Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP-43 Vanesa M. Tomatis¤a, Alejandra Trenchi, Guillermo A. Gomez¤b, Jose L. Daniotti* Centro de Investigaciones en Qu´ımica Biolo´ gica de C o´rdoba (CIQUIBIC, UNC-CONICET), Departamento de Qu´ımica Biolo´ gica, Facultad de Ciencias Qu´ımicas, Universidad Nacional de C o´rdoba, C o´rdoba, Argentina Abstract An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S- acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyl- transferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution. Citation: Tomatis VM, Trenchi A, Gomez GA, Daniotti JL (2010) Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP- 43. PLoS ONE 5(11): e15045. doi:10.1371/journal.pone.0015045 Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received August 31, 2010; Accepted October 13, 2010; Published November 30, 2010 Copyright: 2010 Tomatis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by Grants from Secretar´ıa de Ciencia y Tecnolog´ıa (SECyT)-Universidad Nacional de Co´ rdoba (UNC) (www.secyt.unc. edu.ar), Consejo Nacional de Investigaciones Cient´ıficas y Te´cnicas (CONICET) (www.conicet.gov.ar), Agencia Nacional de Promocio´ n Cient´ıfica y Tecnolo´ gica (ANPCyT) (www.agencia.gov.ar) and Ministerio de Ciencia y Tecnolog´ıa de la Provincia de C o´rdoba, Argentina (www.mincyt.cba.gov.ar). V.M.T., A.T. and G.A.G. were recipients of CONICET (Argentina) Fellowship. J.L.D is a career investigator of CONICET (Argentina). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: daniotti@dqb.fcq.unc.edu.ar €a Current address: Queensland Brain Institute and School of Biomedical Sciences, The University of Queensland, Queensland, Australia €b Current address: Institute for Molecular Bioscience, Division of Molecular Cell Biology, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia Introduction Fatty-acylated peripheral proteins such as members of the small G-protein Ras family, heterotrimeric G-proteins, the neuronal proteins PSD-95 and growth-associated protein-43 (GAP-43) [1–4] are synthesized in the cytosol and posttranlationally modified by different lipid moieties [5–9]. These lipid modifications govern their membrane association and membrane subdomain segregation as well as their trafficking, function and stability [8,10,11]. Among all posttranslational lipid modification of proteins, including isoprenylation and myristoylation, the addition of fatty acid to the sulfhydryl group of a cysteine to form a thioester bond (S-acylation, often referred to as palmitoylation) is the only readily reversible linkage having a much shorter half-life than that of the protein [12–15]. Consequently, S-acylation can operate as a switch regulating not only protein-membrane binding affinity and segregation but also modulating its biological activities [16–18]. S- acylation is catalyzed by protein acyltransferases (PATs) while deacylation by acyl-protein thioesterases (APTs), and continuous cycles of de- and reacylation reactions accounts for the specific subcellular distribution of peripheral proteins as the small GTPases H- and N-Ras [19–22]. PATs have been identified both in yeast and mammalian [18]. These proteins share a common zinc finger-like sequence, containing a cysteine-rich domain with an aspartate-histidine- histidine-cysteine (DHHC) motif, which mediates the PAT activity. Although S-acylation was reported to occur in several membrane compartments [22–24] and with apparent substrate selectivity, a recent work from Bastiaens and coworker demon- strated that S-acylation of semisynthetic substrates is detectable only in the Golgi complex and, that substrate specificity is not essential for the reacylation step [21]. Despite the progress that has been made in identifying and characterizing PATs much less is known about the thioesterases that deacylate proteins. So far, only two APT has been described: palmitoyl-protein thioesterase 1 (PPT1) and acyl-protein thioesterase 1 (APT-1). PPT1 was discovered based on its ability to deacylate H-Ras [25]. Further research revealed that PPT1 is a lysosomal enzyme involved in protein degradation [26] discarding the possibility to play a role in deacylation of cytoplasmic proteins. APT-1, originally isolated PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e15045