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Vol. 20, Issue 6, 1845-1854, March 15, 2009
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*Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom; Division of Membrane Physiology, National Institute for Physiological Sciences, Okazaki 444-8787, Japan; PRESTO, Japan Science and Technology Agency, Tokyo 102-0075, Japan; and INSERM U845, Faculte de Medecine Paris Descartes–Site Necker, 75730 Paris Cedex 15, France
Submitted September 17, 2008;
Revised December 11, 2008;
Accepted January 12, 2009
Monitoring Editor: Thomas F.J. Martin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Greaves and Chamberlain, 2007; Linder and Deschenes, 2007; Nadolski and Linder, 2007). The study of protein palmitoylation has been severely hindered in the past due to a lack of knowledge about the palmitoylating enzymes. However, 23 mammalian and seven yeast palmitoyl transferases (PATs) containing a signature DHHC-CRD (cysteine-rich domain) were recently identified (Lobo et al., 2002; Roth et al., 2002; Fukata et al., 2004; Huang et al., 2004; Keller et al., 2004; Mitchell et al., 2006) and shown to be responsible for the large majority of cellular palmitoylation, in yeast at least (Roth et al., 2006). Mutation of residues in the DHHC domain of these enzymes blocks activity, suggesting that this region of the proteins may form part of the catalytic site.
DHHC proteins have four or more predicted transmembrane domains (TMDs), with the DHHC-CRD putative catalytic domain predicted to be cytosolically exposed; this topology has been confirmed for the yeast enzyme Akr1p (Politis et al., 2005). The large number of DHHC proteins and the association of several of these proteins with the same intracellular compartments (predominantly ER/Golgi) suggest likely differences in substrate specificity and regulation. Indeed, some yeast enzymes were shown to exhibit preferences for certain types of substrate, and although some proteins were modified by more than one enzyme, others required a specific enzyme for palmitoylation (Hou et al., 2005; Smotrys et al., 2005; Roth et al., 2006). The identification of the DHHC family of PATs has reinvigorated the study of protein palmitoylation and provides essential information and tools to dissect the outcome(s) of protein palmitoylation in a cellular context.
Although no general consensus sequence specifying palmitoylation exists, a compelling factor that decides whether a cysteine residue is palmitoylated is undoubtedly its proximity to the membrane. As previously discussed, the exclusive membrane localization of DHHC proteins implies that palmitoylated proteins require specific membrane-targeting signals to facilitate initial membrane interaction (Greaves and Chamberlain, 2006, 2007; Greaves et al., 2008). A number of studies have highlighted the importance of palmitoylation for correct sorting of proteins containing TMDs, e.g., (Hayashi et al., 2005; Lam et al., 2006; Abrami et al., 2008). In addition to TMD proteins, a number of signaling molecules utilize isoprenyl or myristoyl modifications (which are added in the cytosol) to mediate transient membrane interactions. For example, the farnesyl group of H- and N-Ras provides the proteins with a weak membrane affinity that allows the protein to "sample" a variety of intracellular membranes (Magee et al., 1987; Choy et al., 1999; Goodwin et al., 2005; Rocks et al., 2005). Palmitoylation only occurs when Ras associates with a membrane compartment (Golgi/ER) containing the Ras PAT (Swarthout et al., 2005); palmitoylation "traps" Ras on that membrane, facilitating its forward transport (Rocks et al., 2005; Roy et al., 2005).
