Differential Palmitoylation Regulates Intracellular Patterning of SNAP25

Research Article 1351 Differential palmitoylation regulates intracellular patterning of SNAP25

Jennifer Greaves and Luke H. Chamberlain* Centre for Integrative Physiology, School of Biomedical Sciences, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, UK *Author for correspondence ([email protected])

Summary SNAP25 regulates membrane fusion events at the plasma membrane and in the endosomal system, and a functional pool of the protein is delivered to recycling endosomes (REs) and the trans Golgi network (TGN) through an ARF6-dependent cycling pathway. SNAP25 is a peripheral membrane protein, and palmitoylation of a cluster of four cysteine residues mediates its stable association with the membrane. Here, we report that palmitoylation also determines the precise intracellular distribution of SNAP25, and that mutating single palmitoylation sites enhances the amount of SNAP25 at the RE and TGN. The farnesylated CAAX motif from Hras was ligated onto a SNAP25 mutant truncated immediately distal to the cysteine-rich domain. This construct displayed the same intracellular distribution as full-length SNAP25, and decreasing the number of cysteine residues in this construct increased its association with the RE and TGN, confirming the dominant role of the cysteine-rich domain in directing the intracellular distribution of SNAP25. Marked differences in the localisations of SNAP25-CAAX and Hras constructs, each with two palmitoylation sites, were observed, showing that subtle differences in palmitoylated sequences can have a major impact upon intracellular targeting. We propose that the cysteine- rich domain of SNAP25 is designed to facilitate the dual function of this SNARE protein at the plasma membrane and endosomes, and that dynamic palmitoylation acts as a mechanism to regulate the precise intracellular patterning of SNAP25.

Key words: SNAP25, Palmitoylation, Acylation, Intracellular trafficking

Introduction cargo to REs (Aikawa et al., 2006a). Although this cycling pathway Intracellular-membrane-fusion events in eukaryotes are regulated is central to the intracellular functions of SNAP25, it is not clear by members of the soluble N-ethylmaleimide-sensitive fusion how entry into this pathway is achieved. protein-attachment protein receptor (SNARE) protein family (Jahn The majority of SNARE proteins are anchored to membranes by

Journal of Cell Science and Scheller, 2006; Sollner et al., 1993), and these proteins are the transmembrane domains. SNAP25, however, is an interesting minimal membrane fusion machinery in vitro (Weber et al., 1998). exception and the membrane binding of this protein is regulated by One of the most extensively characterised membrane fusion palmitoylation, a common post-translational modification, most pathways is regulated exocytosis, which involves the controlled often involving the attachment of palmitic acid groups (C16:0) to fusion of intracellular secretory vesicles with the plasma membrane. cysteine residues (Resh, 2006). Previous work defined the minimal Regulated exocytosis mediates the secretion of a wide variety of palmitoylation domain of SNAP25b, the major SNAP25 isoform essential molecules, including neurotransmitters, peptides and expressed in adult brain (Bark et al., 1995); this region comprises hormones, in response to specific intracellular signals, most often residues 85–120 and includes four palmitoylated cysteine residues a rise in cytosolic Ca2+ levels. In neurons and neuroendocrine present at amino acid positions 85, 88, 90 and 92 (Gonzalo et al., cells, regulated exocytosis is dependent upon the interaction of the 1999). It is important to note that palmitoylation is not simply a plasma membrane SNAREs syntaxin 1 and SNAP25 with the hydrophobic membrane anchor. Recent work, in different systems, vesicle SNARE VAMP2 (Blasi et al., 1993; Gary et al., 1994; has highlighted the diverse array of effects that palmitoylation can Sadoul et al., 1995; Schiavo et al., 1993; Sollner et al., 1993). have on proteins, including regulating intracellular sorting, Although SNAP25 has been intensively studied as an exocytotic mediating association with membrane microdomains, regulating SNARE protein, more recent work has shown that SNAP25 protein–protein interactions and modulating protein stability depletion also inhibits the trafficking of cargo from sorting (Greaves and Chamberlain, 2007; Greaves et al., 2009b; Linder endosomes to recycling endosomes (REs) in PC12 cells (Aikawa and Deschenes, 2007; Resh, 2006). Furthermore, the versatility et al., 2006a). Consistent with this, previous work has also of palmitoylation as a protein regulator is enhanced by its highlighted an important role for SNAP23, a ubiquitous homologue reversibility, with many proteins undergoing continuous cycles of of SNAP25, in endosomal recycling of internalised transferrin to depalmitoylation and repalmitoylation (Rocks et al., 2005). the basolateral plasma membrane in Madin–Darby canine kidney Intracellular palmitoylation reactions are mediated by a large (MDCK) cells (Leung et al., 1998). The endosomal function of family of more than 23 DHHC (for aspartate-histidine-histidine- SNAP25 is supported by an ARF6-dependent cycling pathway, cysteine) palmitoyl transferases; these proteins are defined by the operating between the plasma membrane and the RE and trans presence of an ~50-amino-acid cysteine-rich domain containing a Golgi network (TGN) compartments, which ensures a sufficient DHHC motif (Fukata et al., 2004; Mitchell et al., 2006; Putilina et pool of SNAP25 to support endosomal membrane fusion (Aikawa al., 1999). All DHHC proteins are predicted polytopic membrane et al., 2006b); disruption of this pathway inhibits trafficking of proteins, associating with distinct intracellular compartments, 1352 Journal of Cell Science 124 (8)

