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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.

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Related in: MedlinePlus

Genetic and Electrophysiological Evidence That Neuronal Overexpression of the Wild-Type Syntaxin 1A Specifically Rescues the syx3–69 Mutant Phenotype(A and B) The increase in mini frequency and evoked EPSP amplitude persists in larvae carrying only one copy of the syx3–69 mutant gene. This mutant fly (syx3–69/syxΔ229) is generated by placing one mutant gene in trans to the  mutation (syxΔ229) in the syntaxin locus. These electrophysiological defects are nearly identical to those found in the syx3–69/syx3–69 homozygote, but different in mini frequencies from the syx3–69/+ heterozygote. These results demonstrate that the mutant phenotype is specifically caused by the syx3–69 mutation. **, p < 0.01; ***, p < 0.001.(C and D) Neuronal overexpression rescues the physiological defects observed in syx3–69 mutants. In C155 Gal4 background, the syx3–69/syxΔ229 mutant only expresses one copy of the T254I mutant protein. Under such circumstances, the C155 Gal4; syx3–69/syxΔ229 larvae display an extraordinarily high frequency of minis and enhanced amplitude of EPSPs. Neuronal overexpression of the wild-type syntaxin 1A (UAS-Syx driven by C155 Gal4) in the syx3–69/syxΔ229 mutant background dramatically reduces mini frequency to a level slightly higher than that in the wild type (i.e., C155 Gal4; UAS-Syx or C155 Gal4 flies) (C). The EPSP amplitude is also similarly reduced to the wild-type level (D). Importantly, overexpression of the wild-type syntaxin 1A in the wild-type background (i.e., C155 Gal4; UAS-Syx) has little effect on both the mini frequency and evoked EPSP amplitude compared to the C155 Gal4 flies. Thus, the rescuing effect on vesicle fusion by the wild-type syntaxin 1A is specific to the T254I mutant syntaxin 1A in the syx3–69 mutant. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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pbio-0050072-g010: Genetic and Electrophysiological Evidence That Neuronal Overexpression of the Wild-Type Syntaxin 1A Specifically Rescues the syx3–69 Mutant Phenotype(A and B) The increase in mini frequency and evoked EPSP amplitude persists in larvae carrying only one copy of the syx3–69 mutant gene. This mutant fly (syx3–69/syxΔ229) is generated by placing one mutant gene in trans to the mutation (syxΔ229) in the syntaxin locus. These electrophysiological defects are nearly identical to those found in the syx3–69/syx3–69 homozygote, but different in mini frequencies from the syx3–69/+ heterozygote. These results demonstrate that the mutant phenotype is specifically caused by the syx3–69 mutation. **, p < 0.01; ***, p < 0.001.(C and D) Neuronal overexpression rescues the physiological defects observed in syx3–69 mutants. In C155 Gal4 background, the syx3–69/syxΔ229 mutant only expresses one copy of the T254I mutant protein. Under such circumstances, the C155 Gal4; syx3–69/syxΔ229 larvae display an extraordinarily high frequency of minis and enhanced amplitude of EPSPs. Neuronal overexpression of the wild-type syntaxin 1A (UAS-Syx driven by C155 Gal4) in the syx3–69/syxΔ229 mutant background dramatically reduces mini frequency to a level slightly higher than that in the wild type (i.e., C155 Gal4; UAS-Syx or C155 Gal4 flies) (C). The EPSP amplitude is also similarly reduced to the wild-type level (D). Importantly, overexpression of the wild-type syntaxin 1A in the wild-type background (i.e., C155 Gal4; UAS-Syx) has little effect on both the mini frequency and evoked EPSP amplitude compared to the C155 Gal4 flies. Thus, the rescuing effect on vesicle fusion by the wild-type syntaxin 1A is specific to the T254I mutant syntaxin 1A in the syx3–69 mutant. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Mentions: The possibility remains that the dominant positive effects we have seen in the heterozygote could result from a second site mutation elsewhere rather than the T254I mutation in the syntaxin locus. To address this concern, we generated transheterozygous flies (syx3–69/syxΔ229) in which the syx3–69 mutant chromosome was placed in trans to a syntaxin mutation (syxΔ229) [23]. In syx3–69/syxΔ229 mutants, the mini frequency was 22.3 Hz (n = 9), which is significantly higher than that in the wild-type larvae (3.2 Hz, n = 8; p < 0.0001) (Figure 10A). At 0.8 mM Ca2+, the evoked EPSP amplitude was also significantly increased to 37.9 mV (n = 9) from 29.5 mV (n = 8) in the wild-type larvae (p < 0.01) (Figure 10B). The resting potential of the mutant animal (−72.4 mV) was similar to that (−73.8 mV) in the wild-type larvae. These results are highly similar to those found in the syx3–69/syx3–69 homozygote. Along with the molecular evidence presented earlier, these results provide further genetic and electrophysiological evidence that the effects we have observed in the syx3–69 mutant is specifically caused by the T254I mutation in the syntaxin gene.


