APT-2-Mediated Deacylation of Peripheral Proteins Figure 4. Analysis of acyl-protein thioesterase expression in CHO-K1 and HeLa cells. A) PCR analysis of gene expression in CHO-K1 (upper panels) and HeLa cells (lower panels). For each indicated gene (see gene number at the top), PCRs were performed using specific primers and the following templates: reaction mixture [Ctrl (-)]; genomic DNA (gDNA) and first-strand cDNA obtained from RT of mRNA (RT-PCR). B) PCR analysis of isoform expression coded by gene # 11313. For each isoform indicated at the top, PCRs were performed using specific primers and the following templates: first-strand cDNA obtained from RT of HeLa cells purified mRNA (HeLa RT-PCR); CHO-K1 genomic DNA (CHO-K1 gDNA) and first-strand cDNA obtained from RT of CHO-K1 cells purified mRNA (CHO-K1 RT-PCR). doi:10.1371/journal.pone.0015045.g004 cell confocal microscopy as described in Fig. 2. As control, we also measured N13GAP-43(C3S) deacylation in cells which did not overexpress APT-2 (Fig. 5A). As previously determined, the half- time of the decay of TGN-membrane associated GAP-43 at the TGN after 2-BP addition in cells which only expresses N13GAP- 43(C3S) was 6 min (see Fig. 2). In a clear contrast, we observed that in cells expressing APT-2, the half-time of N13GAP-43(C3S) deacylation was reduced to 3 min (Fig. 5B), demonstrating that APT-2 catalyze in vivo deacylation of N13GAP-43(C3S). To further extent our research, we investigated the effect of APT-2 overexpression on GAP-43full and N13GAP-43 deacylation. For this, we analyzed the effect of 2-BP treatment on GAP-43 subcellular distribution in cells expressing GAP-43 alone or coexpresing GAP-43 and APT-2 (Fig. 5C and D). We observed that in the absence of APT-2, GAP-43 (N13GAP-43 and GAP- 43full) deacylation was partially evident after 3 h of 2-BP treatment (compare Control with 2-BP condition), maintaining a significant amount of plasma membrane-associated GAP-43. In contrast, after 6 h of 2-BP treatment, we observed nuclear accumulation of soluble GAP-43 and a clear reduction of GAP-43 at the plasma membrane, which indicate that it is highly deacylated under this condition. On the other hand, the overexpression of APT-2 caused a significant reduction in the plasma membrane content of GAP- 43full at 3 h of 2-BP treatment (Fig. 5C). In addition, it was also observed at this time an appreciable accumulation of soluble GAP- 43 in the nuclei when compared to cells expressing only GAP-43. A similar result was also observed when we analyzed the effect of APT-2 overexpression on N13GAP-43 deacylation (Fig. 5D). Altogether, these results indicate that APT-2 is a novel acyl- protein thioesterase which is able to deacylate GAP-43. Effect of APT-2 Overexpression on the Deacylation Rate of H-RasG12V After identifying GAP-43 as a substrate of APT-2, we investigated the effect of APT-2 expression on H-RasG12V deacylation and subcellular distribution (Fig. 6). Similar to results obtained in CHO-K1 cells coexpressing APT-1 and H-RasG12V (Fig. 3E), APT-2 expression caused a total H-RasG12V redistri- PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e15045