Directed control of secretory vesicle fusion

控制体积和渗透压--以及植物和真菌中的膨压--是所有真核生物细胞内稳态的核心。在植物和真菌中，强烈的H+-ATPase及其培养的大量膜电压驱动溶质积累，产生陡峭的渗透梯度和细胞生长所需的膨胀压力。囊泡运输增加了细胞扩张的表面积，并有助于细胞生长时壁的重塑。为了生存和决定生物体的形态，必须控制溶质的运输(尤其是K+离子)和分泌物。尽管它们具有基本的重要性，但我们对细胞如何协调膜运输和溶质运输的速率知之甚少。这一提议建立在发现分泌和运输蛋白质的新亚集的基础上，这些蛋白质出现在迄今为止所描述的所有植物的基因组中，并且在所研究的少数物种中，已知它们之间可以相互作用。在拟南芥中，这些相互作用有助于运输调节、渗透溶质吸收和影响生长；在烟草中，这些过程解偶联导致营养不良的细胞生长和不受控制的组织扩张，类似于一些植物疾病(如甘蓝的根腐病、马铃薯的赤霉病)。这些发现指出了分泌和转运之间的基本协调水平，以共同调节这两个过程。这一发现也表明了可能控制分泌的潜在机制。转运体结合伙伴-离子通道的子集-包括随电压移动的半自主电压传感器结构域(VSD)。众所周知，这种运动激活/去激活这些通道，使它们与膜上的所有其他转运蛋白的活动相协调。分泌蛋白结合发生在VSD上的保守位置，这表明电压可能直接影响分泌。与膜电压的耦合尤其重要，因为电压报告了所有溶质在植物质膜上的运输活动，同时控制着溶质的积累，从而控制细胞的膨胀和扩张。我对这些发现感到非常兴奋。他们提供了分子机制的关键证据，这显然将有助于解开植物生长中离子运输和分泌之间的联系。此外，他们支持一种全新的调节分泌交通的模型，该模型将改写有关真核细胞膜交通的教科书。直到最近，VSD还被认为是原核生物和真核生物中几个研究得很好的离子通道家族的独特成分和调节器，以及海洋被囊动物中一小群电压敏感和膜结合的磷酸酶的成分和调节器。然而，在每一种情况下，VSD都被合并为天然蛋白质结构的组成部分；到目前为止，还没有通过直接VSD结合进行电压相关控制的例子出现。到目前为止，我们的证据表明，电压驱动的VSD运动已经被“劫持”，以充当这一植物诱捕器子集的电压传感器。因此，我的工作假设是，VSD通过与分泌蛋白伙伴结合来管理囊泡运输，就像它们管理通道的活动一样。我现在提议测试这一假设的关键要素。本项目将全面描述VSD结合的电压依赖性，以评估其与电压依赖性通道活动和分泌的关系。我还建议有选择地操纵和分析蛋白质之间的相互作用，修改VSD运动和分泌蛋白质结合，以确定对分泌的影响。这些结果不仅将进一步加深我们对渗透溶质运输、细胞膨大控制和植物生长之间的联系的理解，而且还将提供关于植物膜运输与其他生理和病理过程之间的全新机制的关键信息。

Control of volume and osmolarity - and of turgor in plants and fungi - lies at the very core of cellular homeostasis in all eukaryotes. In plants and fungi, strongly electrogenic H+-ATPases, and the substantial membrane voltages they foster, drive solute accumulation to generate steep osmotic gradients and turgor pressure for cell growth. Vesicle traffic adds surface area for cell expansion and contributes to wall remodelling as the cell grows. The transport of solutes (especially of K+ ions) must be controlled in concert with secretion for survival and to determine organismal form. Despite their fundamental importance we know little of how cells coordinate the rates membrane traffic and solute transport.This proposal builds on the discovery of new subsets of secretory and transport proteins that occur in the genomes of all plants described to date and, in the few species examined, are known to interact with one another. In Arabidopsis these interactions contribute to transport regulation, osmotic solute uptake and affect growth; in tobacco uncoupling these processes leads to hypotrophic cell growth and uncontrolled tissue expansion similar to that of a number of plant diseases (e.g. clubroot in Brassicas, scab disease in potato). The findings point to a basal level of coordination between secretion and transport for co-regulation of the two processes.The findings also indicate a potential mechanism by which secretion may be controlled. The transporter binding partners - a subset of ion channels - include semi-autonomous voltage-sensor domains (VSDs) that move in response to voltage. This movement is known to activate/deactivate the channels, coordinating their activity with all other transporters in the membrane. Secretory protein binding occurs at a conserved site on the VSDs, suggesting that voltage may affect secretion directly. Coupling to membrane voltage is especially significant, because voltage reports on the activity of all solute transport across the plant plasma membrane while governing solute accumulation and, hence, cell turgor and expansion.I am very excited by these findings. They offer critical evidence of a molecular mechanism that clearly will help unravel the connection between ion transport and secretion in plant growth. Furthermore, they support an entirely new model for regulated secretory traffic that will rewrite the textbooks on membrane traffic in eukaryotic cells. Until recently VSDs were thought unique as components and modulators of a few, well-studied families of ion channels in prokaryotes and eukaryotes, and of a small group of voltage-sensitive and membrane-bound phosphatases in marine tunicates. In each of these instances, however, the VSDs are incorporated as an integral part of the native protein structure; no examples of voltage-related control through direct, VSD binding have surfaced until now. Our evidence to date suggests that voltage-driven movement of the VSDs have been 'hijacked' to function as voltage sensors for this subset of plant SNAREs. Thus my working hypothesis is that the VSDs, through their binding to the secretory protein partners, govern vesicle traffic much as they do the activity of the channels. I now propose to test key elements of this hypothesis. This project will fully characterize the voltage-dependence of VSD binding in order to assess its association with voltage-dependent channel activity and secretion. I also propose selective manipulation and analysis of the interactions between the proteins, modifying VSD movement and secretory protein binding to determine the effects on secretion. Not only will the the results further our understanding of the link between osmotic solute transport and control of cell turgor and growth in plants, but they will also yield crucial information about what is clearly an entirely new mechanism linking membrane traffic with other physiological and pathological processes in plants.