Impact of N-terminal acetylation on the structure and molecular interactions of the "GRASP65 homolog protein 1" from Saccharomyces cerevisiae

• Grant number: 25/19163-4
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: November 01, 2025
• End date: January 31, 2029
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Luis Felipe Santos Mendes
• Grantee: Mayra Tamires Santos Silva
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 22/06006-0 - Elucidating the impact of functional protein liquid and solid condensates in the secretory pathway, AP.GR

Abstract

The spatial organization of the Golgi complex is essential for the efficient processing and transport of proteins within the secretory pathway. In eukaryotic organisms, this arrangement depends on matrix proteins associated with the cytoplasmic face of cisternae, including intrinsically disordered proteins capable of interacting with lipids and forming dynamic supramolecular structures. Recent studies suggest that liquid-liquid phase separation (LLPS) may represent a central mechanism in the functional organization of these structures. The Saccharomyces cerevisiae protein Grh1, a homolog of human GRASPs, is a promising model for investigating these phenomena, particularly due to its involvement in unconventional secretion processes and its intrinsically disordered behavior in solution. Data obtained during the proponent's master's research demonstrated that Grh1 acquires distinct structural properties when N-terminally acetylated (AcGrh1), promoting its anchoring to negatively charged lipid membranes. Building on these findings, the present direct PhD project aims to investigate the formation and properties of the supramolecular AcGrh1/Bug1 complex, proposing that this interaction can modulate the physicochemical properties of membranes and form organizing compartments via phase separation. To this end, four main approaches will be pursued: (i) expression and purification of the Golgin Bug1 in alternative eukaryotic systems, such as Kluyveromyces lactis; (ii) structural and functional characterization of the AcGrh1/Bug1 complex in different membrane contexts; (iii) assessment of biomolecular condensate formation in solution and at membrane interfaces; and (iv) analysis of the functional impact of the complex on curvature, fluidity, and topological organization of lipid bilayers. The project integrates advanced biophysical approaches, such as circular dichroism, calorimetry, confocal microscopy, and cryo-TEM, and will be carried out in collaboration with Dr. Ivan Rosa e Silva's group at LNBio/CNPEM, as well as with the Thematic Project coordinated by Prof. Antonio José da Costa Filho (Proc. 2023/04532-9) at FFCLRP/USP. Ultimately, this study aims to elucidate the molecular mechanisms by which the AcGrh1/Bug1 complex contributes to Golgi organization, offering new paradigms on the functional role of intrinsically disordered proteins in the formation of dynamic cellular structures driven by LLPS.