title: SNAP-tag based proteomics approach for the study of the retrograde route.
abstract: Proteomics is a powerful technique for protein identification at large scales. A number of proteomics approaches have been developed to study the steady state composition of intracellular compartments. Here, we report a novel vectorial proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans-Golgi network (TGN). This strategy is based on the covalent modification of the plasma membrane proteome with a membrane impermeable benzylguanine derivative. Benzylguanine-tagged plasma membrane proteins that are subsequently targeted to the retrograde route are covalently captured by a TGN-localized SNAP-tagged fusion protein, which allows for their identification. The approach was validated step-by-step using a well explored retrograde cargo protein, the B-subunit of Shiga toxin. It was then extended to the proteomics format. Among other hits we found one of the historically first identified cargo proteins that undergo retrograde transport, which further validated our approach. Most of the other hits were kinases, receptors or transporters. In conclusion, we have pioneered a vectorial proteomics approach that complements traditional methods for the study of retrograde protein trafficking. This approach is of generic nature and could in principle be extended to other endocytic pathways.
authors.affiliation: Traffic, Signaling, and Delivery Laboratory, Institut Curie-Centre de Recherche, 26 Rue d'Ulm, 75248 Paris Cedex 05, France.
authors.forename: Getao
authors.initials: G
authors.lastname: Shi
authors.forename: Michel
authors.initials: M
authors.lastname: Azoulay
authors.forename: Florent
authors.initials: F
authors.lastname: Dingli
authors.forename: Christophe
authors.initials: C
authors.lastname: Lamaze
authors.forename: Damarys
authors.initials: D
authors.lastname: Loew
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Florent
authors.forename: Ludger
authors.initials: L
authors.lastname: Johannes
mesh_terms: D000818:Animals; D004705:Endocytosis / Q000502:physiology; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D021381:Protein Transport; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D011990:Receptors, Transferrin / Q000032:analysis; D050600:SNARE Proteins / Q000235:genetics; D022621:Shiga Toxin / Q000032:analysis
chemical_list: D008565:Membrane Proteins; D020543:Proteome; D011990:Receptors, Transferrin; D050600:SNARE Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D022621:Shiga Toxin