The role of Cornichon Homologue proteins in cytosolic calcium homeostasis through sorting and activity of plant ionotropic- Glutamate Receptor-Like channels

项目概要 钙信号传导是所有真核细胞的基础。它的存在依赖于体内平衡的维持 胞质钙 ([Ca2]cyt) 的亚微摩尔水平。该基础水平的异常扰动会触发 从癌症到神经变性和细胞凋亡的多种病理学。该提案将重点关注 植物 CORNICHON 同源物 (CNIH) 的囊泡运输和蛋白质靶向的挑衅性假设 蛋白质在 [Ca2] 细胞色素稳态和信号传导中具有直接调节作用。我的小组是这方面的先驱 显示谷氨酸受体样 (GLR) 蛋白是植物中的 Ca2 渗透离子通道。 CORNICHON 蛋白是 ER 货物适配器，介导整合膜蛋白的招募 COPII 囊泡。在这里，我们提供的证据表明，成对的 CNIH 是选择性的必要条件 将 GLR 靶向特定的内膜区室，导致其差异定位 不同的 Ca2 存储。这些结果使我们推测 CNIH 本身在以下方面具有反馈作用： 通过控制针对这些商店的渠道的数量和类型来实现 Ca2 稳态。这个 我们的发现进一步证实了这一假设：GLR 和 CNIH 之间的相互作用门控 在没有配体的情况下产生大量离子电流。我们将通过组合来检验这个假设 遗传学、定量 Ca2 成像、数学建模、电生理学和蛋白质结构 分析，重点关注三个具体目标。 (1) 我们将通过过度表达来操纵CNIH的行动，通过 改变 5 个 CNIH 中货物分类和结构域交换的分子决定因素 拟南芥。这将使我们能够将特定的 GLR 重新定位或保留到不同的亚细胞位置。我们 预测这将通过跨越[Ca2]细胞色素稳态边界产生生长表型，这将告知 我们了解受影响的贩运机制的职能层级。 (2)我们将发展数学 模型来模拟每个亚细胞位置与 [Ca2]cyt 的相关性。我们将通过以下方式校准这些模型 筛选 GLR/CNIH 家族中大量的多重组合突变，并量化其 Ca2+ 编排更改和 GLR 本地化。这种方法将产生的表型将揭示 每个 GLR/位置集的层次贡献。最后（3）我们将研究物理相互作用 CNIH 和 GLR 在哺乳动物细胞中异源表达后通过电生理学和冷冻电镜进行分析。 结果应该使我们能够建立一种基于囊泡运输的 [Ca2] 细胞色素调节的新模型 由 CNIH 调解。我们认为这种机制在植物中可能更加明显和相关，因为 它们缺乏动物细胞进化来协调小配体操作的 Ca2 的所有分子机制 储存（如 IP3 和兰尼定受体、环化酶和磷酸二酯酶）。然而，类似的 这两类蛋白质之间的功能相互作用存在于酵母和动物细胞中，因此我们 认为它们可能在动物细胞生理学和病理学中发挥重要作用，因此具有潜在的挑战性 当前真核细胞中[Ca2]细胞色素调节的范例。

Project Summary Calcium signaling is fundamental in all eukaryotic cells. Its existence relies on the homeostatic maintenance of sub-micromolar levels of cytosolic calcium ([Ca2+]cyt). Abnormal perturbation of this basal level triggers a number of pathologies, from cancer to neurodegeneration and apoptosis. This proposal will focus on the provocative hypothesis that vesicular traffic and protein targeting by plant CORNICHON-homologue (CNIH) proteins have a direct regulatory role in [Ca2+]cyt homeostasis and signalling. My group was pioneer in showing that GLUTAMATE RECEPTOR-like (GLR) proteins are Ca2+ permeable ion channels in plants. CORNICHON proteins are ER cargo adaptors mediating the recruitment of integral membrane proteins into COPII vesicles. Here we present evidence that pairs of CNIHs are a necessary condition for the selective targeting of GLRs to specific endomembrane compartments, resulting in their differential localization to different Ca2+ stores. These results made us hypothesize that CNIHs themselves have a feed-back role in Ca2+ homeostasis by controlling the quantity and types of channels that are targeted to these stores. This hypothesis was further substantiated by our finding that the interaction between GLRs and CNIHs gate substantial ion currents in the absence of a ligand. We will test this hypothesis by a combination of genetics, quantitative Ca2+ imaging, mathematical modelling, electrophysiology and protein structural analysis, focusing on three specific aims. (1) We will manipulate CNIH action by over-expression, by changing molecular determinants of cargo sorting and domain swaps within the 5 CNIHs expressed in Arabidopsis. This will allow us to re-address or retain specific GLRs to different subcellular locations. We predict this will produce growth phenotypes by crossing [Ca2+]cyt homeostasis boundaries, which will inform us of the functional hierarchy of the trafficking mechanisms affected. (2) We will develop mathematical models to simulate the relevance of each sub-cellular location to [Ca2+]cyt. We will calibrate these models by screening a vast array of multiple, combined mutations in the GLR/CNIH families, and quantify their Ca2+ choreography changes and GLR localization. This approach will result in phenotypes that will reveal hierarchical contributions of each GLR/location set. Finally (3) we will study the physical interaction of CNIHs and GLRs by electrophysiology after heterologous expression in mammalian cells and by Cryo-EM. Results should enable us to establish a novel model of [Ca2+]cyt regulation based on vesicular trafficking mediated by CNIHs. We argue that this mechanism may be more visible and relevant in plants because they lack all the molecular machinery that animal cells evolved for coordinating small ligand operated Ca2+ stores (such as IP3 and ryanodine receptors, cyclases and phosphodiesterases). However, similar functional interactions between these two classes of proteins exist in yeast and animal cells, and thus we posit they may have an important role in animal cell physiology and pathology, thus potentially challenging the current paradigm of [Ca2+]cyt regulation in eukaryotic cells.