Limits...
Vesicle fusion probability is determined by the specific interactions of munc18.

Smyth AM, Rickman C, Duncan RR - J. Biol. Chem. (2010)

Bottom Line: Mammalian-regulated secretion is absolutely dependent on four evolutionarily conserved proteins: three SNARE proteins and munc18.Dissecting the functional outcomes of the spatially organized protein interactions between these factors has been difficult because of the close interrelationship between different binding modes.Here, we investigated the spatial distribution of single munc18 molecules at the plasma membrane of cells and the underlying interactions between syntaxin and munc18.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh, George Square, Edinburgh EH8 9XD, United Kingdom.

ABSTRACT
Mammalian-regulated secretion is absolutely dependent on four evolutionarily conserved proteins: three SNARE proteins and munc18. Dissecting the functional outcomes of the spatially organized protein interactions between these factors has been difficult because of the close interrelationship between different binding modes. Here, we investigated the spatial distribution of single munc18 molecules at the plasma membrane of cells and the underlying interactions between syntaxin and munc18. Disruption of munc18 binding to the N-terminal peptide motif of syntaxin did not alter munc18 localization on the plasma membrane but had a pronounced influence on the behavior of secretory vesicles and their likelihood to undergo fusion. We therefore conclude that interaction with the syntaxin N-peptide can confer differential release probabilities to secretory vesicles and may contribute to the delineation of secretory vesicle pools.

Show MeSH

Related in: MedlinePlus

Targeted disruption of mode 2/3 interaction both in vitro and in live cells. A, structural alignment of munc18 (based on crystal structure PDB 3C98) (27) bound to syntaxin (gray helices) with amino acid conservation are shown on a color-coded scale (red, low; blue, high conservation; left panel). Amino acids Asp112, Glu132, and Ile127, predicted to disrupt N-terminal binding, are highlighted on an enlarged view (right panel). B, truncation of the N terminus [Syx7–261] (to inhibit mode 2/3 binding and/or removal of the ionic layer [Syx1–225] of syntaxin (to inhibit mode 1 binding) did not eliminate binding to native munc18. The combination of these truncations or removal of the entire SNARE helix of syntaxin [Syx1–213] eliminated detectable binding to munc18. Mode 2/3 binding to syntaxin [Syx1–255] was sensitive to a high ionic strength wash. Bound material was analyzed by SDS-PAGE and Coomassie staining or Western blotting. C, GST-Syx7–261 and GST-Syx1–225 were immobilized on glutathione-Sepharose beads and incubated with bacterial lysate containing His6-munc18 or its mutant forms. Bound material was analyzed by SDS-PAGE and Coomassie staining. D, EYFP-munc18–1[I127A] resulted in a significant change in the fluorescence co-variance with mCer-syntaxin. Wild type or EYFP-munc18-1[I127A] (red) and mCer-Syx (green) were expressed in munc18-1 silenced PC-12 cells (KD43) and imaged by confocal laser scanning microscopy and subsequent image data deconvolution. The merge image shows areas of coincidence in yellow hues. The two-dimensional histogram represents the intensity for each channel in each voxel with a color scale representing frequency. The residual map displays weighted residuals from the line fit to the histogram, thus indicating fluorescence channel co-variance. The hue is from −1 to 1 with cyan corresponding to a 0 residual. Scale bar, 5 μm. E, cerulean-Syx1–288, in the presence of EYFP-munc18, in munc18-1-silenced PC-12 cells (KD43), exhibited a plasma membrane and intracellular membrane distribution. The color scale represents the fluorescence lifetime, and brightness represents intensity. The weighted mean fluorescence lifetime values were plotted as a frequency distribution histogram (right panels) with a mean fluorescence lifetime of 1568 ± 131 ps (mean ± S.E., n = 6). Munc18[I127A] resulted in a quenching of the fluorescence lifetime to a lesser extent compared with wild type munc18 (lower panels). These data are plotted (lower right panel) and reveal a single lifetime of 2047 ± 133 ps (mean ± S.E., n = 5). The dashed line on both graphs represents the mean fluorescence lifetime of cerulean-Syx1–288 in the presence of unfused munc18 and EYFP (2321 ± 40 ps (mean ± S.E., n = 10; supplemental Fig. 1B). Scale bar, 5 μm, fluorescence lifetime color bar 1250 ps (red)–2250 ps (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2992247&req=5

