Supporting Information Figure S1 In vivo deacylation of single acylated GAP- 43full(C3S). A) CHO-K1 cells transiently expressing GAP- 43full(C3S)-YFP [GAP-43full(C3S)] were treated with 50 mM 2- BP (2-BP) or vehicle (Control) for 2 h. Then, cells were lysed, ultracentrifuged and the S (soluble) and P (pellet) fractions isolated. Buffer containing 1% v/v TX-114 was added to samples and the phase separation was induced at 37uC. Proteins from the A (aqueous) and D (detergent) phases were Western blotted with an antibody to GFP. The lowest panels show the Western blot using antibody to a-tubulin. B) Quantification of Western blot showed in A (see Materials and Methods). Kp = K1/K2, where K1 = P GAP-43/S GAP-43 and K2 = P tubulin/S tubulin. Kp con- trol = 100%. (TIF) Figure S2 APT-1 expression analysis in COS-7 and CHO-K1 cells. PCR analysis of APT-1 expression using the following templates: first-strand cDNA obtained from RT of CHO-K1 cells purified mRNA (RT-PCR CHO-K1); the reaction mixture [Ctrl (-)]; total mRNA from CHO-K1 cells (Total RNA CHO-K1); first-strand cDNA obtained from RT of COS-7 cells purified mRNA (RT-PCR COS-7); genomic DNA from CHO-K1 cells (gDNA CHO-K1) and from COS-7 cells (gDNA COS-7). Genomic DNA and mRNA were extracted from CHO-K1 and COS-7 cells 2 day after seeding in 100 mm Petri dishes. (TIF) Figure S3 APT-1 overexpression did not significantly modify membrane association and deacylation kinetic of diacylated N13GAP-43 and GAP-43full. 60 h after transient transfection, CHO-K1 cells coexpressing GAP-43full and APT-1 (A) or N13GAP-43 and APT-1 (B) 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 the cells expressing APT-1 (cherry fluorescence, pseudocolored gray). Scale bar: 5 mm. (TIF) References 1. Takai Y, Sasaki T, Matozaki T (2001) Small GTP-binding proteins. Physiol Rev 81: 153–208. 2. Marrari Y, Crouthamel M, Irannejad R, Wedegaertner PB (2007) Assembly and trafficking of heterotrimeric G proteins. Biochemistry 46: 7665–7677. 3. el-Husseini Ael D, Bredt DS (2002) Protein palmitoylation: a regulator of neuronal development and function. Nat Rev Neurosci 3: 791–802. 4. Huang K, El-Husseini A (2005) Modulation of neuronal protein trafficking and function by palmitoylation. Curr Opin Neurobiol 15: 527–535. 5. Resh MD (1999) Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta 1451: 1–16. 6. Resh MD (2004) Membrane targeting of lipid modified signal transduction proteins. Subcell Biochem 37: 217–232. 7. Newman CM, Magee AI (1993) Posttranslational processing of the ras superfamily of small GTP-binding proteins. Biochim Biophys Acta 1155: 79– 96. 8. Resh MD (2006) Trafficking and signaling by fatty-acylated and prenylated proteins. Nat Chem Biol 2: 584–590. 9. Bhatnagar RS, Gordon JI (1997) Understanding covalent modifications of proteins by lipids: where cell biology and biophysics mingle. Trends Cell Biol 7: 14–20. 10. Resh MD (2006) Palmitoylation of ligands, receptors, and intracellular signaling molecules. Sci STKE 2006: re14. 11. Smotrys JE, Linder ME (2004) Palmitoylation of intracellular signaling proteins: regulation and function. Annu Rev Biochem 73: 559–587. 12. Magee AI, Gutierrez L, McKay IA, Marshall CJ, Hall A (1987) Dynamic fatty acylation of p21N-ras. EMBO J 6: 3353–3357. APT-2-Mediated Deacylation of Peripheral Proteins Figure S4 Analysis of acyl-protein thioesterase expres- sion in SH-SY5Y and SK-Mel 28 cells. A) PCR screening of acyl-protein thioesterase gene expression in SH-SY5Y, treated (RA) or not (Ctrl) with retinoic acid (middle and upper panels, respectively), and SK-Mel 28 human cells (lower panels). For each indicated gene (see gene accession number at the bottom), PCRs were performed using specific primers and the following templates: reaction mixture [Ctrl (-)]; SH-SY5Y or SK-Mel 28 genomic DNA (gDNA) and first-strand cDNA obtained from RT of SH-SY5Y or SK-Mel 28 cells purified mRNA (RT-PCR). B) PCR analysis of isoform expression coded by gene # 11313. For each isoform indicated at the right (with the corresponding accession number), PCRs were performed using specific primers and the following templates: first-strand cDNA obtained from RT of SH-SY5Y [treated (RA) or not (Ctrl) with retinoic acid] or SK-Mel 28 cells purified mRNA (RT-PCR); SH-SY5Y or SK-Mel 28 genomic DNA (gDNA). (TIF) Table S1 Bioinformatic data of genes potentially coding acyl-protein thioesterases and PCR primer sequences. (DOC) Table S2 Aminoacid and nucleotide sequence align- ments of APT-2 from CHO-K1 and Hela cells. (DOC) Acknowledgments We thank Rodrigo Quiroga (CIQUIBIC, Cordoba, Argentina) for excellent assistance with bioinformatic tools, comments and discussions. The authors also thank the technical assistance of C. Sampedro, G. Schachner and S. Deza. Author Contributions Conceived and designed the experiments: VMT GAG JLD. Performed the experiments: VMT GAG. Analyzed the data: VMT GAG JLD. Contributed reagents/materials/analysis tools: JLD. Wrote the paper: VMT GAG JLD. Performed GAP-43 constructs, helped with experiments and reviewed the final manuscript: AT. 13. Duncan JA, Gilman AG (1998) A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J Biol Chem 273: 15830–15837. 14. Yeh DC, Duncan JA, Yamashita S, Michel T (1999) Depalmitoylation of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca(2+)-calmodulin. J Biol Chem 274: 33148–33154. 15. Baker TL, Zheng H, Walker J, Coloff JL, Buss JE (2003) Distinct rates of palmitate turnover on membrane-bound cellular and oncogenic H-ras. J Biol Chem 278: 19292–19300. 16. Hancock JF (2003) Ras proteins: different signals from different locations. Nat Rev Mol Cell Biol 4: 373–384. 17. Baekkeskov S, Kanaani J (2009) Palmitoylation cycles and regulation of protein function (Review). Mol Membr Biol 26: 42–54. 18. Zeidman R, Jackson CS, Magee AI (2009) Protein acyl thioesterases (Review). Mol Membr Biol 26: 32–41. 19. Goodwin JS, Drake KR, Rogers C, Wright L, Lippincott-Schwartz J, et al. (2005) Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway. J Cell Biol 170: 261–272. 20. Kanaani J, Patterson G, Schaufele F, Lippincott-Schwartz J, Baekkeskov S (2008) A palmitoylation cycle dynamically regulates partitioning of the GABA- synthesizing enzyme GAD65 between ER-Golgi and post-Golgi membranes. J Cell Sci 121: 437–449. 21. Rocks O, Gerauer M, Vartak N, Koch S, Huang ZP, et al. (2010) The palmitoylation machinery is a spatially organizing system for peripheral membrane proteins. Cell 141: 458–471. 22. Rocks O, Peyker A, Kahms M, Verveer PJ, Koerner C, et al. (2005) An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307: 1746–1752. PLoS ONE | www.plosone.org 14 November 2010 | Volume 5 | Issue 11 | e15045