Functional Analysis of ENTH Proteins in Vacuolar Transport

ENTH 蛋白在液泡运输中的功能分析

• 批准号: 281762085
• 负责人: Dr. Michael Sauer
• 金额: --
• 依托单位: Arbeitsgruppe Pflanzenphysiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/281762085?language=en
• 关键词: Functional; Analysis; ENTH; Proteins; Vacuolar

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a special CCV generating mechanism at the TGN with an important role in stress tolerance, growth and senescence (Sauer, unpublished).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data indicate an unexpected direct participation also in late events of CCV genesis and vesicle scission (Sauer, unpublished).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.

植物细胞的液泡是许多生理过程中必不可少的，但液泡蛋白运输的分子机制是复杂的，并没有完全理解。我第一次表明，液泡货物可以通过网格蛋白包被的囊泡（CCV）从trans-Golgi网络（TGN）运输，并且该途径需要新的蛋白质修饰的向液泡的运输1（MTV 1）（Sauer et al.，2013年）。MTV 1含有ENTH结构域，结构上类似于EPSIN蛋白，其与接头复合物（AP）的亚基相互作用，形成CCV生成早期所需的辅助蛋白。然而，MTV 1在遗传学上与EPSIN非常遥远，形成了在整个植物界保守的独特外群（Zouhar和Sauer，2014）。初步的功能和生物化学数据表明，MTV 1在TGN中定义了一种特殊的CCV产生机制，在胁迫耐受性、生长和衰老中起重要作用（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MTV 1途径的独特性质及其在植物界中的严格保守性，深入的功能和分子表征将对植物液泡运输产生重要的见解，此外，还将为CCV生成的一般过程提供新的分子细节。该项目旨在阐明MTV 1途径的独特特性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，因此可以根据实验课程灵活调整，以确保可持续的结果。在功能部分，我们将解决的要求MTV 1的特定生理功能应用遗传和生理学的方法，特别强调胁迫反应和功能关系的MTV 1与其他三个EPSINS在拟南芥。此外，MTV 1和EPSIN之间的功能差异和潜在冗余将得到解决。到目前为止，仅对EPSIN 1进行了有限的生理学研究，而EPSIN 2和3尚未进行功能分析。在分子方面，将测试MTV 1与AP-4复合物的假设相互作用，如果呈阳性，则进行更详细的表征，因为AP-4复合物在植物中仍然知之甚少。此外，假定的直接相互作用的MTV 1与组件的囊泡断裂机械将被验证和详细分析，使用分子和遗传学的方法，也涉及高和超分辨率显微镜。到目前为止，还没有报道过EPSIN样蛋白与囊泡断裂组分之间的直接相互作用，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。