title: Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.
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.
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard MC 156-29, Pasadena, CA 91125, USA.
authors.forename: Claire N
authors.initials: CN
authors.lastname: Bedbrook
authors.affiliation: 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.
authors.forename: Mihoko
authors.initials: M
authors.lastname: Kato
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard MC 156-29, Pasadena, CA 91125, USA.
authors.forename: Sripriya
authors.initials: S
authors.lastname: Ravindra Kumar
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard MC 156-29, Pasadena, CA 91125, USA.
authors.forename: Anupama
authors.initials: A
authors.lastname: Lakshmanan
authors.affiliation: 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.
authors.forename: Ravi D
authors.initials: RD
authors.lastname: Nath
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Sun
authors.affiliation: 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.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Sternberg
authors.affiliation: 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.
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
authors.affiliation: 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.
authors.forename: Viviana
authors.initials: V
authors.lastname: Gradinaru
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D014162:Transfection
chemical_list: D018829:Adhesins, Bacterial; D002352:Carrier Proteins; C470631:FbaB protein, Streptococcus pyogenes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins