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

SNARE蛋白是一个明确的机制的中心成分，该机制用于在真核细胞内的隔室之间运送携带膜和可溶性货物的囊泡。囊泡运输有助于神经递质的释放，促进酵母细胞壁的运输和发芽，对植物细胞的动态平衡、生长和发育至关重要。同源的SNARE蛋白定位于囊泡和靶膜上，而功能SNARE复合体的组装足以驱动膜融合。SNARES还结合其他蛋白质伙伴，专门在特定的生理角色内对齐小泡融合。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的分泌，我们已经证明，拟南芥质膜SNARE SYP121和离子通道亚单位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，SNARs和离子通道之间的结合在调节囊泡融合方面是重要的。尽管这一过程具有基本的性质，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的分子机制知之甚少。事实上，有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--无论是可逆的，如保卫细胞中的，还是在不可逆的扩张生长期间--仍然是一个相当有争议的问题。这一建议基于我们最近对SYP121-Kc1蛋白复合体及其在K+通道调节中的作用所做的重要发现：(1)我们已经确定了SNARE蛋白SYP121上与通道亚单位Kc1结合的位置，以及(2)我们已经证明Kc1与Sec1/Munc18(SM)蛋白小键竞争与SYP121结合。SM蛋白是所有真核生物膜泡融合的关键调节因子，但它们在控制SNARE介导的膜融合中的确切作用仍不清楚，也是一个热门的研究课题。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。他们不仅提供了直接将渗透溶质运输与囊泡运输相结合来控制有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。我们的工作假说是，SYP121和Kc1的SNARE-K+通道相互作用起到‘分子调节器’的作用，类似于James Watt在调节蒸汽机周转率时使用的机械发明，以协调囊泡运输(和细胞扩张)与渗透活性K+离子的吸收。我们现在建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地破坏SYP121、Kc1和Keule之间的相互作用对SNARE复合体组装、膜交通和通道介导的K+转运的影响。我们的多学科方法不仅将加深我们对渗透溶质运输和细胞体积控制之间的联系的理解，而且还可能为将膜运输与其他生理过程联系起来提供一个新的范例。

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.