RUNDC1 Regulates Proinsulin/Insulin Trafficking and TGN Exit

Abstract

Part 1: The regulated secretion of insulin from pancreatic β-cells is essential for maintaining systemic glucose homeostasis, relying on the highly coordinated sorting and maturation of insulin-containing secretory granules (SGs) at the trans-Golgi network (TGN). However, little information has been obtained about the cytoplasmic regulatory mechanisms controlling the kinetic efficacy of proinsulin export at the TGN. Herein, we have identified RUNDC1, a highly conserved member of the RUN domain family of proteins, as a major factor in regulating proinsulin trafficking in pancreatic β cells. Through RNA interference mediated knockdown of RUNDC1 in INS-1 derived insulinoma lines, we demonstrate that loss of RUNDC1 significantly impairs glucose-stimulated insulin secretion and reduces fractional insulin release while having no effect on either overall intracellular insulin synthesis or steady state levels of stored hormone. The secretory defect seen with loss of RUNDC1 is not due to alterations in cargo processing since the expression, localization and maturation of processing enzymes such as carboxypeptidase E (CPE) and proprotein convertases PC1/3 (PC1/3) are normal. Immunofluorescence studies show that depletion of RUNDC1 results in an accumulation of newly synthesized proinsulin at the TGN. Using a SNAP-tagged based pulse chase imaging technique to monitor in real time the kinetic trafficking patterns of newly synthesized cargo, we found that depletion of RUNDC1 delays the kinetic exit of newly formed proinsulin from the TGN. Our collective data indicate that RUNDC1 functions as a critical molecular traffic controller at the TGN and demonstrates that kinetic efficacy of nascent granule formation is required to support the sustained exocytic response to glucose stimulation. Part 2: The Rab GTPase RAB-2 is a master regulator of secretory cargo sorting and maturation, yet the precise biochemical mechanisms controlling its molecular switch remain poorly defined. In C. elegans, RAB-2 must actively cycle between GTP- and GDP-bound states to facilitate secretory granule (SG) maturation, as both hyperactive and inactive variants phenocopy null mutations. Here, we characterize the biochemical regulation of this cycle by the putative GTPase-activating protein (GAP) TBC-8 and the RAB-2-specific interactor RUND-1. Using in vitro malachite green assays, we provide the first direct biochemical evidence that TBC-8 functions as a potent GAP for RAB-2. We further demonstrate that the TBC-8 TBC domain is structurally sufficient for this activity, forming a stable 1:1 heterodimer with RAB-2 at nanomolar concentrations. We find that RUND-1 serves as a potent inhibitor of TBC-8 GAP activity. Mass photometry confirms that RUND-1 and RAB-2 assemble into complexes, providing a biophysical basis for a model where RUND-1 modulates the accessibility of the GAP to the GTPase. Our results suggest a role for RUND-1 in fine-tuning RAB-2 signaling longevity by shielding it from TBC-8-mediated hydrolysis.