GRASP55 PHASE SEPARATION COUPLES CELLULAR STRESS TO MEMBRANE REMODELING

Golgi Reassembly and Stacking Proteins (GRASPs) are peripheral membrane proteins involved in Golgi organization and unconventional protein secretion (UPS), but the molecular mechanisms linking their stress-responsive behavior to membrane remodeling remain poorly understood. Here, we investigated the phase-separation properties and membrane interactions of human GRASP55 and GRASP65 using complementary biochemical, biophysical, and fluorescence imaging approaches. In living HeLa cells, GRASP55 formed dynamic condensate-like assemblies upon nutrient starvation and redistributed from the Golgi toward lysosome-associated compartments, whereas GRASP65 remained predominantly Golgi-associated and did not form comparable large assemblies. Hyperspectral imaging combined with phasor analysis with the environment-sensitive probe ACDAN revealed increased water dipolar relaxation within GRASP55 assemblies under stress, indicating changes in local hydration and molecular organization. LAURDAN imaging further showed stress-dependent alterations in cellular membrane fluidity. Using the high-throughput microfluidic PhaseScan platform, we established phase diagrams demonstrating a strong condensation propensity for GRASP55 but not GRASP65. Domain-swapping experiments indicated that GRASP55 condensation requires the cooperative contribution of its structured GRASP domain and intrinsically disordered SPR domain. Moreover, phosphomimetic substitutions at mTORC1-associated sites modulated GRASP55 phase behavior, with the T232D/T249D/T250D/T257D mutant abolishing condensation, while inclusion of T264D restored phase separation, identifying phosphorylation as an important regulatory mechanism. Complementary hyperspectral imaging, fluorescence lifetime imaging, electron spin resonance, and FRET measurements revealed substantial molecular and conformational heterogeneity within GRASP55 condensates and a progressive transition toward more crowded, solid-like environments during condensate aging. The membrane interaction experiments showed that GRASP55, but not non-condensing GRASP variants, promoted protein engulfment and pronounced membrane remodeling in giant unilamellar vesicles, including tubulation and protein-rich assemblies. Our results support a model in which stress-regulated GRASP55 condensation is coupled to membrane remodeling and may facilitate the organization of trafficking intermediates involved in unconventional protein secretion.