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Vol. 17, Issue 11, 4748-4759, November 2006
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Henry Wellcome Laboratory of Cell Biology, Division of Biochemistry and Molecular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom
Submitted March 7, 2006;
Revised August 14, 2006;
Accepted August 18, 2006
Monitoring Editor: Sean Munro
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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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; Huang and El-Husseini, 2005). Although palmitoylation can occur via an amide linkage to glycine or cysteine residues, S-palmitoylation is more common, where the palmitate group is joined to a cysteine residue via a thioester linkage (Pepinsky et al., 1998; Kleuss and Krause, 2003). Palmitoylation can occur spontaneously in vitro, but it is generally believed that the majority of palmitoylation events occur catalytically in vivo. Recent work has identified a conserved DHHC domain that is present in palmitoyl transferases (PATs) in both yeast and mammalian cells (Bartels et al., 1999; Putilina et al., 1999; Lobo et al., 2002; Roth et al., 2002). In mammalian cells, more than 20 DHHC proteins have been cloned, and several of these proteins have been shown to exhibit substrate-specific PAT activity (Fukata et al., 2004; Huang et al., 2004; Keller et al., 2004; Swarthout et al., 2005). Palmitoylation is thought to occur at various locations in the cell because PAT enzymes and activities have been found to be associated with a range of intracellular organelles, including the Golgi, ER, and plasma membrane (Dunphy et al., 1996; Huang et al., 2004; Swarthout et al., 2005).
Because PATs are membrane-bound enzymes, substrate proteins must have a mechanism to facilitate their initial membrane binding to localize in close proximity to the appropriate PAT. A number of mechanisms have been shown to underlie the initial membrane binding of palmitoylated proteins; examples include C-terminal farnesylation of H-Ras and N-Ras, which targets the proteins to the endoplasmic reticulum and Golgi (Choy et al., 1999), with palmitoylation being required for subsequent intracellular sorting (Apolloni et al., 2000); myristoylation of Src family protein tyrosine kinases on an N-terminal glycine, which mediates initial membrane insertion before palmitoylation of adjacent cysteines (Koegl et al., 1994); and the transmembrane domain of the Influenza fusion protein hemagglutinin, which integrates into membranes allowing the palmitoylation of three membrane proximal cysteine residues (Naeve et al., 1990; Steinhauer et al., 1991; Veit et al., 1991). In contrast to proteins containing these primary membrane anchoring signals, some palmitoylated proteins contain no obvious membrane-binding motif, and it is unclear how these proteins associate with membranes. This group of proteins include molecules such as PSD-95 (Craven et al., 1999), an essential scaffolding protein of the postsynaptic density, SNAP-25, an important regulator of exocytic membrane fusion (Lane and Liu, 1997), and cysteine-string protein (CSP), a molecular chaperone of the Hsp40 protein family that has a range of important cellular functions (see below; Gundersen et al., 1994).
CSP is extensively palmitoylated on a central cysteine-rich "string" domain; this domain contains 14 cysteines in a span of 25 amino acids, and the majority of these cysteines are thought to be palmitoylated in vivo (Gundersen et al., 1994). CSP plays an essential role in regulated exocytosis pathways in neuronal and nonneuronal cells (Umbach et al., 1994; Zinsmaier et al., 1994; Brown et al., 1998; Chamberlain and Burgoyne, 1998b; Zhang et al., 1998; Graham and Burgoyne, 2000) and exhibits a strong neuroprotective function, with CSP null mice displaying progressive, lethal neurodegeneration (Fernandez-Chacon et al., 2004; Chandra et al., 2005). In addition, CSP has also been demonstrated to regulate the intracellular folding and maturation of the cystic fibrosis transmembrane conductance regulator (CFTR; Zhang et al., 2002, 2006). The functions of CSP in regulated exocytosis, neuroprotection, and maturation of CFTR are thought to center on its molecular chaperone activity: CSP forms a "chaperone machine" with HSC70 (heat shock cognate protein of 70 kDa) and SGT (small glutamine-rich tetratricopeptide repeat domain protein) that prevents aggregation and refolds model denatured proteins, suggesting that stabilization and refolding of specific substrate proteins may be an essential and general facet of CSP function (Chamberlain and Burgoyne, 1997; Tobaben et al., 2001).
