APT-2-Mediated Deacylation of Peripheral Proteins details of the boxed area at a higher magnification with the corresponding pseudocoloured signal from each channel. Scale bars: 5 mm. B) CHO-K1 cells transiently expressing GAP-43full-YFP (GAP-43full), GAP-43full(C3S)-YFP [GAP-43full(C3S)], N13GAP-43-YFP (N13GAP-43) or N13GAP-43(C3S)-YFP [N13GAP-43(C3S)] were lysed, ultracentrifuged and the S (soluble) and P (pellet) fraction isolated. Buffer containing 1% v/v Triton X-114 was added to samples and phase separation was induced at 37uC. Proteins from the A (aqueous) and D (detergent) phases were Western blotted with an antibody to GFP. To obtain nonacylated GAP-43full and N13GAP-43, CHO-K1 cells were transfected with the corresponding plasmids in presence of CHX. 30 min before CHX withdrawal, cells were incubated with 50 mM 2-BP until 3 h after CHX withdrawal. S and P fractions were isolated and analyzed by Western blot as explained above. The lower panels show the Western blot using antibody to a-tubulin. C) 60 h after transient transfection, CHO-K1 cells coexpressing N13GAP-43(C3S) and N27GalNAc-T were treated with 50 mM 2-BP (2-BP, lower panels) or vehicle (Ctrl, upper panels) in presence of CHX and protein degradation inhibitors during 30 min before imaging and during all the experiment. GAP-43 subcellular distribution was analyzed by live cell confocal microscopy. Panels show YFP (pseudocolored gray) and CFP (pseudocolored gray), and merge images from YFP (pseudocolored green) and CFP (pseudocolored red) signals at 0, 5, 10 and 15 minutes after 2-BP or vehicle addition. Scale bars: 5 mm. D) Quantification of images showed in 2C (see Materials and Methods). E) Upper, 24 h after transfection, CHO-K1 cells transiently expressing N13GAP-43(C3S)-YFP [N13GAP- 43(C3S)] were lysed, ultracentrifuged and the S and P fraction were recovered. Buffer containing 1% v/v Triton X-114 was added to samples and phase separation was induced at 37uC. Proteins from the A and D phases were Western blotted with an antibody to GFP and a-tubulin. Lower, quantification (as described in Materials and Methods) of Western blot showed in upper panels. F) Left, 60 h after transient transfection, CHO-K1 and HeLa cells coexpressing N13GAP-43(C3S)-YFP and N27GalNAc-T-CFP were treated with 50 mM 2-BP (2-BP, lower panels) or vehicle (Control, upper panels) in presence of protein degradation inhibitors during 30 min. GAP-43 subcellular distribution was analyzed by live cell confocal microscopy. Each panel shows image from YPF (pseudocoloured gray) channel at 0, 5, 10 and 15 min from the beginning of the experiment. The insets in each panel show (from left to right): YFP fluorescence (pseudocolored green), CFP fluorescence (pseudocolored red) fluorescence and the merge of both fluorescence signals. Scale bars: 5 mm. Right, quantification of images showed in left panels (see Materials and Methods). doi:10.1371/journal.pone.0015045.g002 occurred diffusely throughout the cells, characteristic of cytosolic proteins (Fig. 2A). The membrane association and the extent of posttranslational modification of these constructs was further characterized by ultracentrifugation and Triton X-114 (TX-114) partition assays (Fig. 2B) [47]. More than 70% of GAP-43full and N13GAP-43 were found to be associated with the pellet (P) fraction after ultracentrifugation. 20% and 70% of P fraction-associated GAP- 43full and N13GAP-43, respectively, were found enriched in the TX-114 detergent (D) phase, reflecting a higher hydrophobic character for N13GAP-43. In contrast, a drastic reduction in membrane association (,5% in P fraction) of newly synthesized GAP-43 was observed when cells were incubated with the PAT inhibitor 2-BP (see nonacylated N13GAP-43 and nonacylated GAP-43full in Fig. 2B). As additional control, we demonstrated that soluble a-tubulin is mostly associated to the A (aqueous) phase of the soluble fraction, in agreement to the biochemical properties of this protein. 