A redox-sensitive luciferase assay for determining the localization and topology of endoplasmic reticulum proteins

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 deter...

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Bibliographic Details
Published in:PloS one 2012-04, Vol.7 (4), p.e35628-e35628
Main Authors: Li, Hai-Yin, Zheng, Xue-Ming, Che, Mei-Xia, Hu, Hong-Yu
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Assaying
Biochemistry
Biology
Bioluminescence
Cell Line
Chemical bonds
Compartments
Control systems
Cytoplasm
E coli
Endoplasmic reticulum
Endoplasmic Reticulum - metabolism
Enzymes
Escherichia coli
Fluorescence
Fluorometry - methods
Gaussia
Gaussia princeps
Genes, Reporter
Humans
Journalists
Laboratories
Localization
Luciferase
Luciferases - chemistry
Luciferases - genetics
Luciferases - metabolism
Membrane proteins
Membrane Proteins - chemistry
Membrane Proteins - metabolism
Microscopy, Fluorescence
Molecular biology
Molecular Sequence Data
Oxidation-Reduction
Peptides
Protein Transport
Proteins
Quality control
Quality management
Retention
Topology
Trends
Ubiquitin-Protein Ligases - chemistry
Ubiquitin-Protein Ligases - metabolism
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0035628