SNARE status regulates tether recruitment and function in homotypic COPII vesicle fusion
SNARE status regulates tether recruitment and function in homotypic COPII vesicle fusion
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DOI：
10.1074/jbc.m606044200
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发表时间：
2006-12-15
影响因子：
4.8
通讯作者：
Hay, Jesse C.
中科院分区：
文献类型：
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作者：
Bentley, Marvin;Liang, Yingjian;Hay, Jesse C.
在哺乳动物中，外壳复合物II（COPII）包被的运输囊泡将分泌性货物运送到囊泡管状簇（VTC），促进货物分选和运输到高尔基体。我们记录了内质网来源的COPII转运囊泡的体外拴系和SNARE依赖性同型融合，以形成VTC特征性的较大货物容器（Xu，D.，和Hay，J.C.等（2004）J. Cell Biol.167，997-1003）。因此，COPII囊泡似乎包含所有必需的组件同型拴系和融合，提供了一个途径从头VTC生物合成。在这里，我们证明了针对内质网/高尔基体SNARE突触融合蛋白5的抗体抑制COPII囊泡同型拴系以及融合，这意味着SNARE融合上游的一个意想不到的作用。抑制SNARE复合物的访问和/或拆卸与显性负α-可溶性NSF附着蛋白（SNAP）也抑制拴系，暗示SNARE状态作为COPII囊泡拴系的关键决定因素。在显性负性α-SNAP存在下产生的束缚缺陷囊泡特别缺乏Rab 1效应子p115和GM 130，但不缺乏其他外周膜蛋白。此外，Rab效应，包括p115，被证明是所需的同型COPII囊泡拴系。因此，我们的研究结果表明，在COPII囊泡融合中需要SNARE依赖的系链募集和功能。我们预计，通过上游SNARE信号募集系链分子可确保系链事件仅在含有适当平衡的融合机制的焦点位点启动。
In mammals, coat complex II (COPII)-coated transport vesicles deliver secretory cargo to vesicular tubular clusters (VTCs) that facilitate cargo sorting and transport to the Golgi. We documented in vitro tethering and SNARE-dependent homotypic fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers characteristic of VTCs (Xu, D., and Hay, J. C. (2004) J. Cell Biol. 167, 997-1003). COPII vesicles thus appear to contain all necessary components for homotypic tethering and fusion, providing a pathway for de novo VTC biogenesis. Here we demonstrate that antibodies against the endoplasmic reticulum/Golgi SNARE Syntaxin 5 inhibit COPII vesicle homotypic tethering as well as fusion, implying an unanticipated role for SNAREs upstream of fusion. Inhibition of SNARE complex access and/or disassembly with dominant-negative alpha-soluble NSF attachment protein (SNAP) also inhibited tethering, implicating SNARE status as a critical determinant in COPII vesicle tethering. The tethering-defective vesicles generated in the presence of dominant-negative alpha-SNAP specifically lacked the Rab1 effectors p115 and GM130 but not other peripheral membrane proteins. Furthermore, Rab effectors, including p115, were shown to be required for homotypic COPII vesicle tethering. Thus, our results demonstrate a requirement for SNARE-dependent tether recruitment and function in COPII vesicle fusion. We anticipate that recruitment of tether molecules by an upstream SNARE signal ensures that tethering events are initiated only at focal sites containing appropriately poised fusion machinery.