Palmitoylation of the SNAP25 protein family: specificity and regulation by DHHC palmitoyl transferases

Abstract

SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b. Despite the conspicuous differences within the palmitoylated domains of these secretory proteins, there is no information on their comparative interactions with palmitoyl transferases. We report that membrane association of all SNAP25/23 proteins is enhanced by Golgi-localized DHHC3, DHHC7, and DHHC17. In contrast, DHHC15 promoted a statistically significant increase in membrane association of only SNAP25b. To investigate the underlying cause of this differential specificity, we examined a SNAP23 point mutant (C79F) designed to mimic the cysteine-rich domain of SNAP25b. DHHC15 promoted a marked increase in membrane binding and palmitoylation of this SNAP23 mutant, demonstrating that the distinct cysteine-rich domains of SNAP25/23 contribute to differential interactions with DHHC15. The lack of activity of DHHC15 toward wild-type SNAP23 was not overcome by replacing its DHHC domain with that from DHHC3, suggesting that substrate specificity is not determined by the DHHC domain alone. Interestingly, DHHC2, which is closely related to DHHC15, associates with the plasma membrane in PC12 cells and can palmitoylate all SNAP25 isoforms. DHHC2 is, thus, a candidate enzyme to regulate SNAP25/23 palmitoylation dynamics at the plasma membrane. Finally, we demonstrate that overexpression of specific Golgi-localized DHHC proteins active against SNAP25/23 proteins perturbs the normal secretion of human growth hormone from PC12 cells.

FIGURE 1.

Minimal membrane targeting domain of SNAP25b. A, SNAP25b contains a minimal membrane targeting sequence that is located between amino acids 85 and 120. Palmitoylated cysteines in this domain are shown in bold and underlined. The QPARV motif, which is underlined with a dashed line is important for DHHC interactions of SNAP25b. B, comparison is shown of the cysteine-rich domains of SNAP25a, SNAP25b, and SNAP23. The QPARV motif in SNAP25b is conserved in SNAP25a, whereas mouse SNAP23 contains the sequence QPSRI. Amino acid numbers are shown.

FIGURE 2.

Regulation of SNAP25/23 membrane binding by DHHC proteins. HEK293T cells were co-transfected with EGFP-tagged versions of SNAP25a, SNAP25b, or SNAP23 together with HA-tagged constructs encoding each of the 23 murine DHHC constructs (or empty HA vector as a control). Twenty-four hours post-transfection, the cells were fractionated into cytosol (C) and membrane (M) fractions, which were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis with anti-GFP. The left panel of the figure shows representative immunoblots (top, SNAP25a; middle, SNAP25b; bottom, SNAP23). The numbering corresponds to the specific DHHC protein that was co-transfected with SNAP25/23. The right panel shows averaged data and S.E. (n = 3) for the increase in % membrane association of the SNAP25/23 proteins in the presence of each of the DHHCs. For clarity, the basal level of membrane association in the absence of DHHC co-expression was subtracted for each condition. The data were analyzed using a one-way ANOVA, which revealed significant increases in membrane association with DHHC3/7/17 for each SNAP25/23 protein and for DHHC15 specifically with SNAP25b when compared with transfections in the absence of DHHCs (*, p < 0.05).

FIGURE 3.

A point mutation in SNAP23 enhances interaction with DHHC15. HEK293T cells were co-transfected with EGFP-tagged SNAP23 or a SNAP23(C79F) mutant together with HA-tagged DHHC15 or empty vector (control). Twenty-four hours post-transfection the cells were fractionated into cytosol (C) and membrane (M) fractions, which were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis with anti-GFP and anti-HA. A, representative immunoblots are shown. B, averaged data and S.E. (n = 4) for the increase in % membrane association of the SNAP23 proteins in the presence of DHHC15 are shown. ** indicates a statistically significant increase in membrane binding (p < 0.01, Student's t test) compared with wild-type SNAP23. C, HEK293T cells were transfected with EGFP-SNAP23 wild-type or C79F mutant together with pEFBOS-HA (control) or HA-DHHC15. Cells were labeled with [³H]palmitic acid, lysed, and resolved by SDS-PAGE. The top panel shows [³H]palmitate incorporation, the middle panel shows expression levels of EGFP-SNAP23, and the bottom panel shows HA-DHHC15 expression. Molecular weight markers are shown on the left of all figure panels.

FIGURE 4.

A DHHC15 protein containing the DHHC-CR domain from DHHC3 is inactive against SNAP23. A, schematic highlighting the make-up of the DHHC15/3 chimeric protein is shown. B, HEK293T cells were transfected with EGFP-SNAP23 (or GFP) together with pEFBOS-HA (control), HA-DHHC3, HA-DHHC15, or HA-DHHC15/3. Cells were labeled with [³H]palmitic acid acid, lysed, and resolved by SDS-PAGE. The top panel shows [³H]palmitate incorporation, the middle panel shows expression levels of EGFP-SNAP23, and the bottom panel shows DHHC protein expression. Molecular weight markers are shown on the left. C, HA-DHHC15/3 was expressed in HEK293T cells, labeled with Alexa Fluor 488-conjugated anti-HA, and visualized using confocal microscopy. The scale bar represents 5 μm.

FIGURE 5.

