Limits...
Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.

Lagow RD, Bao H, Cohen EN, Daniels RW, Zuzek A, Williams WH, Macleod GT, Sutton RB, Zhang B - PLoS Biol. (2007)

Bottom Line: At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure.Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain.Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Section of Neurobiology and Institute for Neuroscience, University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT
Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.

Show MeSH

Related in: MedlinePlus

Conservation and Divergence of Threonine 254 among Different Syntaxin Orthologs(A) Proposed model of SNARE complex assembly and disassembly in a synaptic vesicle cycle (adapted from [5]). (1) Synaptobrevin forms a partial trans SNARE complex with syntaxin 1A and SNAP-25. (2) By zippering in an N- to C-termini direction, the SNARE proteins form a trans complex and bring the synaptic vesicle close to the plasma membrane. SNARE-mediated synaptic vesicle exocytosis occurs either spontaneously (3) or evoked by Ca2+ (4). (5) cis SNARE complexes are thought to be disassembled by NSF ATPase prior to vesicle recycling. ER, endoplasmic reticulum; PM, plasma membrane; SV, synaptic vesicle.(B) Alignment of amino acids (aa) around position T254 in the Drosophila syntaxin 1A or equivalent residues in syntaxin orthologs from a variety of animals, yeast, and the plant Arabidopsis. The top panel shows a cartoon of syntaxin 1A and the region of the alignment. Syntaxins are organized as “plasma membrane” or “intracellular compartments” according to their cellular distributions. With the exception of syntaxin 4, most plasma membrane syntaxins are known to function in presynaptic terminals or neurosecretory cells for Ca2+-regulated exocytosis. Note that T254 is highly conserved among “presynaptic” syntaxin 1A, 2, and 3A molecules. We call all other syntaxin orthologs shown here “constitutive” syntaxins because they are used for constitutive secretion on the plasma membrane (PM) and intracellular compartments, such as the endosome and the lysosome, the cis and trans Golgi network (Golgi network), and endoplasmic reticulum (ER) [1]. The yeast plasma membrane syntaxin orthologs SSO1 and SSO2, and syntaxins 4 and 131 from Arabidopsis are also shown here. (A more complete alignment can be see in Figure S1.) Unlike the synaptic syntaxins, syntaxin 4 and most syntaxin 11s have a valine (V) at the 254 equivalent position, syntaxins 6, 7, 12, 16, and 17 a leucine (L), and syntaxin 5 an isoleucine (I). The isoleucine found in the syx3–69 mutant resembles some of the wild-type syntaxin orthologs used for constitutive secretion. The core complex layers from 0 to +8 are identified at the bottom. The aa sequence was obtained from the NIH's National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) and aligned using the software DNAStar.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1808484&req=5

pbio-0050072-g001: Conservation and Divergence of Threonine 254 among Different Syntaxin Orthologs(A) Proposed model of SNARE complex assembly and disassembly in a synaptic vesicle cycle (adapted from [5]). (1) Synaptobrevin forms a partial trans SNARE complex with syntaxin 1A and SNAP-25. (2) By zippering in an N- to C-termini direction, the SNARE proteins form a trans complex and bring the synaptic vesicle close to the plasma membrane. SNARE-mediated synaptic vesicle exocytosis occurs either spontaneously (3) or evoked by Ca2+ (4). (5) cis SNARE complexes are thought to be disassembled by NSF ATPase prior to vesicle recycling. ER, endoplasmic reticulum; PM, plasma membrane; SV, synaptic vesicle.(B) Alignment of amino acids (aa) around position T254 in the Drosophila syntaxin 1A or equivalent residues in syntaxin orthologs from a variety of animals, yeast, and the plant Arabidopsis. The top panel shows a cartoon of syntaxin 1A and the region of the alignment. Syntaxins are organized as “plasma membrane” or “intracellular compartments” according to their cellular distributions. With the exception of syntaxin 4, most plasma membrane syntaxins are known to function in presynaptic terminals or neurosecretory cells for Ca2+-regulated exocytosis. Note that T254 is highly conserved among “presynaptic” syntaxin 1A, 2, and 3A molecules. We call all other syntaxin orthologs shown here “constitutive” syntaxins because they are used for constitutive secretion on the plasma membrane (PM) and intracellular compartments, such as the endosome and the lysosome, the cis and trans Golgi network (Golgi network), and endoplasmic reticulum (ER) [1]. The yeast plasma membrane syntaxin orthologs SSO1 and SSO2, and syntaxins 4 and 131 from Arabidopsis are also shown here. (A more complete alignment can be see in Figure S1.) Unlike the synaptic syntaxins, syntaxin 4 and most syntaxin 11s have a valine (V) at the 254 equivalent position, syntaxins 6, 7, 12, 16, and 17 a leucine (L), and syntaxin 5 an isoleucine (I). The isoleucine found in the syx3–69 mutant resembles some of the wild-type syntaxin orthologs used for constitutive secretion. The core complex layers from 0 to +8 are identified at the bottom. The aa sequence was obtained from the NIH's National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) and aligned using the software DNAStar.

