GRASPs are proteins involved in cell processes that seem paradoxical: responsible for shaping the Golgi cisternae and involved in unconventional secretion mechanisms that bypass the Golgi. Despite its physiological relevance, there is still a considerable lack of studies on full-length GRASPs. Our group has previously reported an unexpected behavior of the full-length GRASP from the fungus C. neoformans: its intrinsically-disordered characteristic. Here, we generalize this finding by showing that it is also observed in the GRASP from S. cerevisae (Grh1), which strongly suggests it might be a general property within the GRASP family. Furthermore, Grh1 is also able to form amyloid-like fibrils either upon heating or when submitted to changes in the dielectric constant of its surroundings, a condition that is experienced by the protein when in close contact with membranes of cell compartments, such as the Golgi apparatus. Intrinsic disorder and fibril formation can thus be two structural properties exploited by GRASP during its functional cycle.

Among all proteins localized in the Golgi apparatus, a two-PDZ (PSD95/DlgA/Zo-1) domain protein plays an important role in the assembly of the cisternae. This Golgi Reassembly and Stacking Protein (GRASP) has puzzled researchers due to its large array of functions and relevance in Golgi functionality. We report here a biochemical and biophysical study of the GRASP55/65 homologue in Cryptococcus neoformans (CnGRASP). Bioinformatic analysis, static fluorescence and circular dichroism spectroscopies, calorimetry, small angle X-ray scattering, solution nuclear magnetic resonance, size exclusion chromatography and proteolysis assays were used to unravel structural features of the full-length CnGRASP. We detected the coexistence of regular secondary structures and large amounts of disordered regions. The overall structure is less compact than a regular globular protein and the high structural flexibility makes its hydrophobic core more accessible to solvent. Our results indicate an unusual behavior of CnGRASP in solution, closely resembling a class of intrinsically disordered proteins called molten globule proteins. To the best of our knowledge, this is the first structural characterization of a full-length GRASP and observation of a molten globule-like behavior in the GRASP family. The possible implications of this and how it could explain the multiple facets of this intriguing class of proteins are discussed.

The Golgi complex is part of the endomembrane system and is responsible for receiving transport cargos from the endoplasmic reticulum and for sorting and targeting them to their final destination. To perform its function in higher eukaryotic cells, the Golgi needs to be correctly assembled as a flattened membrane sandwich kept together by a protein matrix. The precise mechanism controlling the Golgi cisternae assembly is not yet known, but it is widely accepted that the Golgi Reassembly and Stacking Protein (GRASP) is a main component of the Golgi protein matrix. Unlike mammalian cells, which have two GRASP genes, lower eukaryotes present only one gene and distinct Golgi cisternae assembly. In this study, we performed a set of biophysical studies to get insights on the structural properties of the GRASP domains (DGRASPs) from both human GRASP55 and GRASP65 and compare them with GRASP domains from lower eukaryotes (Saccharomyces cerevisiae and Cryptococcus neoformans). Our data suggest that both human DGRASPs are essentially different from each other and that DGRASP65 is more similar to the subgroup of DGRASPs from lower eukaryotes in terms of its biophysical properties. GRASP55 is present mainly in the Golgi medial and trans faces, which are absent in both fungi, while GRASP65 is located in the cis‐Golgi. We suggest that the GRASP65 gene is more ancient and that its paralogue GRASP55 might have appeared later in evolution, together with the medial and trans Golgi faces in mammalians.