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Chem Biol. 2015 Aug 20;22(8):1108-21. doi: 10.1016/j.chembiol.2015.06.020. Epub 2015 Jul 23.

Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

Author information

1
Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard MC 156-29, Pasadena, CA 91125, USA.
2
Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard MC 156-29, Pasadena, CA 91125, USA; Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA.
3
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
4
Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard MC 156-29, Pasadena, CA 91125, USA; Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
5
Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard MC 156-29, Pasadena, CA 91125, USA. Electronic address: viviana@caltech.edu.

Abstract

Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering.

PMID:
26211362
PMCID:
PMC4546540
DOI:
10.1016/j.chembiol.2015.06.020
[Indexed for MEDLINE]
Free PMC Article

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