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

剖析 SNARE 回收和植物生长中一个新的重要检查点

• 批准号: BB/N006909/1
• 负责人: Michael Blatt
• 金额: $ 62.63万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN006909%2F1
• 关键词: Dissecting; new; vital; checkpoint; SNARE

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.

在本提案中，我寻求解决融合后如何调节驱动分泌囊泡运输的蛋白质回收的基本问题。 SNARE 蛋白是一种明确机制的核心组成部分，用于在细胞内的区室之间传递携带膜和可溶性物质的囊泡，并有助于所有真核生物的体内平衡和信号传导。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 Sec1/Munc18 (SM) 蛋白可以调节这一过程。 SM 形成类似衣夹的结构，在囊泡融合过程中“夹紧”并稳定复合体中的 SNARE。 SNARE 复合物的融合后分解对于回收同源 SNARE 蛋白和维持囊泡运输至关重要。分解是通过 NSF ATP 酶实现的，该酶将 SNARE 复合物与接头蛋白 α-SNAP 结合。从逻辑上讲，SM 脱脂是 SNARE 复杂拆卸的先决条件，但完全不了解如何调节此过程。事实上，大多数真核生物只表达一种或两种 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协调，以确保 NSF 活动有效分配。该提案建立在我的实验室在将 SEC11 确定为 SYP121 的 SM 伙伴及其组装的 SNARE 复合体之后的重要最新发现的基础上。 SYP121 和 SYP122 是在植物模型拟南芥质膜上的囊泡融合中占主导地位的两个 Qa-SNARE。我们发现，通过第二位点操纵 SEC11 与 SYP121 的结合（以前被认为在融合之前束缚 SM），可以通过 SYP121 和 SYP122 介导的途径阻断囊泡运输，即使 SEC11 不与 SYP122 相互作用。两个 Qa-SNARE 共享其他同源（Qb-、Qc- 和 R-）SNARE，使我们观察到 SEC11 通过其第二位点与 SYP121 结合是融合后所必需的，以促进 SNARE 分解并回收这些结合伴侣。简而言之，我们发现了一个以前未被识别的检查点以及 SM 蛋白在融合后 SNARE 回收中的新作用。这些发现为探索 SM 蛋白的这种全新功能提供了第一个机会。它们不仅提供了 SM-SNARE 结合先前未被认识的作用的证据，而且还支持了 SM 膜流量调节的新模型。我的工作假设是 SEC11 脱结合是一个关键的检查点，并作为 SYP121 SNARE 复合物的分解及其与质膜上平行运输途径的协调的分子“离合器”。我现在建议检验这个假设的各个方面。我的目标是充分表征 SEC11 与 SYP121 的结合，以及它们在 SNARE 复合体拆卸过程中与 α-SNAP 和 NSF 融合后的关联。我还建议检查选择性操纵 SEC11-SYP121 相互作用对囊泡运输、细胞扩增和生长的影响。这里概述的多学科方法将进一步我们对 SM 功能、SNARE 回收的理解，并且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。

项目成果

New Faces behind the Scenes.

幕后新面孔。

• DOI: 10.1104/pp.18.00140
• 发表时间: 2018
• 期刊: Plant physiology
• 影响因子: 7.4
• 作者: Blatt MR

Light-Driven Chloride Transport Kinetics of Halorhodopsin.

• DOI: 10.1016/j.bpj.2018.06.009
• 发表时间: 2018-07
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Hasin Feroz;Bryan H Ferlez;Cécile Lefoulon;Tingwei Ren;Carol S. Baker;John P. Gajewski;D. J. Lugar;Sandeep Gaudana;P. Butler;Jonas Hühn;M. Lamping;W. Parak;J. Hibberd;C. Kerfeld;N. Smirnoff;M. Blatt;J. Golbeck;Manish Kumar

Plant Physiology Launches Associate Features Editors.

植物生理学推出副专题编辑。

• DOI: 10.1104/pp.18.00113
• 发表时间: 2018
• 期刊: Plant physiology
• 影响因子: 7.4
• 作者: Blatt MR

What can mechanistic models tell us about guard cells, photosynthesis, and water use efficiency?

• DOI: 10.1016/j.tplants.2021.08.010
• 发表时间: 2022-01-12
• 期刊: TRENDS IN PLANT SCIENCE
• 影响因子: 20.5
• 作者: Blatt, Michael R.;Jezek, Mareike;Hills, Adrian
• 通讯作者: Hills, Adrian

Evolutionary Conservation of ABA Signaling for Stomatal Closure

气孔关闭 ABA 信号的进化保守

• DOI: 10.1104/pp.16.01848
• 发表时间: 2017-06-01
• 期刊: PLANT PHYSIOLOGY
• 影响因子: 7.4
• 作者: Cai, Shengguan;Chen, Guang;Chen, Zhong-Hua
• 通讯作者: Chen, Zhong-Hua