The GRASP domain in Golgi Reassembly and Stacking Proteins: differences and similarities between lower and higher Eukaryotes

Batista

et al. 2021

Preprint

Self Cite

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 and, 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.

Section: Resultssupporting

confidence: 88%

Section: Methodsmentioning

confidence: 99%

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

Kava

Batista

et al. 2021

Preprint

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“…Saccharomyces cerevisiae (ScGRASP, Grh1) revealed the high propensity of ID regions (IDRs) in both the SPR and the GRASP domains [29][30][31]. Nevertheless, the roles of the intrinsically disordered domains in the functional cycle of both CnGRASP and Grh1 are still unknown.…”

Section: Results From Our Group On Grasps From Both Cryptococcus Neofmentioning

confidence: 99%

Exploring Structural Aspects of the Human Golgi Matrix Protein GRASP55 in Solution

Reddy

Fontana

et al. 2019

Preprint

In mammalian cells, the Golgi apparatus is a central hub for intracellular trafficking, sorting and post-translational modifications of proteins and lipids. The Golgi reassembly and stacking proteins (GRASPs) are somehow involved in the Golgi stacking, which is significant for the proper function of the Golgi apparatus, and also in unconventional protein secretion.However, the structural details on how GRASPs accomplish those tasks are still elusive. In this context, we have explored the biochemical and biophysical properties of the human fulllength GRASP55 in solution. Sequence-based analyses and circular dichroism spectroscopy suggest that GRASP55 presents multiple intrinsically disordered sites, although keeping considerable contents of secondary structure. Size exclusion chromatography coupled with multiple-angle light scattering (SEC-MALS) studies show that GRASP55 are monomers in solution. Urea denaturation of GRASP55 suggests that the transition to the unfolded state is a cooperative process. Differential scanning calorimetry (DSC) analysis displays two endothermic transitions for GRASP55, indicating the existence of an intermediate state prior to unfolding. Thioflavin T fluorescence shows that GRASP55 can form protein aggregates/fibrils at the intermediate state. Transmission electron microscopy and fluorescence lifetime imaging microscopy prove that GRASP55 forms large amorphous aggregates but not amyloid-like fibrils in the intermediate state. The significance of these results could be helpful in discussing the proper function of human GRASP55 in the Golgi organization as well as unconventional secretion of proteins.

“…The human DGRASP55 and DGRASP65 were expressed and purified as described elsewhere [ 17 ]. The protocol for the ancestor proteins production was the same as the one used for modern human proteins.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…GRASPs constitute a family of peripheral membrane-associated proteins, which were first suggested as an essential factor in the Golgi cisternae reassembly after mitotic times [ 15,16 ]. GRASPs are observed in all the branches of the eukaryotic tree of life, although not equally spread in all supergroups [ 17 ]. In Metazoans, evolution led to the appearance of two GRASP paralogue genes called gorasp1 and gorasp2 (translating the proteins GRASP65 and GRASP55, respectively) [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

Resurrecting Golgi proteins to grasp Golgi ribbon formation and self-association under stress

Batista

Kava

et al. 2021

Preprint

Self Cite

The Golgi complex is a membranous organelle located in the heart of the eukaryotic secretory pathway. A subfamily of the Golgi matrix proteins, called GRASPs, are key players in the stress-induced unconventional secretion, the Golgi dynamics during mitosis/apoptosis, and Golgi ribbon formation. The Golgi ribbon is vertebrate-specific and correlates with the appearance of two GRASP paralogs (GRASP55/GRASP65) and two coiled-coil Golgins (GM130/Golgin45), which interact with each other in vivo. Although essential for the Golgi ribbon formation and the increase in Golgi structural complexity, the molecular details leading to their appearance only in this subphylum are still unknown. Moreover, despite the new functionalities supported by the GRASP paralogy, little is known about the structural and evolutionary differences between these paralogues. In this context, we used ancestor sequence reconstruction and several biophysical/biochemical approaches to assess the evolution of the GRASP structure, flexibility, and how they started anchoring their Golgin partners. Our data showed that the Golgins appeared in evolution and were anchored by the single GRASP ancestor before gorasp gene duplication and divergence in Metazoans. After the gorasp divergence, variations inside the GRASP binding pocket determined which paralogue would recruit each Golgin partner (GRASP55 with Golgin45 and GRASP65 with GM130). These interactions are responsible for the protein's specific Golgi locations and the appearance of the Golgi ribbon. We also suggest that the capacity of GRASPs to form supramolecular structures is a long-standing feature, which likely affects GRASP's participation as a trigger of the stress-induced secretory pathway.