真核细胞通过双层磷脂分子内膜转运系统实现膜内外大分子与颗粒性物质有序地跨膜运输与信号转导，包括细胞胞吞、胞吐作用及细胞自噬等多种途径。内膜转运过程通常包括囊泡形成，运输，囊泡与目的膜拴系及膜融合四个关键步骤，其中囊泡运输特异性由拴系蛋白复合体家族负责调控。本论文从结构生物学入手研究细胞胞吐过程中拴系蛋白Exocyst复合体的作用机制。Exocyst复合体是介导细胞胞吐作用中囊泡与细胞膜拴系过程的主要蛋白复合体之一，与肿瘤，糖尿病，神经疾病均相关。该复合体由八个亚基组成，其中Sec3、Exo70两个亚基特异识别细胞膜上PtdIns(4,5)P2，并通过与Sec5、Sec6、Sec8、Sec10、Sec15、Exo84六个亚基组装而介导分泌囊泡锚定在质膜上，以及促进SNARE蛋白复合体组装。到本工作完成为止，其整体构架及八个亚基之间的组装机制仍不清楚。我们运用酿酒酵母表达体系，提纯了高纯度的Exocyst复合体，利用冷冻透射电子显微镜对蛋白复合体天然结构进行研究。课题攻关期间，我摸索出加入低浓度交联剂与变换蛋白标签位置两套实验方案分别用于克服冷冻样品蛋白颗粒不进孔及样品优势取向两项技术难题，成功制备出高质量完整Exocyst复合体的冷冻样品。最终，我们运用单颗粒重构技术解析得到整体5.4?，核心4.4?的Exocyst复合体三维电子密度图，在此基础上运用化学交联质谱技术及从头建模的方法，成功搭建了Exocyst复合体的三维原子模型。经过结构分析，我们发现Exocyst复合体的八个亚基通过每个亚基中的一段结构类似的coiled-coil基序互相缠绕，进行逐级地组装，最终形成一个镂空的完整复合体。我们将这段具有介导复合体组装功能的关键基序命名为CorEx。在进一步的酵母遗传学和细胞生物学研究中，我们发现Sec3蛋白的CorEx结构域对Exocyst复合体剩余亚基招募及囊泡定位发挥了关键作用。因此，我们提出Exocyst可能通过其N端PH结构域结合质膜，并由C端CorEx基序介导与其它亚基的招募与组装，最终通过Exo70亚基与质膜上PI(4,5)P2结合而介导囊泡拴系在细胞质膜上的作用机制。综上，我们的工作首次报道了完整组装的膜拴系Exocyst的复合体结构，并发现了复合体装配过程中一种由CorEx基序介导的逐级组装模型，同时证明了Sec3亚基与剩余亚基在质膜上的组装是由其CorEx基序介导完成。这些发现为高等细胞栓系机制研究提供了重要的信息。
In eukaryotic cells, transmembrane transporting or signal transduction are orderly regulated by the bilayerd phospholipid membrane transport system, including various membrane transport pathways such as endocytosis, exocytosis and autophagy. Membrane transport consist of four steps: vesicle formation, transportation, vesicle-organelle membrane tethering and membrane fusion. During this process, tethering complex family members help vesicles transport to the specified cell parts. Here we use structural biology methods to study the tethering mechanism of Exocyst complex during cell exocytosis.The Exocyst complex is one of the major complexes that mediate membrane tethering of exocytic vesicles，which is related to tumor formation, diabetic, neuropathy processing. It is composed of eight subunits, including two subunits named Sec3, Exo70, that specially recognize PtdIns(4,5)P2 on cell membrane, and Sec5, Sec6, Sec8, Sec10, Sec15, Exo84, which assembled as a complex to promote membrane fusion process mediated by SNARE protein complex. The overall structure of Exocyst complex and the assembly mechanism of the eight subunits are still unclear. In this research work, we isolated Exocyst complex by Saccharomyces cerevisiae endogenous expression system，and took the advantage of electron microscope trying to solve the structure of intact naturally assembled Exocyst complex. During the structure determination, we used two methods to overcome two technical problems respectively. First, we added low concentration crosslinking agent to facilitate the protein complex staying in the hole. And then, we translocated the affinity tag position to overcome the preferred orientation in ice. By these approaches, we obtained a three-dimensional electron density map of whole Exocyst complex at the resolution of 5.4? and the core component at the resolution of 4.4?. Then an atomic model of Exocyst complex was built based on the EM map with the assistance of the chemical cross-linked mass spectrometry pairs and the crystal structure information on PDB Database. The structure revealed that the eight subunits of the Exocyst complex are intertwined with each other through a similar coiled-coil motif, and each subunit is assembled hierarchically to form a hollowed complete complex. We named this key complex assembling motif CorEx. Futher yeast genetics and cell biology studies proved that the Sec3 CorEx motif plays a key role in the integrated complex packing and vesicle localization. Therefore, we proposed that Sec3 may bind on the plasma membrane through its PH domain and recruit the remaining subunits through the Sec3 CorEx motif. Finally, the subsequently interaction between Exo70 and PtdIns(4,5)P2 may mediate the membrane tethering. In conclusion, we reported the first structure of the intact membrane tethering complex Exocyst, and uncovered a complex hierarchically assembling mechanism mediated by CorEx motif. Meanwhile, we demonstrated that Sec3 assembles with the remaining subunits on plasma membrane by CorEx motif. This work provided important information for the vesicle tethering mechanism in eukaryotic cells.