CSP has been predominantly characterized as a secretory vesicle protein; it is present on synaptic vesicles (Mastrogiacomo et al., 1994), chromaffin granules (Kohan et al., 1995; Chamberlain et al., 1996), pancreatic zymogen granules (Braun and Scheller, 1995), insulin-containing granules (Brown et al., 1998; Zhang et al., 1998), and granules of the neurohypophysis (Pupier et al., 1997). In addition, CSP is localized almost exclusively on the plasma membrane (PM) in adipocytes (Chamberlain et al., 2001), partially on the apical PM of Calu-3 lung epithelial cells (Zhang et al., 2002), and exhibits a strong enrichment at the PM after overexpression in PC12 cells (Chamberlain and Burgoyne, 1998b). A small amount of CSP was also suggested to colocalize with an ER marker in Calu-3 cells, consistent with its proposed function in CFTR maturation/folding (Zhang et al., 2002).
Despite the key intracellular functions of CSP, the molecular determinants that mediate the membrane binding and targeting of this protein are largely unknown. Chemically depalmitoylated CSP remains membrane associated, suggesting that palmitoylation is not essential for membrane binding; the cysteine-string domain was suggested to preserve membrane association after depalmitoylation (Van de Goor and Kelly, 1996; Mastrogiacomo et al., 1998). However, another study reported that a CSP mutant with the central "core" of seven cysteines in the cysteine-string domain replaced with serine residues was not palmitoylated and was entirely cytosolic, prompting the conclusion that although palmitoylation may not be required for stable membrane association of CSP, it is required for initial membrane targeting (Chamberlain and Burgoyne, 1998a). Despite these observations, the mechanisms involved in CSP membrane targeting, binding, and trafficking are still largely unknown. In this study, we provide a detailed analysis of CSP membrane interactions that, as well as providing essential data on CSP membrane trafficking, may also serve as an important paradigm for membrane targeting of other palmitoylated proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of Mutant Constructs
For cloning into pEGFP-C2, CSP was PCR amplified with an HindIII site incorporated at the 5' end and a BamHI site at the 3' end. The EGFP-CSP construct was used as a template for generating all CSP mutants. Site-directed mutagenesis was performed using the Quickchange system (Stratagene). CSP N-terminal deletion mutants were generated by PCR and cloned into BamHI- and HindIII-digested pEGFP-C2. CSP C-terminal deletion mutants were generated by introducing a premature stop codon by site-directed mutagenesis. All constructs were verified by sequencing of both strands.
PC12 Cell Culture and Transfection
Rat pheochromocytoma-12 (PC12) cells were cultured in suspension in RPMI-1640 (Invitrogen) supplemented with 10% (vol/vol) horse serum and 5% (vol/vol) FCS (Invitrogen) at 37°C in a humidified atmosphere containing 5% CO2. For subcellular fractionation experiments, 
2 x 106 cells were seeded onto poly-D-lysine–coated 6-well plates. For immunofluorescence, 1 x 106 cells were plated onto poly-D-lysine–coated coverslips. Cells were transfected with 1 µg plasmid DNA 24 h after seeding using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. All cells were analyzed 48 h after transfection.
Subcellular Fractionation
Transfected PC12 cells were separated into cytosolic and membrane fractions using a ProteoExtract Subcellular Proteome Extraction Kit according to the manufacturer's protocol (Calbiochem). For detection of proteins in each fraction, equal volumes of the samples were subjected to SDS-PAGE and immunoblotting.
Chemical Depalmitoylation of PC12 Cell Membranes
PC12 cells were washed twice with ice-cold PBS, resuspended in HES homogenization buffer (0.32 M sucrose, 20 mM HEPES, 1 mM EDTA, pH 7.4, plus protease inhibitors) and homogenized with a Dounce homogenizer. The homogenized cells were centrifuged at 500 x g for 5 min at 4°C, and the postnuclear supernatant was removed and centrifuged at 196,000 x g for 30 min at 4°C to pellet cell membranes. The recovered membrane fraction was incubated in either 1 M hydroxylamine (pH 7) or 1 M Tris (pH 7) for 20 h at room temperature. The treated membranes were recovered by centrifugation at 196,000 x g for 30 min and examined by immunoblotting.
