from rat liver as a lysophospholipase [27], is a cytosolic protein with a widespread tissue distribution. Several proteins have been identified as APT-1 substrates, like heterotrimeric G protein a subunits, endothelial nitric-oxide synthase, SNAP-23 and H-Ras as well as viral proteins [18]. In opposite to acylation, the deacylation step seems to occurs everywhere in the cell and no specific consensus sequence or substrate specificity has been described for this enzymatic reaction so far [21]. In this work, we demonstrated for the first time that lysopho- spholipase II or APT-2 is a cytosolic protein thioesterase involved in GAP-43 deacylation. GAP-43 was early identified as a functional growth cone marker participating in the mechanisms of axonal outgrowth and regeneration [28–31]. Then, it was also identified in peripheral and central glia cells and developing muscle cells [32,33], which points to a fundamental role for GAP- 43 in cellular processes. After synthesis in the cytosol, GAP-43 binds to trans Golgi network (TGN) membranes through a process that requires acylation of cysteine residues at positions 3 and 4 [34,35]. This posttranslational modification is necessary for GAP- 43 inclusion into exocytic and endocytic vesicular carriers. Although several groups have described the molecular deter- minants involved in the polarized sorting of GAP-43 in neural cells [34,36–39], it is still unclear how this subcellular distribution is maintained. An attractive hypothesis is that cycles of deacylation/ acylation controls GAP-43 localization and sorting [4,40,41]. In this sense, it has been recently described that the fast kinetics and topography of the acylation cycle allows the continuous redirection of mislocalized proteins via the post-Golgi sorting apparatus [21]. In addition, the presence of PATs at the plasma membrane has been also demonstrated [3,34]. Using in vivo confocal fluorescence microscopy, biochemical analyses and bioinformatic approaches, we characterized the deacylation kinetics of GAP-43 and identified the thioesterase involved in this process. It was found that GAP-43 is deacylated in vivo when expressed in Chinese hamster ovary (CHO)-K1 and human HeLa cells through a mechanism mediated by APT-2, but not by APT-1. Inhibition of deacylation caused GAP-43 to accumulate in perinuclear structures while APT-2 overexpression increased the cytoplasmic pool of GAP-43. Additionally, it was also demonstrated that APT-2 overexpression affected the steady- state subcellular localization of acylated H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution. Results The Full-Length and N-terminal Domains of GAP-43 are Deacylated In Vivo with Relatively Slow Kinetics Previously, we characterized the acylation, membrane associa- tion and intracellular trafficking of full-length (GAP-43full) and the acylation motif (MLCCMRRTKQVEK, N13GAP-43) of GAP-43 fused to spectral variants of green fluorescent protein (GFP) [cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP)] [34,42]. We demonstrated that both constructs are acylated at the TGN and subsequently transported to the plasma membrane by vesicular carriers. At the plasma membrane, these proteins are endocytosed and directed to the recycling endosomes (RE) in an Arf6-dependent manner. Thus, this intracellular itinerary deter- mines the presence of GAP-43 at the TGN, plasma membrane and RE at steady-state conditions [34,42]. In experiments carried out to investigate GAP-43 deacylation in CHO-K1 cells, it was observed that PATs inhibition with 100 mM 2-bromopalmitate (2- BP) completely abolished acylation of newly synthesized GAP-43 APT-2-Mediated Deacylation of Peripheral Proteins and therefore its binding to the membranes. However, at steady- state conditions 2-BP treatment did not modify the acylation state and membrane binding properties of GAP-43 [34], strongly suggesting that membrane-associated GAP-43 is not being deacylated. However, it should be also considered that deacylated GAP-43 could be a preferred substrate for proteasome degrada- tion [43,44], avoiding cytosolic accumulation, and/or that 2-BP not only inhibits PATs but also APT activity similarly to that observed using APT-1 inhibitors [45]. To assess these alternatives, cells expressing GAP-43 at steady-state conditions were treated with 2-BP and cycloheximide (CHX) in the presence (+Inh) or absence (-Inh) of proteasomal and lysosomal inhibitors during 0, 3 and 6 h (Fig. 1). As shown in Figure 1A, a significant degradation of GAP-43 was observed along the time in the absence of inhibitors. Contrary and supporting the previous assumption, GAP-43 proteolysis was abolished when cells were incubated with 2-BP and CHX in the presence of protein degradation inhibitors. Next, we investigated the in vivo deacylation kinetic of GAP- 43full and N13GAP-43 (Fig. 1B). Cells were treated with 2-BP during 0, 3 and 6 h and the subcellular distribution analyzed by in vivo confocal fluorescent microscopy. CHX and inhibitors of protein degradation were incorporated to the culture medium 1 h before 2-BP addition. All experiments were performed using 50 mM 2-BP to inhibit PATs without affecting APT activity (Tomatis, Gomez and Daniotti, unpublished results) [34]. At 0, 3 and 6 h, both N13GAP-43 and GAP-43full localized at the plasma membrane and perinuclear structures in control cells, as previously reported [34]. In contrast, in 2-BP-treated cells there was a significant amount of soluble, nonacylated GAP-43 protein at 3 and 6 h, being this more evident for GAP-43full than N13GAP-43. We discarded accumulation of newly synthesized GAP-43 since CHX was present during all the experiment. Altogether, these results support the existence of an acylation/deacylation cycle which operates in the membrane binding and subcellular distribution of GAP-43. Additionally, the observed deacylation kinetic for GAP-43full and N13GAP-43 was found to be comparable to that previously described for H-Ras [19]. Single Acylated Mutant of N13GAP-43 is Deacylated In Vivo Faster than its Double Acylated Counterpart Diacylated GAP-43 requires a double deacylation event for its membrane dissociation. To investigate single deacylation events occurring on GAP-43 and to set a more direct method for deacylation readout, we decided to carry out our experiments using single acylated mutants of GAP-43. Importantly, it should also be mentioned that at steady-state conditions the monopalmi- toylated fraction of GAP-43 represent the 60% of the total GAP- 43 protein, indicating the importance of the monoacylated specie in GAP-43 function [46]. We generated single and double cysteine mutants at the acylation motif of N13GAP-43 [N13GAP-43(C3S), N13GAP- 43(C4S), N13GAP-43(C3,4S)] and GAP-43full [GAP-43full(C3S), GAP-43full(C4S), GAP-43full(C3,4S)] which were then character- ized by in vivo confocal microscopy and biochemical analyses. As previously observed, GAP-43full and N13GAP-43 localized, at steady-state, at the RE, plasma membrane and TGN (Fig. 2A). The point mutation at Cys3 in N13GAP-43 caused an accumula- tion at the cytosol and TGN, and disrupted RE association, but did not affect their plasma membrane association. In contrast, the point mutation at Cys3 in GAP-43full disrupted the association with the plasma membrane, drastically reduced their TGN membrane association, and resulted in an accumulation at the cytosol. Double mutants at Cys3 and Cys4 of GAP-43full and N13GAP-43 were not able to associate to any membranes and PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e15045