including the endoplasmic reticulum (ER), Golgi and plasma Studies analysing whether SNAP25 palmitoylation is constitutively membrane (Ohno et al., 2006). As DHHC proteins are membrane- dynamic have produced conflicting results, probably owing to the associated, peripheral proteins that undergo palmitoylation require limitations of the techniques employed (Heindel et al., 2003; Kang additional primary membrane-targeting signals that mediate et al., 2004; Lane and Liu, 1997). However, there is good evidence membrane interactions before palmitoylation. Primary membrane- that SNAP25 palmitoylation is subject to dynamic regulation under targeting signals include N-myristoylation (e.g. certain G subunits some conditions, and palmitate turnover is notably enhanced and SRC family kinases) and prenylation of C-terminal CAAX following inhibition of synaptic activity in cortical neurons (Kang motifs (e.g. Hras and Nras). SNAP25 is not modified by either N- et al., 2004). myristoylation or prenylation, and we have recently shown that the Although palmitoylation plays a dominant role in defining the cysteine-rich domain of this protein is important for initial intracellular localisation of proteins such as Ras, far less is membrane interactions before palmitoylation. In particular, cysteine understood about how multiple palmitoylation of cysteine-rich hydrophobicity and the hydrophobic character of the surrounding domains coordinates the trafficking of soluble proteins such as amino acids are required for efficient membrane association SNAP25. Similarly, the potential for dynamic interplay between (Greaves et al., 2009a). Single cysteine-to-alanine mutations lead palmitoylated cysteine residues in the regulation of protein sorting to a marked reduction in SNAP25 membrane association, whereas is poorly understood (Roy et al., 2005). As regulation of SNAP25 replacement of single cysteine residues with the more hydrophobic intracellular targeting is central to its dual function at the plasma leucine residue preserves membrane binding (Greaves et al., 2009b). membrane and endosomes, we have investigated how multiple However, double cysteine replacement mutants dramatically inhibit palmitoylation impacts upon endosomal targeting and whether the membrane association of SNAP25, even when leucine is the changes in SNAP25 palmitoylation might function as a switch to replacement amino acid. In addition to cysteine residues and the regulate this sorting pathway. surrounding hydrophobic amino acids, membrane binding of SNAP25 also requires residues at the C-terminus of the minimal- Results membrane-targeting domain (in particular, Q116 and P117) The minimal palmitoylated domain regulates plasma (Gonzalo et al., 1999; Greaves et al., 2009a). The residue P117 membrane and RE and TGN targeting of SNAP25 appears to play an important role in determining the specificity of A previous study, in PC12 cells, described an intracellular pool of the interaction with DHHC proteins: in human embryonic kidney SNAP25 on REs and the TGN that cycles between these (HEK)-293 cells, mutation of this residue prevented palmitoylation compartments and the plasma membrane (Aikawa et al., 2006b). by DHHC17 but not by DHHC3. There are several ways that P117 This intracellular pool is readily observed for both endogenous might regulate the interaction with DHHC17, including by directly SNAP25 and eGFP-tagged SNAP25b (Fig. 1A). As a first step participating in protein–protein interaction, by modulating the towards defining the role of palmitoylation in regulating SNAP25 strength of initial membrane interaction before palmitoylation or localisation, we sought to confirm the identity of the intracellular by allowing SNAP25 to associate with specific membrane domains SNAP25 pool by quantitative colocalisation analysis. For this, that facilitate an interaction with DHHC17. PC12 cells were transfected with eGFP–SNAP25b and stained The majority of evidence suggests that newly synthesised with antibodies against markers of specific intracellular

Journal of Cell Science SNAP25 is palmitoylated at the Golgi (Gonzalo and Linder, 1998; compartments: GM130 (Golgi), TGN38 (TGN) and Rab11 (RE). Greaves et al., 2010). Interestingly, recent studies have defined an Clear colocalisation of eGFP–SNAP25b with both Rab11 and intracellular cycling pathway operating between the plasma TGN38 was observed, whereas we did not detect any overlap with membrane and the Golgi; this pathway is followed by a number of GM130 (Fig. 1B), in good agreement with previous work (Aikawa other peripheral palmitoylated proteins, including Hras and Nras et al., 2006b). Analysis of covariance of the fluorescence signals (Goodwin et al., 2005; Rocks et al., 2010; Rocks et al., 2005). provided a quantitative measure of the colocalisation of SNAP25b Following synthesis, Ras proteins are farnesylated on a C-terminal with both Rab11 and TGN38 (Fig. 1B). Triple-labelling experiments CAAX motif, which mediates transient interactions with further confirmed the overlap of eGFP–SNAP25b with the RE and intracellular membranes. When the farnesylated protein associates TGN compartments and its absence from the Golgi (Fig. 1C). An with the Golgi, it is palmitoylated by resident DHHC proteins, intracellular pool that colocalised with Rab11 was also observed promoting stable membrane attachment and delivery to the plasma for both eGFP–SNAP25a- and eGFP–SNAP23-transfected cells membrane through vesicular transport. Subsequent depalmitoylation (supplementary material Fig. S1), confirming that association with releases Ras from membranes, and the process of palmitoylation this intracellular compartment is a conserved feature of all SNAP25 at the Golgi is repeated. This cycling between the plasma membrane protein isoforms. and the Golgi is thus mediated by a combination of vesicular As the intracellular functions of SNAP25 depend upon its transport and cytosolic diffusion, and is regulated by accumulation on endosomal membranes (Aikawa et al., 2006a), palmitoylation–depalmitoylation dynamics. It has been proposed we sought to determine the unique features of SNAP25 that allow that rapid depalmitoylation and membrane release prevents it to traffic to RE and TGN membranes. Membrane binding of excessive accumulation of Ras proteins on endosomal membranes SNAP25 is mediated by multiple palmitoylation of a cysteine-rich (Rocks et al., 2005). cluster (Veit et al., 1996); the minimal palmitoylation domain for By contrast, there is no evidence that SNAP25 cycles on and off SNAP25b has been mapped to residues 85–120 (Gonzalo et al., membranes, or that cytosolic diffusion plays any role in trafficking 1999). The palmitoylation and membrane-binding domain of or localisation of mature SNAP25. This difference between SNAP25b is sufficient for plasma membrane targeting; however, SNAP25 and Ras proteins might arise owing to a slower rate of its role in RE and TGN localisation is not clear. SNAP25 depalmitoylation or might reflect the fact that SNAP25 PC12 cells were co-transfected with eGFP–SNAP25(85–120) has more palmitoylation sites and is therefore less likely to be in a and full-length SNAP25b fused to the mCherry fluorescent protein. fully depalmitoylated (and hence soluble) state at any given time. This co-transfection strategy allows the localisation of the minimal Palmitoylation and SNAP25 trafficking 1353

