The SNARE motif contributes to rbet1 intracellular targeting and dynamics independently of SNARE interactions
The SNARE motif contributes to rbet1 intracellular targeting and dynamics independently of SNARE interactions
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DOI：
10.1074/jbc.m300659200
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发表时间：
2003-04-18
影响因子：
4.8
通讯作者：
Hay, JC
中科院分区：
文献类型：
--
作者：
Joglekar, AP;Xu, DL;Hay, JC
内质网/高尔基体陷阱rbet 1在内质网和高尔基体之间循环，并且对于分泌途径中的货物运输是必需的。虽然已知含有rbet 1的四元SNARE复合物在膜融合中起作用，但rbet 1的结构作用尚不清楚。此外，还没有对rbet 1目标及其周期性路线的结构决定因素进行研究。我们利用蛋白质相互作用试验来证明rbet 1 SNARE基序在突触SNARE复合物中起着与SNAP-25的羧基末端螺旋相似的结构作用，并证明了rbet 1和sec 22 b之间的保守盐桥对SNARE复合物组装的重要性。我们还研究了rbet 1陷阱基序和陷阱的相互作用在rbet 1本地化和动力学的潜在作用。我们发现，与对syntaxin 5 '观察到的相反。rbet 1 SNARE基序对于正确的靶向是必需的。为了测试SNARE相互作用对于SNARE基序的靶向功能是否重要，我们在保守的盐桥位置使用电荷排斥突变，使得rbet 1对于二元、三元和四元SNARE相互作用有缺陷。我们发现，异源SNARE相互作用是不需要在rbet 1靶向或动力学的任何步骤。此外，SNARE基序的异聚体状态不影响其与COPI外壳的相互作用或有效募集到转运囊泡上。我们的结论是，蛋白质靶向是一个完全独立的功能的rbet 1陷阱基序，这是能够不同类别的蛋白质相互作用。
The endoplasmic reticulum/Golgi SNARE rbet1 cycles between the endoplasmic reticulum and Golgi and is essential for cargo transport in the secretory pathway. Although the quaternary SNARE complex containing rbet1 is known to function in membrane fusion, the structural role of rbet1 is unclear. Furthermore, the structural determinants for rbet1 targeting and its cyclical itinerary have not been investigated. We utilized protein interaction assays to demonstrate that the rbet1 SNARE motif plays a structural role similar to the carboxyl-terminal helix of SNAP-25 in the synaptic SNARE complex and demonstrated the importance to SNARE complex assembly of a conserved salt bridge between rbet1 and sec22b. We also examined the potential role of the rbet1 SNARE motif and SNARE interactions in rbet1 localization and dynamics. We found that, in contrast to what has been observed for syntaxin 5'. the rbet1 SNARE motif was essential for proper targeting. To test whether SNARE interaction's were important for the targeting function of the SNARE motif, we used charge repulsion mutations at the conserved salt bridge position that rendered rbet1 defective for binary, ternary, and quaternary SNARE interactions. We found that heteromeric SNARE interactions are not required at any step in rbet1 targeting-or dynamics. Furthermore, the heteromeric state of the SNARE motif does not influence its interaction with the COPI coat or efficient recruitment onto transport vesicles. We conclude that protein targeting is a completely independent function of the rbet1 SNARE motif, which is capable of distinct classes of protein interactions.