Abstract
In mammals, the Golgi apparatus is the central hub for intracellular trafficking, sorting and post-translational modifications of proteins and lipids. Golgi reassembly and stacking proteins (GRASPs) are somehow involved in Golgi stacking, which is relevant for its proper function, and also in unconventional protein secretion. However, the structural details on how GRASPs accomplish those tasks are still elusive. Here, we have explored the biochemical and biophysical properties of human full-length GRASP55 in solution. Sequence-based analyses and circular dichroism spectroscopy suggest that GRASP55 presents multiple intrinsically disordered sites, although keeping considerable contents of regular secondary structure. Size exclusion chromatography and multiple-angle light scattering show that GRASP55 are monomers in solution. Urea denaturation of GRASP55 suggests the transition to the unfolded state is a cooperative process. Differential scanning calorimetry analysis displays two endothermic transitions for GRASP55, indicating the existence of an intermediate state prior to unfolding. Thioflavin T fluorescence suggests GRASP55 intermediate can be aggregates/fibrils. Transmission electron microscopy and fluorescence lifetime imaging microscopy prove GRASP55 forms large amorphous aggregates but not amyloid-like fibrils in the intermediate state. These results could be helpful in discussing the proper function of human GRASP55 in the Golgi organization as well as unconventional secretion of proteins.
Highlights
In mammalian cells, the Golgi apparatus is a central hub for intracellular trafficking and postranslational modifications of proteins and lipids in the secretory pathway [1,2,3]
The results from CDSSTR deconvolution showed that full-length GRASP55 has ∼21% of α-helix, ∼20% of βsheet, ∼17% of turns, and ∼42% of disordered regions with the normalized root mean square deviation (NRMSD) of 0.19, which indicates, as already mentioned above, the presence of multiple disorder sites alongside well-structured regions
The protein disorder prediction tools and Circular dichroism (CD) spectroscopy suggest that full-length GRASP55 show intrinsically disordered (ID) behavior in solution
Summary
The Golgi apparatus is a central hub for intracellular trafficking and postranslational modifications of proteins and lipids in the secretory pathway [1,2,3]. The Golgi apparatus is formed by laterally linking the stacks of flattened cisternae into ribbon-like structures. This unique architecture is essential for its proper functioning. The molecular mechanism underlying how the Golgi stacks of cisternae turn into a ribbon-like structure is still obscure. GRASP65 and GRASP55 are the two paralogue proteins responsible for the complementary stacking of the Golgi [6]. GRASPs are involved in Golgi remodeling in migrating cells [9], and protein secretion [10]. The cleavage of GRASPs is a necessary event for the fragmentation of the Golgi during apoptosis [11]
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