[3H]Palmitic Acid Labeling
Transfected PC12 cells (3 x 106) were cultured for 48 h and then incubated in RPMI1640 medium containing 10 mg/ml BSA for 30 min at 37°C. After this, the cells were incubated in RPMI1640/BSA containing 1 mCi/ml [3H]palmitic acid for 4 h at 37°C. GFP-tagged constructs were isolated using magnetic separation after incubation of cell lysates with magnetic microbeads coupled to GFP antibody (Miltenyi Biotech). Immunoprecipitated samples were recovered from the microbeads in SDS-PAGE sample buffer prewarmed to 95°C, and the recovered samples were separated by SDS-PAGE and transferred to duplicate nitrocellulose membranes. One membrane was processed for immunoblotting analysis using a monoclonal GFP antibody. The duplicate membrane was dried and sprayed with En3hance fluorographic spray (Perkin Elmer-Cetus) according to the manufacturer's instructions and exposed to light-sensitive film for 10–14 d at –80°C.
Immunofluorescence
Transfected PC12 cells growing on poly-D-lysine–coated coverslips were washed three times in PBS and fixed in PBS containing 4% formaldehyde for 30 min. The cells were washed twice in PBS and incubated for 30 min in PBTA (0.1% Triton X-100 and 0.3% BSA in PBS). The permeabilized cells were then incubated in either anti-calreticulin (1:200) or anti-CSP (1:400) for 1 h and washed three times in PBTA. For detection of immunogens, cells were incubated with anti-rabbit-Alexa-594 antibody for 1 h. The cells were then washed three times in PBTA, and coverslips were mounted onto slides using Mowiol 4–88 Reagent. Imaging was performed using a Zeiss LSM 5 Pascal laser scanning microscope (Zeiss, Oberkochken, Germany). For analysis of the C4-7L mutant, cells were washed and incubated in PBS containing 20 µM digitonin for 20 min at room temperature. The permeabilized cells were then fixed in 4% formaldehyde for 30 min and analyzed as described above.
Analysis of Protein–Membrane Interactions
Cells, 10 x 106, were transfected with 10 µg of either EGFP-CSP, CSP(1-136), or CSP(C4-7L). Forty-eight hours after transfection, the cells were washed in PBS and resuspended in HES buffer. The cells were then homogenized using a Dounce homogenizer, and membranes were recovered by centrifugation at 196,000 x g for 30 min. The recovered membranes were incubated in HES buffer, 1 M NaCl (in PBS), 0.1 M sodium carbonate (pH 11.5), or 1% Triton X-100 (in PBS; all containing protease inhibitors) for 30 min at 4°C, and supernatant and pelleted membrane fractions were separated by centrifugation at 196,000 x g for 30 min.
Proteinase-K Digestion Analysis
PC12 cells (5 x 106) transfected with EGFP-CSP or C4-7L were Dounce homogenized (x20) in HES buffer containing 5 mM DTT (without protease inhibitors). The membranes were recovered by centrifugation at 196,000 x g for 30 min and resuspended in 0.6 ml HES/DTT, and the samples were then divided into four aliquots, which were incubated with or without 2 µg proteinase K or 0.2% Triton X-100 (final concentration, vol/vol) for 30 min on ice. The reactions were stopped by the addition of PMSF, and SDS-PAGE sample buffer was added before warming the samples at 95°C for 5 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CSP Mutants Lacking the C-Terminal Domain Are Not Palmitoylated and Colocalize with ER Markers
Expressed EGFP-CSP in PC12 cells is predominantly membrane-bound, with a smaller pool located in the cytosol (Figure 3A, left panel). The apparent molecular weight of the membrane-bound fraction of CSP is 8 kDa greater than that of the cytosolic CSP; this difference reflects the extensive palmitoylation of the cysteine-string domain in membrane-bound CSP (Gundersen et al., 1994; van de Goor and Kelly, 1996; Chamberlain and Burgoyne, 1998a). As expected, chemically induced depalmitoylation of CSP using 1 M hydroxylamine (HA), pH 7.0, resulted in a large decrease in the size of membrane-bound CSP (Figure 3A, left panel); note that depalmitoylated CSP in the membrane fraction now migrates at the same speed as cytosolic CSP and that HA treatment promotes complete depalmitoylation of CSP (Chamberlain and Burgoyne, 1998a). This HA-induced band-shift in CSP serves as a robust assay for