The on–off story of protein palmitoylation

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The on –off story of protein palmitoylation Marie-Jose´ Bijlmakers1 and Mark Marsh2 1

Peter Gorer Department of Immunobiology, Kings College London, 3rd Floor New Guy’s House, Guy’s Hospital, London SE1 9RT, UK 2 MRC Cell Biology Unit, MRC-LMCB and Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, UK

Palmitoylation is one of the most frequent post-translational modifications found on proteins. It contributes to membrane association, protein sorting and many other processes. Through its reversibility, palmitoylation also provides mechanisms to regulate the functional activities of integral and peripheral membrane proteins. Here we discuss evidence that proteins can be palmitoylated at different locations in the cell, how targeting to these locations might be directed, and aspects of the proposed functions of palmitoylation. The covalent attachment of lipid moieties is an essential modification found on many proteins. In eukaryotic and viral systems, four major forms of lipid modification have been recognized so far: the co-translational amino (N)-terminal myristoylation of cytosolic proteins; the modification of plasma membrane (PM) proteins with glycophosphatidyl inositol (GPI); the carboxy (C)-terminal isoprenylation of cytoplasmic proteins; and the most common, and apparently most versatile, modification – the post-translational addition of palmitic acid to many integral and peripheral membrane proteins [1 –3]. In most proteins palmitic acid is esterified to the free thiol of cysteines, but other saturated (such as myristic and stearic) and unsaturated (such as oleic and arachidonic) fatty acids can also be used [3]. Hence S-acylation is the more appropriate term, although ‘palmitoylation’ is used more frequently and is used in this review. The mechanisms involved in palmitoylation are understood poorly. The relevant enzymes are mostly uncharacterized, although some candidates have been reported recently (Box 1). The wide range of substrates, absence of clear consensus motifs, and the fact that palmitoylation occurs at various cellular locations also contribute to the murky picture. Palmitoyl acyltransferase (PAT) activity has been partially purified and found to be tightly membrane associated [4,5]. Non-enzymatic palmitoylation is possible, but, apart from modifications reported in mitochondria, these reactions might only occur in vitro (Box 2). Palmitoylation motifs The features required for palmitoylation are poorly understood. Cysteines that are close to membrane-interCorresponding author: Marie-Jose´ Bijlmakers ([email protected]).

acting domains [transmembrane domains (TMDs) or membrane-associated domains in non-integral membrane proteins] seem to be preferred sites, possibly because of their accessibility to membrane-associated PAT. Additional factors must also be involved because not all cysteines that are near to TMDs are palmitoylated, and some cysteines that are not obviously close to membrane-interacting domains can be acylated. Transmembrane proteins Many viral and cellular integral membrane proteins are palmitoylated on cysteines that are either close to the TMD/cytoplasmic domain (CD) boundary, or located in their CD (Figs 1,2). In some cases the TMD itself can influence palmitoylation. Structural models for the TMD of influenza virus hemagglutinin A (HA) predict a helix with nonhydrophobic residues aligned on one side (Fig. 1). Sequence changes that insert hydrophobic residues into this face reduce palmitoylation on cysteines in the CD [6]. Cysteines introduced into the CD of the Sendai virus F protein, which has a very hydrophobic TMD (Fig. 1), are not palmitoylated, but inserting the TMD from HA into F protein promotes palmitoylation. Thus, nonhydrophobic residues in a TMD can favor palmitoylation of transmembrane proteins. Whether these residues affect transport and/or assembly of oligomeric proteins, or influence interactions directly involved in palmitoylation, is unknown. A need for specific residues around the palmitoylation sites in the CDs of several heptahelical G-proteincoupled receptors (GPCRs) has been suggested. In these proteins, clusters of hydrophobic and positively charged amino acids often precede and follow, respectively, one or more palmitoylated cysteines (Fig. 1). The non-enzymatic in vitro palmitoylation (Box 2) of peptides corresponding to the C-terminal domain of the b2-adrenergic receptor (b2-AR) suggests that this arrangement is optimal for acylation [7]. However, this sequence pattern is not conserved in all palmitoylated GPCRs (Fig. 1), and the relevance of the in vitro acylation is unclear. For the a2-AR, the deletion of positively charged residues from the CD has no effect on palmitoylation [8]. In the coxsackie and adenovirus receptor (CAR), which contains a single TMD, palmitoylation does not seem to require any CD

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Box 1. Purification of palmitoyl acyltransferase activity Attempts to isolate palmitoyl acyltransferases (PATs) using standard protein purification techniques have been frustrated by the extreme instability of the activities. Membrane-associated PAT activities have been partially purified from bovine brain. These activities are resistant to extraction with high salt or high pH, but are sensitive to protease treatment, boiling and detergent extraction, suggesting that the PATs are membrane-integrated enzymes. A protein of 70 kDa that catalyzes the addition of palmitate to the cortical cytoskeletal protein spectrin has been isolated from erythrocytes, but no further characterization of this activity was reported [a]. A dimer of 260 and 270 kDa proteins, which enhances palmitoylation of Drosophila Ras in vitro, has been cloned from the silkworm Bombyx mori [b]. This protein complex is expressed only during embryogenesis and is probably not normally involved in palmitoylating Ras. A genetic screen in Drosophila identified a protein required for palmitoylating Sonic Hedgehog (Shh). Shh is attached to the outer leaflet of the plasma membrane through an amide-linked palmitoyl moiety (although this might be attached initially as a thioester) [c], as well as a covalently linked cholesterol moiety [d]. Loss of the newly identified skinny hedgehog (ski ) [e], which is also known as sightless [f] or rasp [g], leads to an impairment of Shh function that is concomitant with loss of Shh palmitoylation [e,f]. Skinny hedgehog is localized in the lumen of organelles of the secretory pathway and is unlikely to palmitoylate cytosolic or transmembrane proteins. A significant breakthrough has been achieved recently with the identification of two enzyme activities in yeast that mediate C-terminal palmitoylation, the Erp2p –Erp4p complex [h] and Akr1p [i]. Mutations in the genes ERF2 and ERF4 (also known as SHR5 ) were previously found to diminish palmitoylation of Ras2p. A purified complex of Erf2p and Erf4p has now been shown to mediate Ras2p palmitoylation in vitro. Ras2p, which is farnesylated at its C-terminus, is a preferred substrate over the similarly modified mammalian H-Ras and the myristoylated Gai1 subunit. Erf2p is an integral membrane protein that localizes to the endoplasmic reticulum and contains a conserved Asp-His-His-Cys

