The secretory pathway employs vesicle transport to provide a linear pathway for export of cellular products and distribution of membrane and organelle components throughout the cytoplasm. Many diseases, including neurodegeneration, involve disruption of the biosynthetic secretory pathway and unresolved secretory stress--making it essential to understand how secretion is up- and down-regulated under different physiological conditions. While the basic engines of vesicle budding, docking and fusion have been identified, little is known of how they are tuned to respond to physiological conditions and stresses. In mammals, ER-to-Golgi transport, which represents the rate-limiting step in the secretory pathway and the step most relevant to transport-related diseases, has been extensively characterized in vivo and reconstituted in vitro. In broad terms, ER-to-Golgi transport has been shown to comprise: 1) cargo sorting and vesicle budding mediated by the COPII vesicle coat; 2) homotypic COPII vesicle tethering and fusion mediated by tethers and SNAREs to form pre-Golgi organelles called vesicular tubular clusters (VTCs); and 3) VTC-mediated cargo sorting and transport along microtubules leading to fusion with the Golgi. Little is known about how these processes are adjusted dynamically to match secretory output rates with the needs to enforce secretory quality control, avoid ER stress, and keep pace with secretory protein biogenesis and cell growth. One key aspect to regulation of ER-to-Golgi transport that has become apparent in recent years is the role of ER luminal calcium. Calcium, when released from the ER by channel proteins appears to interact with penta-EF hand proteins (PEFs) in the cytoplasm that bind to the COPII coat and modulate its assembly and the rate of cargo egress from the ER. However, the mechanisms of these proteins to produce different secretory outcomes and how they are integrated with ER calcium homeostasis and calcium channels are not understood. This project will employ kinetic assays of ER-to-Golgi transport in intact mammalian cell lines, live-cell calcium measurements, and in vitro studies of PEF protein interactions with vesicle machinery to elucidate how how calcium channels, PEF proteins, and COPII components are integrated to dynamically regulate secretion rate. These studies will have wide significance because proper regulation of secretion is fundamental to key cell survival and stress pathways.
Multiple diseases, including severe neuropathies and neurodegeneration, result from improper trafficking of secretory proteins in ER-to-Golgi transport. Vesicular tubular clusters (VTCs), a key sorting and transport organelle formed by secretion from the ER, have recently been implicated in the biogenesis of viral particles including SARS and poliovirus. The human bleeding disorder combined deficiency of coagulation factor V and VIII (F5F8D) is caused by failure of a luminal Ca2+-dependent sorting event that occurs in ER exit sites and VTCs, and Hailey-Hailey disease is caused by disruption of unknown luminal Ca2+-dependent events in the early secretory pathway. A firm mechanistic understanding of membrane trafficking and its regulation by luminal Ca2+ during ER-to-Golgi transport is central to an understanding of many transport-related pathologies.
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