While primary membrane-targeting information in some proteins is obvious, such as TMDs or isoprenyl/myristoyl modifications, other palmitoylated proteins lack obvious membrane-targeting signals (Greaves and Chamberlain, 2006). This is the case with SNAP25, an essential component of the neuronal/neuroendocrine SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) complex that mediates exocytosis. In contrast to the large majority of SNAREs, SNAP25 lacks a TMD, and mutations within the palmitoylated cysteine-rich domain have been reported to block stable membrane association and targeting (Vogel et al., 2000; Washbourne et al., 2001). The mechanisms regulating SNAP25 membrane targeting before palmitoylation have been much debated. In particular, some studies have suggested a key role for syntaxin 1 (the SNARE partner of SNAP25) in driving initial membrane interaction of SNAP25 (Vogel et al., 2000; Washbourne et al., 2001); however, others (Gonzalo et al., 1999; Loranger and Linder, 2002) have refuted this idea. In particular, a minimal fragment of SNAP25 containing amino acids 85-120 (which includes the palmitoylated cysteines) was shown to target enhanced green fluorescent protein (EGFP) to the plasma membrane (Gonzalo et al., 1999). This region of SNAP25 lacks the SNARE motifs and hence syntaxin-binding sites, providing evidence that SNAP25 traffics independently of syntaxin (Loranger and Linder, 2002). In addition, a recent study reported that syntaxin 1A perturbed plasma membrane delivery of SNAP25 when expressed in the absence of the syntaxin 1A chaperone, munc18 (Medine et al., 2007).
To probe the mechanisms involved in SNAP25 membrane interactions further, we have performed a detailed mutagenic study of the 85-120 minimal membrane-targeting domain present within full-length SNAP25 and examined how specific DHHC enzymes regulate SNAP25 membrane interaction. Cysteine residues and specific surrounding amino acids are implicated in initial membrane binding, most likely via hydrophobic interactions with membranes. Initial access to the membrane interface is important to allow interaction with specific DHHC proteins, which palmitoylate SNAP25 and promote stable membrane attachment. Interestingly, conserved amino acids (in particular proline-117) appear to be important in determining the specificity of DHHC interaction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This construct (lacking the initiating ATG of SNAP25B) was used as a template to produce all the SNAP25B substitution and deletion mutants used in this study by site-directed mutagenesis. This plasmid was also used as a template to amplify the nucleotide sequence coding for amino acids 93-120, which was then cloned into pEGFPC2 using complimentary restriction sites that were present within the oligonucleotide primers used in the PCR reaction. SNAP25B(85-120) with a C-terminal EGFP tag was kindly provided by Dr. Maurine Linder (Washington University at St. Louis) (Gonzalo et al., 1999). Hemagglutinin (HA)-tagged mouse DHHC3, DHHC7, and DHHC17 clones in pEFBOS-HA were as previously described (Fukata et al., 2004). DHHC-to-DHHS mutations were introduced into the respective plasmids by site-directed mutagenesis (Greaves et al., 2008). The fidelity of all mutant constructs was confirmed by DNA sequencing (University of Dundee DNA sequencing service, Dundee, Scotland). Anti-GFP mAb (JL8) was purchased from Clontech (Palo Alto, CA). Anti-SNAP25 and anti-syntaxin 1A antibodies were supplied by Synaptic Systems (Göttingen, Germany). Anti-HA mAb and COMPLETE protease inhibitor cocktail were from Roche (Lewes, East Sussex, United Kingdom). Subcellular proteome extraction kit (SPEK) was purchased from Merck Biosciences (Nottingham, United Kingdom). All other reagents were of an analytical grade from Sigma (Poole, Dorset, United Kingdom).
Cell Culture and Cell Transfection
PC12 cells were grown in RPMI1640 media with 10% horse serum and 5% fetal calf serum containing penicillin/streptomycin. Human embryonic kidney (HEK293) cells were cultured in DMEM with 10% fetal calf serum with penicillin/streptomycin. All reagents used for maintenance of cells were purchased from Invitrogen (Paisley, United Kingdom). Cells were maintained in a humidified atmosphere containing 5% CO2.
For all experiments, cells were plated onto 24-well plates or coverslips precoated with poly-D-lysine. Cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions; the ratio of lipofectamine to DNA used was 2:1. PC12 cells were analyzed 
40 h after transfection, and HEK293 cells were used 20 h after transfection.
