Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

Abstract

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.

Figure 1.

Munc13-4 reconstitutes Ca²⁺-dependent granule exocytosis in multiple cell types. (A) Coomassie-stained SDS gels of wild-type (WT) and C2A*, C2B*, and C2A*B* Munc13-4 proteins. A minor degradation fragment was present in some preparations. Molecular mass is indicated in kilodaltons. (B) A schematic representation of the Munc13-4 sequence showing central DUF1041 and MHD1 homology domains present in all CAPS/Munc13 proteins and the MHD2 homology domain conserved in Munc13 proteins. A pileup of sequences from the β1-β2 regions of the C2A and C2B domains of hMunc13-4 and hSynaptotagmin-1 is shown with the conserved aspartate residues in boxes. Positions of the asparagine substitutions in C2A (D127N and D133N) and in C2B (D941N and D947N) are indicated. (C) Ca²⁺ binding to Munc13-4 measured by changes of intrinsic tryptophan fluorescence. 2 µM wild-type, C2A*, C2B*, and C2A*B* Munc13-4 proteins were excited at 280 nm, and emission spectra were collected from 290–390 nm. Normalized plots of the percentage change in fluorescence intensity are shown. These indicated an ∼5 µM [Ca²⁺]_1/2 for wild-type Munc13-4, whereas [Ca²⁺]_1/2 values for C2A*, C2B*, and C2A*B* Munc13-4 proteins exceeded 300 µM. Mean ± SD values are shown (n = 3). (D) [³H]serotonin release (as a percentage of the total) from preloaded permeable platelets is shown in the absence or presence of 10 µM Ca²⁺ at the indicated concentrations of added wild-type or mutant Munc13-4 proteins. The dotted line shows release in the absence of added Munc13-4. Results are representative of three similar experiments. (E) Release of ANF-GFP (as a percentage of the total) from mechanically permeabilized RBL-2H3 cells is shown for incubations without or with 10 µM Ca²⁺, 0.5 mg/ml cytosol, or 10 nM Munc13-4 or C2 domain mutants. Munc13-4 addition without Ca²⁺ was not stimulatory. Results shown are the mean ± SD for triplicate determinations in a single experiment. The inset shows 120-kD Munc13-4 extracted from intact or permeabilized washed RBL-2H3 cells analyzed by SDS-PAGE and immunoblotting. (F) Release of norepinephrine from mechanically permeabilized PC12 cells detected by voltammetry. Incubations at 35°C were conducted in the presence of 20 nM CAPS, 100 nM Munc13-4, or Munc13-4 mutants, as indicated. No release occurs until Ca²⁺ (to ∼1 µM) was injected (at time 0). Each curve represents a single sample. Results are representative of three similar experiments.

Figure 2.

Munc13-4 binds to the H3 region of syntaxins. (A) Bound and free Munc13-4 were determined by buoyant density flotation of liposomes. (B) 1 µM Munc13-4 was incubated with PC/PS liposomes containing syntaxin-1, -2, -3, or -4 (left) or protein-free PC/PS liposomes (right) for 30 min at room temperature. Bound (fraction 2) and free (fractions 6–8) liposomes were separated on Accudenz gradients and analyzed for Munc13-4 by immunoblotting. Results are representative of two similar experiments. Densitometry with ImageQuant software indicated that bound/free liposomes corresponded to 0.11, 0.06, 0.03, and 0.17 for syntaxin-1, -2, -3, and -4, respectively. (C) Syntaxin-1 contains Habc(28–144), SNARE motif/H3(191–258), and transmembrane(266–288) (TM)domains. (D) Surface plasmon resonance experiments of syntaxin-1 fragments (1–266, 1–177, and 191–266) binding to Munc13-4. Specific binding to Munc13-4 was determined by subtracting background binding to the control MBP. Mean values ± SEM are shown (n = 3). (E) Surface plasmon resonance experiments of the H3 domains of syntaxin-1, -3, -4, -5, -6, and -11. Specific binding to Munc13-4 was determined by subtracting background binding to the control MBP. Results are representative of two similar experiments. (F) 1 µM Munc13-4 was incubated with syntaxin-1–containing liposomes composed of PC/PS/PIP₂ (85:12:3) in the presence of EGTA or 100 µM free Ca²⁺. Accudenz gradient fractions were analyzed by SDS-PAGE and stained with SYPRO ruby.

Figure 3.

