Functional Analysis of ENTH Proteins in Vacuolar Transport

植物细胞的液泡是许多生理过程所必需的，但液泡蛋白运输的分子机制很复杂，还不完全清楚。我首次展示了液泡货物可以通过笼蛋白包裹的小泡(CCV)从跨高尔基网络(TGN)运输，这一路线需要新的蛋白质修饰的VACUOLE1(MTV1)运输(Sauer等人，2013年)。MTV1含有第10个结构域，在结构上与EPSIN蛋白相似，EPSIN蛋白与接头复合体(AP)的亚基相互作用，形成CCV生成早期所需的辅助蛋白。然而，MTV1在系统发育上与EPSIN非常遥远，形成了一个独特的外群，在整个植物界都是保守的(Zouhar和Sauer，2014)。初步的功能和生化数据表明，MTV1在TGN定义了一种特殊的CCV产生机制，在胁迫耐受、生长和衰老中发挥重要作用(Sauer，未发表)。此外，相互作用数据表明，在CCV发生和囊泡破裂的晚期事件中，也有意想不到的直接参与(Sauer，未发表)。由于MTV1途径表面上的独特性质及其在植物界中的严格保守性，深入的功能和分子特征将对植物液泡运输产生重要的见解，并提供关于CCV产生的一般过程的新的分子细节。这项拟议的项目旨在阐明MTV1途径的独特性质并从分子上对其进行定义。它由很大程度上独立的模块组成，因此可以根据实验过程灵活调整，以确保结果可发布。在功能部分，我们将利用遗传学和生理学方法解决MTV1对特定生理功能的需求，特别是在拟南芥中的胁迫反应以及MTV1与其他三个EPSIN的功能关系。此外，还将讨论MTV1和EPSIN之间的功能差异和潜在的冗余。到目前为止，仅对EPSIN1进行了有限的生理学研究，而EPSIN2和EPSIN3根本没有进行功能分析。在分子方面，MTV1与AP-4复合体的假想相互作用将得到测试，如果是阳性的，将进行更详细的表征，因为AP-4复合体在植物中仍然知之甚少。此外，还将使用分子和遗传学方法，也包括高分辨率和超分辨率显微镜，详细验证和分析MTV1与囊泡破裂机械组件的假定直接相互作用。EPSIN类蛋白和囊泡裂解组分之间的直接相互作用到目前为止还没有报道，因此这样的发现也将在植物领域之外引起广泛的普遍兴趣。

Vacuoles of plant cells are essential for a multitude of physiological processes, but the molecular mechanisms of vacuolar protein transport are complex and not fully understood. I showed for the first time that vacuolar cargo can be transported via clathrin coated vesicles (CCVs) from the trans-Golgi network (TGN), and that this route requires the novel protein MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) (Sauer et al., 2013). MTV1 contains an ENTH domain and is structurally similar to EPSIN proteins, which interact with subunits of the adaptor complexes (APs) and form accessory proteins required in early stages of CCV generation. However, MTV1 is phylogenetically very distant from the EPSINs, forming a unique outgroup that is conserved throughout the plant kingdom (Zouhar and Sauer, 2014). Preliminary functional and biochemical data suggest that MTV1 defines a special CCV generating mechanism at the TGN with an important role in stress tolerance, growth and senescence (Sauer, unpublished). Moreover, interaction data indicate an unexpected direct participation also in late events of CCV genesis and vesicle scission (Sauer, unpublished). Because of the apparently unique properties of the MTV1 pathway and its strict conservation within the plant kingdom, an in depth functional and molecular characterization will yield important insights into plant vacuolar transport and, moreover, give novel molecular details about the general process of CCV generation. The proposed project aims to elucidate the unique properties of the MTV1 pathway and define it molecularly. It consists of largely independent modules, so it can be adjusted flexibly depending on the experimental course to ensure a publishable outcome. In a functional part, we will address the requirement for MTV1 for specific physiological functions applying genetic and physiological approaches, with a particular emphasis on stress responses and the functional relation of MTV1 with the other three EPSINs in Arabidopsis. Further, the functional differences and potential redundancy among MTV1 and the EPSINs will be addressed. So far, only limited physiological studies have been performed on EPSIN1, while EPSIN2 and 3 have not been functionally analyzed at all. On the molecular side, the hypothetical interaction of MTV1 with the AP-4 complex will be tested and, if positive, characterized in greater detail, as the AP-4 complex is still little understood in plants. Furthermore, the putative direct interactions of MTV1 with components of the vesicle scission machinery will be verified and analyzed in detail, using molecular and genetic approaches, also involving high and super-resolution microscopy. A direct interaction between EPSIN-like proteins and vesicle scission components has not been reported so far, therefore such a finding would be of broad general interest also beyond the plant field.