The potential role of liquid-liquid phase separation in the cellular fate of the compartments for unconventional protein secretion

Eukaryotic cells have developed sophisticated mechanisms for biomolecule transport, particularly under stress conditions. This study focuses on the unconventional protein secretion (UPS) pathways activated during starvation stress, facilitating the export of soluble leaderless proteins. Specifically, we examine the role of the GRASP family, which is crucial in these processes but has largely unexplored mechanisms, especially regarding the biogenesis and cargo recruitment for the vesicular-like compartment for UPS. Our results reveal that under the circumstances mimicking starvation conditions in yeast, liquid-liquid phase separation (LLPS) is instrumental in the self-association of Grh1, the yeast homologue of GRASP. This association is likely a precursor to the biogenesis of the so-called Compartment for Unconventional Protein Secretion. We demonstrate that Grh1’s self-association involves complex interactions and is regulated by electrostatic, hydrophobic, and hydrogen-bonding forces. Importantly, our study shows that phase-separated states of Grh1 can recruit UPS cargo under starvation-like situations, a significant finding given the minimal interaction between these proteins under normal conditions. Additionally, we address how droplet ageing might affect the ability of cells to return to normal post-stress states. Our findings can potentially enhance our understanding of intracellular protein dynamics and their adaptive responses to stress in eukaryotic cells.