Abstract
Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the plasma membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins initiate membrane fusion through the formation of the SNARE complex, a process tightly regulated by Sec1/Munc18-1 (SM) proteins. The emerging trend is that SM proteins promote SNARE-mediated membrane fusion by binding to a Syntaxin N-terminal motif. Here we report that mutations in the hydrophobic pocket of Munc18-1 (F115E and E132A), predicted to disrupt the N-terminal Sx1a interaction have a modest effect on binding to Sx1a in its free state, but abolish binding to the SNARE complex. Overexpression of the Munc18-1 mutant in PC12 cells lacking Munc18-1 rescues both neuroexocytosis and the plasma membrane localization of Syntaxin. However, total internal reflection fluorescence microscopy analysis reveals that expression of a Munc18-1 double mutant reduces the rate of vesicle fusion, an effect only detectable at the onset of stimulation. The Munc18-1 hydrophobic pocket is therefore critical for SNARE complex binding. However, mutations abrogating this interaction have a limited impact on Ca(2+)-dependent exocytosis in PC12 cells.
Highlights
Following stimulation of neurons, a number of well orchestrated protein/protein [1] and protein/lipid [2] interactions underpin the fusion of secretory vesicles with the presynaptic plasma membrane
Other SM proteins have been shown to bind to their cognate syntaxins via an N-terminal motif (16 –19), allowing interactions that are associated with a positive role for SM proteins in sensitive fusion protein attachment protein receptor (SNARE)-mediated membrane fusion [20]
We used a model of the Munc18-1-Syntaxin 1a (Sx1a) N-terminal interaction and compared interactions with the crystal structure of the closely related SM protein, Munc18c (Fig. 1B, top panel)
Summary
Munc18-1-Sx1a Model—Coordinates for the Munc18c-Sx4(1–29) complex (Protein Data Bank code 2PJX) and the Munc18-1:Sx1a complexes (PDB code 1DN1) were obtained from the Protein Data Bank. Pulldown Binding Assays—Pulldown assays were performed by first isolating tagged proteins on affinity beads, incubating with a molar excess of purified protein in solution. For the pulldown assay with Munc18-1-WT point and truncated mutants, a 3-fold molar excess of detagged Munc was incubated with Sx1a-His beads in standard buffer containing 20 mM imidazole, pH 7.4, and 0.1% Triton X-100. For SNARE complex pulldown assays, SNARE complex-bound beads were incubated with a 3-fold molar excess of Munc18-1-WT, Munc181-F115E, or Munc18-1-F115E/E132A at 4 °C in standard buffer containing 0.1% Triton X-100 and 20 mM imidazole, pH 7.4. 72 h after transfection, PC12-KD43 cells were briefly washed with PSS and either stimulated with PSS or high Kϩ PSS buffer (containing 81 mM NaCl and 70 mM KCl) for 20 min at 37 °C. Values are expressed as mean Ϯ S.E., and data are considered significant at: *, p Ͻ 0.05; **, p Ͻ 0.01
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