A New Method for Determining the Topology of ER Proteins

Eukaryotic endoplasmic reticulum (ER) serves many essential functions, including protein folding, disulfide bond formation, transport and secretion, glycosylation, and membrane integration. The ER resident proteins must adopt specific topology to achieve their proper functions. However, the techniques currently available for determining ER protein location and topology are limited, widespread knowledge of the location and topology of eukaryotic ER proteins demands rapid and convenient approaches.

 

LI Haiyin and her colleagues have developed a rapid and convenient assay to determine the localization or topology of ER proteins. The assay applies the redox-sensitive luciferase from Gaussia princeps (Gluc) for monitoring the redox state in different compartments and green fluorescence protein (GFP) for estimating the amounts of fusion proteins. Using the tandem Gluc-GFP reporter fused to different positions of a target protein, this research firstly confirmed the known topology of single-pass transmembrane ER proteins with different sizes and orientations (such as calnexin, Sec61g and CD3δ), and then successfully characterized the topologies of two ER transmembrane proteins Herp and HRD1 that are involved in the ER quality control system. This method does not require cell extraction or biochemical treatment, and eliminates the need for appropriate and available antibodies or microscopic imaging. These advantages make the approach a promising application to high-throughput screening and applicable to the proteins in secretory pathway, plasma membrane, and other compartments of cells.

 

This work entitled “A Redox-Sensitive Luciferase Assay for Determining the Localization and Topology of Endoplasmic Reticulum Proteins” was published in PLoS ONE on April 18^th, 2012.

 

This study was supported by grants from the National Basic Research Program of China, the Sino-Swiss Joint Research Projects, and the National Natural Science Foundation of China.

 

AUTHOR CONTACT:

HU Hongyu

Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China

Phone:86-21-54921121; E-mail: hyhu@sibs.ac.cn