SNAP-25 palmitoylation and plasma membrane targeting require a functional secretory pathway

Abstract

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.

Figure 1

Palmitoylation of SNAP-25 causes a mobility shift of the protein on SDS-PAGE. PC12 cells were labeled with [³⁵S]methionine or [³H]palmitate for 1 h. SNAP-25 was immunoprecipitated with an affinity-purified polyclonal antibody directed against a C-terminal peptide of SNAP-25. The incorporation of radiolabel was analyzed by SDS-PAGE and fluorography. Incorporation of [³H]palmitate into SNAP-25 was sensitive to treatment of the gels with 1 M hydroxylamine (pH 7.5) after SDS-PAGE, but insensitive to treatment with 1 M Tris (middle and left panels). [³⁵S]methionine incorporation into SNAP-25 was unaffected by soaking the gel in hydroxylamine or Tris (lanes 1 and 3). Treatment of [³⁵S]methionine-labeled SNAP-25 immunoprecipitates with 1 M hydroxylamine before SDS-PAGE removed palmitate, and the protein migrated as a single band (right panel).

Figure 2

Maturation of SNAP-25 into the palmitoylated form occurs within 20 min of its synthesis. PC12 cells were metabolically labeled with [³⁵S]methionine for various times. Cell lysates were either collected immediately (lanes 1–5) or collected following a chase period of 2 h (lane 6). SNAP-25 was immunoprecipitated and the state of palmitoylation was assessed by the mobility of the protein on SDS-PAGE.

Figure 3

Only the palmitoylated form of SNAP-25 associates with membranes. PC12 cells were metabolically labeled with [³⁵S]methionine or [³H]palmitate for 1 h, followed by homogenization and separation into particulate and soluble fractions by high-speed centrifugation. SNAP-25 was immunoprecipitated from the total cell lysate (TCL), particulate fractions (P100), and soluble fractions (S100). The presence of radiolabeled SNAP-25 in each of the fractions was evaluated by SDS-PAGE and fluorography.

Figure 4

Treatment of PC12 cells with BFA results in inhibition of palmitoylation and membrane association of newly synthesized SNAP-25. PC12 cells were labeled with [³⁵S]methionine or [³H]palmitate for 1 h in the presence or absence of 10 μg/ml BFA. (A) Radiolabeled SNAP-25 was immunoprecipitated and analyzed by SDS-PAGE and fluorography. (B) [³⁵S]methionine-labeled cells were homogenized and fractionated into particulate (P100) and soluble (S100) fractions. The presence of SNAP-25 in each of the fractions as well as in the total cell lysate (TCL) was detected by immunoprecipitation followed by SDS-PAGE and fluorography.

Figure 5

Effect of transport inhibitors on palmitoylation of SNAP-25. (A) PC12 cells were labeled with [³H]palmitate or [³⁵S]methionine for 1 h in normal media (control) or in media containing 10 μg/ml BFA, 10 μM monensin, 20 μg/ml nocodazole, nocodazole and BFA together, or 50 mM NH₄Cl. Radiolabeled SNAP-25 was immunoprecipitated from cell lysates. The effect of these drugs on palmitoylation of SNAP-25 was assessed by incorporation of [³H]palmitate and by analysis of the mobility of the [³⁵S]methionine-labeled protein on SDS-PAGE. (B) PC12 cells were pulse labeled for 20 min with [³⁵S]methionine at 37°C and then chased without label for 0.5, 1, or 2 h at 37°C, 20°C, or 15°C. Radiolabeled SNAP-25 was immunoprecipitated and analyzed by SDS-PAGE and fluorography. Note the inhibition of maturation of SNAP-25 at 15 and 20°C.

Figure 6

Immunofluorescence of PC12 cells showing the subcellular distribution of SNAP-25 after treatment with BFA. PC12 cells cultured in four-chamber slides were differentiated with NGF for 2 d and subjected to the following treatments: A, DMSO for 1 h; B, BFA for 1 h; C, DMSO for 1 h; D, cycloheximide for 4 h; E, BFA for 4 h; and F, cycloheximide and BFA for 4 h. The cells were then processed for immunofluorescence and examined by confocal microscopy. SNAP-25 immunostaining was specific, as demonstrated by the lack of labeling in the presence of the SNAP-25 peptide (C) during the incubation with the primary antibody. The staining was unaffected by the presence of an unrelated peptide (A). SNAP-25 was localized to the plasma membrane in cells incubated in the presence (B) or absence (A) of BFA. A 4-h treatment of the cells with BFA (E) resulted in an accumulation of intracellular SNAP-25 when compared with control cells (D). The intracellular accumulation was also apparent when both protein synthesis and transport were inhibited (F).

Figure 7

BFA and monensin do not inhibit palmitoylation of all cellular proteins. (A) PC12 cells were labeled with [³H]palmitate for 1 h in the absence (lane 1) or presence of 10 μg/ml BFA (lane 2) or 10 μM monensin (lane 3). Cell lysates were resolved by SDS-PAGE, and the incorporation of [³H]palmitate into proteins was detected by fluorography. (B) N2A cells were labeled with [³⁵S]methionine or [³H]palmitate for 1 h in the absence or presence of BFA. G_oα and G_iα subunits labeled with [³⁵S]methionine (lanes 1 and 2) or [³H]palmitate (lanes 5 and 6) were immunoprecipitated. Note how palmitoylation of G protein α subunits is not affected by treatment with BFA. SNAP-25 labeled with [³⁵S]methionine (lanes 3 and 4) or [³H]palmitate (lanes 7 and 8) was immunoprecipitated. Inhibition of SNAP-25 palmitoylation by BFA is apparent from reduced labeling with [³H]palmitate (lane 8). (C) NG108 cells were labeled with [³H]palmitate for 1 h in the presence or absence of BFA, and SNAP-25 (lanes 3 and 4) and GAP-43 (lanes 1 and 2) were immunoprecipitated from total cell lysates. Equal amounts of protein in each lane was demonstrated by immunoblotting an aliquot of the immunoprecipitated samples (bottom panel). Palmitoylation of both GAP-43 and SNAP-25 were inhibited by BFA in NG108 cells.

Figure 8

Depalmitoylation of SNAP-25 does not release the protein from membranes. (A) PC12 cells were labeled with [³H]palmitate or [³⁵S]methionine for 90 min and fractionated into membranes and cytosol. Membranes (Mb) or Cytosol (Cy) or the combined fractions (Mb + Cy) were incubated in 0.5 M hydroxylamine (HA) to remove palmitate or 0.5 M Tris as a control. Each sample was then subjected to a high-speed centrifugation to separate a particulate (P) from a soluble fraction (S). SNAP-25 was immunoprecipitated from each of the samples and analyzed by SDS-PAGE and fluorography. Depalmitoylation of the protein was assessed by loss of [³H]palmitate labeling (upper panel) and by faster mobility of [³⁵S]methionine-labeled protein in SDS-PAGE (lower panel). (B) Brain membranes were treated with hydroxylamine or Tris. A sucrose step gradient was used to separate membranes from soluble proteins. SNAP-25 in membranes (lanes 1 and 2) or soluble fractions (lanes 3 and 4) was detected by immunoblotting. Note the presence of depalmitoylated SNAP-25 in the membrane fraction (lane 2). (C) SNAP-25 was immunoprecipitated from brain membranes treated with hydroxylamine or Tris. Co-immunoprecipitating syntaxin was detected by blotting the SNAP-25 immunoprecipitates with antibodies against syntaxin.