title: SNAP-tagging the retrograde route.
abstract: We have developed a chemical biology strategy to identify proteins that follow the retrograde transport route from the plasma membrane to the Golgi apparatus, via endosomes. The general principle is the following: plasma membrane proteins are covalently tagged with a first probe. Only the ones that are then transported to trans-Golgi/TGN membranes are covalently bound to a capture reagent that has been engineered into this compartment. Specifically, the first probe is benzylguanine (BG) that is conjugated onto primary amino groups of plasma-membrane proteins. The capture reagent includes an O(6)-alkylguanine-DNA alkyltransferase-derived fragment, the SNAP-tag, which forms a covalent linkage with BG. The SNAP-tag is fused to the GFP-tagged Golgi membrane anchor from galactosyl transferase for proper targeting to trans-Golgi/TGN membranes. Cell-surface BG-tagged proteins that are transported to trans-Golgi/TGN membranes (i.e., that are retrograde cargoes) are thereby covalently captured by the SNAP-tag fusion protein. For identification, the latter is immunopurified using GFP-Trap, and associated retrograde cargo proteins are identified by mass spectrometry. We here provide a step-by-step protocol of this method.
authors.affiliation: Institut Curie-Centre de Recherche, Trafic, Signaling and Delivery group, Paris Cedex 05, France.
authors.forename: Ludger
authors.initials: L
authors.lastname: Johannes
authors.forename: Massiullah
authors.initials: M
authors.lastname: Shafaq-Zadah
mesh_terms: D004705:Endocytosis; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D022621:Shiga Toxin / Q000096:biosynthesis / Q000235:genetics; D013194:Staining and Labeling
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D022621:Shiga Toxin
keywords: Benzylguanine; Golgi; NHS; PEG; Proteomics; Retrograde transport; SNAP-tag; Shiga toxin