Role of acyl-protein thioesterases on membrane association and spatial organization of H-Ras

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

Puerto Varas

Congreso; XXVIII Annual Meeting, Chilean Society For Cell Biology; 2014

Chilean Society For Cell Biology

Introduction: 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 steady-state subcellular distribution and activity of S-acylated peripheral proteins, such as members of the small G-protein Ras family (H- and N-Ras). Due to the importance of a correct H-Ras subcellular localization to maintain the connection with downstream signaling molecules, we wanted to gain further insight into the acylation of this GTPase. Materials and Methods: Experiments were performed by using biochemical and cell imaging techniques. Results: H-Ras is dually palmitoylated (Cys181,184) and arrives to plasma membrane (PM) using the secretory pathway. Mono- and non-palmitoylated H-Ras mutants expressed in CHO-K1 cells showed notorious differences on their subcellular location and the pharmacological inhibition of H-Ras acylation dramatically reduced its PM association but maintained the Golgi localization when expressed de novo. Next, we focused to investigate the in vivo deacylation process of H-Ras by analyzing substrate accessibility of single acylation mutants to APT1 and APT2. No significant deacylation over the time (2 h) was observed both for H-Ras and H-Ras-C181S associated to PM and Golgi complex, respectively. However, PM deacylation rate on H-Ras-C184S mutant was clearly enhanced in the presence of APT1 or APT2. Discussion: Results support the fact that each distinct 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.