Mentions: Soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) proteins are thought to mediate vesicle fusion in all eukaryotes [1–4]. In nerve terminals, there are two target-SNAREs (t-SNAREs, also called Q-SNAREs), syntaxin 1A and synaptosome-associated protein-25 kDa (SNAP-25) on the plasma membrane, and one vesicle-associated SNARE (v-SNARE, also called R-SNARE), synaptobrevin 2 on synaptic vesicles [2]. Prior to exocytosis, the t- and v-SNAREs are thought to form a trans complex composed of a four-stranded helical bundle with one helix each from syntaxin and synaptobrevin and two helices contributed by SNAP-25 [5–9] (Figure 1A). As vesicles undergo fusion, the SNARE complex rearranges from a trans to a cis configuration such that all the SNARE proteins are localized to one membrane. The cis complex is then thought to be rapidly disrupted by the ATPase NSF [5,10–12], allowing the v-SNARE to be recycled into synaptic vesicles [13]. Although the specific mechanism of vesicle fusion is still in debate, it is now widely accepted that the formation of this four-helix bundle is essential for the fusion of the vesicle phospholipid bilayer with the plasma membrane phospholipid bilayer [3].


Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.

Lagow RD, Bao H, Cohen EN, Daniels RW, Zuzek A, Williams WH, Macleod GT, Sutton RB, Zhang B - PLoS Biol. (2007)

Conservation and Divergence of Threonine 254 among Different Syntaxin Orthologs(A) Proposed model of SNARE complex assembly and disassembly in a synaptic vesicle cycle (adapted from [5]). (1) Synaptobrevin forms a partial trans SNARE complex with syntaxin 1A and SNAP-25. (2) By zippering in an N- to C-termini direction, the SNARE proteins form a trans complex and bring the synaptic vesicle close to the plasma membrane. SNARE-mediated synaptic vesicle exocytosis occurs either spontaneously (3) or evoked by Ca2+ (4). (5) cis SNARE complexes are thought to be disassembled by NSF ATPase prior to vesicle recycling. ER, endoplasmic reticulum; PM, plasma membrane; SV, synaptic vesicle.(B) Alignment of amino acids (aa) around position T254 in the Drosophila syntaxin 1A or equivalent residues in syntaxin orthologs from a variety of animals, yeast, and the plant Arabidopsis. The top panel shows a cartoon of syntaxin 1A and the region of the alignment. Syntaxins are organized as “plasma membrane” or “intracellular compartments” according to their cellular distributions. With the exception of syntaxin 4, most plasma membrane syntaxins are known to function in presynaptic terminals or neurosecretory cells for Ca2+-regulated exocytosis. Note that T254 is highly conserved among “presynaptic” syntaxin 1A, 2, and 3A molecules. We call all other syntaxin orthologs shown here “constitutive” syntaxins because they are used for constitutive secretion on the plasma membrane (PM) and intracellular compartments, such as the endosome and the lysosome, the cis and trans Golgi network (Golgi network), and endoplasmic reticulum (ER) [1]. The yeast plasma membrane syntaxin orthologs SSO1 and SSO2, and syntaxins 4 and 131 from Arabidopsis are also shown here. (A more complete alignment can be see in Figure S1.) Unlike the synaptic syntaxins, syntaxin 4 and most syntaxin 11s have a valine (V) at the 254 equivalent position, syntaxins 6, 7, 12, 16, and 17 a leucine (L), and syntaxin 5 an isoleucine (I). The isoleucine found in the syx3–69 mutant resembles some of the wild-type syntaxin orthologs used for constitutive secretion. The core complex layers from 0 to +8 are identified at the bottom. The aa sequence was obtained from the NIH's National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) and aligned using the software DNAStar.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808484&req=5

