Role of calcium dependent RabGAPs in endocytic trafficking pathways - 1 Abstract. Role of Calcium-dependent RabGAPs in membrane trafficking 2 Membrane trafficking pathways are regulated by a family of Rab GTPases that act as molecular switches 3 based on their GDP/GTP status. Rab-GDP is the inactive cytosolic form, while Rab-GTP associates with 4 membranes and recruits effectors to mediate trafficking events. The membrane-bound active Rab-GTP must 5 interact with a GTPase-activating protein (RabGAP) to hydrolyze its GTP, thereby terminating its activity and 6 allowing dissociation from membranes. Because RabGAPs control Rab inactivation, they ensure the precise 7 timing, directionality, and specificity of vesicle transport, coordinate sequential trafficking steps, and maintain 8 organelle identity and homeostasis. Perturbations in RabGAP expression, localization, and/or activity disrupt 9 intracellular trafficking pathways and contribute to a broad spectrum of human diseases, including nephrotic 10 syndrome, neurodegeneration, and cancer. RabGAPs are characterized by a highly conserved catalytic Tre2– 11 Bub2–Cdc16 (TBC) GAP domain. A distinct subfamily, consisting of TBC1D8, TBC1D8B, TBC1D9, and 12 TBC1D9B, contains an EF-hand calcium-sensing domain, suggesting that these EF-RabGAPs may respond to 13 Ca²⁺ signaling. In exciting preliminary studies, we discovered that the EF-RabGAP TBC1D9 localizes to 14 endolysosomal compartments known to contain high luminal Ca²⁺, binds Ca²⁺ through its EF-hand domain to 15 undergo a conformational shift, and functions as a Ca²⁺-dependent GAP to deactivate the endolysosomal 16 Rab12. Based on these results, we posit a central hypothesis that TBC1D9 (and possibly other EF-RabGAPs) 17 responds to Ca²⁺ signals originating from intracellular stores high in luminal Ca²⁺ to deactivate Rabs, thereby 18 ensuring Rab dissociation from membranes and terminating Rab-mediated downstream trafficking events. This 19 central hypothesis establishes a novel regulatory paradigm in which EF-RabGAPs link Ca²⁺ signaling to Rab- 20 mediated membrane trafficking.I will test this hypothesis by focusing on TBC1D9 to address three key 21 questions: 1) which Rabs are controlled by TBC1D9 in a Ca²⁺-dependent manner and which steps of 22 endolysosomal traffic TBC1D9 regulates; 2) whether TBC1D9 responds to Ca²⁺ released from endolysosomal 23 compartments to regulate Rabs; and 3) the mechanism by which TBC1D9 is activated by Ca²⁺. I will combine 24 biochemical and functional in vitro and in vivo assays, proximity labeling, high-resolution microscopy, cross- 25 linking mass spectrometry, and hydrogen-deuterium exchange to elucidate how TBC1D9 integrates Ca²⁺ 26 signaling with membrane trafficking to sustain cellular homeostasis. This research will significantly enhance our 27 understanding of the spatial and temporal control of membrane trafficking by EF-RabGAPs. Moreover, 28 because EF-RabGAPs are implicated in various diseases, uncovering how dysregulated RabGAP activity 29 contributes to disease pathogenesis may inform the development of novel therapeutic strategies.
