Liquid liquid phase separation of the intrinsically disordered protein JPT2 compartmentalizes components of NAADP-evoked Ca 2+ signaling

ABSTRACT Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca 2+ -releasing second messenger that activates two-pore channels (TPCs) on endosomes and lysosomes. Rather than binding TPCs directly, NAADP acts through cytoplasmic NAADP-binding proteins (NAADP-BPs) which are essential for endolysosomal Ca 2+ release. Here we characterized the properties of two recombinant, purified NAADP-BPs: Jupiter Microtubule Associated Homolog 2 (JPT2) and like-Sm protein 12 (LSM12). In contrast to LSM12, JPT2 is predicted to be an intrinsically disordered protein, a feature confirmed by circular dichroism and NMR spectroscopy. Under conditions of low Na + concentration or molecular crowding, JPT2 underwent phase separation, as demonstrated by multiple orthogonal approaches. JPT2 condensates displayed liquid-like behavior and efficiently recruited LSM12, a novel fluorescent NAADP analog, as well as tubulin. JPT2 condensates also interacted with polymerized microtubules and lysosomes isolated from human cell lines. These findings reveal an unexpected capability of NAADP-BPs to undergo phase separation, and segregate with components needed for NAADP-dependent Ca 2+ release. We speculate that these signaling condensates dictate cellular NAADP sensitivity, desensitization of NAADP responses, as well as NAADP targeting to TPCs at membrane contact sites between acidic organelles and the endoplasmic reticulum. Graphical Abstract