Subcellular Fractionation
Cells were fractionated into cytosol, and membrane fractions using selected buffers from an SPEK kit (Merck), which isolates defined cell fractions by differential detergent extraction (Ramsby et al., 1994; Greaves and Chamberlain, 2006; Greaves et al., 2008). Briefly, cells were washed 2x in PBS and then incubated on ice in 150 µl of buffer 1 containing protease inhibitors for 10 min. The buffer was then removed and centrifuged at 2000 x g to remove cell debris; this fraction contained cell cytosol. The remaining cell material was solubilized in SDS-dissociation buffer. As an alternative fractionation procedure, cells were homogenized in HES buffer (20 mM HEPES, 1 mM EDTA, and 250 mM sucrose, pH 7.4) using a Dounce homogenizer. The homogenate was centrifuged at 190,000 x g for 30 min to separate cytosol (supernatant) and membrane (pellet) fractions. Comparison of this method of cell fractionation to the SPEK procedure confirmed the validity of the SPEK approach for cell fractionation (Supplementary Figure S1).
Small Interfering RNA Transfection
PC12 cells growing on poly-D-lysine coated 24-well plates were transfected with either 100 nM random small interfering RNA (siRNA) or a mixture of two siRNAs against syntaxin 1A (50 nM of each) using Dharmafect reagent. siRNAs and transfection reagent were supplied by Dharmacon Research (Boulder, CO). Approximately 70 h after transfection, the cells were lysed in SDS-dissociation buffer or fractionated using SPEK.
Palmitate Labeling
HEK293 cells plated on six-well plates were transfected with EGFP-SNAP25B or the SNAP25B(117-120A) mutant together with HA-DHHC3 or HA-DHHC17. Approximately 20 h after transfection, the cells were incubated with 0.5 mCi/ml [3H]palmitic acid (Perkin Elmer-Cetus, Beaconsfield, United Kingdom) for 4 h, and EGFP-SNAP25B or the 117-120A mutant were subsequently immunoprecipitated using anti-GFP antibody coupled to magnetic microbeads (Miltenyi Biotech, Bisley, United Kingdom). The precipitated samples were resolved on duplicate gels and transferred to nitrocellulose membranes that were then either subjected to immunoblotting using anti-GFP or were exposed to film with the aid of a Kodak Biomax Transcreen LE intensifier screen (Eastman Kodak, Rochester, NY) for detection of [3H]palmitate incorporation.
Immunofluorescence
PC12 cells growing on poly-D-lysine–coated coverslips were fixed in 4% formaldehyde (in PBS) for 30 min at room temperature, washed in PBS, and mounted onto slides using Mowiol 4-88 reagent. Imaging was performed using a Zeiss LSM 5 Pascal laser scanning microscope (Zeiss, Oberkochen, Germany).
Quantification and Statistical Analysis
Quantification of band density on immunoblots was determined using ImageJ software (http://rsb.info.nih.gov/ij/). Data are expressed as average % membrane association ± SEs. Statistical analysis was performed using unpaired Student's t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, as expression levels of EGFP-SNAP25B were increased, there was a gradual loss of relative membrane association (Figure 1A). This suggests that a factor(s) required for membrane targeting of SNAP25B is present in only limiting concentrations in HEK293 cells, and this cell type thus provides a useful model to examine proteins that regulate SNAP25B membrane interaction.
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). As SNAP25B is palmitoylated by the same enzymes as CSP (Fukata et al., 2006), we reasoned that inefficient membrane association of SNAP25B in HEK293 cells may also reflect limiting expression of these DHHC proteins. To test this idea, we examined the effects on SNAP25B membrane interaction of coexpressing DHHC3, DHHC7, or DHHC17. All three DHHC proteins promoted a significant increase in the level of SNAP25B membrane binding (Figure 1B). This effect was specific, as mutations within the putative catalytic domains (DHHC to DHHS mutations) of DHHC3 and DHHC7 completely abolished the ability of the enzymes to enhance membrane binding of SNAP25B, and indeed both mutants decreased SNAP25B membrane association (Figure 1, C and D).
