Influence of thioesterase activity on the dynamics of organellar targeting of mono and diacylated H-ras

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

Puerto Iguazú, Misiones

Conferencia; 56th Meeting of the International Conference on the Bioscience of Lipids (ICBL); 2015

International Conference on the Bioscience of Lipids (ICBL)

S-acylation is a reversible post-translational modification, catalyzed by palmitoyltransferases (PATs) and acyl-protein thioesterases (APTs), by which a palmitic acid become attached to a cysteine residue. The acylation/deacylation cycle is necessary to maintain the subcellular distribution of S-acylated peripheral proteins, such as members of the Ras family. In particular, H-Ras is dually acylated in cysteines 181 and 184 and arrives to plasma membrane (PM) from the Golgi complex using the secretory pathway. Due to the importance of a proper H-Ras membrane localization to connect with downstream signaling molecules, we investigated the influence of acylation/deacylation cycle on activity and organellar targeting of this small GTPase. Besides differences on their subcellular localization, monoacylated H-Ras mutants, as well as the diacylated counterpart, were able to trigger ERK1/2 signaling. Inhibition of de novo H-Ras acylation dramatically reduced its PM association but maintained the Golgi complex localization. FRET analysis indicated closeness between APT1 and APT2 with H-Ras and this putative physical interaction diminished when the cells were incubated with a specific APTs inhibitor. PM deacylation rate on H-Ras(C184S) mutant was enhanced in presence of APT1 or APT2 while no significant deacylation over time was observed for H-Ras and H-Ras(C181S) associated to PM and Golgi, respectively. Fluorescence photobleaching experiments (FRAP and FLIP) revealed the contribution of vesicular vs diffusion transport on the retrograde journey of monoacylated H-Ras from PM to Golgi complex. Overall, the results indicate 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 APT modulates the stoichiometry of H-Ras palmitoylation and hence its biological function.