pbio-0050072-g001: Conservation and Divergence of Threonine 254 among Different Syntaxin Orthologs(A) Proposed model of SNARE complex assembly and disassembly in a synaptic vesicle cycle (adapted from [5]). (1) Synaptobrevin forms a partial trans SNARE complex with syntaxin 1A and SNAP-25. (2) By zippering in an N- to C-termini direction, the SNARE proteins form a trans complex and bring the synaptic vesicle close to the plasma membrane. SNARE-mediated synaptic vesicle exocytosis occurs either spontaneously (3) or evoked by Ca2+ (4). (5) cis SNARE complexes are thought to be disassembled by NSF ATPase prior to vesicle recycling. ER, endoplasmic reticulum; PM, plasma membrane; SV, synaptic vesicle.(B) Alignment of amino acids (aa) around position T254 in the Drosophila syntaxin 1A or equivalent residues in syntaxin orthologs from a variety of animals, yeast, and the plant Arabidopsis. The top panel shows a cartoon of syntaxin 1A and the region of the alignment. Syntaxins are organized as “plasma membrane” or “intracellular compartments” according to their cellular distributions. With the exception of syntaxin 4, most plasma membrane syntaxins are known to function in presynaptic terminals or neurosecretory cells for Ca2+-regulated exocytosis. Note that T254 is highly conserved among “presynaptic” syntaxin 1A, 2, and 3A molecules. We call all other syntaxin orthologs shown here “constitutive” syntaxins because they are used for constitutive secretion on the plasma membrane (PM) and intracellular compartments, such as the endosome and the lysosome, the cis and trans Golgi network (Golgi network), and endoplasmic reticulum (ER) [1]. The yeast plasma membrane syntaxin orthologs SSO1 and SSO2, and syntaxins 4 and 131 from Arabidopsis are also shown here. (A more complete alignment can be see in Figure S1.) Unlike the synaptic syntaxins, syntaxin 4 and most syntaxin 11s have a valine (V) at the 254 equivalent position, syntaxins 6, 7, 12, 16, and 17 a leucine (L), and syntaxin 5 an isoleucine (I). The isoleucine found in the syx3–69 mutant resembles some of the wild-type syntaxin orthologs used for constitutive secretion. The core complex layers from 0 to +8 are identified at the bottom. The aa sequence was obtained from the NIH's National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) and aligned using the software DNAStar.
Mentions: Soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) proteins are thought to mediate vesicle fusion in all eukaryotes [1–4]. In nerve terminals, there are two target-SNAREs (t-SNAREs, also called Q-SNAREs), syntaxin 1A and synaptosome-associated protein-25 kDa (SNAP-25) on the plasma membrane, and one vesicle-associated SNARE (v-SNARE, also called R-SNARE), synaptobrevin 2 on synaptic vesicles [2]. Prior to exocytosis, the t- and v-SNAREs are thought to form a trans complex composed of a four-stranded helical bundle with one helix each from syntaxin and synaptobrevin and two helices contributed by SNAP-25 [5–9] (Figure 1A). As vesicles undergo fusion, the SNARE complex rearranges from a trans to a cis configuration such that all the SNARE proteins are localized to one membrane. The cis complex is then thought to be rapidly disrupted by the ATPase NSF [5,10–12], allowing the v-SNARE to be recycled into synaptic vesicles [13]. Although the specific mechanism of vesicle fusion is still in debate, it is now widely accepted that the formation of this four-helix bundle is essential for the fusion of the vesicle phospholipid bilayer with the plasma membrane phospholipid bilayer [3].

Bottom Line: At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure.Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain.Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Section of Neurobiology and Institute for Neuroscience, University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT
Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.

Show MeSH
Related in: MedlinePlus