Itri, R. | Search bioRxiv

bioRxiv2026

Exploring the effects of Golgi Reassembly and Stacking Proteins in lipid membranes

Golgi Reassembly and Stacking Proteins (GRASP) have been associated with Golgi-ribbon structure and unconventional vesicular protein secretion. In performing these functions, GRASPs consistently interact with membranes. The presence of lipid modifications, such as myristoylation, is a crucial consideration for obtaining detailed information about the interactions between GRASPs and membranes. Nonetheless, it has been overlooked in the literature so far. Here, we describe a reconstitution protocol for myristoylated human GRASP65 and GRASP55 in lipid model membranes, enabling investigation of their interactions using techniques ranging from structural characterization to spectroscopy and microscopy. Our results showed that myristoyl-anchored GRASPs can influence membrane dynamics, suggesting a possible role for their disordered SPR domain in this interaction.

biophysics↗

bioRxiv2019

Repositioning septins within the core particle

Septins are GTP binding proteins considered to be a novel component of the cytoskeleton. They polymerize into filaments based on hetero-oligomeric core particles which, in humans, are either hexamers or octamers composed of two copies each of either three or four different septins from the 13 available. Not all combinations are possible as it is believed that these must obey substitution rules which determine that different septins must be derived from four distinct and well-established sub-groups. Here, we have purified and characterized one such combinations, SEPT5-SEPT6-SEPT7, and used TEM to derive the first structural information concerning its assembly. The full complex was purified using an affinity tag attached to only one of its components (SEPT7) and was able to bind to and perturb lipid bilayers. Although the complex assembled into elongated hexameric particles, the position of SEPT5 was incompatible with that predicted by the reported structure of SEPT2-SEPT6-SEPT7 based on the substitution rules. MBP-fusion constructs for SEPT5 and SEPT2 and immuno-staining clearly show that these septins occupy the terminal positions of the SEPT5-SEPT6-SEPT7 and SEPT2-SEPT6-SEPT7 hexamers, respectively. In so doing they expose a so-called NC interface which we show to be more susceptible to perturbation at high salt concentrations. Our results show that the true structure of the hexamer is inverted with respect to that described previously and, as such, is more compatible with that reported for yeast. Taken together, our results suggest that the mechanisms involved in spontaneous self-assembly of septin core particles and their filaments deserve further reflection.

biochemistry↗