A Redox-Sensitive Luciferase Assay for Determining the Localization and Topology of Endoplasmic Reticulum Proteins

Hai-Yin Li,Mei-Xia Che,Hong-Yu Hu,Xue-Ming Zheng,F Gisou Van Der Goot

doi:10.1371/journal.pone.0035628

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.

Highlights

Eukaryotic endoplasmic reticulum (ER) serves many essential functions, including protein folding, disulfide bond formation, transport and secretion, glycosylation, and membrane integration [1,2]
Determining the topology of Herp, an ER transmembrane protein. To apply this Gaussia luciferase (Gluc)-green fluorescence protein (GFP) assay to multi-pass transmembrane proteins involved in the ER quality control system, we investigated the topology of Herp, a membrane protein induced by ER stress [16]
Taking GG-ER and GlucGFP to the cytoplasm (GG-Cyto) as examples, the Gluc-GFP reporter has been successfully applied to investigate the localization of ER proteins

Summary

Introduction

Eukaryotic endoplasmic reticulum (ER) serves many essential functions, including protein folding, disulfide bond formation, transport and secretion, glycosylation, and membrane integration [1,2]. The techniques currently available for determining ER protein location and topology are limited. Protease protection assay is to engineer epitopes, such as Myc and FLAG, into a protein of interest, and to assess its sensitivity to protease digestion in the absence or presence of detergents [3]. Another approach is glycosylation scanning, in which different glycosylation sites can be introduced into the fulllength protein [4]. Widespread knowledge of the location and topology of eukaryotic ER proteins demands rapid and convenient approaches

Methods

Results

Conclusion