APT-2-Mediated Deacylation of Peripheral Proteins CHO-K1 cells transiently expressing N13GAP-43(C3S) was incubated with P fraction from nontransfected CHO-K1 cells during 1 h at 4uC. Then, the P fraction was isolated by ultracentrifugation and incubated in the presence (APT-1) or the absence (Control) of recombinant APT-1. Then, S and P fractions were separated by ultracentrifugation and proteins from each fraction Western blotted using an antibody to GFP. Scale bars: 5 mm. doi:10.1371/journal.pone.0015045.g003 was confirmed by DNA sequencing and results showed identical amino acid sequence between human and hamster APT-1 (results not shown). Thus, results demonstrated that APT-1 is not expressed in CHO-K1 cells. To discard the possibility that very low endogenous expression level of APT-1 could be deacylating GAP-43, we evaluated the deacylation kinetics of N13GAP-43(C3S) in CHO-K1 cells overexpressing or not human APT-1 fused to mCherry fluorescent protein (hereafter APT-1, Fig. 3C and D). The deacylation kinetics of GAP-43 was evaluated by analyzing the rate of N13GAP-43(C3S)-TGN-membrane association decay- ment after 2-BP treatment. Results clearly indicated that APT-1 overexpression in CHO-K1 cells did not significantly modify the decay rate of N13GAP-43(C3S) at the TGN compared to cells only expressing N13GAP-43(C3S). Additionally, we also found that APT-1 overexpression did not significantly modify membrane association and deacylation kinetic of diacylated N13GAP-43 and GAP-43full (Fig. S3). To further analyze the effect of APT-1 activity on N13GAP-43(C3S), we performed an in vitro deacylation assay (Fig. 3F). For these experiments, cytosolic fractions were prepared from CHO-K1 cells expressing N13GAP-43(C3S) or a fusion protein containing the C-terminal hypervariable region of K-Ras fused to YFP (K-RasC14), and particulate fractions from nontransfected cells [47]. Soluble fractions from transfected cells were incubated with membranes from nontransfected cells for 1 h at 4uC and then ultracentrifuged to separate soluble and particulate fractions. The isolated particulate fraction containing monoacylated GAP-43 or prenylated K-Ras was incubated for 1 h at 30uC in the presence (+APT-1) or the absence (-APT-1) of recombinant APT-1 [13]. Next, soluble and particulate fractions were separated by ultracentrifugation and presence of N13GAP- 43(C3S) in the fractions evaluated by Western blot. Results indicated that the incubation with recombinant APT-1 did not significantly increase the amount of N13GAP-43(C3S) recovered in the soluble fraction. As expected, the amount of nonpalmitoylated K-Ras isoform present in the soluble fraction was independent of the presence of APT-1 (result not shown). Thus, these results strongly support the concept that APT-1 does not catalyze both in vitro and in vivo N13GAP-43(C3S) deacylation. As additional control of APT-1 activity, we evaluated the effect of ectopically expressed APT-1 on H-RasG12V deacylation and its consequences on subcellular distribution. APT-1 has been identified as one of the thioesterases in the acylation cycle of H- Ras both in vivo [21,50] and in vitro [13]. In addition, it was demonstrated that oncogenic H-RasG12V is deacylated faster than its wild-type counterpart [15]. As shown in Figure 3E, H- RasG12V localizes at the plasma membrane and perinuclear structures previously identified as RE [42]. Treatment of CHO- K1 cells with 2-BP for 3 h led to a redistribution of H-RasG12V to perinuclear structures, which resembles the Golgi complex and the endoplasmic reticulum in agreement with subcellular distribution observed for nonpalmitoylated H-Ras [19,22,51]. A total redistri- bution of H-RasG12V to endomembranes was observed at 6 h after 2-BP treatment. By the contrary, when CHO-K1 cells coexpressing APT-1 and H-RasG12V were incubated with 2-BP, a faster and marked H-Ras subcellular redistribution was evident at 3 h. This result demonstrates that APT-1 is able to participate in the acylation cycle of H-RasG12V, which is consistent with already published data [50]. Altogether, these results indicate that APT-1 is not the enzyme responsible for GAP-43 deacylation in CHO-K1 cells. As CHO-K1 cells do not naturally express APT-1, the results strongly suggest the existence of other uncharacterized acyl-protein thioesterase. Bioinformatic Search and Expression Profiles Define Acyl- Protein Thioesterase 2 (APT-2) as the Enzyme Responsible for GAP-43 Deacylation in CHO-K1 Cells To identify the enzyme/s responsible for GAP-43 deacylation in CHO-K1 cells, we performed a bioinformatic search based on shared properties of the only two proteins described with APT activity, APT-1 and PPT-1. First, we performed a Blast search for nonredundant human protein sequences which align to PPT-1 and/or APT-1 protein sequences. In addition, we searched for members of APT-1 and/or PPT-1 families in Pfam databases (http://pfam.sanger.ac.uk/) [#PF02230 (APT-1) and #PF02089 (PPT-1)]. After careful classification and removal of redundant sequences, the in silico analysis allowed us to identify 21 potential acyl thioesterase transcripts, which are codified for 5 different genes (Table S1). To simplify the experimental screening, we first determined transcript expression in CHO-K1 and HeLa cells by RT-PCR using specific primers for each gene (without discrim- ination of mRNA isoforms, Fig. 4A). In parallel, we also determined transcription profiles in the neuroblastoma cell line SH-SY5Y (undifferentiated and retinoic acid-differentiated) and SK-Mel28 cells (Fig. S4). We found that transcripts coded by gene ID 11313 were expressed in much higher amount in HeLa, SH-SY5Y and SK- Mel-28 cells than in CHO-K1 cells. Interestingly, we observed that the expression level of transcripts coded for this gene increased in retinoic acid-treated SYSH-5Y cells when compared to control conditions. In addition, transcripts coded by gene ID 127018 and gene ID 339168 were only found in neuronal (SH- SY5Y) and epithelial (CHO-K1) cells, respectively. Finally, transcripts coded by gene ID 10434 were found in all analyzed cell lines, except CHO-K1 cells. Thus, the transcription profile of genes ID 127018, 339168 and 10434 did not explain for the presence of acyl-protein thioesterase activity both in CHO-K1 and HeLa cells. Consequently, results strongly suggest that gene ID 11313 potentially code for an acyl-protein thioesterase for GAP- 43. To identify transcripts derived from gene ID 11313 potentially coding for GAP-43 acyl-protein thioesterases, we evaluated their expression both in CHO-K1 and HeLa cells by RT-PCR using specific primers for each transcript (Fig. 4B). Results show that APT-2 is the only isoform expressed both in CHO-K1 and HeLa cells, and suggest that it could be a potential candidate involved in GAP-43 deacylation. Sequence analysis revealed a high degree of conservation between APT-2 from CHO-K1 and HeLa cells (aminoacid identity: 99%; nucleotide sequence 93%) (Table S2). APT-2 Overexpression Increases the Deacylation Rate of GAP-43 Bioinformatic search and mRNA expression experiments suggested that APT-2 is a potential acyl-protein thioesterase for GAP-43. To test this hypothesis, we evaluated the effect of APT-2, fused to mCherry fluorescent protein (hereafter APT-2), overex- pression on N13GAP-43(C3S) deacylation in CHO-K1 cells by live PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e15045