Summary The vast majority of the ~7,000 proteins that traffic through the mammalian secretory are modified by one or more glycans. These alterations include the modifications of Asn (N-linked) and Ser/Thr (O-linked) residues. Carbohydrates appended to proteins can assist with protein folding, quality control and trafficking, or control their activity and function. In this proposal, we will study the mechanism and role of the addition of the hexose epimers of glucose and mannose in the endoplasmic reticulum (ER). Protein maturation is monitored by a quality control process that evaluates the structural integrity of maturing nascent chains, and permits the passage of properly folded proteins. Alternatively, non-native proteins are marked for ER retention so that the defect can be repaired, or if irreparable, targeted for degradation. Calnexin and calreticulin are ER carbohydrate binding molecular chaperones that direct the folding and trafficking of secretory pathway cargo by selectively binding to monoglucosylated side chains on maturing proteins. Therefore, to a large extent the glucosylation state of a glycoprotein controls its flow proteins in the secretory pathway. The glucosylation state is controlled by the UGGT family members UGGT1 and UGGT2 that appear to selectively modify immature or non-native clients to support persistent chaperone binding. These soluble UGGTs transfer glucose from UDP-glucose to maturing cargo in the early secretory pathway. In the first two aims of this proposal, we will test the hypothesis that the UGGTs are central quality control gatekeepers that control the flux of proteins through the ER by examining their specificity and the role of reglucosylation in the cell. The hexose epimer mannose is also added to proteins in the ER. In contrast to reglucosylation, mannosylation involves the transfer of mannose by a membrane embedded transferase from a dolichol-P- precursor directly to Ser/Thr residues to form an O-glycosidic bond. There are two families of putative O- mannosyltransferases that reside in the ER membrane in mammalian cells, the POMTs (POMT1 and 2) and the recently discovered TMTCs (TMTC1-4). Little is known about their mechanisms of action and the function of adding a mannose to maturing proteins in the ER. We have recently found that TMTC3 is involved in the O-mannosylation of E-cadherin and that E-cadherin’s O-mannosylation aids in cell adhesion and neurodevelopment. Mutations in TMTC3 are associated with the neurodevelopment diseases, underscoring the biological significance of this post-translational modification. Specific aim 3 is to understand the mechanism and significance of O-mannosylation in the ER. The long-term goal of this project is to understand the process of hexose addition including the substrate selection and modification steps, and how these post-translational modifications control the trafficking and functions of proteins.