Project summary Protein secretion plays a central role in developing and maintaining multicellular organisms. Specialized cell types in tissues secrete proteins by regulated or constitutive secretion. Regulated secretion occurs in response to an extracellular stimulus that elicits the release of signaling molecules, while constitutive secretion facilitates the deposition of extracellular matrix components that provide tissue integrity. Even though these processes are highly significant for human health, features that determine whether a protein is secreted by regulated or constrictive secretion remain unknown. A central regulator of intracellular protein distribution is the trans-Golgi Network (TGN), which sorts and packages secretory proteins into specific vesicular carriers targeting them to intracellular storage granules (regulated secretion) or the cell surface (constitutive secretion). The identification of the mannose-6-phosphate receptor (M6P-R) that recognizes M6P tags of lysosomal led to the idea that specific sorting receptors also sort secretory proteins. However, conserved recognition signals or cargo receptors remain unknown. How are these molecules recognized and sorted for targeting the correct destination? The concept of concentrating macromolecules into biomolecular condensates by liquid-liquid phase separation (LLPS) has revolutionized modern cell biology. Human cells use this principle to organize biochemical processes spatially without a membrane. Our recent research raises the novel possibility that the segregation of secretory proteins in the TGN lumen follows this concept. Our work has shown that purified chromogranins (CGs) or Cab45 undergo liquid-liquid phase separation (LLPS) in the milieu of TGN. Both proteins have been suggested to co-aggregate with secreted proteins (clients) to facilitate their sorting and packaging. We show that CG or Cab45 liquids, not solid aggregates, are essential for client sorting and packaging. Nonetheless, the underlying mechanisms of LLPS-dependent client packaging remain elusive. Therefore, our long-term goal is to understand the molecular basis of LLPS-dependent cargo sorting for regulated (by CGs) and constitutive (by Cab45) secretion. Our proposal aims at identifying the mechanisms of LLPS-dependent sorting in reconstituted systems that recreate the milieu of the TGN lumen. We will include model membranes to examine if and how these condensates associate with the luminal leaflet of the TGN. We will use cell culture models of regulated (P12 cells) or constitutive (skin fibroblasts) secretion to validate our in-vitro results in living cells. Our concept will establish the molecular requirements for condensate formation, the mechanisms of client recognition and vesicular formation in regulated and constitutive secretion. These results will provide a fundamental understanding of an exciting new paradigm in cell biology and impact the research of pathologies caused by defective protein secre...