Reconstitution of transport to recycling endosomes in vitro.

Using an in vitro assay instead of an approach in vivo greatly facilitates the analysis of complex intracellular mechanisms. This becomes particularly important for studying vesicular trafficking in both the endocytic and exocytic pathways with multiple transport routes connecting cellular organelles. Our chapter describes a novel cell-free assay that reconstitutes endosomal transport to recycling endosomes. The method measures transport of transferrin, a marker for endocytosis/recycling, from an endosome-enriched donor fraction to immunoisolated Rab11-positive acceptor recycling endosomes. Transfer of acridinium-labeled transferrin is detected by a highly sensitive chemiluminescence reaction using a luminometer.