As a further test of DHHC specificity, we examined the effects on SNAP25B membrane binding of coexpressing DHHC2 or DHHC4, enzymes that are not known to palmitoylate SNAP25B (Fukata et al., 2006). These enzymes had no stimulatory effect on SNAP25B membrane binding (Figure 1E), despite being expressed at similar levels to DHHC3. These results clearly demonstrate that specific palmitoyl transferases are sufficient to drive stable membrane interaction of SNAP25B in HEK293 cells.
Note that palmitoylation of SNAP25 leads to a decreased rate of migration by SDS-PAGE that is visible under specific gel conditions (Gonzalo and Linder, 1998). This band-shift was visible for EGFP-SNAP25B when samples were resolved on 10% SDS-PAGE gels (as shown in Figure 1, A and B). This observation shows that the increase in membrane binding of SNAP25B promoted by DHHC3/DHHC7/DHHC17 in HEK293 cells correlates with increased palmitoylation. The visualization of a palmitoylation-dependent band-shift in SNAP25 gives a more meaningful readout of palmitoylation status than [3H]palmitate labeling. For example, it is not easy to determine whether a change in [3H]palmitate incorporation into a multiply palmitoylated protein such as SNAP25 reflects: 1) an increase in the number of protein molecules that are palmitoylated, 2) an increased extent of palmitoylation of a fixed pool of protein, or 3) a combination of these factors. By analyzing SNAP25 migration in purified cytosol and membrane fractions, we can be confident that the number of SNAP25 molecules that are palmitoylated is increased by DHHC coexpression.
Cysteine Residues and Flanking Hydrophobic Amino Acids Are Important for SNAP25B Membrane Interactions
The previous section clearly demonstrated that the expression level of specific DHHC proteins regulates stable membrane binding of SNAP25B in HEK293 cells. However, as DHHC proteins are membrane associated, these results do not offer any insight into the mechanism of initial membrane interaction of SNAP25B. Syntaxin 1 has been proposed by several groups to regulate membrane binding of SNAP25, and indeed the two proteins were reported to interact in the cytosol of PC12 cells after synthesis (Vogel et al., 2000). However, other groups have suggested that SNAP25 membrane trafficking occurs independently of syntaxin (see e.g., Gonzalo et al., 1999; Loranger and Linder, 2002; Medine et al., 2007), and we have shown that DHHC proteins are sufficient to drive stable membrane attachment of SNAP25 in HEK293 cells in the absence of syntaxin 1 expression. The major isoform of syntaxin 1 in PC12 cells is syntaxin 1A, and to directly examine the role of this protein in SNAP25 membrane binding in PC12 cells, we used siRNA specific for syntaxin 1A to deplete cellular expression levels. Reduction of syntaxin 1A by 70% (Figure 2A) was found to have no effect on SNAP25 membrane association (Figure 2B), despite reports that endogenous SNAP25 is expressed at several-fold excess above syntaxin 1 in this cell type (Xiao et al., 2004).
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; Greaves et al., 2008), we reasoned that perhaps the hydrophobicity of cysteine residues is important for initial membrane contact of SNAP25B. A number of studies have examined the effects on SNAP25 membrane interactions of mutating individual cysteine residues to alanine, serine, or glycine. The general consensus from these studies is that mutation of an individual cysteine reduces membrane binding/palmitoylation by 50%, whereas mutation of any two cysteines results in an 90% reduction (Lane and Liu, 1997; Gonelle-Gispert et al., 2000; Washbourne et al., 2001). However, no comprehensive analysis of the individual cysteine residues has been performed in a cell type that endogenously expresses SNAP25. Given that SNAP25 has four potential palmitoylation sites and that (in simplistic terms, at least) stable membrane binding should be bestowed by two palmitates (Shahinian and Silvius, 1995), it is not clear why individual cysteine mutations have such a large effect on membrane binding. A possibility could be that the presence of multiple cysteines increases the likelihood that one cysteine will be palmitoylated, which would enhance membrane affinity and subsequently increase the likelihood that other cysteines will be modified. Notwithstanding this, one possible problem with previous analyses is that alanine, serine, and glycine are all less hydrophobic than cysteine, and hence defects in membrane binding caused by such mutations could reflect an inhibition of initial hydrophobic membrane interactions rather than a loss of cysteine palmitoylation.
