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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

Received: 2013-09-02　　Accepted: 2013-12-05

Corresponding author: 蔡时青　　E-mail: sqcai@ion.ac.cn

Citing This Article：

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).