内质网是众多真核生物蛋白合成、加工修饰及成熟的重要场所。新合成蛋白从内质网到高尔基体的选择性运输是确保其准确高效分泌的前提。丝状真菌里氏木霉具有强大的纤维素酶合成分泌能力，但其高效的内质网分选机制并不清楚。本项申请拟围绕里氏木霉p24家族等内质网分选受体在纤维素酶高效分泌过程中的内质网分选与脱离机制展开研究。首先在确定不同分选受体在相应纤维素酶分泌过程中作用的基础上，利用免疫共沉淀结合蛋白近距离生物素标记等技术，解析分选受体分别在内质网腔内外与纤维素酶和囊泡包被复合物识别互作的分子机制；进一步利用基于RUSH的脉冲示踪技术，系统分析分选受体介导的纤维素酶识别捕获与囊泡复合物组装促进纤维素酶从内质网脱离的过程机制，从而明晰内质网分选受体介导纤维素酶高效分选运输的机理。研究结果将不仅拓展真核生物内质网蛋白质分选装置的结构功能研究，还将为构建高产纤维素酶菌株或蛋白质生产底盘提供新思路和新策略。

The endoplasmic reticulum (ER) is responsible for the synthesis, modification, and maturation of great diversity of the secretory products that are transported to different destinations. Transport of newly synthesized proteins from the ER to the Golgi apparatus is highly selective, which secures the efficiency, fidelity, and control of this crucial transport step for a broad spectrum of secretory proteins. The filamentous fungus Trichoderma reesei is characterized by its outstanding ability to biosynthesize a large amount of cellulase. Unfortunately, the precise mechanism underlying the efficient and selective capture of cellulases from the ER is not clear yet. The present application aims to focus on the T. reesei ER sorting receptors, p24 and Erv29, for the major cellulase components. Systematic studies will be carried out to elucidate how the sorting receptor specifically recognizes and captures its corresponding cargo in the ER lumen, as well as how the receptor interacts with and therefore facilitates the assembly of the vesicle coat protein complex on the cytoplasmic side of the ER membrane. Combining co-imunoprecipitation and proximity-dependent biotin labelling (PDB) techniques, attempts will first be made to dissect the molecular mechanisms underlying the specific interactions between the luminal part of the receptor and the corresponding cellulase as well as interactions between the cytoplasmic part of the receptor and the vesicle coat subunits. The role of the sorting receptor-mediated cargo capture and the assembly of the antrograde vesicle coat in contributing to the exit of cellulase carrier vesicle from the ER will then be analyzed systematically by a pulse-chase based on the technique of retention using selective hooks (RUSH). The results are anticipated not only to extend our understanding of the structural and functional aspects of the elaborate eukaryotic protein sorting machinery at the ER, but also to help provide new strategies in designing highly efficient T. reesei strains for enhanced cellulases or heterologous protein production.