One acetyl group, two interfaces: N-terminal acetylation controls Grh1 partitioning and membrane remodelling

N-terminal acetylation is one of the most prevalent protein modifications in eukaryotes and can regulate stability, molecular interactions and subcellular localisation ^1–3. However, its effects on protein behaviour at membrane-bound and membraneless interfaces remain poorly understood. Here, we investigated how this modification regulates the conformational and interfacial behaviour of Grh1, the yeast homologue of mammalian GRASP proteins ⁴. Fluorescence microscopy, circular dichroism and differential scanning calorimetry were used to compare non-acetylated Grh1 with its N-terminally acetylated form, AcGrh1, in biomolecular condensates and model membranes. N-terminal acetylation altered condensate number, size, hydration and responsiveness. Co-condensation of the two variants produced multiphase condensates with a core-shell architecture, in which Grh1 was enriched in the core and AcGrh1 at the periphery, demonstrating that a single acetyl group is sufficient to modify miscibility, partitioning and interfacial preference ⁵. We next investigated whether this effect extended to lipid interfaces. Zwitterionic DMPC membranes produced minimal changes in protein structure and bilayer thermotropic behaviour. In contrast, increasing the fraction of anionic DMPG progressively shifted the conformational ensembles of both variants. From 0 to 100% DMPG, the spectral parameter R increased by approximately 37% for Grh1 and 36% for AcGrh1. The bilayer response, however, was strongly dependent on acetylation. At 80% DMPG, AcGrh1 shifted the main-transition temperature from 22.63 ± 0.07 to 23.39 ± 0.04 °C and increased the calorimetric enthalpy from 4.8 ± 0.3 to 7.2 ± 0.4 kcal·mol⁻¹, whereas non-acetylated Grh1 caused only minor perturbations. These results reveal an asymmetric form of bidirectional coupling. Anionic membranes reorganise both protein variants, whereas N-terminal acetylation determines whether this interaction is transmitted back to the bilayer as changes in lipid packing, transition enthalpy and cooperativity. Therefore, a single acetyl group coordinates the partitioning and remodelling of Grh1 across membrane-bound and membraneless interfaces.