To test this idea, we individually mutated each cysteine residue in SNAP25B (C85, C88, C90, and C92) to either alanine or leucine. Alanine is less hydrophobic than cysteine, whereas leucine has a similar (or greater) hydrophobicity. These experiments were performed in PC12 cells, in which EGFP-SNAP25B is efficiently palmitoylated and trafficked (see e.g., Salaun et al., 2005). Interestingly, for the alanine substitutions, we observed a marked difference in the effect on membrane binding dependent on which cysteine was mutated. C85A and C88A mutations decreased membrane binding by >50%, whereas C92A had a lesser effect and C90A was almost without effect on membrane interaction (Figure 3B). Thus, the individual cysteines each contribute to stable SNAP25 membrane interaction to different extents. Intriguingly though, the effect of every alanine substitution was almost completely reversed by replacement with leucine (Figure 3B). These results are consistent with an important role for cysteine hydrophobicity in SNAP25B membrane interactions; we propose that this role is to facilitate initial membrane interaction and thus ensure spatial proximity of the cysteines to specific membrane-localized DHHC palmitoyl transferases. As a control to ensure that the introduction of leucine residues to the cysteine-rich domain was not creating an artificial membrane-binding domain, all four cysteine were replaced with leucines (4CL). Figure 3C shows that the SNAP25B(4CL) mutant did not associate appreciably with cell membranes. Thus, although leucines can substitute for individual cysteines, introduction of leucines is not sufficient to promote membrane association in the absence of palmitoylation. The proposal that cysteine hydrophobicity is important for initial membrane association of SNAP25 is also strengthened by the observation that amino acids surrounding the palmitoylated cysteines are predominantly hydrophobic (see Figure 3A). Indeed, the introduction of a double L87A/V89A mutation also significantly inhibited SNAP25 membrane binding (Figure 3D), an effect that was reversed when the hydrophobic character of these amino acids was maintained (L87V/V89L). Thus, the hydrophobicity of the cysteine-rich domain as a whole plays an important role in SNAP25 membrane binding.
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90% in PC12 cells (data not shown). To test the proposal that this dramatic loss of membrane binding of C85A/C92A reflects a loss of initial membrane targeting, we tested whether coexpression of DHHC3 could rescue membrane binding of this mutant in HEK293 cells. The C85A/C92A mutant was present almost entirely in the cytosolic fraction of HEK293 cells and this distribution did not change upon DHHC3 coexpression (Figure 3E). As the C85A/C92A mutant still retains two palmitoylation sites, this result is consistent with a loss of initial membrane binding of C85A/C92A and hence inability to localize in proximity to the DHHC3 protein.