Figure 1: Targeted disruption of mode 2/3 interaction both in vitro and in live cells. A, structural alignment of munc18 (based on crystal structure PDB 3C98) (27) bound to syntaxin (gray helices) with amino acid conservation are shown on a color-coded scale (red, low; blue, high conservation; left panel). Amino acids Asp112, Glu132, and Ile127, predicted to disrupt N-terminal binding, are highlighted on an enlarged view (right panel). B, truncation of the N terminus [Syx7–261] (to inhibit mode 2/3 binding and/or removal of the ionic layer [Syx1–225] of syntaxin (to inhibit mode 1 binding) did not eliminate binding to native munc18. The combination of these truncations or removal of the entire SNARE helix of syntaxin [Syx1–213] eliminated detectable binding to munc18. Mode 2/3 binding to syntaxin [Syx1–255] was sensitive to a high ionic strength wash. Bound material was analyzed by SDS-PAGE and Coomassie staining or Western blotting. C, GST-Syx7–261 and GST-Syx1–225 were immobilized on glutathione-Sepharose beads and incubated with bacterial lysate containing His6-munc18 or its mutant forms. Bound material was analyzed by SDS-PAGE and Coomassie staining. D, EYFP-munc18–1[I127A] resulted in a significant change in the fluorescence co-variance with mCer-syntaxin. Wild type or EYFP-munc18-1[I127A] (red) and mCer-Syx (green) were expressed in munc18-1 silenced PC-12 cells (KD43) and imaged by confocal laser scanning microscopy and subsequent image data deconvolution. The merge image shows areas of coincidence in yellow hues. The two-dimensional histogram represents the intensity for each channel in each voxel with a color scale representing frequency. The residual map displays weighted residuals from the line fit to the histogram, thus indicating fluorescence channel co-variance. The hue is from −1 to 1 with cyan corresponding to a 0 residual. Scale bar, 5 μm. E, cerulean-Syx1–288, in the presence of EYFP-munc18, in munc18-1-silenced PC-12 cells (KD43), exhibited a plasma membrane and intracellular membrane distribution. The color scale represents the fluorescence lifetime, and brightness represents intensity. The weighted mean fluorescence lifetime values were plotted as a frequency distribution histogram (right panels) with a mean fluorescence lifetime of 1568 ± 131 ps (mean ± S.E., n = 6). Munc18[I127A] resulted in a quenching of the fluorescence lifetime to a lesser extent compared with wild type munc18 (lower panels). These data are plotted (lower right panel) and reveal a single lifetime of 2047 ± 133 ps (mean ± S.E., n = 5). The dashed line on both graphs represents the mean fluorescence lifetime of cerulean-Syx1–288 in the presence of unfused munc18 and EYFP (2321 ± 40 ps (mean ± S.E., n = 10; supplemental Fig. 1B). Scale bar, 5 μm, fluorescence lifetime color bar 1250 ps (red)–2250 ps (blue).

Mentions: To determine the mode of munc18-syntaxin binding employed in our experiments we needed to design targeted mutations to disrupt each type of interaction specifically. The approach we used was to examine the amino acid sequence of members of the SM protein family. Evolutionary conservation of amino acids, with relation to the three-dimensional protein structure is indicative of an essential function (34). We aligned the amino acid sequence of 191 predicted SM proteins, mapping the degree of conservation on to the crystal structure of munc18 bound to syntaxin1 (27) (Fig. 1A). This approach highlighted the amino acids lining the mode 2/3 binding pocket on munc18 and indicated Glu132 and Asp112 (expected to form hydrogen bonds to the N-terminal motif of syntaxin1) and Ile127 (forming one side of the hydrophobic pocket (27)) as being potentially important for this interaction. We constructed two-point mutations for each amino acid (I127A/I127F, E132A/E132K, and D112A/D112K) to investigate their individual contributions in binding syntaxin and the downstream functional role this binding mode could play.


