Thioesterase activity influences the dynamic intracellular distribution of mono and diacylated H-Ras

PEDRO MP; VILCAES AA; DANIOTTI JL

San Diego

Congreso; 2015 Cell Biology ASCB Annual Meeting; 2015

ASCB

Endomembrane distribution is a key aspect of Ras biology. In particular, proper acylation/deacylation cycles are necessary to maintain a correct subcellular distribution of the S-acylated H-Ras isoform. This reversible post-translational modification is catalyzed by palmitoyltransferases (PATs) and acyl-protein thioesterases (APTs). The aim of this work is to investigate the role of APT1 and APT2 on the dynamics of organellar targeting of diacylated H-Ras as well as on its monoacylated counterparts (C181, C184). By biochemical approaches, we demonstrated that H-Ras and its acylation mutants were mostly associated with cell membranes and were able to trigger ERK1/2 signaling. However, notorious differences on their subcellular localization were observed. In addition, the inhibition of de novo H-Ras acylation dramatically reduced its plasma membrane (PM) association but maintained the Golgi complex and endoplasmic reticulum localization, in agreement with the subcellular distribution observed for non-palmitoylated H-Ras(C181,184S). FRET analysis indicated closeness between APT1 and APT2 with H-Ras and this putative physical interaction was diminished either when the cells were incubated with a specific APTs inhibitor or when the fluorescence acceptor partner was in the deacylated state. Interestingly, the PM deacylation rate of H-Ras(C184S) was enhanced in cells ectopically expressing APT1 or APT2, while no significant deacylation over time was observed for H-Ras and H-Ras(C181S) associated to PM and Golgi complex, respectively. Fluorescence photobleaching experiments (FRAP and FLIP) revealed a differential contribution of both vesicular and diffusion transport on the retrograde journey of monoacylated H-Ras mutant from PM to Golgi complex. Overall, the results indicate that each fatty acid moiety provides singular information for spatial organization of H-Ras and suggest a differential accessibility of fatty acids to APTs. Thus, modulation of thioesterase activity in a particular cellular context could be relevant to orchestrate the type and dynamic of the intracellular transport of H-Ras, and, in consequence, influence the proper connection of the small GTPase with downstream signaling molecules.