cysteine-rich domain (DHHC-CRD). Akr1p also contains a DHHCCRD but shares no other homology with Erf2p. Purified Akr1p palmitoylates the casein kinase Yck2p in vitro. The predicted protein has six transmembrane domains and has been localized preliminarily to the Golgi. The substrate specificity of these proteins – Akr1p mutants palmitoylate Ras2p normally and Erf2p mutants palmitoylate Yck22p normally – suggests that many more PAT activities might exist.

References a Das, A.K. et al. (1997) Purification and biochemical characterization of a protein – palmitoyl acyltransferase from human erythrocytes. J. Biol. Chem. 272, 11021 – 11025 b Ueno, K. and Suzuki, Y. (1997) p260/270 expressed in embryonic abdominal leg cells of Bombyx mori can transfer palmitate to peptides. J. Biol. Chem. 272, 13519– 13526 c Pepinsky, R.B. et al. (1998) Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem. 273, 14037– 14045 d Porter, J.A. et al. (1996) Cholesterol modification of hedgehog signaling proteins in animal development. Science 274, 255 – 259 e Chamoun, Z. et al. (2001) Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal. Science 293, 2080 – 2084 f Lee, J.D. and Treisman, J.E. (2001) Sightless has homology to transmemebrane acyltransferases and is required to generate active Hedgehog protein. Curr. Biol. 11, 1147– 1152 g Micchelli, C.A. et al. (2002) Rasp, a putative transmembrane acyltransferase is required for Hedgehog signaling. Development 129, 843– 851 h Lobo, S. et al. (2002) Identification of a Ras palmitoyl transferase in Saccharomyces cerevisiae. J. Biol. Chem. 277, 41268– 41273 i Roth, A.F. et al. (2002) The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase. J. Cell Biol. 159, 23 – 28

Box 2. Non-enzymatic S-acylation Spontaneous transfer of palmitate from palmitoyl-CoA to cysteine residues can occur in vitro. A cysteinic SH group, which is a good nucleophile, attacks the thioester bond between CoA and palmitate. This non-enzymatic, autoacylation occurs with several proteins, including Ga subunits, SNAP25, myelin proteolipid and rhodopsin, as well as with peptides representing the myristoylated N-terminal domain of the Src kinase Yes and the C-terminal domain of the b2-adrenergic receptor. The reaction shows similarities to in vivo palmitoylation; for example, autoacylation of Ga subunits at physiological pH occurs exclusively at Cys3, the principal palmitoylation site in vivo, and is both dependent on N-terminal myristoylation and enhanced by the presence of bg subunits (but only when the g subunit is prenylated) [a]. Similarly, as in vivo, the autoacylation of SNAP25 is enhanced by the presence of one of its binding partners, syntaxin [b]. Autoacylation is dependent on time, temperature, concentration and pH, and does not occur when denatured substrates are used. Whether autoacylation on cytosolic cysteines occurs in vivo is unclear. Several differences between the enzymatic and non-enzymatic processes suggest that it does not. In vitro, some Ga subunits are palmitoylated more efficiently than other subunits, although this does not seem to be the case in vivo. Conditions that allow complete Ga palmitoylation in vitro do not support palmitoylation of myristoylated Fyn [a]; in vivo, these proteins are acylated with comparable kinetics. Proteins that are not normally palmitoylated, such as actin, are Sacylated in vitro. http://ticb.trends.com

The most compelling argument against autoacylation comes from considerations of acyl-CoA binding protein (ACBP). This abundant cytosolic protein binds long-chain fatty acids with high affinity and is likely to keep the cytosolic concentration of free long-chain fatty acylCoA below that needed for autoacylation. Autoacylation of Ga subunits, for example, is predicted to take hours rather than minutes at cytosolic fatty acid concentrations [c]. In agreement, autoacylation of Ga is completely inhibited in the presence of ACBP, whereas in vitro Sacylation in the presence of a partially purified PAT is only partially affected [d].

