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.
The regulated release of proteins, such as peptide hormones, neuropeptides, and many growth factors, in response to physiologically appropriate signals depends on their sorting to a type of secretory vesicles capable of regulated exocytosis. This proposal aims at determining the molecular mechanisms controlling the biogenesis of these vesicles and its significance for regulated release. This work will lead to a better understanding of a cell biological process crucial for physiology, behavior, and development.
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