Fig. 1. Association of SNAP25 with REs and TGN. (A)Left-hand panel: localisation of eGFP–SNAP25 (SN25) in PC12 cells. Right-hand panel: endogenous SNAP25 in PC12 cells, detected using an anti-SNAP25 antibody. (B)PC12 cells were transfected with eGFP– SNAP25. The cells were then fixed, permeabilised and stained with antibodies against Rab11 (top panel), TGN38 (middle panel) and GM130 (bottom panel). Representative images are shown. Pearson’s correlation Journal of Cell Science coefficients (R) were calculated to measure the covariance between the fluorescence signals of eGFP and Alex-Fluor-633-labelled secondary antibodies. (C)PC12 cells expressing eGFP–SNAP25 were stained for GM130 and Rab11 (top panel) or TGN38 and Rab11 (bottom panel). Scale bars: 5m.

palmitoylated domain to be directly compared with that of full- farnesylation signal and a polybasic domain, to mediate membrane length SNAP25 in the same cell. Fig. 2 shows that the intracellular binding and plasma membrane delivery (Apolloni et al., 2000) (see localisation of the minimal palmitoylation domain was almost Fig. 2A). This cysteine-less 4CL-KrasMTD SNAP25 mutant was identical to that of full-length SNAP25, demonstrating that all the targeted to the plasma membrane, in a manner similar to wild-type information required for RE and TGN targeting is contained within SNAP25; however, it displayed a clear loss of localisation to RE this region of SNAP25. By extension, we can conclude that and TGN membranes (Fig. 2B,C). Taken together, these data SNAP25 localisation at the plasma membrane and RE and TGN is support the notion that palmitoylation mediates targeting of independent of its interaction with other SNARE proteins. SNAP25 to RE and TGN membranes. The observed localisation of the minimal palmitoylation domain To quantify the localisation of these SNAP25 mutants with is consistent with the idea that palmitoylation regulates RE and respect to the wild-type protein, the intensity of the eGFP and TGN targeting of SNAP25. However, another possibility is that mCherry intracellular (RE and TGN) fluorescence (Fi) and total palmitoylation only functions as a membrane anchor, and that cell fluorescence (Ft) was measured as described in the Materials amino acids downstream of the modified cysteine residues (residues and Methods section. The intracellular RE and TGN fluorescence 93–120) regulate targeting of SNAP25. To examine this possibility was expressed relative to the whole-cell fluorescence for each further, palmitoylation was blocked by mutating all four cysteine fluorescent protein (Fi/Ft–Fi). The average cellular ratio of Fi/Ft– residues in full-length SNAP25 to leucine residues (termed 4CL). Fi(eGFP) to Fi/Ft–Fi(mCherry) was 1.046 for eGFP–SNAP25(85– As this 4CL mutant is cytosolic (Greaves et al., 2009a), we ligated 120) relative to mCherry–SNAP25 (wild-type). This value suggests it to 15 amino acids from the C-terminal membrane-targeting that the amount of targeting of the minimal palmitoylation domain domain of Kras (4CL-KrasMTD). This domain contains a to the RE and TGN is essentially the same as that of full-length 1354 Journal of Cell Science 124 (8)

Fig. 2. The palmitoylated cysteine-rich domain of SNAP25 is necessary and sufficient for endosomal targeting. (A)Schematic diagram depicting the constructs used. The Kras membrane-targeting domain (MTD, red) contained the amino acid sequence GKKKKKKSKTKCVIM (single-letter code). Green, GFP; blue, minimal membrane-binding domain of SNAP25, residues 85–120. (B)PC12 cells co-transfected with wild- type (WT) copies of eGFP–SNAP25 (SN25) and mCherry–SNAP25. (C)PC12 cells co-expressing mCherry–SNAP25 and eGFP–SNAP25(85–120) (top panel) or eGFP–SNAP25(4CL-KrasMTD) (bottom panel). The graph shows the ratio (+s.e.m.) of intracellular RE and TGN fluorescence (Fi) as a fraction of total (Ft) cell fluorescence (Fi/Ft–Fi) for eGFP relative to mCherry (GFP:mCherry). ***P<0.001, as determined by a Student’s t-test, in the intracellular fluorescence of 4CL-KrasMTD (n7) compared with SNAP25(85–120) (n8). Scale bars: 5m.

SNAP25. By contrast, the eGFP–4CL-KrasMTD mutant had an palmitoylation, brought about by cysteine mutagenesis, affect its average value of 0.581 relative to wild-type SNAP25 (Fig. 2B), localisation. The precise stoichiometry of the palmitoylation of the demonstrating that this chimaeric construct has a quantifiable loss cysteine-rich domain of SNAP25 is not known; however, of RE and TGN association. [3H]palmitate incorporation was decreased when each of the four cysteine residues were mutated individually to leucine (Fig. 4A). Dynamic palmitoylation of SNAP25 This suggests that all cysteine residues in SNAP25 are modified to The results shown in Fig. 2 suggest that palmitoylation regulates some degree by palmitoylation. To analyse whether the mutation the intracellular patterning of SNAP25. As palmitoylation is a of the cysteine residues affects SNAP25 localisation, PC12 cells reversible modification (Resh, 2006), it is possible that dynamic were co-transfected with mCherry–SNAP25 wild-type and eGFP-