distinguishing between palmitoylated and unpalmitoylated forms of the protein (Van de Goor and Kelly, 1996; Chamberlain and Burgoyne, 1998a; Mastrogiacomo et al., 1998). To investigate whether the membrane-associated, C-terminally truncated mutants of CSP are correctly palmitoylated, membranes prepared from PC12 cells expressing EGFP-CSP(1-136) and EGFP-CSP(106-136) were treated with 1 M HA or 1 M Tris as a control. Although both EGFP-CSP(1-136) and EGFP-CSP(106-136) are localized to the membrane fraction, there was no detectable molecular mass shift between Tris- and HA-treated samples (Figure 3A, middle and right panels). This result suggests that, in contrast to full-length EGFP-CSP, CSP(1-136) and CSP(106-136) are either unpalmitoylated or their level of palmitoylation is dramatically reduced. To examine this further, cells expressing wild-type CSP or the 1-136 mutant were labeled with [3H]palmitic acid. As shown previously in this study, EGFP-CSP was detected on immunoblots as two bands (Figure 3B, left panel), representing palmitoylated (arrowhead) and nonpalmitoylated (asterisk) forms (Figure 3B, right panel). There was a clear loss of 3H incorporation into CSP(1-136) compared with wild-type protein in this gel run (Figure 3B), demonstrating that palmitoylation of CSP(1-136) is decreased relative to wild-type protein. However, to rule out the possibility that the 1-136 mutant was modified by a small number of palmitates, we repeated this experiment with increased gel loading of CSP(1-136) and with longer exposure of 3H signals (Figure 3C, left panel). When the loading of CSP(1-136) was increased, a very minor (5% of total) higher molecular weight pool of this mutant became visible on immunoblots (arrowhead). Figure 3C (right panel) shows that this higher molecular weight band was modified with [3H]palmitate (arrowhead). In contrast, the major lower molecular weight band (95% of the total protein, denoted by an asterisk) was completely devoid of 3H signal. Note that in several experiments of this type that were performed, the 3H signal always overlayed exactly with the very minor, higher molecular weight pool of CSP(1-136) and was completely distinct from the major CSP(1-136) band (for additional data, see Figure 6B). This experiment demonstrates that CSP(1-136) is not palmitoylated, except for a very minor pool of the protein that is only detected when gel loadings of immunoprecipitated samples are increased. Thus, the major (lower molecular weight) pool of CSP(1-136) associates with membranes in the absence of palmitoylation.
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), protein distribution was examined by immunofluorescence. For this, PC12 cells transfected with EGFP-CSP(1-136) and EGFP-CSP(106-136) were costained for endogenous CSP, using an antibody that recognizes the extreme C-terminus, and examined by confocal imaging. Figure 3D shows that endogenous CSP in PC12 cells has a punctuate (vesicular) distribution with some enrichment at the plasma membrane. However, the localization of the EGFP-CSP(1-136) and EGFP-CSP(106-136) mutants was clearly distinct to that of endogenous CSP, suggesting that these mutants are not localized to the same membrane compartments as full-length CSP (in addition, compare with distribution of full-length EGFP-CSP shown in Figure 3F). Instead, EGFP-CSP(1-136) was found to share significant overlap with calreticulin (Figure 3E), a component of the ER. The presence of CSP(1-136) at the ER was further confirmed by its colocalization with DsRed2-ER, a red fluorescent protein fused to an ER-targeting sequence (Figure 3F, middle panel). The CSP(106-136) also showed significant overlap with DsRed-ER (Figure 3F, bottom panel), whereas transfected EGFP-CSP showed no overlap with the ER marker and was present at the plasma membrane, similar to the staining pattern of endogenous CSP (Figure 3F, top panel, left, compare with endogenous CSP staining in Figure 3D). The results presented in this section show that although EGFP-CSP(1-136) and EGFP-CSP(106-136) associate tightly with cell membranes, these mutant proteins are not palmitoylated and are mislocalized, showing significant overlap with ER marker proteins.
