The fidelity of the protein maturation steps in the endoplasmic reticulum (ER) is monitored by a quality control process that interrogates the structural integrity of the protein byproducts, and allows properly folded proteins to pass further through the secretory pathway. In contrast, non-native proteins are targeted for ER retention so that the aberration can be repaired, or if irreparable, selected for degradation and the recycling of components. A well-organized network of macromolecular complexes controls the processes of the ER. The carbohydrate binding (or lectin) chaperones, calnexin and calreticulin, direct the folding and trafficking of secretory pathway cargo by selectively binding to monoglucosylated side chains on maturing proteins in a region-specific manner. These chaperones can control the trajectory of the folding reaction and the flow of proteins through the secretory pathway. Therefore, the proper maturation and flux of the enormously diverse range of proteins that passage through the ER is in large part controlled by their glucosylation state. How these critical tags are added in the natural setting of the ER is an outstanding question that is currently poorly understood. The glucosylation status of ER-trafficked proteins is controlled by uridine diphosphate-glucose (UDP-Glc): glycoprotein glucosyltransferase 1 (UGGT1), which selectively modifies unglucosylated immature and non-native clients to support persistent chaperone binding and ER retention. Our main hypothesis is that UGGT1 serves as the central quality control gatekeeper that controls folding and the passage of thousands of substrates in the mammalian secretory pathway. Although UGGT1 has been studied extensively in isolation using purified and engineered components, little is known about it, and its homologue (UGGT2), activity in live cells. The focus of this proposal is to understand the mechanism of action and the roles of the UGGT proteins in their natural environment, the ER lumen. The long-term goal of this project is to understand the mechanism by which the quality control system aids the folding of complex proteins and evaluates the fidelity of the maturation process to regulate trafficking through the secretory pathway. The studies proposed in three aims will provide a deeper understanding of how the UGGTs select cargo for modification and how they control their flux through the secretory pathway (aim 1). These studies will include how clients are extracted from the lectin chaperone binding cycle and selected for destruction (aim 2), and how the quality control machinery is organized within the ER to maintain cellular homeostasis (aim 3).
Successful completion of this project will lead to a better understanding of the fundamental mechanism of protein quality control, and have a potential impact on the development of therapeutic strategies directed towards the growing number of conformational diseases.
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