An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins, such as members of the small G-protein Ras family. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. So far, only two acyl-protein thioesterases has been described: APT-1 and APT-2. To gain further insight into the mechanisms of deacylation, both thioesterases were expressed in E. coli and subjected to biochemical and enzymatic studies using palmitoyl-CoA as substrate (P-CoA). APT-1 and APT-2 hydrolyzed P-CoA with different Km and showed no preference for the substrate in its monomeric or micellar form (CMC ~100 µM). Next, we evaluated the effect of ectopically expressed APT-1 and APT-2 on H-RasG12V deacylation and its consequences on subcellular distribution in CHO-K1 cells. H-RasG12V normally localizes at the plasma membrane and perinuclear structures identified as recycling endosomes. When cells were incubated with 2-BP (PAT inhibitor), it redistributed to Golgi complex and endoplasmic reticulum. If we coexpressed APT-1 or APT-2 with H-RasG12V, the redistribution of the small GTPase resulted much faster. This localization is in agreement with subcellular distribution of mono and nonpalmitoylated H-Ras mutants, indicating a role of APT-1 and APT-2 in H-Ras deacylation. Due to the importance of a correct H-Ras subcellular localization to maintain the connection with downstream signaling molecules, the insights gained here might be of particular value for future studies to investigate how APTs level modulate H-Ras signaling.
