PROJECT SUMMARY Most newly synthesized proteins destined for the cell surface or secretion must transit through the endoplasmic reticulum (ER) for folding and post-translational processing. Grp94, the ER resident member of the hsp90 family of chaperones, is required for the maturation of various membrane and secreted proteins. Grp94's control over the fate of these diverse clients makes it an emerging therapeutic target for a number of chronic diseases including type 2 diabetes, familial hypercholesterolemia, and sepsis, as well as cancers such as HER2-positive breast cancer and multiple myeloma. An unanswered question is how Grp94 adapts to different needs within a dynamic ER folding environment to ensure efficient client capture and folding. Despite being the most abundant protein in the ER, Grp94 is even further upregulated under conditions of cell stress or disease. The increased overexpression of Grp94 correlates with its hyperglycosylation, the utilization of the five minor glycosylation sites that are sparingly occupied in nonstressed cells and tissues. Recent studies have shown that hyperglycosylation imparts new functions to Grp94 that are essential for the successful response to stress or maintenance of the pathological state. Our central hypothesis is that differential glycosylation serves as a tuning mechanism that alters the functional status of Grp94. A systematic analysis of the effects of glycosylation on Grp94 has been hampered by the difficulty in producing site-specific, homogeneous glycoproteins that would allow for the clear interpretation of functional and biochemical assays. Here we propose to study how the activity of Grp94 is regulated by glycosylation and to visualize the capture of a client protein. We will make use of a novel in-cell expression system developed in our lab that allows us to selectively control the glycosylation patterns of Grp94 at all available sequons. We will employ these defined glycosylated species in both in vivo and in vitro Grp94-specific assays that allows for the interrogation of Grp94 activity as a function of attached glycans. Combined with structural and biochemical tools, we expect to determine how glycosylation regulates the structure and function of Grp94 and how a client protein is captured by the chaperone.