A conserved motif downstream of the cysteine-rich domain is important for membrane targeting of all SNAP25/23 proteins. A, comparison is shown of the cysteine-rich domains and downstream QPARV motif in SNAP25/23. B, analysis of the importance of the PARV/PSRI sequences in SNAP25/23 for membrane binding is shown. Wild-type (wt) and mutant EGFP-tagged SNAP25/23 proteins were transfected into PC12 cells before cell fractionation into cytosol (C) and membrane (M) fractions. The recovered fractions were probed with a GFP antibody. The left panel shows representative immunoblots, whereas the right panel is averaged data (n = 4). The reduction in membrane binding of mutant proteins compared with the respective wild-type control was statistically significant for all SNAP25/23 proteins (p < 0.00002 for SNAP25b, p < 0.02 for SNAP25a, and p < 0.0005 for SNAP23, Student's t test). C, PC12 cells were co-transfected with mcherry-tagged versions of wild-type SNAP25a, SNAP25b, or SNAP23 together with EGFP-tagged proteins in which the PARV and PSRI sequences were mutated to either ALAA or AAAA, respectively. Scale bars represent 5 μm.

FIGURE 6.

Comparison of SNAP25b membrane binding and palmitoylation in the presence and absence of DHHC3 overexpression. A, HEK293T cells were transfected with EGFP-SNAP25b together with HA-DHHC3 or empty HA vector (control). Twenty-four hours post-transfection the cells were fractionated into cytosol (C) and membrane (M) fractions (top panel). Alternatively, the cells were incubated with [³H]palmitic acid for 4 h. Samples were then resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting with anti-GFP or detection of the ³H signal with the aid of an intensifier screen (bottom panel). B, -Fold increase in membrane binding and palmitoylation in the presence of DHHC3 was calculated (n = 3).

FIGURE 7.

SNAP25b is palmitoylated by DHHC2. A, a subset of DHHC proteins are localized at the plasma membrane in PC12 cells. Cells were transfected with HA-tagged or GFP-tagged DHHCs and fixed 48 h later. Localization of HA-tagged proteins was visualized by staining with an HA antibody conjugated to Alexa Fluor 488. The DHHC2(CA)-GFP protein has a cysteine-to-alanine mutation in the DHHC domain (C156A). The scale bar represents 5 μm. B, EGFP-SNAP25b was co-transfected into HEK293T cells together with each of the indicated DHHC proteins (empty HA vector was used for control). Twenty-four hours after transfection, the cells were incubated in [³H]palmitic acid for 4 h, washed, and lysed. The resulting lysates were resolved by SDS-PAGE and transferred to nitrocellulose, and the ³H signal was detected using an intensifier screen. Duplicate nitrocellulose membranes were probed with anti-GFP and anti-HA antibodies. Other bands labeled with ³H are DHHC proteins, which undergo autopalmitoylation. C, shown are mean values and S.E. for the ³H signal intensity of EGFP-SNAP25b as a fraction of the signal detected with the GFP antibody (n = 4). *** denotes a p value of <0.001 for the level of palmitoylation of EGFP-SNAP25b when co-expressed with DHHC2 compared with control, analyzed by one-way ANOVA. D, cells were transfected with EGFP-SNAP25b together with pEFBOS-HA (control), HA-DHHC2, or HA-DHHC2 (C156A). Cells were labeled with [³H]palmitic acid acid, lysed, and resolved by SDS-PAGE. The top panel shows [³H]palmitate incorporation, and the bottom panel shows expression levels of EGFP-SNAP25b. E, RT-PCR analysis of DHHC2 mRNA expression in rat brain and PC12 cells. Controls used were HA-DHHC plasmid (+ control) and no template (− control). Position of molecular weight markers are shown on the left side of all immunoblots.

FIGURE 8.

Palmitoylation of SNAP25a and SNAP23 by DHHC2. EGFP-SNAP25a and EGFP-SNAP23 were co-transfected into HEK293T cells together with HA-DHHC2 or empty vector (control). Twenty-four hours after transfection, the cells were incubated in [³H]palmitic acid for 4 h, washed, and lysed. The resulting lysates were resolved by SDS-PAGE and transferred to nitrocellulose. The ³H signal was detected using an intensifier screen, and duplicate nitrocellulose membranes were probed with anti-GFP. The top panel shows representative blots, with the molecular weight standards indicated on the left side. The bottom panel shows averaged data ± S.E. (n = 6) for palmitoylation in the presence and absence of DHHC2. The palmitoylation level in the absence of DHHC co-expression was set as 1.

FIGURE 9.

Effects of DHHC overexpression on exocytosis in PC12 cells. A, PC12 cells were co-transfected with a plasmid encoding hGH together with HA-DHHC3, DHHC7, DHHC17, or empty HA plasmid (control). Forty-eight hours post-transfection, the cells were washed and incubated in Krebs buffer in the absence (basal) and presence of 300 μm ATP. The secreted and cell-associated hGH was assayed using an enzyme-linked immunosorbent assay kit, and secretion determined as a percentage of the total cell content. Results from three independent experiments (n = 3 for each experiment) were averaged, and the means ± S.E. are presented. One-way ANOVA tests revealed that DHHC7 and DHHC17 significantly reduced ATP-stimulated secretion compared with control transfected cells. The basal secretion level for DHHC7- and DHHC17-transfected cells was significantly increased compared with control transfected cells. *, indicates a p value of < 0.05; **, denotes a p value of < 0.01; ***, is p < 0.001. B, PC12 cells transfected with the HA-DHHC constructs were lysed, separated by SDS-PAGE, and transferred to nitrocellulose for immunoblotting analysis with anti-HA. Molecular weight standards are shown on the left. C, PC12 cells transfected with HA-DHHC3, HA-DHHC7, or HA-DHHC17 were fixed and permeabilized. The cells were incubated with anti-SNAP25 (SMI81) and anti-mouse antibody conjugated to Alexa Fluor 647 to stain endogenous SNAP25 and anti-HA conjugated to Alexa Fluor 488 to stain the HA-tagged DHHC constructs. Scale bars represent 5 μm.