Cedric Asensio, PhD - US grants

? DESCRIPTION (provided by applicant): The ability to release signaling molecules, such as peptide hormones, neuropeptides, and many growth factors, in response to an appropriate extracellular stimulus, is central to physiology, behavior, and development. The secretory vesicles mediating this regulated secretion are called secretory granules or large dense core vesicles (LDCVs). They form at the trans-Golgi network (TGN) where their soluble cargo aggregates to create a dense core, but the cellular mechanisms, and in particular, the cytosolic machinery that produces these secretory vesicles is not well understood. Recently, we have performed an RNAi library screen and identified the adaptor protein AP-3 and VPS41 as part of the first cytosolic components that are necessary for biogenesis of LDCVs. In mammalian cells, loss of AP-3 leads to defects in neuroendocrine secretion. Vesicles with a dense core can still form, but they show altered size, morphology, and protein composition. In particular, we found that proteins required for regulated exocytosis such as synaptotagmin are redistributed away from LDCVs. More recently, we also found that loss of VPS41 dysregulates neuroendocrine secretion and leads to very similar defects in LDCV formation. VPS41 has previously been implicated in delivery of proteins to the lysosome as a subunit of the homotypic fusion and protein sorting (HOPS) tethering complex, but we found that VPS41 contributes to LDCV formation independently of HOPS. In addition, VPS41 interacts genetically and biochemically with AP-3, and this interaction is required for regulated secretion. We also observed that recombinant VPS41 can form clathrin-like lattice in vitro, and this depends on the presence of a clathrin-heavy chain repeat (CHCR) in its C-terminus. This motif is also required for regulated secretion. Our work thus suggests that AP-3 recruits and concentrates specific transmembrane proteins onto LDCVs, and that VPS41 might function as a coat protein for AP-3, but we still do not understand how these components are regulated, how they cooperate in the cell, and how they influence the properties of regulated release. To better understand the molecular mechanisms that enable the formation of LDCVs, we will now 1) determine how VPS41 influences LDCV formation using a new biochemical assay to monitor membrane protein sorting at the TGN, 2) determine in vitro the mechanisms controlling the polymerization of VPS41 into a lattice, and its significance for LDCV biogenesis, and 3) identify how VPS41 can function independently of the HOPS complex. Extending on previous work, these studies will provide invaluable information about the biology and biochemistry of a novel self-assembling protein, and will serve as a framework for future studies to explore its significance for normal physiology and disease.

Abstract The ability to release signaling molecules, such as peptide hormones, neuropeptides, and many growth factors, in response to an appropriate extracellular stimulus, is central to physiology, behavior, and development. The secretory vesicles mediating this regulated secretion are called large dense core vesicles (LDCVs). They bud from the trans-Golgi network (TGN) where their soluble cargo aggregates to form a dense core, but the cellular mechanisms, and in particular, the cytosolic machinery that produces these secretory vesicles are still not well understood. Recent published studies in C. Elegans have identified HID-1 as a cytosolic factor involved in neuropeptide sorting, but its function remains unknown. HID-1 is a peripheral membrane protein associated with the TGN and its expression is restricted to neuroendocrine cells, suggesting that it might contribute to LDCV biogenesis. Our preliminary loss-of-function experiments with rat neuroendocrine HID-1 knockout (KO) cells generated by genome-editing have confirmed that HID-1 is indeed required for mammalian neuroendocrine secretion. We will now 1) determine how HID-1 contributes to LDCV formation using a combination of biochemical and imaging assays, 2) establish how HID-1 targets specifically to the TGN, and 3) identify the mechanism regulating the reversible binding of HID-1 to the TGN and assess its significance for LDCV formation. These studies will provide invaluable information about the cell biology and biochemistry of a novel peripheral membrane protein involved in membrane trafficking with important implications for our understanding of neuroendocrine secretion.