Dynamin-catalyzed membrane fission requires coordinated GTP hydrolysis.
Dynamin-catalyzed membrane fission requires coordinated GTP hydrolysis.
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DOI：
10.1371/journal.pone.0055691
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Schmid SL
中科院分区：
文献类型：
--
作者：
Liu YW;Mattila JP;Schmid SL
发动蛋白是研究最多的膜裂变机制，并已作为其他裂变GTP酶的研究范式;然而，关于其功能的几个关键问题仍未解决。特别是，因为大多数动力蛋白GTdR结构域突变体研究到目前为止同样损害基础和组装刺激的GTdR活性，它一直难以区分各自的作用，网格蛋白介导的内吞作用（CME）或动力蛋白催化的膜分裂。在这里，我们比较了一个新的发动蛋白突变体，Q40 E，这是选择性受损的装配刺激GT3活性与S45 N，一个GTP结合突变体同样缺陷的基础和装配刺激GT3活性。这两种突变体都能有效地抑制CME，并有效地招募其他内吞辅助蛋白到停滞的被膜纹孔中。然而，Q40 E突变体在比S45 N更晚的步骤阻断，提供了额外的证据，证明在整个网格蛋白包被的纹孔成熟过程中需要发动蛋白的GTP结合和/或基础GTP酶活性。重要的是，使用体外测定组装刺激的GTdR活性和膜分裂，我们发现，后者是更有力地抑制两个显性负突变体比前者。这些研究确定，从具有过量膜储库（SUPER）模板的支撑双层的有效裂变需要跨越组装的发动蛋白领的两个梯级的协调GTP水解。
Dynamin is the most-studied membrane fission machinery and has served as a paradigm for studies of other fission GTPases; however, several critical questions regarding its function remain unresolved. In particular, because most dynamin GTPase domain mutants studied to date equally impair both basal and assembly-stimulated GTPase activities, it has been difficult to distinguish their respective roles in clathrin-mediated endocytosis (CME) or in dynamin catalyzed membrane fission. Here we compared a new dynamin mutant, Q40E, which is selectively impaired in assembly-stimulated GTPase activity with S45N, a GTP-binding mutant equally defective in both basal and assembly-stimulated GTPase activities. Both mutants potently inhibit CME and effectively recruit other endocytic accessory proteins to stalled coated pits. However, the Q40E mutant blocks at a later step than S45N, providing additional evidence that GTP binding and/or basal GTPase activities of dynamin are required throughout clathrin coated pit maturation. Importantly, using in vitro assays for assembly-stimulated GTPase activity and membrane fission, we find that the latter is much more potently inhibited by both dominant-negative mutants than the former. These studies establish that efficient fission from supported bilayers with excess membrane reservoir (SUPER) templates requires coordinated GTP hydrolysis across two rungs of an assembled dynamin collar.
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影响因子：
9.8
作者：
Loerke D;Mettlen M;Yarar D;Jaqaman K;Jaqaman H;Danuser G;Schmid SL
通讯作者：
Schmid SL
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作者：
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作者：
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通讯作者：
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