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。它的存在有赖于动态平衡的维持 亚微摩尔水平的胞浆钙([Ca~(2+)]Cyt)。这一基础水平的异常扰动会触发 许多病理疾病，从癌症到神经变性和细胞凋亡。这项提案将重点放在 挑衅性假说：囊泡运输和植物角蛋白同系物靶向蛋白质(CNIH) 蛋白质在细胞动态平衡和信号传递中起着直接的调节作用。我的团队是 表明谷氨酸受体样蛋白(GLR)在植物体内是钙离子通道。 Cornichon蛋白是ER货物适配器，介导完整的膜蛋白募集到 COPII囊泡。在这里，我们提出的证据表明，CNIH对是选择性的必要条件 将GLRs靶向于特定的内膜室，导致其差异定位于 不同的钙库。这些结果使我们假设CNIH本身在 通过控制针对这些商店的渠道的数量和类型，实现CA2的动态平衡。这 我们发现GLRs和CNIHs Gate之间的相互作用进一步证实了这一假说 在没有配基的情况下产生大量的离子电流。我们将通过以下组合来检验这一假设 遗传学、定量钙成像、数学模型、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的作用，通过 表达的5个CNIH中货物分类和结构域交换的分子决定因素的变化 拟南芥。这将使我们能够将特定的GLR重新寻址或保留到不同的亚细胞位置。我们 预测这将通过跨越[Ca~(2+)]细胞动态平衡的边界产生生长表型，这将告诉 美国对受影响的贩运机制的职能等级进行了评估。(2)我们将发展数学 模拟每个亚细胞位置与[钙]细胞的相关性的模型。我们将通过以下方式校准这些型号 在GLR/CNIH家族中筛选大量多个复合突变，并对其钙离子进行量化 编舞变化和GLR定位。这种方法将产生表型，从而揭示 每个GLR/位置集合的分层贡献。最后(3)我们将研究物理相互作用。 在哺乳动物细胞中异源表达CNIHs和GLRs后，通过电生理学和Cryo-EM进行检测。 结果应该使我们能够建立一个新的基于囊泡运输的[钙]细胞调节模型 由CNIHs调解。我们认为，这种机制在植物中可能更明显和相关，因为 它们缺乏动物细胞进化来协调小配体操作的钙离子的所有分子机制。 储存(如IP3和Ryanodine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 假设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 真核细胞中细胞内[钙]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.