EXPLORING THE PHASE SEPARATION AND MEMBRANE INTERACTION OF HUMAN GOLGI REASSEMBLY AND STACKING PROTEINS

Recent studies have associated the Golgi Reassembly and Stacking Proteins family (GRASPs) with vesicular and unconventional protein secretion pathways. In performing these functions, GRASPs interact with membranes in some manner. The presence of lipid modifications, such as myristoylation, is a crucial factor to consider when investigating the interaction processes between GRASPs and membranes. Here, we describe a reconstitution protocol for the myristoylated human GRASP65 and GRASP55 with model lipid membranes, employing a combination of techniques ranging from structural characterization to the study of lipid-bound GRASPs and their interactions with biological membrane models using spectroscopy and microscopy. In the context of supramolecular arrangement formation, such as phase separation, we investigated the capacity of human GRASPs to undergo phase separation under various conditions using a microfluidic system called Phase Scan, which enables high-throughput analysis of biomolecular condensates. We observed that the phase separation propensity of GRASP55 can be modulated by mimicking phosphorylated residues at the SPR domain and through its interaction with Golgin45, which has previously been shown to interact with GRASP55 at the Golgi apparatus. Furthermore, we explored the effects of the phase separated GRASP55 in contact with giant unilamellar vesicles (GUVs) and examined the properties of the biomolecular condensates inside HeLa cells.