STRUCTURAL ANALYSIS AND INTERACTIONS 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 to Golgi. The Golgi matrix has some membrane proteins that have a potential importance to the correct functioning of Golgi, the transmembrane emp24 domain-containing protein (TMED), was identified as major transmembrane components of vesicles recycling between the ER and Golgi complex. The TMEDs are regulators of vesicular trafficking, functioning as cargo receptors and participating primarily in the early stages of the secretory pathway. 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. It has previously been reported that Golgi reassembly and stacking proteins (GRASPs) are capable of interacting with three members of the TMED family (TMED2, 9, and 10). This interaction is important for their function, a disruption in the binding signal should perturb their localization or transport. In this scenario, some proteins appear at the cell surface even at low expression levels. This project aims to investigate the human TMED2–TMED10 heterocomplex and elucidate the molecular mechanisms. To this end, we generated constructs for both co-expression of the two TMED proteins and their individual expression. The purified proteins were characterized by circular dichroism spectroscopy and size-exclusion chromatography coupled to multi-angle light scattering (SEC–MALS) to assess their secondary structure, oligomeric state, and complex formation. Their interactions with GRASP proteins were also quantified using microscale thermophoresis. Our current efforts are focused on optimizing the expression and purification protocols to obtain homogeneous, high-quality samples suitable for high-resolution structural studies, particularly cryo-electron microscopy.