Residues Downstream of the Cysteine-String Domain Are Required for Palmitoylation and Correct Sorting of CSP
Because the major pool of the CSP(1-136) mutant protein is membrane-bound but not palmitoylated, we investigated whether regions in the C-terminal domain of CSP are required for palmitoylation. As an initial step to examine this, CSP(1-146), CSP(1-156), and CSP(1-166) truncation mutants were constructed. PC12 cells transfected with these constructs were separated into membrane and cytosolic fractions, and protein distribution was determined by immunoblotting. Efficient palmitoylation of the mutant proteins was determined by the presence of a more slowly migrating immunoreactive band in the membrane fraction compared with the cytosolic fraction; by this criterion, the CSP(1-146), CSP(1-156), and CSP(1-166) mutants were all efficiently palmitoylated, in contrast to the CSP(1-136) mutant (Figure 4A, top panel). Thus, residues between amino acids 136-146 of CSP are required for efficient palmitoylation of the cysteine-string domain. To examine this in more detail, we constructed the following mutants: CSP(1-137), CSP(1-138), CSP(1-139), CSP(1-140), CSP(1-141), CSP(1-142), CSP(1-143), CSP(1-144), and CSP(1-145). An immunoblot showing the profile of these mutant proteins in cytosol and membrane fractions prepared from PC12 cells is shown in Figure 4A, (middle and bottom panels). There is a gradual increase in palmitoylation (indicated by a band-shift in membrane-bound CSP) as the number of residues downstream of the cysteine-string domain is increased, with the membrane-bound fraction of CSP showing an almost complete shift to a slower migrating band in the CSP(1-146) mutant. In addition to enhancing palmitoylation of CSP, the addition of extra amino acids to the C-terminus of the cysteine-string domain also increased the soluble pool of protein (Figure 4A). The reason for this is not clear, but may be a consequence of an altered membrane orientation of the cysteine-string domain that we speculate is important for palmitoylation (see below).
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To investigate the mechanism whereby addition of downstream residues facilitates palmitoylation of the cysteine-string domain, we made a series of point mutations in full-length EGFP-CSP. The following mutants were transfected into PC12 cells, and their cytosol/membrane localization examined by immunoblotting: K137A, P138A, K139A, A140L, P141A, E142A, and G143A. Interestingly, mutation of lysine 137 to alanine (K137A) was found to significantly reduce membrane binding and palmitoylation of CSP (Figure 4C), implying that this residue is important for efficient membrane binding and/or palmitoylation of the cysteine-string domain. To further investigate the role of lysine 137 in membrane binding/palmitoylation, we constructed additional point mutants at this site. Lysine-to-arginine (K137R) and lysine-to-glutamic acid (K137E) mutants were also assessed for membrane binding and palmitoylation. Interestingly, both of these mutations were tolerated far better than the K137A mutation and displayed membrane binding and palmitoylation at wild-type levels (Figure 4D). On the basis of these results, we propose that lysine 137 is important for the correct membrane orientation of the cysteine-string domain, rather than being a ligand for a PAT. This region of the protein may require to be localized at the surface of the membrane to allow efficient palmitoylation of the cysteine-string domain; this extra-membrane position would presumably be satisfied by lysine, arginine, or glutamic acid residues, whereas alanine may be more embedded in the lipid bilayer.