Mutational Analysis of the Role of Amino Acids 93-120 in SNAP25B Membrane Binding
The data presented in the previous section support the notion that cysteine residues in SNAP25B play an important role in initial membrane interaction of SNAP25B. However, previous work, analyzing SNAP25 truncation mutants, mapped the minimal membrane-targeting sequence of SNAP25B to residues 85-120 (Gonzalo et al., 1999), which includes the palmitoylated cysteine residues at positions 85, 88, 90, and 92 (Figure 3A) and the downstream 28 amino acids. It is not clear whether residues 93-120 play a direct role in membrane binding of full-length SNAP25B (e.g., by forming an essential part of the DHHC-binding site). As a first step to dissect the role of residues 93-120 in membrane binding of SNAP25B, we examined the possibility that residues downstream of the palmitoylated cysteines mediate initial membrane interactions of SNAP25B. Thus, PC12 cells were transfected with S25(85-120)-EGFP or EGFP-S25(93-120) constructs, and the protein distribution was analyzed by confocal imaging. Figure 4A shows that, in contrast to the S25(85-120) construct that was efficiently targeted to the plasma membrane (Gonzalo et al., 1999), the S25(93-120) construct showed a dispersed localization that was distributed throughout the cytoplasm and the nucleus of PC12 cells. These data demonstrate that region 93-120 of SNAP25B does not contain strong autonomous membrane-targeting information.
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). Residues 93-120 were mutated in blocks of four amino acids to alanine (any alanines were mutated to leucine). The constructs were transfected into PC12 cells, and cytosol and membrane fractions prepared. As shown in Figure 4, B and C, the majority of amino acids between residues 93-120 were dispensable for membrane binding, with the exception of the C-terminal eight amino acids (residues 113-120). We also confirmed these results using a cell homogenization–based fractionation approach (Supplementary Figure S1). These results agree well with the work of Linder's group studying SNAP25B fragments, which showed a decrease in membrane binding when amino acids Q116, P117, and R119 were mutated together within the context of the SNAP25B(85-120) construct (Gonzalo et al., 1999). Note that we consistently observed a lower expression level of the 113-116A and 117-120A mutants compared with wild-type EGFP-SNAP25B in PC12 cells, suggesting that turnover of these mutants may be increased, perhaps because of a loss of membrane binding. As an independent measure of membrane targeting and to detect any changes in intracellular localization of the various SNAP25B mutants, we also examined transfected cells by confocal imaging. All membrane-bound mutants (93-96A, 97-100A, 101-104A, 105-108A, and 109-112A) were strongly enriched at the plasma membrane (Figure 5), demonstrating that these amino acids are not required for either membrane binding or intracellular sorting of SNAP25B. Note that SNAP25B localizes to the plasma membrane and also to an intracellular endosome compartment, as described by Martin's group (Aikawa et al., 2006). The relative level of SNAP25B in this intracellular compartment varied between individual cells, but we did not detect any consistent differences in the intracellular localizations of any of the membrane-bound mutants. In contrast, the 113-116A and 117-120A mutants both displayed a more dispersed cytosolic localization (Figure 5), although some association with the plasma membrane and intracellular membranes was still apparent. Thus, these results are in agreement with the subcellular fractionation data (Figure 4, B and C). These results do not support the proposal that the trafficking of the isolated 85-120 fragment might use a distinct mechanism from full-length SNAP25B (Vogel et al., 2000).
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; Wimley and White, 1996). Interestingly, we found that this P117V mutation had no major effect on SNAP25B membrane binding (Figure 7B). In contrast, replacing P117 with glycine, aspartic acid, lysine, leucine or serine all significantly inhibited membrane binding (Figure 7B).
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101–107) caused a marked loss of SNAP25B membrane binding (Figure 8B). These results show that the spacing between the cysteine-rich domain and residues downstream (P117) is important, suggesting that these two regions of the membrane-targeting domain are functionally coupled.
