蛋白机械酶相似。
Mitochondrial dynamics resulting from competing membrane fusion and fission reactions are required for normal cellular function in eukaryotes. Mgm1p, a dynamin-related protein, is a key component in yeast mitochondrial fusion and is evolutionarily conserved. Previous studies suggest that Mgm1p mediates mitochondrial inner membrane fusion in a manner similar to that of other dynamin proteins that use GTP hydrolysis and oligomerization to induce structural changes in lipid bilayers; however, a direct demonstration of these activities has yet to be presented. Here we show that purified Mgm1p forms low-order oligomers that are dependent on protein concentration, suggesting a dynamic and reversible interaction. We further demonstrate that Mgm1p has GTPase activity and kinetic properties consistent with a mechanoenzyme and with a role in inner membrane mitochondrial fusion. Mutations of key residues in conserved motifs of the GTPase domain show markedly reduced or diminished GTPase activity. A mutation in the GTPase effector domain, involved in assembly and assembly-stimulated GTP hydrolysis, has basal GTPase activity similar to that of wild-type Mgm1p but has a weaker propensity to form oligomers. Finally, our data indicate that Mgm1p interacts specifically with negatively charged phospholipids found in mitochondrial membranes, and point mutations in the predicted lipid-binding domain abrogate these interactions. These findings suggest the presence of a putative lipid-binding domain, providing insight into how this protein mediates inner membrane fusion. Together, these data indicate that Mgm1p mediates fusion through oligomerization, GTP hydrolysis, and lipid binding in a manner similar to those of other dynamin mechanoenzymes.