Project Summary Accurate protein transport in the secretory pathway is vital for cell function and growth. Our research program is focused on coat-dependent sorting mechanisms that drive protein trafficking between the endoplasmic reticulum (ER) and Golgi complex. Nascent secretory proteins are translated at the ER and then fully folded proteins are selectively packaged into COPII coated vesicles for anterograde transport to the Golgi complex. This forward pathway is balanced by retrograde transport from the Golgi, which selectively returns proteins to the ER in COPI coated vesicles. To ensure delivery of only folded secretory proteins, a process known as ER quality control largely retains nascent proteins in the ER until correctly folded or targets terminally misfolded proteins for degradation. The coordinated mechanisms that maintain organelle identity, advance folded biosynthetic cargo and retain misfolded proteins are not well understood. We identified a set of transmembrane cargo receptors that function in coat-dependent sorting and quality control in the early secretory pathway. This research plan will address key questions on how cargo receptors recognize their clients and catalyze net directional traffic of proteins. In the past funding period, we characterized the Erv41-Erv46 complex as a retrograde receptor that retrieves ER-resident proteins through the activity of a conserved thioredoxin-like domain in Erv46. Our recent findings indicate the Erv41-Erv46 receptor also retrieves misfolded secretory proteins that have exited the ER. The specific aims of this proposal will test the molecular model that the Erv46 subunit recognizes hydrophobic features displayed by escaped cargo in the low pH environment of Golgi compartments. After return to the ER, redox activity on the Erv46 thioredoxin-like domain releases bound cargo from the receptor at neutral pH. Through these mechanisms, pH and redox gradients drive efficient protein sorting in the early secretory pathway. This model will be tested in the following experimental aims. Aim 1: Define the molecular mechanisms of Erv41- Erv46 cargo binding at reduced pH of the Golgi compartment. Aim 2: Determine the mechanism by which cargo is efficiently released from Erv41-Erv46 in the ER. Aim 3: Test the model that Erv41-Erv46 recognizes and binds directly to misfolded cargo for active retrieval to the ER. We will rigorously test our models by exploiting iterative genetic, cellular and biochemical approaches to monitor protein function in vivo, in cell free assays and in reconstitution experiments with purified factors. Defining the molecular mechanisms that underlie conserved protein sorting processes will provide fundamental insights on cellular organization and contribute to treatments for human diseases connected to secretory pathway function.

Project Narrative More than one-quarter of translated proteins enter the secretory pathway. Our research is designed to elucidate sorting mechanisms that guide a broad range of secretory proteins through the early stages of this pathway. Defects in biogenesis of secretory proteins have wide-ranging effects on human health and disease including atherosclerosis, blood clotting disorders, kidney disease and cystic fibrosis.