酿酒酵母内质网滞留信号介导的蛋白滞留效应研究

Protein endoplasmic reticulum (ER) retention is an important physiological phenomenon in eukaryotic cells. Through the interaction between the specific ER signal (ERS) sequence at their carboxy terminus with the retention receptor proteins located in the ER membrane, the ER structural and functional proteins could retain in the ER, affecting the process of the post-translational modifications, folding and secretion of proteins. However, due to the lack of systematic studies of ER retention proteins and their retention mechanisms, their importance is often underestimated. In this project, the ERS mediated protein ER retention in Saccharomyces cerevisiae will be further investigated based on our recent progress of the characterization of HDEL-type ERS. Firstly, an ERS mutant library will be constructed and systematically analyzed, obtaining the functional ERS and deciphering their characteristic motifs, which will then be used to identify the endogenous ER retention proteins through genomic sequence alignment of Saccharomyces cerevisiae. Secondly, a new method using the yeast ER as the reaction location will be developed to specifically explore the interactions between proteins located in the yeast ER. Thirdly, the physiological functions of Erd1/Erd2, known as the ER retention receptors, in the process of protein ER retention in yeast will be further studied, and unreported yeast ER retention receptor proteins will be identified. This project will provide guidance for studying the protein ER retention and its related physiological functions in eukaryotic cells.

蛋白内质网滞留是真核细胞中的一种重要生理现象。内质网结构与功能蛋白通过羧基端特异的内质网滞留信号序列与内质网膜上的受体蛋白相互作用在细胞内质网中滞留，影响蛋白的翻译后修饰、折叠和分泌过程。然而，由于缺乏对滞留蛋白及其滞留机制的系统性研究，其重要性经常被低估。本项目拟在前期针对酿酒酵母HDEL模式内质网滞留信号的研究基础上进一步探究酵母羧基端滞留信号序列介导的蛋白内质网滞留效应：一是构建内质网滞留信号文库，筛选有滞留效应的羧基端信号序列，解析滞留信号特征，并利用酿酒酵母全基因组序列对比鉴定内质网滞留蛋白；二是建立以酵母内质网为反应场所的蛋白互作新方法来研究酵母内质网内的蛋白互作；三是进一步解析内质网滞留受体蛋白Erd1/Erd2在蛋白内质网滞留过程中的生理生化功能，并挖掘、鉴定新型酵母内质网滞留受体蛋白。本项目将为真核细胞内质网内发生的蛋白滞留及其相关生理功能研究提供理论基础。

在真核细胞中，内质网和高尔基体驻留蛋白通过羧基端特异的滞留信号肽与对应滞留受体互作产生滞留效应，从而滞留在内质网和高尔基体内，维持了两者结构和功能的完整性。然而，目前缺乏对滞留信号肽的系统性研究，真核蛋白滞留效应机制研究仍停留在初步阶段。.为了系统性分析酿酒酵母内质网滞留信号肽，本研究构建了一个６个氨基酸长度的酿酒酵母内质网滞留信号肽文库，利用高通量筛选、二代测序和生物信息学方法，发现了酿酒酵母蛋白羧基端带正电荷（Lys）的短肽具有一定滞留效应。经过与酿酒酵母全蛋白组数据库比对分析，发现有69种酵母内质网和高尔基体定位蛋白的羧基端含有KK或KXKXX的序列特征。随后利用前期建立的酵母内质网滞留效应检测方法分析了这69种短肽的滞留效应，发现有24种序列与HDEL具有相当的内质网滞留强度。随后，我们共表达了目前报道的4种与内质网滞留相关的内质网膜蛋白Erd1、Erd2、囊泡蛋白复合物COP I的亚基α-COP和β-COP。结果表明，部分序列是与这4个蛋白介导的滞留机制相关，而PETYKK、SNKKNN、NTKKNN和ASKKGN短肽的滞留效应与这四个蛋白都不相关，说明酿酒酵母体内可能存在其他新型的蛋白内质网滞留机制。.此外，本研究基于Split-TEV 蛋白酶、YESS 系统和内质网滞留信号肽，开发了以酿酒酵母为宿主细胞的 YST-PPI系统，可针对性的对内质网蛋白进行互作研究。利用YST-PPI系统，本研究对酿酒酵母带有DEL模式滞留信号肽的 15个蛋白质之间的相互作用进行了全面解析。结果表明，内质网分子伴侣 LHS1可与其他 12 个蛋白质发生强相互作用。随后，通过构建 LHS1 基因缺失的 EBY100（ΔLHS1）菌株和LHS1 过表达的 EBY100（+LHS1）菌株，与 EBY100 菌株对比，来分析内质网蛋白互作结果，发现 LHS1 有可能在内质网滞留蛋白协同作用中起到了重要作用。.综上所述，本研究针对酿酒酵母内质网滞留效应和内质网滞留蛋白互作的系统性解析，不仅有助于人们对真核细胞内质网中蛋白滞留机制的深入了解，也为研究蛋白质折叠修饰和分泌过程中蛋白质之间是如何协同发挥作用提供了理论基础。