Vesicle fusion probability is determined by the specific interactions of munc18.

Smyth AM, Rickman C, Duncan RR - J. Biol. Chem. (2010)

Targeted disruption of mode 2/3 interaction both in vitro and in live cells. A, structural alignment of munc18 (based on crystal structure PDB 3C98) (27) bound to syntaxin (gray helices) with amino acid conservation are shown on a color-coded scale (red, low; blue, high conservation; left panel). Amino acids Asp112, Glu132, and Ile127, predicted to disrupt N-terminal binding, are highlighted on an enlarged view (right panel). B, truncation of the N terminus [Syx7–261] (to inhibit mode 2/3 binding and/or removal of the ionic layer [Syx1–225] of syntaxin (to inhibit mode 1 binding) did not eliminate binding to native munc18. The combination of these truncations or removal of the entire SNARE helix of syntaxin [Syx1–213] eliminated detectable binding to munc18. Mode 2/3 binding to syntaxin [Syx1–255] was sensitive to a high ionic strength wash. Bound material was analyzed by SDS-PAGE and Coomassie staining or Western blotting. C, GST-Syx7–261 and GST-Syx1–225 were immobilized on glutathione-Sepharose beads and incubated with bacterial lysate containing His6-munc18 or its mutant forms. Bound material was analyzed by SDS-PAGE and Coomassie staining. D, EYFP-munc18–1[I127A] resulted in a significant change in the fluorescence co-variance with mCer-syntaxin. Wild type or EYFP-munc18-1[I127A] (red) and mCer-Syx (green) were expressed in munc18-1 silenced PC-12 cells (KD43) and imaged by confocal laser scanning microscopy and subsequent image data deconvolution. The merge image shows areas of coincidence in yellow hues. The two-dimensional histogram represents the intensity for each channel in each voxel with a color scale representing frequency. The residual map displays weighted residuals from the line fit to the histogram, thus indicating fluorescence channel co-variance. The hue is from −1 to 1 with cyan corresponding to a 0 residual. Scale bar, 5 μm. E, cerulean-Syx1–288, in the presence of EYFP-munc18, in munc18-1-silenced PC-12 cells (KD43), exhibited a plasma membrane and intracellular membrane distribution. The color scale represents the fluorescence lifetime, and brightness represents intensity. The weighted mean fluorescence lifetime values were plotted as a frequency distribution histogram (right panels) with a mean fluorescence lifetime of 1568 ± 131 ps (mean ± S.E., n = 6). Munc18[I127A] resulted in a quenching of the fluorescence lifetime to a lesser extent compared with wild type munc18 (lower panels). These data are plotted (lower right panel) and reveal a single lifetime of 2047 ± 133 ps (mean ± S.E., n = 5). The dashed line on both graphs represents the mean fluorescence lifetime of cerulean-Syx1–288 in the presence of unfused munc18 and EYFP (2321 ± 40 ps (mean ± S.E., n = 10; supplemental Fig. 1B). Scale bar, 5 μm, fluorescence lifetime color bar 1250 ps (red)–2250 ps (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992247&req=5