Analysis of the Role of Specific Cysteine Residues in Palmitoylation and Sorting of CSP
Our finding that membrane association of CSP can occur in the absence of efficient palmitoylation appears at odds with a previous observation showing that mutating seven of the cysteine residues in the cysteine-string domain to serines abolished palmitoylation and membrane binding (Chamberlain and Burgoyne, 1998a). To better define the relationship between specific cysteine residues and palmitoylation/membrane binding of CSP, we performed a more detailed mutational analysis of the cysteine-string domain. Figure 5A shows a time course of hydroxylamine-induced depalmitoylation of endogenous CSP; note that several immunoreactive bands can be detected, consistent with the notion that CSP is multiply palmitoylated (Gundersen et al., 1994; Van de Goor and Kelly, 1996). The cysteine-string region of CSP contains 14 cysteine residues, which we designate 1-14 (see Figure 1A). In a first series of experiments, we constructed the following cysteine-to-serine mutants in EGFP-CSP: C1-3S, C4-7S, C8-10S, and C11-14S. These constructs were transfected into PC12 cells, and their distribution in recovered cytosol and membrane fractions was examined by immunoblotting. Figure 5B shows that although C8-10S and C11-14S retain efficient membrane binding, the C1-3S and C4-7S mutants were largely recovered in the cytosol. Furthermore, the C8-10S and C11-14S mutants exhibited a significant band-shift upon HA-induced depalmitoylation, indicating that these mutants are modified by palmitoylation (Figure 5C). Note that the HA-induced band-shift in the C8-10S and C11-14S mutants is smaller than for wild-type CSP, suggesting that cysteines 8-14 are likely palmitoylated in wild-type CSP. In addition to being dispensable for membrane binding, cysteines 8-14 were not required for membrane sorting of CSP, because the C8-10S and C11-14S mutants showed a strong enrichment at the PM, similar to wild-type EGFP-CSP (Figure 5D). In contrast, C1-3S and C4-7S mutants were dispersed throughout the cytosol of PC12 cells, consistent with their lack of membrane binding (Figure 5D). These results demonstrate that although CSP is extensively palmitoylated, cysteines in the C-terminal half of the cysteine-string domain are dispensable for membrane binding, global palmitoylation, and intracellular sorting of CSP.
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). Because serine is a polar amino acid, we also generated mutants in which the cysteines were replaced with alanines. Interestingly, in contrast to the C1-3S mutant, a C1-3A mutant associated with cell membranes and was robustly palmitoylated, as indicated by a band-shift in the membrane-bound pool of this mutant protein (Figure 6A, left panel). This result shows that palmitoylation of cysteines 1-3 is not required for membrane association, but that polar residues introduced at these positions inhibit membrane binding. We found that the replacement of any single cysteine residue with serine in the C1-3 region preserved membrane association, whereas any combination of two cysteine-to-serine mutations in this region reduced membrane association (unpublished data). In contrast to C1-3A, a C4-7A mutant behaved essentially the same as C4-7S and was only weakly associated with membranes (Figure 6A, right panel). However, although alanine is more hydrophobic than serine, it is still less hydrophobic than cysteine. Therefore, we also replaced the cysteines at positions 4-7 with leucine (C4-7L), a strong hydrophobic amino acid; in contrast to C4-7S and C4-7A mutants, a large fraction of C4-7L associated with cell membranes (Figure 6A, right panel), again emphasizing that the hydrophobicity of cysteine residues in the N-terminal half of the cysteine-string domain is likely to play a key role in membrane binding.
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As before, we sought to confirm this observation using [3H]palmitate-labeling experiments. For this, C4-7L was compared with wild-type CSP, CSP(1-136), and C1-3A. As shown in Figure 6B, the major band detected for the C4-7L mutant (asterisk) does not incorporate the 3H label, whereas a very minor (5%) higher molecular weight pool of the protein is clearly modified with [3H]palmitate (Figure 6B, right panel, arrowheads). In several experiments, the 3H signal detected for the C4-7L mutant always overlayed exactly with the minor higher molecular weight band denoted by the arrowheads. Thus, the major pool of membrane-bound C4-7L mutant is unpalmitoylated. In addition, we also examined palmitoylation of the C1-3A mutant to ensure that the HA-dependent migration of other mutant proteins examined in this study gave a clear indication of their palmitoylation status. As expected, the labeling pattern of C1-3A was similar to that of EGFP-CSP, demonstrating that this protein is palmitoylated in the absence of the first three cysteines in the string domain. For both C1-3A and wild-type CSP, note that the lower molecular weight band incorporating the 3H label is clearly distinct from the nonpalmitoylated pool of this protein (asterisk) and likely represents a degraded form of palmitoylated CSP that was visible after longer exposure of the immunoblot.
