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

项目摘要 钙信号是所有真核细胞的基础。它的存在依赖于 亚微摩尔水平的胞质钙（[Ca 2 +]cyt）。这个基础水平的异常扰动触发了一个 许多病理，从癌症到神经变性和细胞凋亡。本提案将侧重于 植物CORNICHON同源物（CNIH）囊泡运输和蛋白质靶向的挑衅性假说 蛋白质在[Ca 2 +]细胞内稳态和信号传导中具有直接调节作用。我的团队是 表明谷氨酸受体样（GLR）蛋白是植物中的Ca 2+渗透性离子通道。 CORNICHON蛋白是ER货物衔接子，其介导整合膜蛋白募集到细胞中。 COPII囊泡。在这里，我们提出的证据表明，对CNIH是一个必要条件的选择性 GLR靶向特定的内膜隔室，导致其差异定位， 不同的Ca 2+储存。这些结果使我们假设CNIH本身在 通过控制针对这些商店的通道的数量和类型来维持Ca 2+稳态。这 我们发现GLR和CNIH门之间的相互作用进一步证实了这一假设 在不存在配体的情况下的大量离子电流。我们将通过以下组合来检验这一假设： 遗传学、定量Ca 2+成像、数学建模、电生理学和蛋白质结构 分析，重点是三个具体目标。(1)我们将通过过度表达， 改变表达在细胞中的5个CNIH内的货物分选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[Ca 2 +]cyt稳态边界来产生生长表型，这将告知 我们需要了解受影响的贩运机制的职能等级。(2)我们将发展数学 模型来模拟每个亚细胞位置与[Ca 2 +]cyt的相关性。我们将校准这些模型， 在GLR/CNIH家族中筛选大量的多个组合突变，并定量其Ca 2 + 编排变化和GLR定位。这种方法将导致表型， 每个GLR/位置集的分层贡献。最后（3）我们将研究的物理相互作用 通过在哺乳动物细胞中异源表达后的电生理学和通过Cryo-EM测定CNIH和GLR。 结果使我们能够建立一个新的模型，[Ca 2 +]细胞调节的基础上囊泡运输 由CNIH介导。我们认为，这种机制可能在植物中更明显，更相关，因为 它们缺乏动物细胞进化来协调小配体操作的Ca 2+的所有分子机制 储存（如IP 3和ryanodine受体、环化酶和磷酸二酯酶）。但类似 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 因此，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。 [Ca 2 +]cyt在真核细胞调节的当前范例。

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.