References a Duncan, J.A. and Gilman, A.G. (1996) Autoacylation of G protein a subunits. J. Biol. Chem. 271, 23594– 23600 b Veit, M. (2000) Palmitoylation of the 25-kDa synaptosomal protein (SNAP-25) in vitro occurs in the absence of an enzyme, but is stimulated by binding to syntaxin. Biochem. J. 345, 145 – 151 c Leventis, R. et al. (1997) Acyl-CoA binding proteins inhibit the nonenzymic S-acylation of cysteinyl-containing peptide sequences by long-chain acyl-CoAs. Biochemistry 36, 5546– 5553 d Dunphy, J.T. et al. (2000) Differential effects of acyl-CoA binding protein on enzymatic and non-enzymatic thioacylation of protein and peptide substrates. Biochim. Biophys. Acta 1485, 185 – 198

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TRANSMEMBRANE PROTEINS Viral Proteins Flu HA Sendai F

..VILWFSFGASCFLLLAIAMGLVFICVKNGNMRCTICI-COOH .. V I T I I V V M V V I L V V I I V I V I V L Y R L K R S M L M G N P . . ..VITIIVVMVVILVVIIVIVIVLYRLKRCMLMCNP..

7TM G-protein-coupled receptors α2A-adrenergic R . . F F W F G Y C N S S L N P V I Y T I F N H D F R R A F K K I L C R G D R K R I V - COOH β2-adrenergic R ..EVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQ.. Vasopressin V2 R . . L L M L L A S L N S C T N P W I Y A S F S S S V S S E L R S L L CC A R G R T P P S L G P Q D E S C T T A S S . . Luteinizing hormone R .. VL LVL FY PINS CAN PFLYA IFT KTF QRDFF LL LSKFGC C KR RAELYRR KDFS AYT SN.. Serotonin R ..AFLWLGYINSGLNPFLYAFLNKSFRRAFLIILCCDDERYRRPSILGQTVPCSTTTINGS..

Endothelin B R Rhodopsin Bradykinin B2 R Dopamine D(1) R

..VLDYIGINMASLNSCINPIALYLVSKRFKNCFKSCLCCWCQSFEEKQSLEEKQSCLKFK.. ..IFMTIPAFFAKSAAIYNPVIYIMMNKQFRNCMLTTICCGKNPLGDDEASATVSKTETSQ.. ..VITQIASFMAYSNSCLNPLVYVIVGKRFRKKSWEVYQGVCQKGGCRSEPIQMENSMGTLR.. ..FDVFVWFGWANSSLNPIIYAFNADFRKAFSTLLGCYRLCPATNNAIETVSINNNG.. ..VTETLGMTHCCINPIIYAFVGEKFRNYLLVFFQKHIAKRFCKCCSIFQQEAPERAS..

CCR5 T-cell specific CD4 LAT pTα CD8β CD8α

.. F L A C V L G G S F G F L G F L G L C I L C CV R CR H Q Q R Q A A R M S Q I K R L L S E K K T C Q C . . ..LSPVGLGLLLLPFLVTLLAALCVRCRELPVSYDSTSTESLYPRSILIKPP.. ..LWLSLLRLLLFKLLLLDVLLTCSHLRLHVLAGQHLQPPPSRKSLPPTHRIWT-COOH . . T L S L L V V C I L L L L A F L G V A V Y F Y C V R R R A R I H F M K Q F H K - COOH ..W A P L A G I C V A L L L S L I I T L I C Y H R S R K R V C K C P R P L V R Q E G K P R P S E K I V

CYTOSOLIC PROTEINS Myristoylated and palmitoylated proteins Lck MGCGCSSHPEDDWMENIDVCENCHYPIVPL.. Fyn MGCVQCKDKEATKLTEERDGSLNQSSGYRY.. Fgr MGCVFCKKLEPVATAKEDAGLEGDFRSYGA.. Yrk MGCVHCKEKISGKGQGGSGTGTPAHPPSQY.. Yes MGCIKSKENKSPAIKYRPENTPEPVSTSVS.. Lyn MGCIKSKGKDSLSDDGVDLKTQPVRNTERT.. Hck MGCVKSRFLRDGSKASKTEPSANQKGPVYV.. Gαo Gα z Gαi1

MGCTLSAEERAALERSKAIEKNLKEDGISA.. MGCRQSSEEKEAARRSRRIDRHLRSESQRQ.. MGCTLSAEDKAAVERSKMIDRNLREDGEKA..

eNOS Vac8p

MGNLKSVAQEPGPPCGLGLGLGLGLCGKQGPATPAP.. MGSCCSCLKDSSDEASVSPIADNEREAVTLLLGYLE..

Palmitoylated only PSD-95 MDCLCIVTTKKYRYQDEDTPPLEHSPAHLP.. MGCLGNSKTEDQRNEEKAQREANKKIEKQL.. Gαs GAP-43 MLCCMRRTKQVEKNDDDQKIEQDGIKPEDK.. SCG10 MAKTAMAYKEKMKELSMLSLICSCFYPEPRNINI.. GRIP1b MPGWKKNIPICLQAEEQER--------------------------EEFKG.. GRIP1a MIAVSFKCRCQILRRLTKDESPYTKSASQTKPPDGALAVRRQSIPEEFKG.. GAD-65 MASPGSFWSFGSEDGSGDPENPGTARAWCQVAQKFTGGIGNKLCALLYGDSEK PAESGGDVTSRAATRKVACTCDQKPCSCPKGDVNYALLHAT.. GAD-67 MASSTPSPATSSNAGADPNTTNLRPTTYDTWCGVAHGCTRKLGLKICGFLQRT NSLEEKSRLVSAFRERQASKNLLSCENSDPGARFRRTETDFSNLFAQ.. TRENDS in Cell Biology

