Phase separation as a concentrating mechanism of protein/peptide hormones for the secretory granule storage
Protein/peptide hormones in the regulated secretory pathway are stored within secretory granules (SGs) as densely packed aggregates for extended periods of time until their stimulated release. These aggregated dense cores are reported as functional amyloids for several pituitary hormones. However, the molecular events initiating hormone aggregation and SG biogenesis remain poorly understood. Using a diverse set of protein/peptide hormones with different sizes and structures, we demonstrated that all these hormones undergo phase separation in trans-Golgi network (TGN)-relevant conditions (including low pH, the presence of glycosaminoglycans, etc.), which rapidly transitioned into solid-like structures and eventually form amyloid fibrils. Further, the cellular model expressing two protein hormones (GH-EGFP and PRL-EGFP) showed the highly dynamic, liquid-like condensate formation at the TGN volume, which was transported via microtubule to the cellular periphery/tips and formed a ready pool of mature SGs containing amyloid-rich hormone aggregates. Importantly, both in vitro and cellular studies confirmed that solid-like amyloid aggregates are reversible and can release bioactive monomeric hormones, recapitulating regulated exocytosis. Together, our findings suggest hormone phase separation at the TGN as a common molecular principle for concentrating cargo, driving rapid functional aggregation, and ensuring efficient storage within SGs.