Methods for analysis of AP-3/Rabin4' in regulation of lysosome distribution

The position of lysosomes in the cytoplasm is important for their ability to fuse with the plasma membrane and release of proteases that are involved in tissue remodeling. Motor-directed bidirectional transport along microtubules is a critical process determining the distribution of lysosomes. How lysosomes are tethered to microtubules is incompletely understood, but a role for small GTPases of rab and arl families has been documented. We recently found that the rab5 and rab4 effector rabip4' interacts with the adaptor complex AP-3 in a rab4-dependent manner on tubular endosomes. We here describe the assays that led to the identification of AP-3 as a rabip4' partner and the role of the complex in regulating the spatial distribution of lysosomes.