Fig. 1. Amino acid sequences surrounding palmitoylation sites. The gray box indicates predicted TMDs. Cysteines known to be palmitoylated are depicted in green; cysteines known not to be palmitoylated are underlined. The glycines (G) to which myristic acid is attached are underlined. Hydrophobic amino acids are in bold, basic residues in red and acidic ones in blue. The proteins in italics are not palmitoylated and are depicted for comparison with closely related palmitoylated counterparts, or, in the case of Sendai F protein, to illustrate the difference in hydrophobicity of the transmembrane domain (TMD) compared with that of the Influenza HA (Flu HA) protein, as discussed in the text. Sendai F protein does not normally contain cysteines in its cytoplasmic domain, the lower sequence shows introduced cysteines [8].

determinants at all, although the possible role of TMD sequences in this protein has not been assessed [9]. The problem of identifying palmitoylation signals is illustrated by the CD8ab heterodimer – the T-cell antigen receptor (TCR) co-receptor on cytotoxic T cells. This heterodimer assembles before exit from the endoplasmic reticulum (ER). Both CD8a and CD8b have cysteines located close to the TMD/CD boundary, but only CD8b is palmitoylated (Fig. 1). The hydrohttp://ticb.trends.com

phobicity of the TMD is similar in the two proteins, and positively charged residues are found close to the cysteines in both. Most palmitoylated cysteines are found within ten residues of either side of the TMD/CD boundary (Fig. 1). However, the acylation of CD cysteines that are further from a TMD occurs on several proteins, including the cation-independent mannose 6-phosphate receptor (MPR), in which palmitoylation occurs 34

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(a)

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HIV-Env

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Fig. 2. Examples of different types of palmitoylated proteins. (a) Integral membrane proteins with palmitate attached close to the TMD–CD boundary. Proteins with a single TMD or with several membrane-spanning domains can be palmitoylated on either their CD or their TMD. Proteins with a single TMD can be of type I or II orientation. Palmitoylated multiple-membrane-spanning proteins with two (e.g. caveolin), four (e.g. CD9 and CD151) and seven (GPCR) TMDs have been described. (b) Integral membrane proteins with palmitate at cysteines that are distant from the TMD –CD boundary. Palmitoylation could position CD elements close to the membrane. (c) Palmitoylated and myristoylated cytosolic proteins. Myristic acid is added during translation through an amide bond to Gly2 (after removal of the N-terminal methionine). In most cases, posttranslational palmitoylation at one or more nearby cysteines is required for stable membrane association. (d) Palmitoylated and prenylated cytosolic proteins. Prenylation occurs shortly after translation at a C-terminal cysteine in the Cys-Aaa-Aaa-Xaa motif, where Aaa is an aliphatic residue and Xaa is any residue. Either farnesyl or geranylgeranyl are attached through a thioether bond. Palmitoylation at a nearby cysteine follows prenylation and strengthens membrane association. (e) Cytosolic proteins that are only palmitoylated. Palmitate can be attached at the N-terminus or at other sites in the protein. Abbreviations: CD, cytoplasmic domain; TMD, transmembrane domain.

residues from the TMD [10], and the envelope (Env) proteins of primate immunodeficiency viruses [11]. For example, HIV-1 HXB2 Env is palmitoylated on cysteines located 59 and 132 residues from the TMD. Of these, the membrane proximal cysteine is conserved in most HIV strains and is located close to a proposed amphipathic helix that might interact with the membrane [11]. Cytosolic proteins In cytosolic proteins, palmitate is found attached either close to myristic acid or prenyl groups, or in the absence of other acylations (Fig. 2). Newly synthesized Src family kinases and Ga subunits are co-translationally myristoylated, but they do not stably associate with membranes until palmitoylation has occurred [12– 15]. Such dually acylated proteins often have positively charged amino acids around the palmitoylation sites (Fig. 1). These residues might be needed to enhance membrane binding before palmitoylation and thus to enhance accessibility to PAT [3]. In agreement with this, the membrane-association kinetics of the Src kinase Fyn, which has lysines at positions 7, 9 and 13, is faster than that of the related kinase Lck, which lacks basic residues in its extreme Nterminus [16,17]. Mutant, nonpalmitoylated Fyn also associates with membranes to a greater extent than does nonpalmitoylated Lck [13– 15]. In fact, basic residues are important for membrane-association of GAP43 [18], a http://ticb.trends.com

palmitoylated but nonmyristoylated protein found in neuronal growth cones (Fig. 1). Hydrophobic residues that neighbor a cysteine can influence palmitoylation in some cytosolic proteins. The endothelial form of nitric oxide synthase (eNOS) is modified by myristoylation and by palmitoylation of Cys15 and Cys26. The sequence between these two cysteines contains five Gly-Leu repeats (Fig. 1). Substitution of the leucine residues with serines abolishes palmitoylation of eNOS [19]. Gly-Leu motifs have not been found in other proteins, but hydrophobic residues enhance palmitoylation of the nonmyristoylated proteins PSD95 and GAP43. PSD95, a scaffolding protein of the MAGUK family that is important for clustering neuronal receptors at postsynaptic densities, is palmitoylated on Cys3 and Cys5 [20]. Replacing Leu4, Ile6 or Val7 with other hydrophobic residues has no effect on palmitoylation, but replacing them with alanine, serine or acidic residues reduces palmitoylation [21]. Similarly, for GAP43, which is palmitoylated on Cys3 and Cys4 [22], replacing both Leu2 and Met5 with serines prevents palmitoylation, whereas replacing Met5 with hydrophobic isoleucine only partially inhibits this modification [21]. As for transmembrane proteins, at present it is difficult to predict whether specific cytosolic proteins are substrates for palmitoylation. Glutamate receptor interacting protein 1 (GRIP1) is synthesized as two splice variants,

