a redox-sensitive luciferase assay for determining the localization and topology of endoplasmic reticulum proteins一个redox-sensitive荧光素酶试验确定内质网的蛋白质的定位和拓扑.pdfVIP

A Redox-Sensitive Luciferase Assay for Determining the Localization and Topology of Endoplasmic Reticulum Proteins Hai-Yin Li, Xue-Ming Zheng, Mei-Xia Che, Hong-Yu Hu* State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Abstract Correct localization and transmembrane topology are crucial for the proteins residing and functioning in the endoplasmic reticulum (ER). We have developed a rapid and convenient assay, based on the redox-sensitive luciferase from Gaussia princeps (Gluc) and green fluorescence protein (GFP), to determine the localization or topology of ER proteins. Using the tandem Gluc-GFP reporter fused to different positions of a target protein, we successfully characterized the topologies of two ER transmembrane proteins Herp and HRD1 that are involved in the ER quality control system. This assay method may also be applicable to the proteins in secretory pathway, plasma membrane, and other compartments of cells. Citation: Li H-Y, Zheng X-M, Che M-X, Hu H-Y (2012) A Redox-Sensitive Luciferase Assay for Determining the Localization and Topology of Endoplasmic Reticulum Proteins. PLoS ONE 7(4): e35628. doi:10.1371/journal.pone.0035628 Editor: F. Gisou van der Goot, Ecole Polytechnique Federale de Lausanne, Switzerland Received November 9, 2011; Accepted March 19, 2012; Published April 18, 2012 Copyright: 2012 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Basic Research Program of China (2012CB911003), the National Natural Science Foundation of China ,