Abstract 2832 RAB-2 GTPase regulation by TBC-8 and RUND-1

Neuronal and endocrine cells store and secrete molecular cargos like neurotransmitters and metabolic hormones through the regulated secretory pathway.Dense-core vesicles (DCVs) originate from the trans-Golgi network and undergo a maturation process involving peptide processing and cargo sorting before being stimulated to release their cargos outside the cell.Dysregulation of this process leads to a wide range of neurological and metabolic disorders like depression, schizophrenia, substance use disorder, and diabetes.Despite the critical importance of this process, the molecular mechanisms underpinning it remain poorly understood.Vesicular traffic are largely coordinated by the Rab family of small GTPase proteins.Previous work identified the conserved proteins TBC-8 and RUND-1 as regulators of DCV maturation in Caenorhabditis elegans; and both proteins bind active GTP-bound RAB-2.TBC-8 is the putative RAB-2 GTPase activating protein (GAP) which promotes conversion of GTP-RAB-2 into GDP-RAB-2, thereby inactivating the Rab.RUND-1 interacts with both active GTP-RAB-2 and TBC-8, yet its precise function remains unknown.This study aims to characterize the biochemical function of TBC-8 and RUND-1 in regulating RAB-2 activity.Using purified proteins, we demonstrate that TBC-8 greatly promotes RAB-2 GTP hydrolysis, indicating it is the bona fide RAB-2 GAP.Additionally, we show that RUND-1 strongly inhibits TBC-8-stimulated RAB-2 GTP hydrolysis, suggesting RUND-1 may compete with TBC-8 for RAB-2 binding.Given this interplay between RUND-1 and TBC-8 in binding RAB-2, we hypothesize that RAB-2 exhibits exclusively pairwise interactions with its partners.To test this, we will use mass photometry to study whether RUND-1 and TBC-8 can bind RAB-2 simultaneously or if one complex is preferentially formed.Based on our current findings, we propose a model where TBC-8 promotes RAB-2 inactivation by stimulating GTP hydrolysis and RUND-1 blocks RAB-2 inactivation by TBC-8, prolonging the activate state of RAB-2.Future research involves generating RUND-1 point mutants to disrupt the RUND-1:RAB-2 interaction and explore the consequences on C. elegans phenotype due to loss of this complex.