62 Biochemical Society Transactions (2013) Volume 41, part 1 Palmitoylation and the trafficking of peripheral membrane proteins Luke H. Chamberlain 1 , Kimon Lemonidis, Maria Sanchez-Perez, Martin W. Werno, Oforiwa A. Gorleku and Jennifer Greaves Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, U.K. Abstract Palmitoylation, the attachment of palmitate and other fatty acids on to cysteine residues, is a common post-translational modification of both integral and peripheral membrane proteins. Dynamic palmitoylation controls the intracellular distribution of peripheral membrane proteins by regulating membrane–cytosol exchange and/or by modifying the flux of the proteins through vesicular transport systems. Introduction S-palmitoylation, the attachment of fatty acids (predomin- antly palmitate) on to cysteine residues, has emerged as an important post-translational modification of a broad range of cellular proteins. Palmitoylation reactions are mediated by the DHHC protein family [1,2]. These proteins are identified by the presence of a 51-amino-acid domain containing a DHHC (Asp-His-His-Cys) motif and a high abundance of cysteine residues. Mammalian genomes encode up to 24 DHHC proteins (excluding splice variants) [3], which localize mainly to the endoplasmic reticulum and Golgi apparatus [4]. Genetic studies in the yeast Saccharomyces cerevisiae have shown that essentially all protein palmitoylation is dependent upon DHHC proteins [5], although there may be a few examples of DHHC protein- independent palmitoylation (e.g. [6]). DHHC proteins are all predicted to be polytopic membrane proteins, with the catalytic DHHC domain present on a cytoplasmic loop [7]. This topological organization restricts palmitoylation reac- tions to the cytoplasmic surface of intracellular membrane compartments. This is an important distinction between palmitoylation and other lipidation reactions, such as N-myristoylation and isoprenylation, which are catalysed by soluble enzymes. Palmitoylated substrates include both transmembrane and soluble proteins (a term used in the present paper to indicate the lack of a transmembrane domain) [8]. The strong membrane affinity of palmitate is essential for supporting the peripheral membrane association of many soluble proteins. For such proteins, the intracellular localization of the partner DHHC protein dictates to which membrane compartment these proteins become stably anchored. However, many soluble proteins do not remain at the compartment where their palmitoylation takes place, but instead are collected as cargo and delivered to other regions of the cell via Key words: cysteine-string protein, DHHC protein, palmitoylation, 25 kDa synaptosome- associated protein (SNAP25). Abbreviations used: APT, acyl protein thioesterase; BFA, brefeldin A; CSP, cysteine-string protein; PM, plasma membrane; RE, recycling endosome; RNAi, RNA interference; SNAP25, 25 kDa synaptosome-associated protein; TGN, trans-Golgi network. 1 To whom correspondance should be addressed (email luke.chamberlain@strath.ac.uk). vesicular transport [9]. Furthermore, palmitoylation is a reversible modification, and the intracellular localization of palmitoylated peripheral membrane proteins is markedly affected by dynamic changes in palmitoylation status; palmitate turnover on some proteins may be as short as a few minutes [10]. The enzymes mediating protein depalmitoylation have not been characterized as extensively as the DHHC palmitoyltransferases; however, APTs (acyl protein thioesterases) 1 and 2 mediate fatty acid turnover on at least some palmitoylated proteins, and are generally considered to be the predominant enzymes mediating protein depalmitoylation in cells [11–13]. Trafficking of palmitoylated Ras proteins The role of palmitoylation in regulating the trafficking of Ras proteins has been extensively studied, and models for the palmitoylation-dependent localization of Ras have served as exemplars for other soluble proteins. The activation status of Ras proteins is controlled by GTP/GDP binding, and the signalling functions of these proteins is central to key processes such as cellular growth and differentiation [14]. Hyperactivating mutations in Ras are present in many cancers, and thus it is important to delineate mechanisms regulating their activity from a clinical/translational per- spective [15]. As Ras function is dependent on membrane interaction, there has been considerable interest in the lipid modifications and membrane-targeting pathways of these proteins. All Ras isoforms undergo farnesylation on a C-terminal CAAX (Cys-Ala-Ala-Xaa) motif [16]. However, a single isoprenyl chain is not sufficient for stable membrane interaction [17] or for trafficking of Ras proteins to the PM (plasma membrane) [18]. Instead, PM targeting of Ras proteins depends on a second signal that is located adjacent to the CAAX motif: a polybasic domain for K-Ras and palmitoylation for N- and H-Ras [16,18,19]. Analyses of the trafficking of palmitoylated Ras isoforms have led to the development of a fascinating molecular description of how dynamic palmitoylation and depalmitoylation events co-ordinate the precise intracellular patterning of these proteins [10,20,21]. It has been recognized for many C  The Authors Journal compilation C  2013 Biochemical Society Biochem. Soc. Trans. (2013) 41, 62–66; doi:10.1042/BST20120243 Biochemical Society Transactions www.biochemsoctrans.org