Direct measurement of ATP13A2 polyamine-dependent ATPase activity following rapid purification of lysosomes

The P5B family P-type ATPase ATP13A2(PARK9) is a bona fide polyamine transporter resident in the endolysosomal compartment where it mediates the import of endocytosed polyamines from the lysosome lumen into the cytosol. Dysfunction of ATP13A2 can negatively impact cellular survival and genetic aberrations its coding gene are linked to a number of neurodegenerative disorders with devastating consequences. While there has been much progress in its structural characterization in vitro, our understanding of ATP13A2′s mechanism of action and regulation in a native lysosomal setting remains incomplete. Here we describe our approach to measure the polyamine-dependent ATPase activity of lysosomal ATP13A2 following our newly developed method to rapidly capture and purify lysosomes from mammalian cells. This strategy enables the targeted functional interrogation of the lysosome-localized population of ATP13A2 specifically.