How Evolution Tunes Stress-Triggered Protein Phase Separation to Promote Cell Fitness During Stress

Abstract

In eukaryotic cells, a variety of stresses trigger the coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these in vitro phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in budding yeast), a defining marker of stress granules, phase-separates and forms hydrogels in vitro during physiological heat stress. Low-complexity regions (LCRs) are thought to cause demixing, yet Pab1's proline-rich LCR tunes rather than causes phase separation. Exploiting evolutionary patterns, we create LCR mutations which systematically alter its biophysical properties and phase separation in vitro and in vivo. Mutations which impede Pab1's heat-triggered phase separation reduce the organism's ability to survive prolonged heat stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely demarcate stress onset, a broadly generalizable mechanism.