Roles of O-fucosyltransferase 1 and O-linked fucose in notch receptor function

O-fucosyltransferase 1 (Ofut1) is a soluble endoplasmic reticulum protein that directly transfers fucose onto serine or threonine residues of EGF domain-containing proteins such as Notch receptors. Genetic analysis indicates that Ofut1 is essential for Notch receptor activation. To explore the molecular basis for the absolute requirement of Ofut1 for Notch function, biochemical and cell biological approaches were used. Ligand-binding assay revealed that Ofut1 is essential for Notch receptors to physically interact with their ligands. In addition, secretion assay and cell surface staining showed that secretion of Notch receptors is impaired in OFUT1-depleted cells, indicating that the structure of Notch receptors is altered. Interestingly, promotion of Notch secretion by OFUT1 does not require its enzyme activity. Together with the fact that OFUT1 physically associates with Notch and that OFUT1 prevents misfolding of Notch mutants, it is proposed that OFUT1 acts as a chaperone that promotes the folding of the EGF repeat of Notch receptors. This chapter focuses on the methods used to analyze the roles of Ofut1 in Notch receptor structure and function.