PROJECT SUMMARY 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 is initiated at ER exit sites (ERES) and 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 at ER exit sites 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 microscopy of protein dynamics at ER exit sites to learn 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 cell function and cell survival during physiological stresses.