APT-2-Mediated Deacylation of Peripheral Proteins Figure 6. Effect of APT-2 overexpression on the deacylation rate of H-RasG12V. 60 h after transient transfection, CHO-K1 cells coexpressing H-RasG12V and APT-2 were treated with 50 mM 2-BP (2-BP) or vehicle (Ctrl) in the presence of CHX and protein degradation inhibitors for 0, 3 and 6 h and H-RasG12V subcellular distribution analyzed by live cell confocal microscopy. Each panel shows image from YFP fluorescence (pseudocolored gray). The insets show cells expressing APT-1 (cherry fluorescence). Scale bar: 5 mm. doi:10.1371/journal.pone.0015045.g006 of fatty acylated species of GAP-43, including, non-, single and double acylated forms [46]. Although in vitro experiments shown that an acylated peptide substrate derived from GAP-43 (MLCCMRR) was efficiently deacylated by PPT-1 at neutral pH [52], the lysosomal localization of this enzyme preclude the possibility that it is involved in cytosolic GAP-43 deacylation [21,53-55]. In opposite, APT-1, originally isolated from rat liver as a lysophospholipase [27], appears to be involved in the regulation of the reversible S-acylation of cytoplasmic proteins in vivo, like heterotrimeric G protein a subunits, endothelial nitric-oxide synthase, SNAP-23 and H-Ras as well as viral proteins [18]. However, evidence also indicates that APT-1 does not deacylate proteins without any discrimination. For instance, caveolin is not deacylated by APT-1 [56] and not all substrates are deacylated with the same efficiency [57]. Regarding GAP-43 deacylation, there is not any direct evidence suggesting the participation of APT-1. Taken into consideration these antecedents, we attempted to evaluate the role of APT-1 in GAP-43 deacylation. First of all, we demonstrated that wild-type or single acylation mutants of GAP- 43 are deacylated in CHO-K1 cells. This result is compatible with those recently published [21] and in agreement with previous reports [40,41]. Interestingly, we demonstrated that APT-1 is not expressed in CHO-K1 cells and that APT-1 overexpression did not significantly increase the deacylation rate of monoacylated or wild-type GAP-43 but did enhance the deacylation rate of H- RasG12V protein. Paradoxically, APT-1 overexpression caused an accumulation of GAP-43full at the Golgi complex, a phenomena which could be compatible with an increase in acylation activity probably mediated by APT-1 since under the experimental conditions used in Figure S3 endogenous PAT activity, but not APT-1, was inhibited at 50 mM 2-BP (Tomatis, Gomez and Daniotti, unpublished results). In this sense, it was previously described that APT-1 may not only function as a thioesterase but also as an acyltransferase. Particularly, it was demonstrated an APT-1-mediated S-palmitoylation of H- and N- Ras [45]. Additionally, the no accumulation of N13GAP-43 at the Golgi complex in APT-1 expressing CHO-K1 cells could be reflecting substrate selectivity or structural preference of APT-1 to exert a proper acylation/reverse hydrolysis. To identify the enzyme/s responsible for GAP-43 deacylation, we performed a bioinformatic search based on shared properties of the only two proteins described with APT activity, APT-1 and PPT-1. Results show that APT-2 is the only isoform expressed both in CHO-K1 and HeLa cells, and suggested that it could be a potential candidate involved in GAP-43 deacylation. Subsequent experiments using live cell imaging analysis confirmed the bioinformatic results and demonstrated for the first time that APT-2 behave as acyl-protein thioesterase which is able to deacylate either GAP-43full or N13GAP-43. Mouse APT-2, which shares 64% identity with rat and mouse APT-1, was found to be ubiquitously expressed in mouse tissues and able to hydrolyze several lipid substrates with varied efficiency [58,59]. Sequence analysis using the PSORTII program (http:// psort.hgc.jp/form2.html) indicates that APT-2 is likely a soluble protein. By fusing the cDNA encoding this protein to fluorescent proteins, we found that APT-2 is mostly cytosolic and there is not any apparent membrane association. This fact strongly suggests that APT-2 can deacylate GAP-43 by transient binding to the membrane-cytosol interface probably in a widely distributed process. In particular, we observed that deacylation of mono- acylated N13GAP-43 occurs as early in the acylation cycle as at the Golgi complex. In agreement with this result, it was recently demonstrated that palmitate removal of H-Ras mutants (mono and double-palmitoylated) takes place at the Golgi complex [21]. Although some patterns of S-acylation exist, there is no specific consensus sequence for S-acylation. Similarly, it has not been defined so far a consensus for the sequences surrounding the thioacyl group which could be recognized by APT-1. In vitro, APT-1 can depalmitoylate structurally different proteins (soluble or integral membrane proteins), having different subcellular distributions and S-acylated either at the N- (i.e. endothelial nitric-oxide synthase and Gia1) or at the C-terminus (i.e. H-Ras) [18]. We observed that, like APT-1, APT-2 has not an easily detected consensus sequence for deacylation. We demonstrated that APT-2 can deacylate two unrelated proteins like H-Ras, which is modified at its C-terminus by both acylation and prenylation and GAP-43, which is acylated on N-terminal cysteines 3 and 4. Moreover, we observed variable substrate preference for APT-2 when compared deacylation of GAP-43full and the acylation motif of GAP-43 fused to YFP. Concerning APT-2 deacylation of H-Ras, it was early speculated it could be occurring in yeast cells where the APT-1 PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e15045