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GRIP1a and GRIP1b, which differ only in their N-terminal regions. Both proteins have N-terminal cysteines and clusters of basic and hydrophobic residues (Fig. 1), but only GRIP1b is palmitoylated [23]. A similar situation exists for the two forms of glutamic acid decarboxylase, GAD65 and GAD67: these proteins have cysteines distributed throughout a central region, but only GAD65 is palmitoylated (Fig. 1) [24]. The lack of a clear consensus sequence and the diverse nature of the amino acids found to influence palmitoylation suggest that common structural features rather than strict sequence requirements are likely to be key factors that specify palmitoylation. Cellular site of palmitoylation PAT activity has been found in fractions containing PM, Golgi and mitochondrial membranes (Box 1) [25]. It is also enriched in sphingomyelin- and cholesterol-rich membrane microdomains [26] – the so-called ‘lipid rafts’ that are associated with both PM and intracellular membrane systems. Kinetic and biochemical experiments have indicated that PAT activities are located on the intermediate compartment (IC) [27] and, in yeast, on the vacuole [28]. The recently identified yeast palmitoyl transferases Erf2p – Erf4p and Akr1p have been localized to the ER and Golgi, respectively [4,5] (Box 1). Like PAT, palmitoylated proteins are found at the PM and on many intracellular membranes. The cellular site of palmitoylation has been investigated for only a few proteins, and assumed for many others. Determining the location where specific proteins are palmitoylated has proved to be difficult. Whether the substrates are integral or peripheral membrane proteins, they can be rapidly relocated after palmitoylation. In many situations, proteins go through cycles of depalmitoylation and repalmitoylation, and thus might be acylated at more than one location. Although many proteins were thought to undergo acylation at the PM, there are now clear examples of proteins that are palmitoylated on intracellular membranes. Transmembrane proteins Palmitoylation of vesicular stomatitis virus glycoprotein (VSV-G) and Sindbis virus glycoprotein E1 occurs early in the exocytic pathway (Fig. 3a). When cells are incubated at 158C, these proteins are not transported from the ER and palmitoylation is blocked. Restoration of transport leads to palmitoylation before aspartic-acidlinked oligosaccharides are trimmed, which implicates the IC or cis-Golgi as the site of palmitoylation [27]. The timing of palmitoylation of influenza HA, relative to its trimerization and carbohydrate-trimming, indicates that palmitoylation of this protein takes place in a similar location [29]. A GPCR, CCR5, is also palmitoylated early in the exocytic pathway. Significantly, CCR5 cysteine mutants are transported inefficiently to the PM and show decreased mobility in compartments of the exocytic pathway [30]. Sensitivity to brefeldin A (BFA) has implicated the Golgi apparatus, or post-Golgi compartments in the palmitoylation of some proteins, such as the tetraspanins http://ticb.trends.com

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CD151 and CD9 [31]. However, palmitoylation of other proteins is unaffected by BFA, although their transport to the PM is inhibited (Fig. 3). In addition, some ER resident proteins that are not normally palmitoylated are found to be palmitoylated in BFA-treated cells, which suggests that a Golgi or IC-associated PAT is redistributed to the ER [32]. For proteins such as CD151, it is unclear whether the BFA-induced inhibition of palmitoylation is due to the loss of a functional Golgi apparatus, the inhibition of a specific PAT, or some other indirect effect. Overall, the results from biochemical and cell fractionation experiments indicate that with the exception of the ER, transmembrane proteins are palmitoylated along the exocytic pathway. Myristoylated and palmitoylated peripheral membrane proteins The biosynthesis of three myristoylated and palmitoylated proteins, Lck, Fyn and the heterotrimeric G-protein subunit (Gaz), has been analyzed in detail. At steady state, these proteins are primarily located at the PM, but their myristoylated precursors are either soluble or only weakly associated with membranes. Despite similarities in their cellular distribution, N-terminal sequences and acyl modifications (Fig. 1), the pathways through which these proteins achieve their distribution differ significantly. Lck, which is expressed primarily in T lymphocytes, interacts with the CDs of the TCR co-receptors CD4 and CD8 [33]. This interaction, which depends on Lck being palmitoylated and stably bound to the membrane, begins within minutes of Lck synthesis and occurs early in the exocytic pathway, possibly in the IC or cis-Golgi [16]. Indeed, Lck might be modified by the same PAT that acylates the co-receptors. Subsequently, membranebound Lck moves to the PM on exocytic transport vesicles (Fig. 3b). Notably, Lck transport to the PM, but not its palmitoylation, is inhibited by BFA. By contrast, newly synthesized Fyn is targeted directly to the PM with no requirement for the exocytic machinery [17] (Fig. 3e). Gaz has properties that are intermediate between those of Lck and Fyn. Gaz associates first with intracellular membranes and subsequently with the PM [34]. Although reminiscent of Lck biosynthesis, palmitoylation of Gaz does not occur on intracellular membranes, and BFA does not affect its transport to the PM. Instead, Gaz seems to sample intracellular membranes, before undergoing palmitoylation and stable binding at the PM (Fig. 3d). Why different proteins use distinct mechanisms is unclear. The main factors that are likely to determine where palmitoylation occurs are the subcellular distribution and substrate specificity of different PATs, and the interactions that influence targeting to PATs. For example, it is possible that Gaz can be palmitoylated only by a PM-restricted PAT. Indeed, Erf2p – Erf4p and Akr1p, which both mediate palmitoylation at C-termini, have diverging substrate specificities and localizations [4,5] (Box 1).