Journal of Cell Science changes in SNAP25 palmitoylation might modulate the RE and TGN targeting without displacing the protein from membranes. Previous pulse–chase experiments, examining incorporation of radiolabelled palmitate, have produced conflicting results on whether palmitoylation of SNAP25 is dynamic under basal conditions (Heindel et al., 2003; Kang et al., 2004; Lane and Liu, 1997); these discrepancies are probably a consequence of the insensitivity of pulse–chase protocols to dynamic changes in multiply palmitoylated proteins. As a more sensitive test of whether SNAP25 palmitoylation is dynamic, PC12 cells were treated with the protein synthesis inhibitor cycloheximide for 2 hours before Fig. 3. Dynamic palmitoylation of SNAP25. (A)PC12 cells transfected with the addition of radiolabelled palmitate. Fig. 3A shows that eGFP–SNAP25 (SN25) were incubated with or without 50g/ml 3 cycloheximide (+CHX and control, respectively) for 2 hours. The cells were incorporation of [ H]palmitate into SNAP25 was still readily then labelled for 4 hours with [3H]palmitate in the continued absence or detected in the presence of cycloheximide, consistent with the presence of cycloheximide. eGFP–SNAP25 was immunoprecipitated by virtue notion that mature SNAP25 is undergoing palmitoylation of the eGFP tag, resolved by SDS-PAGE and duplicate membranes transferred remodelling. The averaged results from three experiments revealed onto nitrocellulose for visualisation of the 3H signal with the aid of an that [3H]palmitate incorporation in the presence of cycloheximide intensifier screen (top panel) or for detection of precipitated protein using an was ~50% that in the absence of the drug (Fig. 3A, right-hand anti-GFP antibody (bottom panel). The position of molecular-mass-markers panel). Fig. 3B confirms that cycloheximide effectively blocked (in kDa) is indicated. The graph shows average values (means+s.e.m.) for 3 3 protein synthesis, as judged by a complete loss of [35S]methionine [ H]palmitate incorporation (n3). The quantified H signal was not incorporation into cellular protein. normalised to protein levels, allowing a more direct assessment of the contribution of dynamic SNAP25 palmitoylation to the total cellular SNAP25 palmitoylation. (B)PC12 cells were incubated with or without 50g/ml RE and TGN targeting of SNAP25 is modulated by cycloheximide for 2 hours. The cells were then incubated with [35S]methionine mutation of specific palmitoylation sites for 1 hour in the continued absence or presence of cycloheximide. Cell lysates As palmitoylation is essential for targeting SNAP25 to RE and were resolved by SDS-PAGE and stained with Coomassie Blue to visualise TGN membranes (Fig. 2), and because this modification is dynamic total cellular protein (left-hand panel); the 35S signal was visualised with the (Fig. 3A), we examined whether changes in SNAP25 aid of an intensifier screen (right-hand panel). Palmitoylation and SNAP25 trafficking 1355

tagged versions of each of the cysteine-to-leucine mutants. Interestingly, mutation of C88 and C90 promoted a marked increase in RE and TGN targeting (Fig. 4B; supplementary material Fig. S2), suggesting that dynamic palmitoylation remodelling might play a key role in regulating SNAP25 localisation. In support of this, quantitative analysis of multiple transfected cells revealed an increase in the RE and TGN targeting of all the SNAP25 cysteine mutants, which was substantially higher for the C88L and C90L proteins (Fig. 4B). Thus, decreasing the number of palmitoylation sites in SNAP25 enhances RE and TGN targeting, and specific cysteine residues (i.e. C88 and C90) appear to be more dominant in directing SNAP25 localisation. Importantly, the increased intracellular level of the cysteine mutants was not related to the fluorescent tags, as enhanced RE and TGN targeting of the C88L mutant was still observed when the tags were switched (Fig. 4C). The results described in Fig. 4 clearly show that RE and TGN accumulation of eGFP–SNAP25b is enhanced when the number of palmitoylated cysteine residues is reduced from four to three. It is interesting to note that SNAP23 has an additional cysteine residue (five rather than four) in its cysteine-rich domain (Fig. 5). To determine whether functional differences between SNAP25b and SNAP23 might arise owing to the effects of the different number of cysteine residues on RE and TGN targeting, we examined a SNAP25b mutant with an extra cysteine (F84C) to mimic SNAP23 (Salaun et al., 2005a) (Fig. 5A). Introduction of this fifth cysteine into SNAP25 had no major effect on the intracellular localisation of SNAP25 (Fig. 5B), and quantitative analysis revealed an average value for Fi/Ft–Fi(eGFP):Fi/Ft–Fi(mCherry) of 1.0±0.022 (n8). To test whether the intracellular targeting of other SNAP25 proteins is also regulated by palmitoylation, we examined the Fig. 4. Effects of single cysteine residue mutations on SNAP25 localisation. localisation of eGFP–SNAP23 with a double cysteine mutation (A)Sequences of the cysteine-rich domains of wild-type (WT) SNAP25 (SN25) (C79F and C83F), mimicking the cysteine configuration of the and the individual cysteine-to-leucine mutants. The graph compares SNAP25b (C88L) mutant. The intracellular localisation of this [3H]palmitate incorporation into the various mutants (means+s.e.m.), assayed as SNAP23 mutant was clearly enhanced compared with that of in Fig. 3 (n3). The level of palmitate incorporated was expressed relative to the protein level detected by immunoblotting. (B) PC12 cells were co-transfected Journal of Cell Science mCherry–SNAP23 (wild-type) (supplementary material Fig. S3).  Thus, palmitoylation regulates the precise intracellular localisation with mCherry–SNAP25 wild-type and eGFP-tagged SNAP25 cysteine mutants of distinct SNAP25 proteins. and examined at 48 hours post transfection. The left-hand panel shows representative images for eGFP-tagged SNAP25(C88L) and SNAP25(C90L) To ensure that the increased intracellular accumulation of the mutants and mCherry–SNAP25 (wild-type). The right-hand panel is a graph SNAP25 cysteine mutants did not reflect association with the showing the ratio (means+s.e.m.) of intracellular RE and TGN fluorescence (Fi) Golgi, PC12 cells expressing eGFP–SNAP25(C88L) were stained as a fraction of the total (Ft) cell fluorescence (Fi/Ft–Fi) for eGFP relative to with anti-GM130 antibodies. No colocalisation of the cysteine mCherry. The data were analysed using one-way ANOVA, which revealed mutant with the Golgi marker was apparent (Fig. 6A). We also significant increases in the intracellular pool of all cysteine mutants compared examined the possibility that the enhanced intracellular with the wild-type SNAP25. *P<0.05; ***P<0.001 (n16–22 cells per mutant). accumulation of the SNAP25(C88L) mutant reflected a lag in the (C)eGFP–SNAP25 wild-type was co-transfected with mCherry- rate of transport of newly synthesised protein to the plasma SNAP25(C88L). The left-hand panels show a representative image. The graph in membrane, rather than a shift in the cycling of mature SNAP25 the right-hand panel shows the ratio (means+s.e.m.) of intracellular RE and TGN between the plasma membrane and the REs and TGN. For this, fluorescence (Fi) as a fraction of total (Ft) cell fluorescence (Fi/Ft–Fi) for eGFP relative to mCherry. The data were analysed using a Student’s t-test, which cells transfected with eGFP–SNAP25(C88L) and mCherry– revealed a significant increase in the intracellular pool of the C88L mutant SNAP25 (wild-type) were incubated in the absence or presence of compared with that of wild-type SNAP25. ***P<0.001 (n11–13 cells). Scale cycloheximide for 3 hours before fixation. Cycloheximide treatment bars: 5m. did not reduce the intracellular accumulation of SNAP25(C88L) compared with that of wild-type SNAP25 (Fig. 6B), suggesting that it is cycling of SNAP25 that is affected by this mutation. interaction of SNAP25 before palmitoylation. Indeed, two palmitoylation sites should be sufficient for tight membrane binding Further analysis of the role of the cysteine-rich domain in (Shahinian and Silvius, 1995). To extend our investigation into directing the intracellular localisation of SNAP25 how palmitoylation regulates SNAP25 localisation, we generated Our analysis of the effects of cysteine mutagenesis on SNAP25 mutant proteins that would permit palmitoylation to be studied localisation is limited to single point mutations, as removal of without confounding effects on primary membrane-targeting more than one cysteine prevents membrane binding (Greaves et signals. For this, SNAP25 was truncated immediately following al., 2009a). It is important to note that this effect of double cysteine the cysteine-rich domain and attached to the farnesylated CAAX mutations is probably attributable to defects in the initial membrane motif (CVLS) of Hras (Fig. 7A). The lysine preceding the CAAX 1356 Journal of Cell Science 124 (8)

