APT-2-Mediated Deacylation of Peripheral Proteins Figure 5. APT-2 overexpression increases the deacylation rate of GAP-43. A) 60 h after transient transfection, CHO-K1 cells expressing N13GAP-43(C3S) (Control, upper panels) or coexpressing N13GAP-43(C3S) and APT-2 (APT-2, lower panels) were treated with 50 mM 2-BP. CHX and protein degradation inhibitors were added 30 min before imaging and maintained in the culture media until the end of each experiment. GAP-43 subcellular distribution was analyzed by live cell confocal microscopy. Each panel shows image from YPF fluorescence (pseudocolored gray) at 0, 5, 10 and 15 min from 2-BP addition. The insets in each panel from left to right show: YFP fluorescence (pseudocolored green), CFP fluorescence (pseudocolored red) and the merge of both fluorescence signals. B) Quantification [N13GAP-43(C3S) associated to TGN membranes] of images showed in panels a (see Materials and Methods). C) 60 h after transient transfection, CHO-K1 cells expressing GAP-43full (upper panels) or coexpressing GAP-43full and APT-2 (lower panels) 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 the GAP-43 subcellular distribution was analyzed by live cell confocal microscopy. Each panel shows image from YFP fluorescence (pseudocolored gray). The insets show cells expressing APT-2 (cherry fluorescence, pseudocolored gray). D) 60 h after transient transfection, CHO-K1 cells expressing N13GAP-43 (upper panels) or coexpressing N13GAP-43 and APT-2 (lower panels) 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 the subcellular distribution was analyzed by live cell confocal microscopy. Each panel shows image from YFP fluorescence (pseudocolored gray). The insets indicate cells expressing APT-2 (cherry fluorescence, pseudocolored gray). Scale bars: 5 mm. doi:10.1371/journal.pone.0015045.g005 bution to the endoplasmic reticulum after 3 h of 2-BP treatment. Thus, results demonstrate that H-RasG12V deacylation rate in cells coexpressing APT-2 is much higher than the observed in cells expressing H-RasG12V alone (see Fig. 3E) and suggest that, like APT-1, APT-2 is also able to deacylate H-Ras in vivo. Discussion The major aim of this study was to gain insight into the mechanisms participating in GAP-43 deacylation. Using confocal and video fluorescence microscopy in living cells, in association with biochemical approaches, we demonstrated in HeLa and CHO-K1 cells that both N13GAP-43 and GAP-43full are completely deacylated in vivo in a scale time of hours. In addition, single acylated mutants of GAP-43 were found to be deacylated faster than their double acylated counterparts. GAP-43 deacylation was not catalyzed by APT-1, the only bona fide cytosolic protein thioesterase described so far. By bioinformatic search, molecular biology techniques and later cell imaging analysis, we identified and characterized the phospholipase like protein APT-2 as the enzyme involved in GAP-43 deacylation. It had been early demonstrated that GAP-43 is deacylated in vivo [21,22,40,41] and that at steady-state conditions there are a variety PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e15045