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(b) Cytosolic proteins Palmitoylation at intracellular compartment Transport blocked by BFA (Lck, N-Ras, H-Ras) (c) Cytosolic proteins Palmitoylation at intracellular compartment Palmitoylation blocked by BFA (SNAP-25, GAP-43)

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BFA

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Intermediate compartment /cis-Golgi Palmitic acid

(e) Cytosolic proteins Palmitoylation and interaction at plasma membrane only No effect of BFA (Fyn)

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Fig. 3. Transport pathways of palmitoylated proteins. (a) Palmitoylated transmembrane (TM) proteins follow the normal route through the exocytic pathway to the plasma membrane (PM). Palmitoylation of some TM proteins is blocked by brefeldin A (BFA). (b –e) Other pathways for cytosolic proteins are distinguished by the effect of BFA on transport (b) or palmitoylation (c) or by the lack of an effect on either (d,e). Direct (e) and indirect (b– d) transport routes to a final destination can be differentiated.

The localization of binding partners might attract proteins to specific membrane compartments. When bg subunits are misdirected to mitochondria, Ga subunits also mislocalize to this compartment [35]. In addition, bg subunits can recruit myristoylated, palmitoylationdeficient Ga to membranes, and enhance the palmitoylation of nonmyristoylated Ga [36 – 38]. Thus, bg subunits could be involved in targeting Ga to PAT. Although not essential for Lck palmitoylation, CD4 or CD8 might influence Lck recruitment to a membrane compartment early in the exocytic pathway. Differences in the N-terminal sequences of Src kinases, and similarly modified proteins, might influence protein interactions that are important for targeting to specific PATs or membrane systems. Analogous to the Rab escort proteins (REPs) that bring Rabs to geranylgeranyltransferases, targeting might also involve chaperones that have yet to be identified [39]. Prenylated and palmitoylated peripheral membrane proteins Ras proteins are prenylated at their C-termini and several, including H-Ras and N-Ras, are also palmitoylated. Cysteines located four residues from the C-terminus are http://ticb.trends.com

prenylated by a cytosolic prenyltransferase [1]; subsequently, the three C-terminal amino acids are removed by prenylcysteine endoprotease (hRce1) and the resulting C-terminal cysteine is methylesterified by a prenylcysteine carboxymethyltransferase (pcCMT). hRce1 and pcCMT are located on the ER [40– 42], indicating that this compartment has a role in Ras transport to the PM. Palmitoylated H-Ras and N-Ras are associated to some extent with the Golgi apparatus, and either BFA treatment or culturing the cells at 158C causes these Ras proteins to accumulate on intracellular membranes without reducing palmitate incorporation [43,44]. By contrast, the nonpalmitoylated K-Ras is not found in the Golgi region and its transport is unaffected by BFA. Thus, palmitoylation of H-Ras and N-Ras determines Golgi targeting and transport to the PM through the exocytic pathway (Fig. 3b). The enzyme that palmitoylates Ras2 in yeast, Erf2p, is localized to the ER, but a mammalian homolog has not been identified as yet. Peripheral membrane proteins modified with palmitate only The transport of two neuronal palmitoylated proteins, SNAP25 and GAP43, has been studied using chimeras of

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green fluorescent protein (GFP) in living cells [45]. SNAP25, a t-SNARE that functions in the fusion and exocytosis of secretory vesicles, is palmitoylated on cysteines located in its central domain [46]. By contrast, GAP43 is palmitoylated at its N-terminus [22]. Both proteins are located in axons but first accumulate at the trans-Golgi network (TGN) and are then transported on vesicles to the PM. Again, this resembles the synthesis of Lck, Ras and Gaz (Fig. 3); however, palmitoylation of SNAP25 and GAP43 is inhibited by BFA [47] (Fig. 3c). The palmitoylation of these proteins thus requires functional Golgi membranes either to deliver the proteins to a specific location or, perhaps, to facilitate the reaction itself. Why BFA inhibits the palmitoylation of these and not other proteins that follow the same transport route is unclear. More information on the distribution of PAT activities in normal and drug-treated cells, and the mechanisms through which PATs interact with substrate proteins, is required. SNAP25 contains a motif, located C-terminal to the palmitoylation sites, that is important for both membrane association and palmitoylation [48]. This motif might be responsible for recruiting newly synthesized SNAP25 to PATs, but the interactions that it mediates remain to be characterized. Nonpalmitoylated SNAP25 can also be recruited to membranes containing syntaxin, another t-SNARE [49]. This interaction might prevent SNAP25 from being released from membranes after depalmitoylation. Several palmitoylated cytosolic proteins are located on Golgi membranes and might be palmitoylated there. GAD65 is palmitoylated on cysteines in the central part of the protein but is targeted to the Golgi by its 27-residue N-terminal domain [24]. The related GAD67 differs in this N-terminal region and is not targeted to the Golgi (Fig. 1). Replacing the N-terminus of GAD65 with that of GAD67 blocks Golgi targeting and palmitoylation, which suggests that GAD65 palmitoylation requires Golgi targeting. For other Golgi-associated palmitoylated proteins, targeting and palmitoylation signals are more difficult to separate. SCG10 concentrates in neuronal growth cones and in the trans-Golgi [50,51]. Its 34-residue N-terminal domain, which includes two acylated cysteines, is sufficient to target GFP to the Golgi region. Palmitoylation mutants of SCG10 associate with the membrane but do not localize to the Golgi or to growth cones. Similarly, palmitoylation of eNOS is not required for membrane binding but is essential for Golgi localization [19]. Thus, Golgi targeting might be required for palmitoylation and, vice versa, palmitoylation might be necessary for retention on Golgi membranes. Functions of palmitoylation In peripheral membrane proteins, palmitoylation can promote or specify membrane interactions. In integral membrane proteins, however, the functions of palmitoylation are less clear. With increasing knowledge of protein trafficking and the structure of the PM and other cellular membranes, roles for palmitoylation are emerging. The use of bromopalmitate as a tool to inhibit palmitoylation also has led to progress in this area [52]. But, as for the http://ticb.trends.com

