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与秀丽隐杆线虫氯离子通道CLH-1相互作用蛋白的构建：GFP-Trap技术和质谱分析的研究

周子量, 姜静, 尹江安, 蔡时青

中国科学院上海生命科学研究院神经科学研究所，上海 200031； 中国科学院大学，上海 200031

摘要

氯离子通道是一类分布广泛的阴离子选择性通道家族蛋白，在细胞容积维持、细胞质内pH调节、蛋白运输、细胞迁移、增殖及分化等重要生理过程中扮演着多种角色。然而，目前有关其功能调控研究较少。本研究构建了表达电压门控氯离子通道CLH-1融合绿色荧光蛋白(CLH-1::GFP)的转基因线虫。CLH-1::GFP主要表达在线虫头部神经元和身体后部肠道细胞中。利用最近发展起来的捕获GFP的GFP-Trap技术，从转基因线虫裂解液中捕获了可能调控氯离子通道CLH-1功能的相互作用蛋白。通过质谱鉴定，共有27个蛋白和CLH-1::GFP一起被捕获下来。生化实验显示其中真核生物延伸因子1 (EEF-1)与CLH-1蛋白直接相互作用；并且，在和EEF-1共表达的情况下，CLH-1的蛋白水平显著上调。本结果表明利用线虫表达氯离子通道GFP融合蛋白，结合GFP-Trap和质谱分析技术，可以识别和电压门控氯离子通道相互作用蛋白，同时也表明EEF-1可以调控线虫CLH-1通道功能。

关键词： 氯通道; 真核生物延伸因子; 绿色荧光融合蛋白; 质谱

分类号：Q25

Identifying interacting proteins of a Caenorhabditis elegans voltage-gated chloride channel Clh-1 using GFP-trap and mass spectrometry

ZHOU Zi-Liang, JIANG Jing, YIN Jiang-An, CAI Shi-Qing

Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China； University of Chinese Academy of Sciences, Shanghai 200031, China

Abstract

Chloride channels belong to a superfamily of ion channels that permit passive passage of anions, mainly chloride, across cell membrane. They play a variety of important physiological roles in regulation of cytosolic pH, cell volume homeostasis, organic solute transport, cell migration, cell proliferation, and differentiation. However, little is known about the functional regulation of these channels. In this study, we generated an integrated transgenic worm strain expressing green fluorescence protein (GFP) fused CLC-type chloride channel 1 (CLH-1::GFP), a voltage-gated chloride channel in Caenorhabditis elegans (C. elegans). CLH-1::GFP was expressed in some unidentified head neurons and posterior intestinal cells of C. elegans. Interacting proteins of CLH-1::GFP were purified by GFP-Trap, a novel system for efficient isolation of GFP fusion proteins and their interacting factors. Mass spectrometry (MS) analysis revealed that a total of 27 high probability interacting proteins were co-trapped with CLHp-1::GFP. Biochemical evidence showed that eukaryotic translation elongation factor 1 (EEF-1), one of these co-trapped proteins identified by MS, physically interacted with CLH-1, in consistent with GFP-Trap experiments. Further immunostaining data revealed that the protein level of CLH-1 was significantly increased upon co-expression with EEF-1. These results suggest that the combination of GFP-Trap purification with MS is an excellent tool to identify novel interacting proteins of voltage-gated chloride channels in C. elegans. Our data also show that EEF-1 is a regulator of voltage-gated chloride channel CLH-1.

Key words: Chloride channels; EEF-1; GFP fusion protein; GFP-trap

收稿日期：2013-09-02　　录用日期：2013-12-05

通讯作者：蔡时青　　E-mail: sqcai@ion.ac.cn

引用本文：

周子量, 姜静, 尹江安, 蔡时青. 与秀丽隐杆线虫氯离子通道CLH-1相互作用蛋白的构建：GFP-Trap技术和质谱分析的研究[J]. 生理学报 2014; 66 (3): 341-348.

ZHOU Zi-Liang, JIANG Jing, YIN Jiang-An, CAI Shi-Qing. Identifying interacting proteins of a Caenorhabditis elegans voltage-gated chloride channel Clh-1 using GFP-trap and mass spectrometry. Acta Physiol Sin 2014; 66 (3): 341-348 (in Chinese with English abstract).