Having shown that mutation of cysteines at positions 8-10 and 11-14 could be tolerated without affecting intracellular sorting of CSP, we next examined the intracellular distribution of the C1-3A and C4-7L mutants (i.e., the mutants in the N-terminal half of the cysteine-string domain that were membrane-bound). Similar to the C8-10S and C11-14S mutants, the C1-3A mutant was sorted correctly, showing a strong enrichment at the plasma membrane (Figure 6C, top panel). In contrast, the C4-7L mutant was completely excluded from the plasma membrane and (similar to the unpalmitoylated CSP(106-136) and CSP(1-136) mutants) exhibited significant overlap with the ER marker, calreticulin (Figure 6C, bottom panel). Note that cells expressing CSP(C4-7L) were permeabilized with digitonin before fixation to remove the cytosolic pool of protein (see Materials and Methods; Chamberlain et al., 1996). As a control, we found that this same treatment had no effect on the plasma membrane localization of the C1-3A mutant (unpublished data). Thus, three separate mutants generated in this study, each displaying defective palmitoylation, colocalize with ER markers. This finding raises the possibility that CSP associates initially with the ER and that subsequent palmitoylation is required for ER exit and correct intracellular sorting of CSP.
Membrane Association of EGFP-CSP, CSP(1-136), and CSP(C4-7L)
To examine the mechanism of membrane binding of wild-type CSP, CSP(1-136), and CSP(C4-7L), membranes were purified from PC12 cells expressing these constructs. The membranes were incubated in HES buffer, 1 M NaCl, 0.1 M sodium carbonate, pH 11.5 (Na2CO3), or 1% Triton X-100 for 30 min, and protein release from membranes was assessed by immunoblotting. Figure 7A shows that the peripheral membrane proteins munc18-1 and 
SNAP were released to different extents from membranes treated with NaCl or sodium carbonate. In contrast, wild-type CSP, CSP(1-136), and CSP(C4-7L) were all tightly associated with membranes and were only released into the supernatant after treatment with Triton X-100. This result is consistent with the idea that initial membrane association of CSP (before palmitoylation) is mediated by hydrophobic interactions between the cysteine-string domain and the membrane.
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35 kDa protease-resistant fragment (Figure 7B, left panel). However, the CSP epitope (in the last 8 amino acids of the proteins) is completely lost after proteinase K treatment for both EGFP-CSP and C4-7L proteins (Figure 7B, middle panel). If the C-terminus of C4-7L was intraluminal, then the 113–198 domain of this protein would be inaccessible to protease, leaving a CSP immunoreactive band of 8855 Da. In four separate experiments, we never detected a low molecular weight, protease-resistant CSP fragment, implying that the C-termini of both wild-type CSP and the C4-7L mutant are efficiently cleaved by proteinase K. As a control, we also examined cleavage of an intraluminal protein, the ER chaperone calreticulin. Importantly, this protein was only cleaved in the presence of Triton X-100 (Figure 7B, right panel), confirming that proteinase K digestion experiments reliably report transmembrane topology. Overall, the results shown in Figure 7B strongly suggest that the C4-7L mutant exhibits the same membrane topology as EGFP-CSP, implying that membrane association of this mutant is achieved by a mechanism similar to that of EGFP-CSP. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cysteine-string domain may adopt a helical or beta sheet structure that aligns CSP parallel with the plane of the membrane. Charged residues at either side of this domain (including K137) may be important in ensuring the appropriate membrane orientation of CSP, facilitating efficient palmitoylation. Membrane embedding of the cysteine-string domain may facilitate an energetically favorable association of palmitic acid groups with membrane phospholipids, perhaps aligning carbonyls of palmitate with carbonyls of phospholipids.
Because unpalmitoylated mutants of CSP are only released from membranes treated with Triton X-100, the initial interaction of CSP with membranes is likely to involve direct hydrophobic interactions with membrane lipids rather than with a membrane receptor. This important observation implies that cysteine residues in the cysteine-string domain associate tightly with membranes before palmitoylation. By extension, the membrane interaction of hydrophobic cysteine-rich domains may play a more general role in initial membrane binding of palmitoylated proteins. Of interest, El-Husseini et al. (2000) identified a hydrophobic sequence, 3Cys-Leu-5Cys-Ile-Val, required for palmitoylation and correct targeting of PSD-95; the cysteines at position 3 and 5 of this sequence are both sites for palmitoylation. Mutation of either cysteine at position 3 or 5 of this sequence to serine almost abolished total palmitoylation of PSD-95; however, single leucine mutations (particularly at position 5) allowed robust incorporation of palmitate (Topinka and Bredt, 1998; El-Husseini et al., 2000). Furthermore, the same study showed that the overall hydrophobic character of the sequence Met-Leu-Cys-Cys-Met was essential for the palmitoylation of GAP-43. These results were interpreted to suggest that the PSD-95 and GAP-43 PATs may specifically recognize these hydrophobic sequences. However, based on the results of our study, it is possible that this sequence also plays an important role in membrane binding (independently of PAT) before palmitoylation. Thus, hydrophobic cysteine-rich domains may represent a common motif that directly associate with membranes before palmitoylation. A caveat is that inhibition of PSD-95 palmitoylation increases the soluble pool of this protein (El-Husseini et al., 2002), suggesting that the membrane interaction of this protein in the absence of palmitoylation is weaker than observed for CSP.
