Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis
Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis
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DOI：
10.1038/nsmb893
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发表时间：
2005-02-01
影响因子：
16.8
通讯作者：
Nakano, A
中科院分区：
文献类型：
--
作者：
Sato, K;Nakano, A
COPII 外壳蛋白是将货物和 SNARE 蛋白从内质网 (ER) 直接捕获到运输囊泡中所必需的。货物和 SNARE 捕获发生在由货物、Sar1p-GTP 和 COPII 亚基 Sec23/24p 组成的“出芽前复合体”形成过程中。 ER 膜上出芽前复合物的组装和分解循环与 Sar1p GTPase 循环耦合。使用 FRET 监测单轮 Sec23/24p 与重构脂质体中 SNARE 的结合和解离，我们发现 Sec23/24p 从 v-SNARE 和复合 t-SNARE 解离，其动力学慢于 Sar1p-GTP 水解。一旦 Sec23/24p 与 v-SNARE 或复合 t-SNARE 结合，复合物在 GDP-GTP 交换因子 Sec12p 介导的多轮 Sar1p-GTP 水解过程中保持组装状态。这些数据提出了一个在 Sar1p GTPase 循环期间维持动力学稳定的出芽前复合物的模型，该循环调节货物分选到运输囊泡中。
COPII coat proteins are required for direct capture of cargo and SNARE proteins into transport vesicles from the endoplasmic reticulum (ER). Cargo and SNARE capture occurs during the formation of a `prebudding complex' comprising a cargo, Sar1p-GTP and the COPII subunits Sec23/24p. The assembly and disassembly cycle of the prebudding complex on ER membranes is coupled to the Sar1p GTPase cycle. Using FRET to monitor a single round of Sec23/24p binding and dissociation from SNAREs in reconstituted liposomes, we show that Sec23/24p dissociates from v-SNARE and complexed t-SNARE with kinetics slower than Sar1p-GTP hydrolysis. Once Sec23/24p becomes associated with v-SNARE or complexed t-SNARE, the complex remains assembled during multiple rounds of Sar1p-GTP hydrolysis mediated by the GDP-GTP exchange factor Sec12p. These data suggest a model for the maintenance of kinetically stable prebudding complexes during the Sar1p GTPase cycle that regulates cargo sorting into transport vesicles.