Regulation of membrane fusion by a novel Sec1/Munc18-associated protein

SNARE蛋白是真核细胞内携带膜和可溶性货物的囊泡在隔室之间传递的明确机制的核心组成部分。囊泡运输有助于神经的神经递质释放，酵母的细胞壁传递和出芽，对植物的细胞稳态和生长发育至关重要。同源SNARE蛋白定位于囊泡和靶膜，功能性SNARE复合物的组装足以驱动膜融合。SNAREs还结合其他蛋白质伙伴，专门在某些生理作用下对齐囊泡融合。在动物中，SNARE与离子通道的相互作用似乎促进了电信号和神经内分泌分泌，我们已经证明拟南芥质膜SNARE SYP121与离子通道亚基KC1之间的相互作用影响渗透活性K+离子的运输。一个明显的潜在推论是，SNAREs和离子通道之间的结合在调节囊泡融合中是重要的。尽管这一过程具有基本性质，但我们对植物或其他有壁真核细胞中细胞体积与渗透溶质运输耦合的分子机制知之甚少。事实上，壁细胞如何调节渗透活性溶质（尤其是K+）的运输与细胞体积平行-无论是在保护细胞中可逆的还是在不可逆的扩张生长过程中-仍然是一个相当有争议的问题。该建议建立在我们最近对SYP121-KC1蛋白复合物及其在K+通道调节中的作用的鉴定的重要发现之上：(1)我们已经确定了SNARE蛋白SYP121上与通道亚基KC1结合的位点，(2)我们已经证明KC1与Sec1/Munc18 （SM）蛋白KEULE竞争与SYP121结合。SM蛋白是所有真核生物膜囊泡融合的关键调节因子，但其在控制snare介导的膜融合中的确切作用尚不清楚，是一个深入研究的课题。我们的发现指出了SM蛋白功能和snare通道相互作用之间的联系。它们不仅提供了直接耦合渗透溶质运输和囊泡运输来控制细胞壁真核生物细胞扩张的机制的第一个证据，而且还支持了sm调节膜运输的新模型。我们的工作假设是，SYP121和KC1的SNARE-K+通道相互作用作为“分子调节剂”，类似于James Watt用于调节蒸汽机周转率的机械发明，以协调囊泡交通（和细胞扩张）与渗透活性K+离子的吸收。我们现在提议测试这一假设的各个方面。我们的目标是充分表征SYP121与KC1和KEULE的结合，以建立相互作用的分子图谱。我们还建议研究选择性破坏SYP121、KC1和KEULE之间的相互作用对SNARE复合物组装、膜运输和通道介导的K+运输的影响。我们的多学科方法不仅将进一步加深我们对渗透溶质运输和细胞体积控制之间联系的理解，而且还可能为将膜运输与其他生理过程联系起来提供一种新的范例。

SNARE proteins are central components of a well-defined mechanism for the delivery of vesicles carrying membrane and soluble cargo between compartments within eukaryotic cells. Vesicle traffic contributes to neurotransmitter release in nerves, to cell wall delivery and budding in yeast, and is essential for cellular homeostasis, growth and development in plants. Cognate SNARE proteins localise to vesicle and target membranes, and assembly of functional SNARE complexes is sufficient to drive membrane fusion. SNAREs also bind other protein partners, specialised to align vesicle fusion within certain physiological roles. SNARE interactions with ion channels in animals appear to facilitate electrical signalling and neuroendocrine secretion, and we have shown that interaction between the Arabidopsis plasma membrane SNARE SYP121 and the ion channel subunit KC1 affects transport of the osmotically-active K+ ion. An obvious potential corollary of this is that binding between SNAREs and ion channels is important in regulating vesicle fusion. Despite the fundamental nature of the process, we have little understanding of the molecular mechanisms that couple cellular volume with osmotic solute transport in plants or other walled eukaryotic cells. Indeed, how walled cells regulate transport of osmotically-active solutes (especially of K+) in parallel with cell volume - whether reversible as in guard cells or during irreversible expansive growth - remains a matter of considerable debate. This proposal builds on significant recent findings we have made following on our identification of the SYP121-KC1 protein complex and its role in K+ channel regulation: (1) we have identified the site on the SNARE protein SYP121 that binds the channel subunit KC1, and (2) we have demonstrated that KC1 competes with the Sec1/Munc18 (SM) protein KEULE for binding to SYP121. SM proteins are key regulators of membrane vesicle fusion in all eukaryotes, but their precise role in controlling SNARE-mediated membrane fusion remains unclear and a topic of intense research. Our findings point to a link between SM protein function and a SNARE-channel interaction. Not only do they offer the first evidence of a mechanism that directly couples osmotic solute transport with vesicle traffic to control cell expansion in walled eukaryotes, but they also support a new model for SM-regulated membrane traffic. Our working hypothesis is that the SNARE-K+ channel interaction of SYP121 and KC1 serves as a 'molecular governor', analogous to the mechanical invention James Watt employed in moderating the turnover rate of his steam engines, to coordinate vesicle traffic (and cell expansion) with uptake of the osmotically-active K+ ion. We now propose to test various aspects of this hypothesis. We aim to fully characterize the binding of SYP121 to KC1 and KEULE in order to build up a molecular map of the interactions. We also propose to examine the consequences of selectively disrupting the interactions between SYP121, KC1 and KEULE on SNARE complex assembly, membrane traffic and channel-mediated K+ transport. Our multidisciplinary approach will not only further our understanding of the link between osmotic solute transport and control of cell volume, but is also likely to provide a novel paradigm for linking membrane traffic with other physiological processes.