The highly conserved OST3 proteins have been implicated in the disparate functions of regulating protein glycosylation and transporting magnesium. This project will use integrated genetics, biochemistry and omics to decipher these moonlighting roles. We will detail the role of OST3 proteins in regulating mammalian glycosylation and reconstruct the vertebrate co-evolutionary trajectory of OST3 protein:substrate interactions. We will also identify and characterise the regulation, mechanisms and metabolic consequences of OST3 protein-mediated magnesium transport. These outcomes will provide fundamental insights into eukaryotic biology, and allow advances in engineered systems for glycoprotein production and modulating cellular metabolism.