Dissecting a new and vital checkpoint in SNARE recycling and plant growth

我寻求，在这个建议，以解决如何回收的蛋白质，驱动分泌囊泡交通融合后调节的基本问题。SNARE蛋白是用于在细胞内的隔室之间递送携带膜和可溶性货物的囊泡的明确定义的机制的中心组分，并且有助于所有真核生物中的稳态和信号传导。同源（Qa-、Qb-、Qc-和R-）SNARE蛋白定位于囊泡和靶膜，并组装成复合物以驱动膜融合。已知所谓的Sec 1/Munc 18（SM）蛋白调节这一过程。SM形成衣夹样结构，其在囊泡融合期间“夹紧”并稳定复合物中的SNARE。融合后SNARE复合物的分解对于回收同源SNARE蛋白和维持囊泡运输是必不可少的。通过NSF ATP酶将SNARE复合物与接头蛋白α-SNAP结合实现SNARE。从逻辑上讲，SM脱粘是SNARE复合体解体的先决条件，但对这一过程如何调节的理解是完全缺乏的。事实上，大多数真核生物仅表达一种或两种α-SNAP和NSF蛋白，但通过大量不同的SNARE介导的运输途径维持囊泡运输。显然，贩运途径之间必须进行实质性的协调，以确保NSF活动有效地distributed.This建议建立在我的实验室最近的重大发现，我们确定SEC 11作为SYP 121的SM合作伙伴和它组装的SNARE复合物。SYP 121和SYP 122是在植物模式拟南芥的质膜上的囊泡融合中占主导地位的两个Qa-SNARE。我们发现，操纵SEC 11结合SYP 121通过二级网站，以前认为系链SM融合前，阻断囊泡交通通过SYP 121和SYP 122介导的途径，即使SEC 11不与SYP 122相互作用。这两个Qa-SNARE共享其他同源（Qb-，Qc-和R-）SNARE，导致我们观察到SEC 11通过其二级位点与SYP 121结合是必要的，融合后，以促进SNARE分解和回收这些结合伴侣。简而言之，我们发现了一个以前未被识别的检查点和SM蛋白在融合后SNARE再循环中的新作用。这些发现首次提供了探索SM蛋白这种全新功能的机会。它们不仅提供了以前未被认识到的SM-SNARE结合作用的证据，而且还支持SM调节膜交通的新模型。我的工作假设是，SEC 11脱结合是一个关键的检查点，并作为一个分子的“离合器”的SYP 121陷阱复合物的拆卸和协调与平行的运输途径在质膜。我现在打算从各个方面来检验这个假设。我的目标是充分验证SEC 11与SYP 121的结合，以及它们在融合后与α-SNAP和NSF在SNARE复合物分解中的结合。我还建议研究选择性操纵SEC 11-SYP 121相互作用对囊泡交通，细胞扩增和生长的后果。这里概述的多学科的方法将进一步我们的SM功能，陷阱回收的理解，它很可能提供一个新的范例，了解真核生物内的囊泡交通的协调。

I seek, in this proposal, to address the fundamental question of how recycling of the proteins that drive secretory vesicle traffic is regulated post-fusion. SNARE proteins are central components of a well-defined mechanism for the delivery of vesicles carrying membrane and soluble cargo between compartments within cells and contribute to homeostasis and signaling in all eukaryotes. Cognate (Qa-, Qb-, Qc- and R-)SNARE proteins localize to vesicle and target membranes, and assemble in complex to drive membrane fusion. So-called Sec1/Munc18 (SM) proteins are known to regulate this process. SMs form clothespeg-like structures that 'clamp' and stabilize the SNAREs in complex during vesicle fusion. Post-fusion disassembly of the SNARE complex is essential to recycle the cognate SNARE proteins and maintain vesicle traffic. Disassembly is achieved by the NSF ATPase which binds the SNARE complex with the adaptor protein alpha-SNAP. Logic dictates that SM debinding is prerequisite for SNARE complex disassembly, but an understanding of how this process might be regulated is wholly absent. Indeed, most eukaryotes express only one or two alpha-SNAP and NSF proteins, yet maintain vesicle traffic via a large number of different SNARE-mediated trafficking pathways. Clearly, substantial coordination between trafficking pathways must occur to ensure NSF activity is effectively distributed.This proposal builds on significant recent findings of my laboratory following our identification of SEC11 as the SM partner of SYP121 and the SNARE complexes it assembles. SYP121 and SYP122 are the two Qa-SNAREs that dominate in vesicle fusion at the plasma membrane of the plant model Arabidopsis. We found that manipulating SEC11 binding to SYP121 via a secondary site, previously thought to tether the SM prior to fusion, blocks vesicle traffic via both SYP121- and SYP122-mediated pathways, even though SEC11 does not interact with SYP122. The two Qa-SNAREs share other cognate (Qb-, Qc- and R-)SNAREs, leading us to observe that SEC11 binding to SYP121 via its secondary site is necessary, post-fusion, to promote SNARE disassembly and recycle these binding partners. In short, we have uncovered a previously unrecognized checkpoint and a new role for an SM protein in SNARE recycling post-fusion.The findings offer the first opportunity to explore this, entirely novel function of an SM protein. Not only do they provide evidence of a previously unrecognized role for SM-SNARE binding, but they also support a new model for SM regulation of membrane traffic. My working hypothesis is that SEC11 debinding is a key checkpoint and serves as a molecular 'clutch' for disassembly of the SYP121 SNARE complex and its coordination with parallel trafficking pathways at the plasma membrane. I propose now to test various aspects of this hypothesis. I aim to fully characterise the binding of SEC11 with SYP121 and their association, post-fusion, with alpha-SNAP and NSF in disassembly of the SNARE complex. I propose also to examine the consequences of selectively manipulating SEC11-SYP121 interactions on vesicle traffic, cell expansion and growth. The multidisciplinary approach outlined here will further our understanding of SM function, SNARE recycling, and it is likely to provide a novel paradigm for understanding the coordination of vesicle traffic within eukaryotes.