title: Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms.
abstract: Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines.
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, QLD 4072, Australia.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ariotti
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, QLD 4072, Australia.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Hall
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, QLD 4072, Australia.
authors.forename: James
authors.initials: J
authors.lastname: Rae
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, QLD 4072, Australia.
authors.forename: Charles
authors.initials: C
authors.lastname: Ferguson
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, QLD 4072, Australia.
authors.forename: Kerrie-Ann
authors.initials: KA
authors.lastname: McMahon
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, QLD 4072, Australia.
authors.forename: Nick
authors.initials: N
authors.lastname: Martel
authors.affiliation: Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Robyn E
authors.initials: RE
authors.lastname: Webb
authors.affiliation: Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Richard I
authors.initials: RI
authors.lastname: Webb
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, QLD 4072, Australia.
authors.forename: Rohan D
authors.initials: RD
authors.lastname: Teasdale
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, QLD 4072, Australia; Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, QLD 4072, Australia. Electronic address: r.parton@imb.uq.edu.au.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Parton
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000254:growth & development / Q000378:metabolism*; D060387:Ascorbate Peroxidases / Q000378:metabolism*; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods*; D021381:Protein Transport; D013025:Glycine max / Q000201:enzymology; D013347:Subcellular Fractions; D015027:Zebrafish / Q000254:growth & development / Q000378:metabolism*
chemical_list: D049452:Green Fluorescent Proteins; D060387:Ascorbate Peroxidases