Analysis of the Dual Function of the Exocyst Complex Subunit Exo70 as a Spatiotemporal Mediator of Secretion and Autophagy

分泌途径负责将蛋白质和脂质传递到质膜。分泌活动的速率和空间分布的变化是正常发育所固有的，但也有助于适应由温度变化或感染引起的应激。由免疫反应触发的高分泌活性，或由热引起的细胞应激，通过分泌途径以及降解系统影响蛋白质生物生成的速度、保真度和能力，通常导致蛋白质组失衡（即蛋白质稳态的丧失）。胞囊是一种保守的异八聚体复合物，在胞吐过程中介导高尔基后囊泡与PM的早期拴系。它的Exo70亚基通过介导多种蛋白质和脂质相互作用，作为时空调节剂发挥关键作用。先前的研究和我们的初步数据表明，Exo70亚基具有质膜定位和通过反式高尔基网络转运等典型特征。植物中的Exo70家族已经扩展到许多同源物（拟南芥中有23个），并且一些研究支持特定蛋白质货物运输到质膜的专门化。此外，我们和其他人已经证明exo70可以通过自噬转移到液泡中。我们的初步数据表明，液泡降解可以在蛋白毒性胁迫或高分泌活性的情况下被诱导，并由两个atg8相互作用基序（AIMs）驱动，这些基序在大多数Exo70同源物中是保守的。在酵母ATG8和Exo70之间也观察到相互作用，表明跨王国的进化守恒。因此，我们假设Exo70在分泌途径和自噬中发挥双重作用，抑制分泌活性，从而恢复细胞稳态。迄今为止，对分泌途径中囊泡功能的分子理解仍然具有挑战性，我们对细胞途径之间的交叉点保护和恢复蛋白质稳态的知识是零碎的。在这个项目中，我们的目标是阐明Exo70亚基的双重功能：1)分析决定质膜结合特异性的分子机制；2)自噬降解在维持蛋白质稳态中的作用。

The secretory pathway is responsible for the delivery of proteins and lipids to the plasma membrane. Changes in the rate and spatial distribution of secretory activity are inherent to normal development, but also enable adaptation to stresses resulting from temperature changes or infection. High secretory activity triggered by immune responses, or cellular stress caused by heat, affect speed, fidelity and capacity of protein biogenesis via the secretory pathway, as well as degradation systems, often resulting in proteome imbalances (i.e. loss of protein homeostasis) . The exocyst is a conserved hetero-octameric complex mediating early tethering of post Golgi vesicles to the PM during exocytosis. Its Exo70 subunit plays a critical role as a spatiotemporal regulator by mediating numerous protein and lipid interactions. Previous studies and our preliminary data show that Exo70 subunits display typical traits such as plasma membrane localization and transit through trans-Golgi network. The Exo70 family in plants has expanded into many homologues (23 in Arabidopsis thaliana) and several studies support a specialization in the transport of specific protein cargoes to the plasma membrane. In addition, we and others, have shown that Exo70s can be diverted into the vacuole by autophagy. Our preliminary data shows that vacuolar degradation can be induced in response to proteotoxic stress or high secretory activity and is driven by two ATG8-interacting motifs (AIMs) which are conserved in the majority of Exo70 homologues. Interaction was also observed between the yeast ATG8 and Exo70, indicating evolutionary conservation across kingdoms. We therefore hypothesize that Exo70 plays a dual role in both the secretory pathway, as well as in autophagy to dampen secretory activity and thus restore cellular homeostasis. To date, the molecular understanding of the exocyst function in the secretory pathway remains challenging, and our knowledge regarding the intersection points between cellular pathways to safeguard and restore protein homeostasis is fragmentary. In this project we aim to elucidate the dual function of the Exo70 subunit by i) analysing the molecular mechanisms dictating plasma membrane binding specificities and ii) the role of its degradation by autophagy in maintaining protein homeostasis.