Fig. 5. Localisation of a SNAP25 mutant containing a cysteine-rich domain modelled on SNAP23. (A)The sequences of the cysteine-rich domains of wild-type (WT) SNAP23 and SNAP25b, and the SNAP25(F84C) mutant are shown. Palmitoylated cysteine residues are shown in red. (B)PC12 cells were co-transfected with mCherry–SNAP25 (SN25) wild-type and eGFP-tagged SNAP25(F84C), and were examined at 48 hours post transfection. Scale bars: 5m. The ratio of intracellular RE and TGN fluorescence (Fi) as a fraction of total (Ft) cell fluorescence (Fi/Ft–Fi) for eGFP relative to mCherry was 1.0(±0.022 s.e.m.) (n8).

motif of Hras was also added to the SNAP25(1–92) truncation mutant to maintain the spacing between the farnesylated cysteine residue and the most proximal palmitoylated cysteine residue (Fig. 7A). Removal of amino acids downstream from the cysteine-rich region has previously been shown to prevent the stable membrane association of SNAP25 (Gonzalo et al., 1999; Greaves et al., 2009a), and membrane targeting of SNAP25 requires residues 85– Fig. 6. Enhanced intracellular accumulation of SNAP25(C88L) does not 120 (Gonzalo et al., 1999). Thus, SNAP25(1–92) displayed a reflect association with the Golgi and is not overcome by cycloheximide dispersed localisation, consistent with a loss of membrane binding treatment. (A)PC12 cells were transfected with eGFP–SNAP25(C88L) (Fig. 7B). There appeared to be a small amount of this construct [SN25(C88L)], and were fixed and stained with an anti-GM130 primary associated with membranes, which probably reflects the weak antibody and an Alexa-Fluor-633-conjugated secondary antibody. The merge membrane affinity of the intact cysteine-rich domain. By contrast, zoom image compares the intracellular localization of both proteins. (B)PC12 the SNAP25(1–92)-CAAX construct displayed an almost identical cells expressing eGFP–SNAP25(C88L) and mCherry–SNAP25 wild-type (SN25) were treated with or without 50g/ml cycloheximide for 3 hours

Journal of Cell Science localisation to that of mCherry–SNAP25 (Fig. 7B). Thus, the before fixation and were viewed using confocal microscopy. The dominant role of the SNAP25 cysteine-rich domain in directing representative image demonstrates that the increased intracellular fluorescence intracellular targeting is apparent even when it is placed in a of the SNAP25(C88L) mutant was not reversed by cycloheximide treatment. different sequence context. Furthermore, as observed with the full- The graph in the lower panel shows the ratio of intracellular fluorescence (Fi) length protein (Fig. 4), introduction of a C88L mutation into the as a fraction of total (Ft) cell fluorescence (Fi/Ft–Fi) for eGFP relative to chimaeric protein promoted a significant increase in the association mCherry (n13 cells without cycloheximide; n9 cells with cycloheximide). with RE and TGN membranes, whereas a C85L mutation only had Scale bars: 5m. a minor effect (Fig. 7B). The initial membrane interactions of the SNAP25(1–92)-CAAX mutants will be mediated by farnesylation, allowing greater scope work has shown transient association of the CAAX motif of Ras to investigate whether SNAP25 localisation can be modified further with ER and Golgi membranes, and it is these membranes that are by removing additional palmitoylation sites. Thus, we combined likely to be labelled by the 4CL-CAAX protein (Rocks et al., the C85L and C88L mutations, and observed a substantial increase 2010). in intracellular targeting compared with that for the single cysteine The number and configuration of cysteine residues in the residue mutants (Fig. 7B), further highlighting the negative SNAP25(1–92,C85L,C88L)-CAAX mutant is similar to that of correlation between the number of palmitoylation sites and RE and Hras (Fig. 7A). However, the localisation of the membrane-targeting TGN targeting. Fig. 7C shows that the localisation of the domain of Hras (Fig. 7A, tH) was completely distinct from that of SNAP25(1–92,C85L,C88L)-CAAX mutant did not overlap with this SNAP25 mutant and only a very small fraction of Hras GM130, demonstrating that the intracellular accumulation of this associated with the same intracellular membranes as wild-type mutant does not reflect Golgi targeting. The targeting of the SNAP25 (Fig. 7B). This result demonstrates that subtle differences SNAP25(1–92)-CAAX chimaeras to the plasma membrane and in palmitoylation motifs can have dramatic effects on intracellular defined intracellular compartments was entirely dependent upon targeting. palmitoylation, as removal of all of the palmitoylation sites (4CL) led to a dispersed localisation (Fig. 7B). Some association of the Discussion SNAP25(4CL)-CAAX construct with intracellular membranes was This study highlights a hitherto unknown function for multiple visible, although this was largely distinct from the RE and TGN palmitoylation in regulating the targeting of SNAP25 to RE and membranes that were labelled by wild-type SNAP25. Previous TGN membranes (Fig. 8). It is clear from this analysis that Palmitoylation and SNAP25 trafficking 1357