Ca²⁺-regulated Munc13-4 binding to t-SNAREs and PS requires C2A and C2B domains, respectively. (A–D) All binding experiments were conducted by incubating 1 µM Munc13-4 with the indicated liposomes at room temperature for 30 min with EGTA or Ca²⁺ followed by separation of bound and free protein by buoyant density centrifugation conducted in the absence or presence of Ca²⁺. Bound fractions were analyzed by SDS-PAGE, stained with SYPRO ruby, and quantified by densitometry. Munc13-4 migrated between 100- and 150-kD standards. Syntaxin-1 and SNAP-25 migrated between 25- and 37-kD standards. The extent of binding (right graphs) is shown as means ± SEM for n = 3. (A) Munc13-4 binding to t-SNARE–containing or protein-free PC/PS liposomes in the presence of EGTA or 100 µM Ca²⁺. (B) Munc13-4 binding to t-SNARE–containing or protein-free PC liposomes in the presence of EGTA or 100 µM Ca²⁺. (C) Binding of wild-type (WT), D127N/D133N, D941N/D947N, and D127N/D133N/D941N/D947N Munc13-4 proteins (termed WT, C2A*, C2B*, and C2A*B* Munc13-4, respectively) to PC/PS liposomes in the presence of EGTA or 100 µM Ca²⁺. Binding of WT and C2A* Munc13-4 to PC/PS liposomes in the presence of Ca²⁺ was not significantly different. (D) Binding of wild-type, C2A*, C2B*, and C2A*B* Munc13-4 proteins to t-SNARE–containing PC liposomes. Munc13-4 binding was normalized to syntaxin-1 (Stx1) content of liposomes. Ca²⁺-stimulated binding of C2A* and C2A*B* Munc13-4 proteins was significantly reduced compared with wild type (*, P < 0.05; **, P < 0.002), whereas binding by C2B* Munc13-4 was not significantly different based on an unpaired t test.

Figure 4.

Munc13-4 promotes liposome fusion in a calcium-dependent manner. (A) VAMP-2–containing PC/PS donor liposomes (with NBD-PE and Rh-PE) and t-SNARE–containing PC/PS acceptor liposomes were incubated with 1 µM Munc13-4 in the presence of EGTA or 400 µM free calcium (+Ca²⁺), as indicated. Control reactions with protein-free (Pf) liposomes replacing t-SNARE liposomes were incubated in parallel for background subtraction. Fusion was detected as increased NBD-PE fluorescence relative to maximal dequenched values. (B) Incubations similar to those of A were conducted at 100 µM Ca²⁺ at the indicated Munc13-4 concentrations (left), and initial fusion rates were determined (right). v + t, reactions with v-SNARE–containing liposomes plus t-SNARE–containing liposomes. (C) Incubations similar to those of A were conducted with 1 µM Munc13-4 at the indicated free Ca²⁺ concentrations (left), and initial fusion rates were determined (right). The EC₅₀ for Ca²⁺ was calculated to be 23 ± 4 µM. In all graphs, the curves represent single samples. Results shown are representative of two to four similar experiments.

Figure 5.

Munc13-4 utilizes N-terminally truncated syntaxin-1 and requires Ca²⁺-binding C2 domains in SNARE-dependent liposome fusion. (A) Incubations were conducted in the absence or presence of 1 µM Munc13-4 and 400 µM Ca²⁺, as indicated with DiI-containing VAMP-2 donor liposomes and DiD-containing acceptor liposomes that contained either syntaxin-1(191–288) with SNAP-25 (H3/SNAP-25) or full-length syntaxin-1(1–288) with SNAP-25 (t-SNARE). Fusion was detected as increased DiD fluorescence expressed relative to the lowest fluorescence as a ratio (f/f_o). Curves show individual samples. Results are representative of three similar experiments. (B) 1 µM wild-type (WT), C2A*, C2B*, or C2A*B* Munc13-4 proteins were incubated with donor v-SNARE liposomes (containing NBD-PE and Rh-PE) and acceptor t-SNARE liposomes, as in Fig. 4 A, with 100 µM Ca²⁺. Fusion was detected as increased NBD-PE fluorescence relative to maximal dequenched values. Curves show individual samples. Results are representative of two similar experiments. v + t, reactions with v-SNARE–containing liposomes plus t-SNARE–containing liposomes.

Figure 6.

Munc13-4 stimulation of trans-SNARE complex formation is Ca²⁺ dependent. (A) Munc13-4 stimulates the formation of SDS-resistant SNARE complexes. Incubations with VAMP-2– and syntaxin-1/SNAP-25–containing liposomes were conducted on ice for 30 min with 100 µM Ca²⁺ and 1 µM Munc13-4, as indicated. Reactions terminated in SDS sample buffer without boiling were analyzed by immunoblotting with monoclonal HPC-1 syntaxin-1 antibody. The arrows indicate the positions of ∼70-kD, ∼120-kD, and ∼165-kD SNARE complexes. Similar experiments indicated that VAMP-2 and SNAP-25 comigrated with these complexes. See James et al. (2009) for characterization of SNARE protein complexes. (B) Munc13-4 stimulates the docking of VAMP-2 liposomes onto t-SNARE–containing supported bilayer membranes. VAMP-2 liposomes with Rh-PE were incubated with t-SNARE–containing supported bilayers, and the number of liposomes stably docked for ≥2 min was monitored by total internal reflection fluorescence microscopy. Bar, 5 µm. Incubations contained 1 µM Munc13-4, 200 µM Ca²⁺, or 200 µM Mg²⁺, as indicated. Munc13-4 promoted liposome docking in the presence of Ca²⁺ (b) but not in its absence (c) or with Mg²⁺ (f), nor was liposome docking promoted by Ca²⁺ alone (a). t-SNAREs were required for Munc13-4–promoted docking (d), whereas preincubations of t-SNARE bilayers with 8 µM soluble syntaxin(1–266) (sol. syx.) for 30 min (e) inhibited Munc13-4–promoted docking. Mean values ± SD (n = 3) are shown.