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101–107) were transfected into HEK293 cells either with DHHC3, DHHC7, DHHC17, or empty vector. Recovered cytosol and membrane fractions were resolved on 10% SDS-PAGE gels to allow visualization of the palmitoylation-dependent band-shift. Results presented (Figure 9) clearly show that both DHHC3 and DHHC7 enhanced membrane binding of the 117-120A and 101–107 mutants and also that a band-shift occurs for membrane-bound protein, consistent with palmitoylation. In contrast, cotransfection of DHHC17 only very weakly stimulated membrane binding of the SNAP25 mutants (Figure 9), suggesting that residues 117-120 and the spacing of these residues from the cysteine-rich domain are particularly important for recognition of SNAP25B by DHHC17. Note that experiments with wild-type SNAP25 were performed in parallel, confirming that DHHC17 is active against wild-type SNAP25B under identical conditions (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Vogel et al., 2000; Washbourne et al., 2001; Loranger and Linder, 2002). Our results clearly show that membrane interaction of SNAP25B in HEK293 cells is not inefficient because of low expression of syntaxin isoforms, but instead most likely results from a paucity of specific SNAP25B-palmitoylating enzymes. The finding that DHHC proteins mediate membrane binding of SNAP25B is particularly relevant as, unlike tail-anchored proteins such as syntaxin 1, DHHC proteins are polytopic membrane proteins that are membrane-inserted cotranslationally. This essentially rules out a coordinated interaction of newly synthesized DHHC and SNAP25B in the cytosol that would facilitate initial membrane targeting of SNAP25B.
Mutational analyses of SNAP25B membrane binding are consistent with the notion that membrane association of unpalmitoylated SNAP25B is mediated by hydrophobic interactions of the cysteine-rich domain with the membrane. Replacement of single cysteines with alanine residues had a marked effect on membrane binding, particularly when C85 or C88 were substituted. However, membrane binding was restored when these cysteines were replaced by more hydrophobic leucine residues, demonstrating that cysteines are not just sites for palmitate attachment but that other features of the cysteine residues (presumably hydrophobicity) also contribute to membrane binding. The finding that C85A and C88A mutations had a larger effect on membrane binding than C90A and C92A mutations is consistent with previous work studying palmitoylation and membrane binding in Cos-7 cells, which showed that C85S and C88S mutations resulted in a greater loss in SNAP25B palmitoylation and membrane binding than mutation of C90S or C92S (Lane and Liu, 1997). The cysteine residues in the cysteine-rich domain of SNAP25 are part of an overall hydrophobic domain, and we also found that L87 and V89, which sit adjacent to C88, are important for SNAP25 membrane interaction. In a similar manner to the cysteine residues, the hydrophobicity of L87 and V89 appears to be important for membrane binding, as alanine mutation of these residues inhibits membrane binding, whereas switching the residues (and hence maintaining hydrophobicity) does not have a deleterious effect on membrane interaction. It was interesting that individual cysteine-to-leucine mutants never restored SNAP25 membrane binding exactly to wild-type levels. This might suggest that the presence of multiple closely spaced cysteines plays an additional role in stable membrane binding by increasing the likelihood that palmitoylation of a cysteine residue will occur, and this is likely to be important for proteins with a weak membrane affinity such as SNAP25.
A role for cysteine residues and flanking hydrophobic amino acids in regulating initial membrane interaction of SNAP25 agrees with our previous work implicating cysteine hydrophobicity in initial membrane attachment of CSP (Greaves and Chamberlain, 2006). This close association of cysteine-rich domains with the membrane is an attractive idea as it would ensure that cysteines are in intimate membrane contact, which appears to be an important factor in determining palmitoylation sites. It is not clear whether SNAP25B is self-sufficient for initial membrane interaction or if additional "chaperones" regulate movement of the newly synthesized protein to membranes. If additional molecules are required, then these are likely to be widely expressed (as they are not limiting in HEK293 cells). Thus, we propose that SNAP25B utilizes a similar mechanism of membrane binding/palmitoylation as previously proposed by us for CSP (Greaves and Chamberlain, 2006; Greaves et al., 2008). One interesting difference, however, is that CSP and SNAP25B membrane binding differ in their sensitivity to brefeldin A (BFA; Gonzalo and Linder, 1998; Greaves et al., 2008); membrane binding of SNAP25B is sensitive to BFA, whereas CSP is resistant. As DHHC proteins retain their activity after BFA treatment of both PC12 and HEK293 cells (Greaves et al., 2008), we propose that this difference in sensitivity might reflect a difference in initial membrane interactions; for example, SNAP25B might have a high affinity for intact Golgi membranes, whereas CSP has a more general membrane affinity.
