STRUCTURAL ANALYSIS AND ANCHORING MECHANISM OF THE TRANSMEMBRANE EMP24 DOMAIN-CONTAINING PROTEINS

       Conventional protein secretion refers to the process by which proteins are synthesised within the cell and subsequently transported to their final destinations. Following their synthesis in the endoplasmic reticulum (ER), these proteins are packaged into protein-coated transport vesicles (COPs), which mediate the transfer of proteins and lipids between various cellular compartments. Among these, COPII vesicles are specifically responsible for exporting proteins from the ER to the Golgi complex, where they undergo post-translational modifications. At the ER-Golgi interface, the transmembrane emp24 domain-containing protein (TMED) family plays a central role in mediating selective transport events. TMED proteins have emerged as key regulators of vesicular trafficking, functioning as cargo receptors and participating primarily in the early stages of the secretory pathway. Despite their established importance, the precise molecular mechanisms underlying cargo recognition, anchoring, and delivery remain incompletely understood. Within the TMED family, TMED2 and TMED10 are particularly notable, as they are directly involved in the initial assembly of COPI vesicles and in the COPII vesicle-mediated anterograde transport of GPI-anchored proteins. This project aims to produce the human TMED2–TMED10 heterocomplex and investigate its molecular mechanisms. To achieve this, we subcloned constructs for both the co-expression of the two TMED proteins and their individual expression. We are currently optimising expression and purification protocols to enable structural analyses. These will include Circular Dichroism and Size-Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) to characterise their oligomeric states in membrane-like environments, and cryo-electron microscopy (cryo-EM) to resolve their structures at high resolution.