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.2011 Nov;22(21):4150-60.
doi: 10.1091/mbc.E11-02-0150. Epub 2011 Sep 7.

Dual roles of Munc18-1 rely on distinct binding modes of the central cavity with Stx1A and SNARE complex

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Dual roles of Munc18-1 rely on distinct binding modes of the central cavity with Stx1A and SNARE complex

Lei Shi et al. Mol Biol Cell.2011 Nov.

Abstract

Sec1/Munc18 proteins play a fundamental role in multiple steps of intracellular membrane trafficking. Dual functions have been attributed to Munc18-1: it can act as a chaperone when it interacts with monomeric syntaxin 1A, and it can activate soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) for membrane fusion when it binds to SNARE complexes. Although both modes of binding involve the central cavity of Munc18-1, their precise molecular mechanisms of action are not fully understood. In this paper, we describe a novel Munc18-1 mutant in the central cavity that showed a reduced interaction with syntaxin 1A and impaired chaperone function, but still bound to assembled SNARE complexes and promoted liposome fusion and secretion in neuroendocrine cells. Soluble syntaxin 1A H3 domain partially blocks Munc18-1 activation of liposome fusion by occupying the Munc18-1 central cavity. Our findings lead us to propose a transition model between the two distinct binding modes by which Munc18 can control and assist in SNARE-complex assembly during neurotransmitter release.

