Abstract
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.
Highlights
The Golgi apparatus is a highly dynamic organelle responsible for sorting out proteins and other biomolecules to the cell surface and to the extracellular milieu[1]
Our results indicate an unexpected behavior in solution and provide information that can affect the way one thinks Golgi Reassembly and Stacking Protein (GRASP) accomplishes its plethora of functions
A GRASP orthologue has been described as a regulator of polysaccharide export, through non-conventional mechanisms, and related to pathogenesis in the neuropathogen C. neoformans[17]
Summary
The Golgi apparatus is a highly dynamic organelle responsible for sorting out proteins and other biomolecules to the cell surface and to the extracellular milieu[1]. GRASPs can be dramatically regulated in a limited physical space, such as in between the cisternae of the Golgi complex, and in the cytosol This family of proteins must be able to correctly interact with itself in a trans orientation ( preventing the cis oligomerization)[11] to be accessible to the phosphorylation/ dephosphorylation machinery in the cell cycle[12] and to proteases during apoptotic times[3]. Two-PDZ domains, which form the so-called GRASP domain, and a non-conserved serine and proline rich domain, usually larger than the GRASP domain, compose the overall structure[10] These structural data together with several in vivo studies aiming at functional elucidation[10] show that GRASPs are involved in a large set of functions that likely includes a great number of interacting partners. Our results indicate an unexpected behavior in solution and provide information that can affect the way one thinks GRASP accomplishes its plethora of functions
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