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Title: RFP tags for labeling secretory pathway proteins

Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.
Authors:
;  [1] ; ;  [2] ;  [3] ;  [1] ;  [3]
  1. State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China)
  2. College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China)
  3. Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)
Publication Date:
OSTI Identifier:
22416427
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 447; Journal Issue: 3; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BRIGHTNESS; COLOR; DISSOCIATION; EVALUATION; FLUORESCENCE; LABELLING; LYSOSOMES; MEMBRANE PROTEINS