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

在这项提案中，我试图解决一个根本问题，即在融合后，如何调控推动分泌囊泡运输的蛋白质的循环。SNARE蛋白是一个明确的机制的中心成分，该机制用于在细胞内的隔室之间运送携带膜和可溶性货物的囊泡，并有助于所有真核生物的动态平衡和信号转导。同源(Qa-、Qb-、Qc-和R-)SNARE蛋白定位于囊泡和靶膜，并以复合体的形式组装，驱动膜融合。已知所谓的Sec1/Munc18(SM)蛋白调节这一过程。在囊泡融合过程中，SMS形成类似衣夹的结构，‘夹住’并稳定复合体中的陷阱。融合后拆解SNARE复合体对于回收同源SNARE蛋白和维持囊泡运输是必不可少的。拆解是通过NSF ATPase实现的，它将SNARE复合体与适配蛋白α-SNAP结合。从逻辑上讲，SM脱脂是SNARE复杂拆卸的先决条件，但对这一过程可能如何调控的理解是完全缺乏的。事实上，大多数真核生物只表达一到两个α-SNAP和NSF蛋白，但通过大量不同的SNAR介导的运输途径维持囊泡运输。显然，必须在贩运途径之间进行实质性的协调，以确保NSF的活动得到有效分配。这项建议建立在我的实验室最近的重大发现基础上，我们确定SEC11是SYP121的SM合作伙伴及其组装的SNARE复合体。SYP121和SYP122是在植物模型拟南芥的质膜上控制囊泡融合的两个QA陷阱。我们发现，通过二级位点操纵SEC11与SYP121的结合，即使SEC11不与SYP122相互作用，也可以通过SYP121和SYP122介导的途径阻止囊泡流量。这两个QA-SNARE共享其他同源(QB-、QC-和R-)SNARs，这使得我们观察到SEC11通过其二级位点与SYP121结合是必要的，融合后，为了促进SNARE的分解和循环这些结合伙伴是必要的。简而言之，我们发现了一个以前未被识别的检查点，以及SM蛋白在融合后SNARE循环中的新角色。这一发现为探索SM蛋白的这一全新功能提供了第一次机会。它们不仅为SM-SNARE结合提供了以前未被认识到的作用的证据，而且也支持了SM调节膜交通的新模型。我的工作假设是，SEC11脱脂是一个关键的检查点，是SYP121 SNARE复合体的分解及其与质膜上平行运输途径的协调的分子‘离合器’。我现在提议测试这一假设的各个方面。我的目标是充分描述SEC11与SYP121的结合，以及它们在融合后与α-SNAP和NSF在拆解SNARE复合体过程中的结合。我还建议研究选择性地操纵SEC11-SYP121相互作用对囊泡运输、细胞扩张和生长的影响。本文概述的多学科方法将进一步加深我们对SM功能、SNARE循环的理解，并可能为理解真核生物内囊泡运输的协调提供一个新的范例。

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.