The proposed role of palmitoylation in regulating CSP traffic out of the ER is similar to the previously reported palmitoylation-dependent sorting of Ras proteins from the ER/Golgi to the PM (Apolloni et al., 2000). However, it is not clear how palmitoylation facilitates sorting to post-ER compartments. Palmitoylation has been proposed to increase the affinity of proteins for specific subdomains of the plasma membrane (Melkonian et al., 1999). By analogy, palmitoylation may also promote the lateral segregation of proteins at the ER into domains that regulate vesicle budding. This could occur, for example, if palmitoylation increased the affinity of CSPs for a specific membrane geometry such as that occurring at ER exit sites. Alternatively, because palmitoylation has previously been suggested to regulate protein–protein interactions (e.g., Washbourne et al., 2000), palmitoylation of CSP may enhance its binding to an ER-localized sorting chaperone.
Palmitoylation-dependent membrane sorting may allow CSP to function in diverse cellular pathways. The distinct intracellular localization of palmitoylated CSP (vesicles/PM) is likely required for its function in exocytosis pathways, whereas the ER localization of unpalmitoylated CSP fits better with its function as a CFTR chaperone. Thus, there may be an active mechanism to ensure that a small pool of CSP is retained at the ER; this could be achieved through specific protein–protein interactions or alternatively by regulation of CSP palmitoylation. It will be interesting to determine if the small pool of CSP detected in the ER of Calu-3 cells is palmitoylated or not.
Interestingly, Gundersen's group (Mastrogiacomo et al., 1998) provided evidence that the cysteine-string domain is embedded in the bilayer after chemically induced depalmitoylation; however, it was suggested that it was hydroxylamine treatment that promoted a nonphysiological membrane interaction of the cysteine-string region. Although the work presented in the present study supports the results of Mastrogiacomo et al., our analysis clearly implies that membrane insertion of the cysteine-string domain of CSP is physiological and precedes palmitoylation. Interestingly, intermediate immunoreactive bands corresponding to partially palmitoylated CSP are never detected by SDS-PAGE (this study; Gundersen et al., 1994). Similarly, membrane-associated, unpalmitoylated protein has not consistently been detected for wild-type CSP. These points imply that the full palmitoylation of CSP is a rapid event, is likely enzyme-mediated and occurs in the same compartment (ER) to which CSP initially binds. It is interesting to note however that Drosophila CSP does not associate with membranes and is not palmitoylated when expressed in PC12 cells (Van de Goor and Kelly, 1996). This finding may suggest that membrane integration of the cysteine-string domain requires a specific chaperone factor that is not conserved between Drosophila and mammalian CSPs.
Another interesting point to emerge from this study is that specific cysteine residues in the cysteine-string domain can be mutated without effect on membrane sorting. This suggests that the extensive palmitoylation of CSP is not required for either membrane binding or intracellular sorting. Although extensive palmitoylation of CSP may be important to maintain the appropriate membrane orientation of the protein, it is also possible that this multiple palmitoylation is important for some other aspect of CSP function.
In conclusion, this study has revealed an important dual function of the cysteine-string domain in initial membrane binding and subsequent palmitoylation-dependent sorting of CSP. Future studies of palmitoylation mechanisms will determine if a similar mode of membrane binding and sorting operates for other palmitoylated proteins.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: L. Chamberlain (l.chamberlain{at}bio.gla.ac.uk)
Abbreviations used: CSP, cysteine-string protein; CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum; PAT, palmitoyl transferase
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