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examples discussed above, few clear-cut rules have been established. Localization of cytosolic proteins Palmitoylation can aid the association of cytosolic proteins with membranes and can also facilitate sorting. A N-terminal sequence, specifying myristoylation and palmitoylation, attached to GFP can promote a different distribution to one specifying dual palmitoylation [53]. The precise position of the palmitate can also influence distribution: in Lck, mutation of Cys5 but not Cys3 (Fig. 1) results in accumulation of the protein in the Golgi region of transfected NIH-3T3 cells [54]. In addition, replacing residues around the palmitoylation sites can affect the localization of these proteins. Wild-type Lck localizes to the PM and Golgi in HeLa cells [54], but replacing its N-terminal residues with those of Fyn leads to localization exclusively in the PM (Bijlmakers and Marsh, unpublished). This N-terminal region contains the Fyn palmitoylation sites Cys3 and Cys6 and, in contrast to Lck, three positively charged residues. The targeting of proteins to axons and dendrites in neurons is also influenced by changes around protein palmitoylation sites. PSD95 normally localizes to dendrites, but deleting the amino acid between the palmitoylated cysteines (Fig. 1) allows transport into axons as well [55]. Similarly, introducing positively charged residues around the palmitoylation sites enhances axonal targeting. For GAP43, replacing N-terminal basic residues (Fig. 1) reduces normal axonal targeting and increases targeting to dendrites [55]. Because transport to dendrites and axons can involve the TGN [45], these changes seem to influence post-Golgi sorting. Thus, the presence of palmitate moieties, together with their context, can influence the sorting of some palmitoylated cytosolic proteins. Localization to rafts and signaling Many palmitoylated proteins, including Src family kinases and some Ga subunits, associate with lipid rafts [3]. It has been suggested that signal transduction could be regulated, in part, by sequestering signaling proteins into different PM domains until they are brought together by an activating signal. The strongest support for such a process, coupled with a role for palmitoylation, comes from studies on T cells. Engineered forms of Lck (which is crucial for T-cell activation) that are attached to membranes through a TMD, rather than through acylation, show reduced association with rafts and reduced signaling activity [56]. Similarly, mutation of the palmitoylation sites on LAT, a transmembrane adaptor protein that is also essential for TCR signaling, abrogates both raft localization and T-cell activation [57]. The TCR co-receptors CD4 and CD8 are also palmitoylated and show some propensity to associate with rafts [58 – 60]. For CD8, palmitoylation has been shown to be essential for co-receptor function. Similarly, palmitoylation and raft localization of the Src family kinase Lyn is required for FcRe signaling [61,62]. The activities of some Ga subunits are compromised when their palmitoylation sites are mutated (for reviews,

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see Refs [63 – 67]). Absence of palmitoylation reduces targeting to rafts [68] and/or caveolae [69]. The lack of palmitate and/or altered sorting can further influence Ga interactions with bg subunits, GPCRs and regulators of G-protein signaling (RGS proteins) and, as a result, coupling to specific signaling pathways. In addition, the activity of some RGS proteins is influenced by palmitoylation [70,71]. Similarly, eNOS requires palmitoylation for optimal functioning and targeting to Golgi membranes and caveolae [19]. Nonpalmitoylated eNOS is catalytically indistinguishable from the wild-type enzyme when purified, but produces less NO in stimulated cells [72]. By contrast, the activity of the mitochondrial proteins MMSDH and CPS1 is inhibited by palmitoylation of their active site [73,74] Protein trafficking Palmitoylation influences the trafficking of some transmembrane proteins: effects on endocytosis, recycling, protein stability and transport from ER to the PM have been observed [75,76]. For example, palmitoylation seems to facilitate transport of the newly synthesized chemokine receptor CCR5 to the PM. Non-acylated CCR5 that does reach the cell surface is compromised in its ability to couple to signaling pathways activated by chemokine agonists and in endocytosis through clathrin-coated vesicles [30,77]. How acylation contributes to CCR5 functional activities is unclear. CCR5 might associate transiently and in an agonist-dependent manner with rafts to facilitate coupling to G proteins. CD151 mutants lacking key cysteines have reduced stability, and biosynthetic intermediates can be observed that are barely detected during synthesis of the wild-type protein [31]. For MPR, an acylated cysteine is essential for the activity of the so-called ‘lysosome avoidance motif ’, which enables the protein to recycle from late endosomes to the Golgi [78]. Replacing this cysteine inactivates the signal (a di-aromatic sequence), which causes MPR to be sorted to lysosomes and degraded [10]. Although in these cases palmitoylation is apparently required for transport to the correct cellular location, it is not essential for the transport of all acylated transmembrane proteins. For CD4, replacing the palmitoylation sites fails to influence transport to the cell surface [58]. Palmitoylation of several viral Env proteins has been proposed to facilitate raft-association and virus assembly, because the rafts can provide a membrane platform on which viral structural proteins can concentrate to enhance assembly [79,80]. Palmitoylation of the Env proteins of Sindbis, Semliki Forest, HIV, SIV and Rous sarcoma viruses is required for efficient viral replication [11,81 – 84]. Replacing the CD cysteines in influenza HA has been shown to reduce raft-association and to inhibit infectious virus assembly [85,86]. The CDs of HIV and SIV Envs are palmitoylated on either one or two cysteines that are located distal to the TMD/CD boundary. These palmitoylations have been proposed to stabilize the association of a putative amphipathic helix with the inner leaflet of the PM and might position signals in the CD for http://ticb.trends.com

