Demixing of simultaneously co-expressed four phase-separating proteins in the endoplasmic reticulum lumen

Intracellular protein crystallization represents an intriguing form of biomolecular self-assembly. While the list of such crystallizing proteins is growing and their physiological roles are beginning to be elucidated, the underlying requirements and processes for intracellular protein crystallogenesis remain largely unknown. This study examines how simultaneously co-expressed phase-separating proteins influence each others phase-separation event in the endoplasmic reticulum (ER) lumen by using four cargoes selected based on their ability to produce distinctive crystals and droplet inclusion bodies. The co-expressed model proteins independently reached their respective threshold concentrations and spontaneously phase-separated into inclusions in the ER without losing their signature morphologic characteristics. The fact that protein crystals and droplets continued to grow in size over time suggests that nascent cargo proteins were continuously synthesized and folded in the ER to fuel the growth of corresponding inclusion bodies. Namely, despite the highly crowded molecular environment, overexpressed cargoes find their mates by self-association and propagate into microscale structures in the ER. This study demonstrates that cells can accommodate up to four distinct phase separation events simultaneously in the ER lumen, and the phase separation events proceed without interfering with each other and without morphological or content mixing.