gene was disrupted since the in vitro acyl-protein thioesterase activity against acylated H-Ras was similar to that of wild-type yeast [57]. In this sense, our results agree with this hypothesis and support the fact that both APT-1 and APT-2 mediate H-Ras deacylation. However, bioinformatic search do not indicate that APT-2 homologue exists in yeast, suggesting that in this organism Ras deacylation might be catalyzed by another still unidentified thioesterase protein [18]. Previously, it was demonstrated that proteasome inhibitors increase the cellular GAP-43 level, leading to the accumulation of polyubiquitinated forms of this protein in transfected cells. Moreover, the ubiquitin-proteasome pathway was described to be important in the turnover of this protein in neuron cells [43,44]. In this work, we show that 2-BP treatment caused a significative reduction in the amount of transiently expressed GAP-43. Interestingly, we demonstrated that the reduction in the protein level was abrogated when cells were also treated with proteasome inhibitors (chloroquine, MG132 and lactacystin) which strongly suggest that GAP-43 deacylation is an early and necessary step for its later ubiquitination and degradation by the proteasome. In addition, it also suggests that acyl-protein thioesterase levels not only regulate palmitate turnover but also global protein turnover of GAP-43. Materials and Methods Plasmids The expression vectors pECFP-C1 (where ECFP, enhanced CFP), pEYFP-N1 (where EYFP, enhanced YFP), pECFP-C1, pEYFP-C1, pEGFP-F, pmCherry-C1 and the plasmid encoding GFP-H-RasC20 were from Clontech (CA, USA). Expression plasmids for N13GAP-43-(YFP), GAP-43full-(YFP) and its acylation mutants, (YFP)-K-RasC14, YFP-H-Rasfull, N27GalNAc-T-CFP and N52Gal-T2-CFP have been previously described [34,42,47,60]. The expression plasmid encoding YFP-H-RasG12V was performed by directed mutagenesis of YFP-H-Rasfull using PCR. Both Cherry-APT-1 and Cherry-APT-2 constructs were obtained by RT-PCR using purified mRNA from HeLa and CHO-K1 cells, respectively, and the amplified products were cloned into pmCherry-C1 using EcoRI and SalI restriction sites. Cell Culture and DNA Transfections CHO-K1, HeLa, SK-Mel 28 and SHSY-5Y cells (ATCC, Manassas, VA, USA) were maintained at 37uC, 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (ATB, 100 mg/ml penicillin and 100 mg/ml streptomycin). To induce SH-SY5Y differentiation, cells were incubated with 30 mM retinoic acid for 6 days. Cells grown on Petri dishes were used for both live cell imaging and Western blot experiments. Cells were transfected with 0.6–1.2 mg/35 mm dish of the indicated plasmid using cationic liposomes (Lipofectamine, Invitrogen, CA, USA) or polyethylei- mine (PEI, Sigma-Aldrich, St Louis, MO, USA). At 24 h after cell transfection, cells were processed for Western blot experiments or plated onto coverslips, incubated for 24 h, and used in live cell imaging. 2-BP Treatment The stock solution of 2-BP (0.42 M, Fluka) was prepared in DMSO. CHO-K1 and HeLa cells expressing recombinant proteins were incubated with 50 mM 2-BP or vehicle (DMSO) for control cells for the indicated times. To analyze the effect of 2- BP on the steady state subcellular distribution of fluorescent proteins, CHO-K1 and HeLa cells were treated with 60 or APT-2-Mediated Deacylation of Peripheral Proteins 150 mg/ml CHX (Sigma-Aldrich), respectively, and inhibitors of protein degradation (60 mM cloroquine and 7.5 mM MG132). These inhibitors were added to the culture medium 24 h after cell transfection and 1 h before 2-BP treatment (Western blot experiments) or 36 h after platting transfected cells onto coverslips (live cell imaging). In live cell imaging experiments, coverslips were mounted on the microscope stage and 2-BP was added during time series acquisition. To analyze the effect of acylation inhibition on membrane binding properties of GAP-43, 50 mg/ml CHX was incorporated to the culture medium during the transfection. Then, 5 h after transfection, 10% FBS was added to the medium and 2h later 50 mM 2-BP was also incorporated. After 1 h of 2-BP treatment, CHX was removed by three washes with DMEM containing FBS, antibiotics and 2-BP. Finally, after 4 h of CHX withdrawal, cells were scrapped and used for biochemical experiments. Electrophoresis and Western Blotting Electrophoresis, transfer onto nitrocellulose membranes and protein immunodetection were performed as described by [47]. Anti-GFP polyclonal antibody (Roche Diagnostics, IN, USA) was used at a dilution of 1:700. Antibodies were detected using near- infrared fluorescence (Li-COR Biotechnology, Lincoln, NE, USA) with secondary antibody coupled to IRDye800CW (Li-COR Biotechnology). The relative contribution of individual bands was calculated using the computer software ImageJ (National Institute for Health, Bethesda, MA, USA) on raw nonsaturated digital images and the N13GAP-43 Kp value determined as follow: Kp~ S P ~ ASzDS ðAPzDPÞ Where S and P refer to the amount of GAP-43 present in S [Aqueous (A) plus Detergent (D) fractions] and P (A plus D fraction), respectively, after background subtraction. Values of Kp were normalized to those observed in control conditions. Subcellular Fractionation and TX-114 Partition Assay Cells grown onto 60 mm dishes were washed with cold PBS (140 mM NaCl, 8.4 mM Na2HPO4, 1.6 mM NaH2PO4, pH 7.5); and harvested by scrapping in PBS containing a protease inhibitor mixture (PIM) containing 5 mg/ml aprotinin, 0.5 mg/ml leupeptin, and 0.7 mg/mL pepstatin (PBS-PIM). Extracts were centrifuged at 4uC for 5 min at 13000 g and resuspended in 400 ml of 5 mM Tris-HCl (pH 7.0) (buffer T) in the presence of protease inhibitors (T-PIM). Pellets were dispersed by repeated pipetting and vortexing. After 30 min of incubation in T-PIM, pellets were passed 60 times through a 25-gauge needle. Nuclear fractions and unbroken cells were removed by twice centrifugation at 4uC for 5 min at 600 g. Supernatants were then ultracentrifuged at 4uC for 1 h at 400000 g using a TLA 100.3 rotor (Beckman Coulter, Inc., CA, USA). The supernatant (S1 fraction) was removed, and the pellet (P1 fraction) was resuspended in 400 ml of T-PIM. Both fractions were further ultracentrifuged at 400000 g and the supernatant of S1 fraction was removed (S fraction) and the pellet of P1 fraction was resuspended in 400 ml of T-PIM (P fraction). 100 ml of 5% (v/v) TX-114 in T-PIM was added to S and P fractions and incubated at 4uC during 1 h. Then, samples were incubated at 37uC for 3 min and centrifuged at 13000 g. The aqueous upper phase (A) and the detergent-enriched lower phase (D) were separated and extracted again with detergent and aqueous solutions, respectively. The resulting samples were PLoS ONE | www.plosone.org 11 November 2010 | Volume 5 | Issue 11 | e15045