54% of GAP-43full(C3S) and 35% of N13GAP-43(C3S) were found to be associated with the P fraction. Additionally, 20% and 43% of P fraction-associated GAP-43full(C3S) and N13GAP- 43(C3S), respectively, were found enriched in the D phase, reflecting a higher hydrophobic character for N13GAP-43(C3S) (Fig. 2B). Altogether, these results demonstrate that acylation is necessary for GAP-43 membrane binding and that N13GAP- 43(C3S) is the single acylation mutant having the highest proportion of acylation and, consequently, membrane association. Next, we evaluated the deacylation kinetics of N13GAP-43(C3S) in order to investigate single deacylation events occurring on GAP- 43. CHO-K1 cells transiently coexpressing N13GAP-43(C3S) and N27GalNAc-T (TGN marker) were treated with 50 mM 2-BP (2- BP) or dimethylsulfoxide (DMSO, Control) and GAP-43 subcel- lular distribution monitored at different times by live cell confocal fluorescent microscopy (Fig. 2C). CHX and protein degradation inhibitors were incorporated to the culture medium 1 h before and during 2-BP treatment. In control cells, the amount of TGN- membrane association of GAP-43(C3S) did not significantly change over time. In opposite, TGN-associated GAP-43 signifi- cantly decreased over time in 2-BP treated cells with a half-life of 6 min (Fig. 2C and D) clearly much faster than its wild-type diacylated counterpart (see Fig. 1). The observed decrease of the TGN-membrane association of GAP-43(C3S) in 2-BP conditions was not attributed to TGN membrane redistribution, since the TGN marker N27GalNAc-T was not affected under these experimental conditions, nor to an increase in TGN to plasma membrane vesicular transport of N13GAP-43(C3S) since all experiments were performed at 20uC, a condition which drastically decreases this process [34]. To support the in vivo microscopy experiments, membrane association and extent of posttranslational modification of N13GAP-43(C3S) were determined at the same experimental conditions by subcellular fractionation and TX-114 partition assay. It was found that 2-BP, when compared to control conditions, caused a significant decrease of membrane boun- ded-N13GAP-43(C3S) (Fig. 2E). A similar behavior was observed for GAP-43full(C3S) (Fig. S1). Overall, these biochemical results are in close agreement with those obtained by live cell confocal microscopy analysis. To discard that the observed GAP-43 deacylation process could be a cell type-specific phenomenon, then, we extended our analysis using human HeLa cells and found that N13GAP-43(C3S) is deacylated with a similar kinetics both in HeLa and CHO-K1 cells (Fig. 2F), precluding the possibility that GAP-43 deacylation is a cell type specific process. These results demonstrate that N13GAP-43(C3S) is deacylated in vivo and that GAP-43 deacylation occurs at the TGN, besides the plasma membrane, revealing the importance of an acylation/ deacylation cycle operating in GAP-43 subcellular distribution [17,19,22,48,49]. GAP-43 Deacylation in CHO-K1 Cells is APT-1 Independent Up to now, results indicate that N13GAP-43(C3S) is deacylated with similar kinetic when it is expressed in CHO-K1 and HeLa cells. APT-1 is the unique described cytosolic protein thioesterase able to catalyze the deacylation of several substrates including H- Ras and G alpha subunit proteins [13,18,50]. However, there is no information about the role of this enzyme on GAP-43 deacylation. To investigate whether APT-1 deacylates GAP-43, we first analyzed mRNA expression of APT-1 both in CHO-K1 and HeLa cells by reverse transcriptase-polimerase reaction chain (RT- PCR). The results showed that a PCR product of the expected size was obtained using mRNAs from HeLa cells as template (Fig. 3A). The same result was also obtained using mRNA from African green monkey kidney fibroblast cells (COS-7 line) (Fig. S2). In opposite, no amplification of any product was observed using mRNA purified from CHO-K1 cells. Primer specificity was validated by APT-1 cDNA amplification using genomic DNA from CHO-K1 cells and mRNA purified from hamster brain, liver and ovary tissues (Fig. 3A and B). The identity of all PCR products PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e15045