Fig. 7. Localisation of SNAP25 chimaeric proteins containing a CAAX motif ligated to the cysteine-rich domain. (A)Sequences of the palmitoylated cysteine-rich domains from wild-type (WT) SNAP25 (SN25) and specific cysteine mutants fused to the CAAX motif (KCVLS) from Hras. Palmitoylated cysteine residues are shown in red. (B)PC12 cells were co-transfected with mCherry–

Journal of Cell Science SNAP25 wild-type and eGFP-tagged SNAP25(1–92)-CAAX proteins, and examined at 48 hours post transfection. Representative images for all mutants are shown. The graph shows the ratio of intracellular RE and TGN fluorescence (Fi) as a fraction of total (Ft) cell fluorescence (Fi/Ft–Fi) for (means+s.e.m.) eGFP relative to mCherry. The data were analysed using one-way ANOVA. *P<0.05, compared with SN25(1–92)-CAAX) (n8–13 cells per mutant). (C)Localisation of eGFP–SNAP25(1–92, C85L,C88L)-CAAX compared with that of endogenous GM130 detected using an Alexa- Fluor-633-conjugated secondary antibody. Scale bars: 5m.

palmitoylation does not simply function as a membrane anchor for to the plasma membrane from the Golgi or whether it transits via SNAP25 but also dictates the precise patterning of the protein other compartments such as REs and the TGN. It is clear that across intracellular membranes. By employing cysteine residue SNAP25 levels at the RE and TGN compartments are not mutagenesis, we found that decreasing the number of palmitoylation maintained by new protein synthesis, as cycloheximide had very sites increased RE and TGN targeting. As palmitoylation is a little effect on this localisation (Aikawa et al., 2006b; Gonzalo and reversible process, these observations highlight differential Linder, 1998). Instead, a constitutive cycling pathway replenishes palmitoylation as a mechanism to regulate targeting of SNAP25 to RE and TGN compartments with SNAP25 derived from the plasma specific intracellular membranes. membrane (Aikawa et al., 2006b). The continued enrichment of A recent study suggested that Golgi enzymes play a major role the SNAP25(C88L) mutant at the RE and TGN compartments in regulating the palmitoylation of peripheral membrane proteins following cycloheximide treatment suggests that changes in plasma (Rocks et al., 2010). Consistent with this, Golgi integrity and Golgi membrane to RE and TGN cycling contribute to the increased DHHC proteins are important for palmitoylation of newly intracellular targeting of this mutant. Thus, it appears that synthesised SNAP25 (Gonzalo and Linder, 1998; Greaves et al., palmitoylation facilitates entry of SNAP25 into the plasma 2010; Greaves et al., 2008). Following palmitoylation at the Golgi, membrane–RE-TGN cycling pathway but that a step of this cycling SNAP25 is probably delivered to the plasma membrane by vesicular is refractive to the extent of SNAP25 palmitoylation. Interestingly, transport; however, it is not clear whether SNAP25 traffics directly Martin and co-workers (Aikawa et al., 2006b) previously suggested 1358 Journal of Cell Science 124 (8)

wild-type SNAP25 and the SNAP25(F84C) mutant, mediating a similar association with cholesterol-rich domains and facilitating RE and TGN exit. In this case the different association of SNAP25(F84C) and wild-type SNAP25 with cholesterol-rich domains in vitro could reflect heterogeneous palmitoylation of the proteins and the likelihood that more F84C molecules than wild-type SNAP25 will have at least four palmitate chains. In future studies, it will be interesting to determine the precise stage of trafficking that is perturbed by cysteine mutagenesis; this could be assessed using fluorescence photobleaching/ photoactivation approaches. Although it is clear that many proteins undergo rapid cycles of palmitoylation and depalmitoylation, there has been some debate about whether this is also true for SNAP25. In PC12 cells, the half- life of palmitoylation was reported to be ~3 hours compared with the half-life of the protein of ~8 hours (Lane and Liu, 1997). However, two subsequent reports did not detect constitutive palmitate turnover on SNAP25 (Heindel et al., 2003; Kang et al., 2004). It is important to emphasise that these studies all employed pulse–chase experiments to analyse the rate of palmitate turnover, examining the loss of the 3H radiolabel over time. A significant issue with pulse–chase analyses is the inherent lack of sensitivity to palmitoylation changes in multiply palmitoylated proteins. For example, if one palmitate group is removed from 40% of intracellular SNAP25, this might only amount to a 10% loss of radiolabel (Prescott et al., 2009). This level of change in Fig. 8. Model for palmitoylation-dependent cycling of SNAP25. The colour palmitoylation status might have a major effect on SNAP25 of vesicles depicted in the top panel indicates the donor compartment (e.g. yellow vesicles are formed from the Golgi). Cycling of SNAP25 between the trafficking or function but could not be reliably detected using plasma membrane and the REs and TGN is likely to be regulated by DHHC pulse–chase experiments. Furthermore, recent analyses of proteins and/or thioesterases present at these compartments. The lower panel palmitoylated proteins that undergo dynamic palmitoylation suggest shows the plasma-membrane-to-Golgi cycling pathway previously described that pulse–chase experiments also underestimate palmitate turnover for palmitoylated Ras proteins, for comparison. Although not depicted on the (Rocks et al., 2005). As an alternative approach to test whether figure, there is evidence that Ras proteins can traffic via REs en route to the SNAP25 palmitoylation is dynamic, we examined [3H]palmitate plasma membrane (Misaki et al., 2010). incorporation in the presence or absence of cycloheximide to block

