Arabidopsis endomembrane enrichments: Affinity enrichment of RABF1/ARA6-RFP

Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi Network (TGN), Early Endosomes (EE), secretory vesicles, Late Endosomes (LE), Multivesicular Bodies (MVB) compartments and tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localises to the Golgi, Clathrin Light Chain 2 (CLC2) labelling Clathrin-coated vesicles and pits and the Vesicle Associated Membrane Protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings.