Differential influence of acyl-protein thioesterases on the dynamic intracellular distribution of mono- and diacylated H-Ras

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

San Diego

Congreso; Annual Meeting The American Society for Cell Biology; 2015

The American Society for Cell Biology (USA)

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 was to investigate the role of APT1 and APT2, the only two bona fide thioesterases that have been shown to mediate protein deacylation, on the dynamics of organellar targeting of diacylated H-Ras (C181, 184) as well as on its monoacylated counterparts. 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. 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 effect over time was observed for H-Ras and H-Ras(C181S), which were mainly 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 acylated isoforms of H-Ras, and, in consequence, influence the proper connection of the small GTPase with downstream signaling molecules.