Details

Autor(en) / Beteiligte

• Johannes, Ludger
• Shafaq-Zadah, Massiullah

Titel

Chapter 9 - SNAP-Tagging the Retrograde Route

Ist Teil von

• Methods in cell biology, 2013-01, Vol.118, p.139-155

Erscheinungsjahr

2013

Beschreibungen/Notizen

• 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 O6-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.