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interaction with sorting machineries or viral assembly intermediates. In addition to virus assembly, palmitoylation might also be involved in virus entry. During infection, influenza HA initiates fusion of the viral membrane with an endosomal membrane. Model systems have suggested that fusion, which is induced by low pH, proceeds through an initial hemifusion intermediate, followed by the formation and expansion of a fusion pore. Although acylation is not required for hemifusion, non-acylated HA is compromised in its ability to form and/or expand the fusion pore [87,88]. Similar functions might be attributed to the acyl moieties in other viral fusion proteins. Palmitate turnover For several palmitoylated proteins, the half time of the palmitate moieties is significantly shorter than that of the protein, indicating that the complex goes through cycles of depalmitoylation and repalmitoylation. For example, MPR palmitate turns over with a t1/2 of 2 hours, but the t1/2 of MPR is 40 hours. Given that palmitoylation provides a mechanism for binding cytosolic proteins to membranes, or for segregating proteins to microdomains, depalmitoylation and repalmitoylation could provide a mechanism to regulate membrane association and/or sorting. For proteins involved in signal transduction, these cycles could be induced by activation and, by controlling access to specific substrates, could regulate signaling. Indeed, an agonist-induced increase in palmitate turnover has been observed for b2-AR, Gas and eNOS [65,89,90]. In addition, palmitate cycling on PSD95 has been proposed to modulate synaptic strength by controlling the postsynaptic density of DL -a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) receptors. Palmitoylation allows PSD95 to cluster in the postsynaptic membrane. Blocking PSD95 palmitoylation leads to a loss of AMPA receptors from these domains, and the rapid endocytosis of AMPA receptors requires depalmitoylation of PSD95 [91]. The finding that acylated proteins are palmitoylated and depalmitoylated raises additional issues about PAT activity. For PSD95, repalmitoylation of the depalmitoylated protein seems to occur at the PM, that is, close to the postsynaptic membrane. Thus, some PM proteins might be initially palmitoylated in the exocytic pathway to facilitate their sorting to the PM, but once this cellular localization has been established repalmitoylation might exploit local PATs. In cells such as neurons, this could preclude the need to return proteins to specific exocytic pathway sites for repalmitoylation. Turnover of palmitate on SNAP25 is likely to be important for its activity in vesicle fusion because the nonpalmitoylated protein is defective in vitro [92]. Vac8p, a protein needed for vacuole inheritance and morphology in Saccharomyces cerevisiae, is N-terminally myristoylated and palmitoylated [28]. A recent report implicates Vac8p in a homotypic vacuole fusion reaction that requires palmitoylation of the protein during the priming step. As with several other palmitoylated proteins, acylation of Vac8p is dynamic.

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In addition to the palmitoylation or repalmitoylation reactions mediated by PATs, cycling of palmitate involves depalmitoylation by protein palmitoyl thioesterases that presumably have appropriate locations and the requisite controls to mediate depalmitoylation under specified conditions. So far, an acyl protein thioesterase (APT1) that depalmitoylates Ga subunits, Ras and eNOS in vitro, and Gas in vivo, has been described [93,94]. Concluding remarks As the number of proteins that are known to be palmitoylated continues to grow, so the range of different functions that can be attributed to this modification is expanding. Progress has been made in understanding the targeting of proteins to PATs, and different pathways have been identified even for closely related proteins. However, there is still much to learn and it is likely that subtle differences in sequence and structure have significant implications for the sorting and functional properties of palmitoylated proteins. Sequence motifs for targeting palmitoylated proteins are being identified, but the ways in which these work remain to be elucidated. Nevertheless, understanding the details of these reactions could indicate new ways in which to modify palmitoylation and the functions of specific palmitoylated proteins. Like the farnesylation inhibitors that are being tested as anti-tumor drugs (owing to their ability to inhibit Ras function), inhibitors of palmitoylation could, for example, influence Lck activity and might be used to suppress T-cell functions. The existence of different transport pathways for palmitoylated proteins suggests that some specificity of inhibition might be achievable. Several PAT enzymes with different specificities are likely to exist; indeed, two enzymes that mediate C-terminal palmitoylation have been reported recently. The further characterization of these proteins and identification of novel PAT activities, such as those that mediate N-terminal palmitoylation, will be important steps forward. Genetic screens in yeast and Drosophila are likely to be useful tools in the search for PATs. Acknowledgements We thank our colleagues at Kings College London and University College London for discussion and helpful criticism during the preparation of this review, in particular A. Pelchen-Matthews, N. Signoret, M. Malim, A. Giannini and N. Franc for critically reading the manuscript. M.-J.B. and M.M. are supported by the UK Medical Research Council. We apologize to colleagues whose work has not been cited owing to space limitations.

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