Effect of acyl-protein thioesterases on membrane association and spatial organization of H-ras.

PEDRO M.P.; VILCAES A.; DANIOTTI J.L.

Rosario

Congreso; 50 Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

S-acylation is a reversible post-translational modification catalyzed by palmitoyltransferases and acyl-protein thioesterases (APTs). The acylation/deacylation cycle is necessary to maintain the subcellular distribution of S-acylated peripheral proteins, such as members of the Ras family. Due to the importance of a proper H-Ras localization to connect with downstream signaling molecules, we decided to investigate the acylation of this GTPase. H-Ras is dually acylated (Cys181,184) and arrives to plasma membrane (PM) using the secretory pathway. Mono- and non-acylated H-Ras mutants showed differences on their localization and the inhibition of H-Ras acylation dramatically reduced its PM association but maintained the Golgi location when expressed de novo. Then, we focused to investigate the deacylation process of H-Ras by analyzing substrate accessibility to APT1 and APT2. No significant deacylation over time was observed for H-Ras and H-Ras(C181S) associated to PM and Golgi, respectively. However, PM deacylation rate on H-Ras(C184S) mutant was enhanced in the presence of APT1 or APT2. Results support the fact that each fatty acid moiety provides particular information for spatial organization of H-Ras and suggest a differential accessibility of fatty acids to APTs. The insights gained here might be of particular value to investigate how APTs level modulate H-Ras function.