Figure 1: Targeted disruption of mode 2/3 interaction both in vitro and in live cells. A, structural alignment of munc18 (based on crystal structure PDB 3C98) (27) bound to syntaxin (gray helices) with amino acid conservation are shown on a color-coded scale (red, low; blue, high conservation; left panel). Amino acids Asp112, Glu132, and Ile127, predicted to disrupt N-terminal binding, are highlighted on an enlarged view (right panel). B, truncation of the N terminus [Syx7–261] (to inhibit mode 2/3 binding and/or removal of the ionic layer [Syx1–225] of syntaxin (to inhibit mode 1 binding) did not eliminate binding to native munc18. The combination of these truncations or removal of the entire SNARE helix of syntaxin [Syx1–213] eliminated detectable binding to munc18. Mode 2/3 binding to syntaxin [Syx1–255] was sensitive to a high ionic strength wash. Bound material was analyzed by SDS-PAGE and Coomassie staining or Western blotting. C, GST-Syx7–261 and GST-Syx1–225 were immobilized on glutathione-Sepharose beads and incubated with bacterial lysate containing His6-munc18 or its mutant forms. Bound material was analyzed by SDS-PAGE and Coomassie staining. D, EYFP-munc18–1[I127A] resulted in a significant change in the fluorescence co-variance with mCer-syntaxin. Wild type or EYFP-munc18-1[I127A] (red) and mCer-Syx (green) were expressed in munc18-1 silenced PC-12 cells (KD43) and imaged by confocal laser scanning microscopy and subsequent image data deconvolution. The merge image shows areas of coincidence in yellow hues. The two-dimensional histogram represents the intensity for each channel in each voxel with a color scale representing frequency. The residual map displays weighted residuals from the line fit to the histogram, thus indicating fluorescence channel co-variance. The hue is from −1 to 1 with cyan corresponding to a 0 residual. Scale bar, 5 μm. E, cerulean-Syx1–288, in the presence of EYFP-munc18, in munc18-1-silenced PC-12 cells (KD43), exhibited a plasma membrane and intracellular membrane distribution. The color scale represents the fluorescence lifetime, and brightness represents intensity. The weighted mean fluorescence lifetime values were plotted as a frequency distribution histogram (right panels) with a mean fluorescence lifetime of 1568 ± 131 ps (mean ± S.E., n = 6). Munc18[I127A] resulted in a quenching of the fluorescence lifetime to a lesser extent compared with wild type munc18 (lower panels). These data are plotted (lower right panel) and reveal a single lifetime of 2047 ± 133 ps (mean ± S.E., n = 5). The dashed line on both graphs represents the mean fluorescence lifetime of cerulean-Syx1–288 in the presence of unfused munc18 and EYFP (2321 ± 40 ps (mean ± S.E., n = 10; supplemental Fig. 1B). Scale bar, 5 μm, fluorescence lifetime color bar 1250 ps (red)–2250 ps (blue).
Mentions: To determine the mode of munc18-syntaxin binding employed in our experiments we needed to design targeted mutations to disrupt each type of interaction specifically. The approach we used was to examine the amino acid sequence of members of the SM protein family. Evolutionary conservation of amino acids, with relation to the three-dimensional protein structure is indicative of an essential function (34). We aligned the amino acid sequence of 191 predicted SM proteins, mapping the degree of conservation on to the crystal structure of munc18 bound to syntaxin1 (27) (Fig. 1A). This approach highlighted the amino acids lining the mode 2/3 binding pocket on munc18 and indicated Glu132 and Asp112 (expected to form hydrogen bonds to the N-terminal motif of syntaxin1) and Ile127 (forming one side of the hydrophobic pocket (27)) as being potentially important for this interaction. We constructed two-point mutations for each amino acid (I127A/I127F, E132A/E132K, and D112A/D112K) to investigate their individual contributions in binding syntaxin and the downstream functional role this binding mode could play.

Bottom Line: Mammalian-regulated secretion is absolutely dependent on four evolutionarily conserved proteins: three SNARE proteins and munc18.Dissecting the functional outcomes of the spatially organized protein interactions between these factors has been difficult because of the close interrelationship between different binding modes.Here, we investigated the spatial distribution of single munc18 molecules at the plasma membrane of cells and the underlying interactions between syntaxin and munc18.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Physiology, University of Edinburgh, George Square, Edinburgh EH8 9XD, United Kingdom.

ABSTRACT
Mammalian-regulated secretion is absolutely dependent on four evolutionarily conserved proteins: three SNARE proteins and munc18. Dissecting the functional outcomes of the spatially organized protein interactions between these factors has been difficult because of the close interrelationship between different binding modes. Here, we investigated the spatial distribution of single munc18 molecules at the plasma membrane of cells and the underlying interactions between syntaxin and munc18. Disruption of munc18 binding to the N-terminal peptide motif of syntaxin did not alter munc18 localization on the plasma membrane but had a pronounced influence on the behavior of secretory vesicles and their likelihood to undergo fusion. We therefore conclude that interaction with the syntaxin N-peptide can confer differential release probabilities to secretory vesicles and may contribute to the delineation of secretory vesicle pools.

Show MeSH
Related in: MedlinePlus