Journal of Cell Science protein synthesis. A previous study showed that palmitoylation of SNAP25 was largely complete following 1 hour of labelling and 2 that palmitoylation turnover on SNAP25 might represent a possible hours of chase (Gonzalo and Linder, 1998). We therefore treated mechanism to regulate cycling between the plasma membrane and cells with cycloheximide for 2 hours before the addition of the REs and TGN. [3H]palmitate, in the continued presence of cycloheximide, for a The altered distribution of the SNAP25 cysteine mutants is further 4 hours. It is unlikely that the level of palmitoylation consistent with an increased rate of internalisation or a decreased observed following cycloheximide treatment (50% of control rate of exit from the RE and TGN. Many effects of palmitoylation values) could be due to incorporation into residual SNAP25 that on modified proteins are thought to reflect changes in association had yet to be palmitoylated. Thus, we propose that SNAP25 does with cholesterol-rich membrane domains (Greaves and undergo constitutive palmitoylation remodelling and that this can Chamberlain, 2007; Melkonian et al., 1999). These domains be enhanced further, for example, in response to synaptic activity might also be intricately linked with the effects of differential (Kang et al., 2004). palmitoylation on SNAP25 sorting, for example, by regulating Previous studies have highlighted a dynamic Ras cycling pathway, SNAP25 exit from RE and TGN membranes. It should be noted, operating between the Golgi and plasma membrane, which also however, that we have previously shown that SNAP25(F84C) appears to be followed by other peripherally localized palmitoylated displays a higher in vitro association with cholesterol-rich proteins. The rapid palmitoylation–depalmitoylation dynamics of membranes than that of wild-type SNAP25 (Salaun et al., 2005b). Hras and Nras, which occurs on the time-scale of a few minutes, is The observation that the SNAP25(F84C) mutant did not display proposed to mediate the distribution of the proteins over the Golgi a marked reduction in RE and TGN localisation might imply that and plasma membrane and prevent excessive accumulation on an association with cholesterol-rich domains is not the sole factor endosomal membranes (Rocks et al., 2010; Rocks et al., 2005). In accountable for the effects of palmitoylation on SNAP25 agreement with this idea, we observed that association of Hras with localisation. However, it is also possible that SNAP25 molecules RE and TGN membranes was markedly lower than that of SNAP25. with either four or five palmitoylated cysteine residues display However, in PC12 cells, we did not detect an obvious Golgi pool of the same affinity for cholesterol-rich domains, whereas three Hras, perhaps reflecting the high palmitoylation capacity of PC12 palmitoylated cysteine residues might support a weaker Golgi membranes and rapid Golgi exit dynamics. association with these domains. Thus, DHHC proteins at RE and The results obtained with the SNAP25 truncation mutants TGN membranes might catalyse the complete palmitoylation of fused to the Hras CAAX motif are interesting for several reasons. Palmitoylation and SNAP25 trafficking 1359

The inverse correlation observed between cysteine number and was inserted immediately upstream of the stop codon by site-directed mutagenesis. targeting to RE and TGN membranes was in good agreement A plasmid encoding the membrane-targeting domain of Hras (tH) fused to the C- terminus of eGFP was kindly provided by Ian Prior (University of Liverpool, UK). with our data for full-length SNAP25, and further suggests that SNAP25 mutants containing the KCVLS sequence from Hras were generated by the cysteine-rich domain of SNAP25 is an autonomous unit that site-directed mutagenesis of the pEGFPC2-SNAP25b construct. A GFP–Rab11 is dominant and sufficient for determining SNAP25 localisation. construct, provided by Giampietro Schiavo (London Research Institute, UK), was used to generate mCherry–Rab11. All constructs were verified by DNA sequencing By fusing the SNAP25 cysteine-rich domain to a CAAX motif, (University of Dundee DNA sequencing service, Dundee, UK). we were also able to investigate how combined cysteine mutations affect the localisation of SNAP25. The C85L,C88L-CAAX PC12 cells mutant displayed a substantial increase in intracellular PC12 cells were grown in RPMI1640 with 10% horse serum and 5% fetal calf serum, in a humidified atmosphere containing 7.5% CO2. Lipofectamine 2000 was localisation, far greater than that of either the single C85L-CAAX used for transfections of 0.2 g of each plasmid per coverslip. or C88L-CAAX mutants. This finding suggests that the effects of differential palmitoylation on RE and TGN targeting can be fine- Confocal microscopy tuned by the precise number and distribution of palmitoylated For comparison of the intracellular localisations of SNAP25 wild-type and mutant proteins, PC12 cells were co-transfected with 0.2 g each of the respective mCherry- cysteine residues. The localisation of the C85L,C88L-CAAX and eGFP-tagged plasmids. Cells were fixed in 4% formaldehyde at 48 hours post- mutant was intriguing as it was strikingly different to the transfection. For antibody staining, fixed cells were permeabilised for 6 minutes in localisation of Hras, even though the proteins had very similar 0.25% Triton X-100 (in PBS with 0.3% BSA). Antibodies against GM130, TGN38, Rab11 and SNAP25 were used at a dilution of 1:50, and Alexa-Fluor-633-conjugated palmitoylation motifs (Fig. 7A). On the basis of current models anti-(rabbit Ig) and anti-(mouse Ig) secondary antibodies were used at 1:400. for the trafficking of peripheral palmitoylated proteins (Rocks et Antibody incubations were for 60 minutes at room temperature. al., 2010), it might have been predicted that reducing the number Image data were acquired on Zeiss Axiovert and Leica SP5 confocal microscopes of cysteine residues in the SNAP25-CAAX construct would at Nyquist sampling rates and were deconvolved using Huygens software (Scientific Volume Imaging). Image J software was used to calculate Pearson’s correlation change the distribution so that it was similar to Hras. Thus, this coefficient (R) values for analysis of the co-variance of the fluorescence signals work clearly demonstrates that subtle differences in the between the intracellular pool of eGFP–SNAP25 and endogenous antibody-labelled palmitoylation domains of peripheral proteins can have major GM130, TGN38 and Rab11. To limit the contribution of SNAP25 plasma membrane effects on intracellular trafficking. fluorescence to the calculated R values, this analysis was performed on a region of interest that encompassed the fluorescent signals from the intracellular compartments. Whereas Ras proteins undergo cytosolic diffusion and The ‘zoom’ images shown in Fig. 1 are representative of single slices from such repalmitoylation at the Golgi, mature SNAP25 is not thought to regions of interest. Pearson’s values were calculated from the region of interest undergo membrane–cytosol exchange. This might reflect a slower applied to whole-cell image stacks. To quantify the changes to the localisation of rate of depalmitoylation of SNAP25 or the fact that SNAP25 has defined SNAP25 mutants compared with that of wild-type SNAP25, summed z- projections of eGFP and mCherry fluorescence were acquired. The intracellular more palmitoylation sites than Ras. Different rates of fluorescence intensity (Fi) and total cellular fluorescence intensity (Ft) were measured depalmitoylation might partly explain the distinct localisations from identical regions of the eGFP and mCherry projection images using ImageJ observed for Hras protein and the SNAP25(C85L,C88L)-CAAX software. The intracellular fluorescence was expressed as a fraction of the total mutant. However, SNAP25 could also be subject to modification by fluorescence for each channel (Fi/Ft–Fi) and a ratio was calculated for this value for eGFP:mCherry (a ratio of 1 indicates identical levels of eGFP and mCherry constructs post-Golgi DHHC proteins, preventing complete depalmitoylation in the intracellular endosomal compartment, a ratio of greater than 1 indicates an and membrane release. Indeed, we recently reported that DHHC2, increase in the eGFP fluorescence in the intracellular pool, and a value lower than 1