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Figures

FIGURE 1:
FIGURE 1:
Munc18-4M mutant binds poorly to Stx1A. (A) Cocrystal structure of Munc18 (green)-Stx1A (Habc domain: blue; H3 domain: purple): the C-terminal half of the Stx1A H3 domain inserts deeply into the Munc18 central cavity; the residues R39/K46/M47/K63 (highlighted in yellow) within the Munc18 central cavity make close contacts with residues D231/E234/N236/D242 (red) within the Stx1A H3 domain. (B) coIP experiments using HeLa cells transiently transfected with Stx1A and either Munc18-wt or Munc18-4M mutant (R39A/K46A/M47A/K63A). (C) coIP experiments using HeLa cells transiently transfected with Stx1A-wt or Stx1A-4M mutant and Munc18-wt. Blots are representative of three independent experiments.
FIGURE 2:
FIGURE 2:
Munc18-4M mutant cannot efficiently traffic Stx1A to the plasma membrane in PC12 Munc18-1/-2 double-knockdown cells (Hanet al., 2009). (A–C) Stx1A localized on the plasma membrane in C8 control cells, whereas Stx1A mostly localized within the secretory pathway in DKD16 knockdown cells (E–G). Reintroduction of Munc18-wt rescued the phenotype and relocalized Stx1A back onto plasma membrane (I–K). Expression of Munc18-4M mutant had no rescue function, and Stx1A was still retained inside the cell (M–O). Asterisks, transfected cells; arrows, nontransfected cells. The DsRed-N1vector was used as cotransfection marker. Fluorescence intensity distribution (D, H, L, and P) along the white line in the transfected cell (C, G, K, and O). Quantification of the fluorescence intensity distribution plots calculated as the ratio between: the mean fluorescence intensity at the borders of the cell over the fluorescence intensity at the middle distance of the borders (Q). Results are mean ± SD of two independent experiments (n = 50). (R) hGH secretion assay using C8 or DKD16 cell lines cotransfected with a plasmid coding for hGH-YFP construct and either Munc18-wt, Munc18-4M, Munc18-4M and Stx1, or without Munc18. Cells were stimulated with or without high K+ buffer for 15 min at 37°C. After cells were pelleted, the release of hGH to the medium was quantified by ELISA as percentage of the total content of hGH in each well. Results are mean ± SD of two independent experiments; each condition was run in triplicate.
FIGURE 3:
FIGURE 3:
Munc18-4M mutant can bind the SNARE complexes and promote liposome fusion. (A) Pulldown experiment to test binding of Munc18-wt or Munc18-4M mutant to immobilized His-tagged t-SNARE or SNARE complex. Munc18-wt and Munc18-4M proteins were expressed as His6x-SUMO-construct and purified using Ni-NTA beads following SUMO-protease cleavage. Briefly, for pulldown experiments, His-tagged t-SNARE or SNARE complex was immobilized on the Ni-NTA beads and incubated with either Munc18-wt or Munc18-4M. Bound fractions were analyzed by SDS–PAGE followed by Coomassie staining. Note that the SUMO protein recovered on the bound fractions corresponds to the His-SUMO-tag that copurified as contaminant of Munc18 proteins, as it is also observed in the input lanes of the gel. (B) Standard liposome fusion in the absence or presence of 5 μM Munc18-wt or Munc18-4M mutant. (D) Preassembled liposome fusion assay in which the v- and t-liposomes were preincubated for 3 h at 4°C in absence or presence of 5 μM Munc18-wt or Munc18-4M mutant; subsequently, cdv2 was added before starting the fusion reaction to prevent the assembly of newly formed SNARE complexes.
FIGURE 4:
FIGURE 4:
Stx1A H3 domain binds Munc18 and partially inhibits Munc18 activation of liposome fusion. (A) Equal amounts of either Munc18-wt or Munc18-4M were incubated with the glutathione beads in the presence or absence of GST-Stx1-H3 and in the presence or absence of increasing concentration of soluble SNARE complexes. Beads were washed, and the Munc18 bound fraction was determined by Western blotting using a polyclonal anti-Munc18 antibody. Arrows indicate the expected molecular weight of Munc18. Asterisks mark the expected molecular weight of GST-Stx1-H3 after Ponceau staining. The amount of soluble SNARE complexes added to the reaction (0.5, 1, 2, and 4) is indicated as SNARE complex/Munc18 ratio. (B) Quantification of Munc18 bound fraction in the competition experiment. Results are the mean ± SD of two independent experiments. (C) Standard and preassembled liposome fusion experiments performed in the absence or presence of 5 μM Munc18-wt alone or 5 μM Munc18 and 5 μM Stx1A H3 domain. (D) Fold of activation of the initial rate of liposome fusion reaction in a dose–response experiment. Munc18-wt (5 μM) and different concentrations of Stx1A H3 domain were coincubated with t-/v-liposomes on ice for 3 h, which was followed by the fusion assay. (E) Order of addition experiment in which standard and preassembled liposome fusion experiments were performed as described in Figure 3; in this case, Stx1A-H3 was added either during the preincubation step or after the preincubation.
FIGURE 5:
FIGURE 5:
Stx1A H3 domain inhibition of Munc18 activation depends on the binding of its C-terminal region to the Munc18 central cavity. (A) Standard liposome fusion reactions performed in the absence or presence of 5 μM Stx1A H3 domain and Munc18-wt or Munc18-4M. Both panels show the same data for the “v+t” condition, since they were run in a single experiment for multiple times. (B) Inhibition effect of Stx1A H3 domain to Munc18-wt or Munc18-4M mutant on activation of liposome fusion. The maximum initial speed (within first 20 min) of liposome fusion in the presence of 5 μM Munc18 (wt or 4M) was calculated and set as 100%; the maximum initial speed in the presence of Stx1A H3 was measured and converted to the percentage of Munc18 activation. (C) Standard liposome fusion was carried out in the absence or presence of 5 μM Munc18-wt and 5 μM of Stx H3-wt or Stx1A H3-4M mutant (D231A/E234A/N236A/D242A). (D) Inhibition of Stx1A H3-wt, but not of Stx1A 4M, onto Munc18-wt activation of liposome fusion.
FIGURE 6:
FIGURE 6:
Proposed model of the transition between the distinct binding modes of Munc18/Stx1A and Munc18/SNARE complexes. (A) Munc18 (green) binds the closed form of Stx1A through the Habc domain (blue), the H3 domain (purple), and the N-peptide (blue). The four-helix bundle and the C-terminal half of Stx1A H3 domain are crucial for this high-affinity binding mode. (B) Munc18 binds the “open” form of Stx1A: the Habc domain is flexible and dynamic, and it may transiently move out of the Munc18 central cavity, whereas the H3 domain still binds to the central cavity. “Open” Stx1A is ready to assemble into the SNARE complex with SNAP-25 and VAMP2 by the “N- to C-terminal” direction. N-peptide (not shown) still binds Munc18 and may play an important role at this stage by presumably stabilizing Stx1A bound to Munc18. (C) Munc18 binds SNARE complex, involving both the Munc18 central cavity/SNARE four-helix bundle interaction and Munc18/Stx1A-N-peptide (Stx1A Habc domain and N-peptide are not shown).
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References

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