The GTPase effector domain sequence of the Dnm1p GTPase regulates self-assembly and controls a rate-limiting step in mitochondrial fission
The GTPase effector domain sequence of the Dnm1p GTPase regulates self-assembly and controls a rate-limiting step in mitochondrial fission
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DOI：
10.1091/mbc.12.9.2756
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发表时间：
2001-09-01
影响因子：
3.3
通讯作者：
Shaw, JM
中科院分区：
文献类型：
--
作者：
Fukushima, NH;Brisch, E;Shaw, JM
Dnm1p属于动力蛋白相关的GTP酶家族，需要重塑不同的细胞膜。在萌芽酵母中，含有Drum1p的复合体聚集在线粒体膜外膜的细胞质表面，位于线粒体管分裂的位置。我们之前的遗传学研究表明，Drum1p的GTP酶活性是线粒体分裂所必需的，Dnm1p与自身相互作用。在本研究中，我们证明了细菌表达的Dnm1p在体外可以结合和降解GTP。免疫共沉淀研究和酵母双杂交分析表明，Dnm1p在体内可以寡聚。利用酵母双杂交系统，我们证明了这种Dnm1p寡聚部分是由与Dynamin中GTP酶效应域(GED)相关的C-末端序列介导的。这里描述的Dnm1p相互作用类似于报道的Dynamin和Dynamin相关蛋白在体内形成高阶结构的相互作用，表明Drum1p组装形成环绕线粒体的环或环。小管。根据以前的发现，Dnm1p GED中的K705A突变被预测为干扰GTP的水解，稳定活性的Dnm1p-GTP，并刺激分裂中的限速步骤。在这里，我们证明了DNM1 K705A蛋白在酵母中的表达增强了线粒体的分裂。我们的结果提供了证据，证明动力蛋白相关蛋白的GED区域调节膜分裂中的限速步骤。
Dnm1p belongs to a family of dynamin-related GTPases required to remodel different cellular membranes. In budding yeast, Drum1p-containing complexes assemble on the cytoplasmic surface of the outer mitochondrial membrane at sites where mitochondrial tubules divide. Our previous genetic studies suggested that Drum1p's GTPase activity was required for mitochondrial fission and that Dnm1p interacted with itself. In this study, we show that bacterially expressed Dnm1p can bind and hydrolyze GTP in vitro. Coimmunoprecipitation studies and yeast two-hybrid analysis suggest that Dnm1p oligomerizes in vivo. With the use of the yeast two-hybrid system, we show that this Dnm1p oligomerization is mediated, in part, by a C-terminal sequence related to the GTPase effector domain (GED) in dynamin. The Dnm1p interactions characterized here are similar to those reported for dynamin and dynamin-related proteins that form higher order structures in vivo, suggesting that Drum1p assembles to form rings or collars that surround mitochondrial. tubules. Based on previous findings, a K705A mutation in the Dnm1p GED is predicted to interfere with GTP hydrolysis, stabilize active Dnm1p-GTP, and stimulate a rate-limiting step in fission. Here we show that expression of the Dnm1 K705A protein in yeast enhances mitochondrial fission. Our results provide evidence that the GED region of a dynamin-related protein modulates a rate-limiting step in membrane fission.