Journal of Cell Science which localises to the plasma membrane in PC12 cells, is active highlighted an increased level of mCherry fluorescence in the intracellular pool). Fluorescence images from cells coexpressing wild-type and mutant proteins were against the SNAP25 protein family (Greaves et al., 2010); this presented in pseudocolour using the Image J software. DHHC protein is therefore a prime candidate to regulate SNAP25 palmitoylation dynamics and intracellular sorting. By contrast, the Image analysis was performed in the CALM and IMPACT confocal rapid depalmitoylation and cytosolic exchange of Ras proteins might imaging facilities at the University of Edinburgh. We thank Giampietro indicate that these proteins are not regulated by post-Golgi DHHC Schiavo for the kind gift of GFP–Rab11. This work was supported by proteins but this issue merits further investigation. a senior non-clinical fellowship award to L.H.C. from the Medical This work highlights the evolving view that palmitoylation Research Council. Deposited in PMC for release after 6 months. should not be regarded simply as a mechanism to attach soluble Supplementary material available online at proteins to membranes and emphasises how subtle differences in http://jcs.biologists.org/cgi/content/full/124/8/1351/DC1 the number and configuration of palmitoylated cysteines can have major effects on protein trafficking. We predict that this post- References Aikawa, Y., Lynch, K. L., Boswell, K. L. and Martin, T. F. J. (2006a). A second SNARE translational modification will have many more intricate effects on role for exocytic SNAP25 in endosome fusion. Mol. Biol. Cell 17, 2113-2124. SNAP25 targeting and function than are currently recognised. Aikawa, Y., Xia, X. and Martin, T. F. J. (2006b). SNAP25, but not syntaxin 1A, recycles via an ARF6-regulated pathway in neuroendocrine cells. Mol. Biol. Cell 17, 711-722. Materials and Methods Apolloni, A., Prior, I. A., Lindsay, M., Parton, R. G. and Hancock, J. F. (2000). H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol. Cell. Chemicals and reagents 35 3 Biol. 20, 2475-2487. MP Biomedicals supplied [ S]L-methione and PerkinElmer provided [ H]palmitate. Bark, I. C., Hahn, K. M., Ryabinin, A. E. and Wilson, M. C. (1995). Differential Cycloheximide was purchased from Sigma. Lipofectamine 2000, cell culture media expression of SNAP-25 protein isoforms during divergent vesicle fusion events of and serum, rabbit anti-Rab11 antibody and Alexa-Fluor-conjugated secondary neural development. Proc. Natl. Acad. Sci. USA 92, 1510-1514. antibodies were from Invitrogen. Anti-SNAP25 monoclonal antibody was from Blasi, J., Chapman, E. R., Link, E., Binz, T., Yamasaki, S., Camilli, P. D., Sudhof, T. Covance. Monoclonal antibodies against GM130 and TGN38 were supplied by BD C., Niemann, H. and Jahn, R. (1993). Botulinum neurotoxin A selectively cleaves the Biosciences. synaptic protein SNAP-25. Nature 365, 160-163. Fukata, M., Fukata, Y., Adesnik, H., Nicoll, R. A. and Bredt, D. S. (2004). Identification Plasmids of PSD-95 palmitoylating enzymes. Neuron 44, 987-996. SNAP25b wild-type and cysteine mutants, cloned into pEGFPC2, were as previously Gary, W. L., Ulrich, W. and Dolly, J. O. (1994). Botulinum A and the light chain of described (Greaves et al., 2009a). The region encoding amino acids 85–120 of tetanus toxins inhibit distinct stages of Mg.ATP-dependent catecholamine exocytosis SNAP25b was amplified by PCR and cloned into pEGFPC2. To generate the 4CL- from permeabilised chromaffin cells. Eur. J. Biochem. 222, 325-333. KrasMTD construct, a 4CL mutant was used as a template and an additional 45 Gonzalo, S. and Linder, M. E. (1998). SNAP-25 palmitoylation and plasma membrane nucleotides, encoding the amino acid sequence GKKKKKKSKTKCVIM from Kras, targeting require a functional secretory pathway. Mol. Biol. Cell 9, 585-597. 1360 Journal of Cell Science 124 (8)

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