Myristoylation and its effects on the human Golgi Reassembly and Stacking Protein 55

GRASP55 is a myristoylated protein localized in the medial/trans-Golgi faces and involved in the Golgi structure maintenance and the regulation of unconventional secretion pathways. It is believed that GRASP55 achieves its main functionalities in the Golgi organization by acting as a tethering factor. When bound to the lipid bilayer, its orientation relative to the membrane surface is restricted to determine its proper trans-oligomerization. Despite the paramount role of myristoylation in GRASP function, the impact of such protein modification on the membrane-anchoring properties and the structural organization of GRASP remains elusive. Here, an optimized protocol for the myristoylation in E. coli of the membrane-anchoring domain of GRASP55 is presented. The biophysical properties of the myristoylated/non-myristoylated GRASP55 GRASP domain were characterized in a membrane-mimicking micellar environment. Although myristoylation did not cause any impact on the protein's secondary structure, according to our circular dichroism data, it had a significant impact on the protein's thermal stability and solubility. Electrophoresis of negatively charged liposomes incubated with the two GRASP55 constructions showed different electrophoretic mobility for the myristoylated anchored protein only, thus demonstrating that myristoylation is essential for the biological membrane anchoring. Molecular dynamics simulations were used to further explore the anchoring process in determining the restricted orientation of GRASPs in the membrane.