Ras proteins have been suggested to undergo a dynamic cycle of palmitoylation and depalmitoylation that regulates localization between the plasma membrane and intracellular membranes (Goodwin et al., 2005; Rocks et al., 2005), and a similar cycle was proposed for other palmitoylated peptides (Rocks et al., 2005). In this cycle, depalmitoylation of plasma membrane–localized protein results in release into the cytosol, where the protein can bind to intracellular membranes and be repalmitoylated and transported again to the plasma membrane. Such a palmitoylation cycle could be an intriguing pathway to regulate SNAP25 localization and hence exocytosis efficiency. However, although the half-life of palmitate attachment to SNAP25 was suggested to be shorter than the half-life of the protein in PC12 cells (Lane and Liu, 1997), no turnover of palmitate on SNAP25 was detected in cortical neurons (Kang et al., 2004). Thus, it is unclear at this stage whether a similar palmitoylation cycle might operate for SNAP25, but the membrane-binding properties of the hydrophobic cysteine-rich domain of SNAP25 could be well suited to coordinate such a cycle.
In addition to the cysteine-rich domain, stable membrane binding of SNAP25B in PC12 cells is also dependent on downstream residues (P117) and their spacing from the palmitoylated cysteines. P117 was also identified by Linder and coworkers as part of a group of amino acids important for trafficking of the isolated 85-120 membrane-targeting domain, and it was suggested that this region of SNAP25 might be important to allow binding to a palmitoyl transferase (Gonzalo et al., 1999). The results presented here support these suggestions and, intriguingly, suggest that the region of SNAP25B containing P117 may also play an important role in determining DHHC specificity: at similar DHHC expression levels, this region of SNAP25B was important for palmitoylation and stable membrane binding induced by DHHC17 but not DHHC3 or DHHC7. This observation to our knowledge represents the first description of intrinsic substrate elements that might modulate DHHC specificity. At this stage we do not know why P117 is particularly important for efficient palmitoylation by DHHC17. The most obvious possibility is that this region of SNAP25 supports the direct interaction of SNAP25 and DHHC17. Alternatively, P117 may be important in promoting the association of SNAP25 with specific membranes or membrane domains that sequester DHHC17. Although DHHC3, DHHC7, and DHHC17 are all localized to the Golgi in HEK293 cells (Greaves et al., 2008), it is not known whether the proteins are associated with the same or different Golgi cisternae. Similarly, it is not known whether these DHHC proteins are present in the same Golgi subdomains; for example, the ankyrin repeat region of DHHC17 might target the protein to actin-rich regions of the Golgi. Thus, P117 may also be important in ensuring efficient targeting of SNAP25B to the same membrane compartment or subcompartment that DHHC17 resides in.
The reduced membrane binding of the 117-120A and 101–107 mutants in PC12 cells is consistent with an important role for DHHC17 in regulating SNAP25 palmitoylation in this cell type. Attempts to deplete DHHC proteins by siRNA in PC12 cells have thus far proved unsuccessful. However, it is worth noting that depletion of DHHC17 in Drosophila was recently reported to cause mislocalization of SNAP25 (Ohyama et al., 2007; Stowers and Isacoff, 2007), supporting the notion that DHHC17 regulates SNAP25 palmitoylation in vivo.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Luke H. Chamberlain (Luke.Chamberlain{at}ed.ac.uk)
Abbreviations used: CSP, cysteine-string protein; DHHC, aspartic acid-histidine-histidine-cysteine motif; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; HEK, human embryonic kidney; PAT, palmitoyl transferase; PC12, pheochromocytoma-12; SNAP25, synaptosomal-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide–sensitive factor attachment protein receptor.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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