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)

Genetic and Electrophysiological Evidence That Neuronal Overexpression of the Wild-Type Syntaxin 1A Specifically Rescues the syx3–69 Mutant Phenotype(A and B) The increase in mini frequency and evoked EPSP amplitude persists in larvae carrying only one copy of the syx3–69 mutant gene. This mutant fly (syx3–69/syxΔ229) is generated by placing one mutant gene in trans to the  mutation (syxΔ229) in the syntaxin locus. These electrophysiological defects are nearly identical to those found in the syx3–69/syx3–69 homozygote, but different in mini frequencies from the syx3–69/+ heterozygote. These results demonstrate that the mutant phenotype is specifically caused by the syx3–69 mutation. **, p < 0.01; ***, p < 0.001.(C and D) Neuronal overexpression rescues the physiological defects observed in syx3–69 mutants. In C155 Gal4 background, the syx3–69/syxΔ229 mutant only expresses one copy of the T254I mutant protein. Under such circumstances, the C155 Gal4; syx3–69/syxΔ229 larvae display an extraordinarily high frequency of minis and enhanced amplitude of EPSPs. Neuronal overexpression of the wild-type syntaxin 1A (UAS-Syx driven by C155 Gal4) in the syx3–69/syxΔ229 mutant background dramatically reduces mini frequency to a level slightly higher than that in the wild type (i.e., C155 Gal4; UAS-Syx or C155 Gal4 flies) (C). The EPSP amplitude is also similarly reduced to the wild-type level (D). Importantly, overexpression of the wild-type syntaxin 1A in the wild-type background (i.e., C155 Gal4; UAS-Syx) has little effect on both the mini frequency and evoked EPSP amplitude compared to the C155 Gal4 flies. Thus, the rescuing effect on vesicle fusion by the wild-type syntaxin 1A is specific to the T254I mutant syntaxin 1A in the syx3–69 mutant. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0050072-g010: Genetic and Electrophysiological Evidence That Neuronal Overexpression of the Wild-Type Syntaxin 1A Specifically Rescues the syx3–69 Mutant Phenotype(A and B) The increase in mini frequency and evoked EPSP amplitude persists in larvae carrying only one copy of the syx3–69 mutant gene. This mutant fly (syx3–69/syxΔ229) is generated by placing one mutant gene in trans to the mutation (syxΔ229) in the syntaxin locus. These electrophysiological defects are nearly identical to those found in the syx3–69/syx3–69 homozygote, but different in mini frequencies from the syx3–69/+ heterozygote. These results demonstrate that the mutant phenotype is specifically caused by the syx3–69 mutation. **, p < 0.01; ***, p < 0.001.(C and D) Neuronal overexpression rescues the physiological defects observed in syx3–69 mutants. In C155 Gal4 background, the syx3–69/syxΔ229 mutant only expresses one copy of the T254I mutant protein. Under such circumstances, the C155 Gal4; syx3–69/syxΔ229 larvae display an extraordinarily high frequency of minis and enhanced amplitude of EPSPs. Neuronal overexpression of the wild-type syntaxin 1A (UAS-Syx driven by C155 Gal4) in the syx3–69/syxΔ229 mutant background dramatically reduces mini frequency to a level slightly higher than that in the wild type (i.e., C155 Gal4; UAS-Syx or C155 Gal4 flies) (C). The EPSP amplitude is also similarly reduced to the wild-type level (D). Importantly, overexpression of the wild-type syntaxin 1A in the wild-type background (i.e., C155 Gal4; UAS-Syx) has little effect on both the mini frequency and evoked EPSP amplitude compared to the C155 Gal4 flies. Thus, the rescuing effect on vesicle fusion by the wild-type syntaxin 1A is specific to the T254I mutant syntaxin 1A in the syx3–69 mutant. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Mentions: The possibility remains that the dominant positive effects we have seen in the heterozygote could result from a second site mutation elsewhere rather than the T254I mutation in the syntaxin locus. To address this concern, we generated transheterozygous flies (syx3–69/syxΔ229) in which the syx3–69 mutant chromosome was placed in trans to a syntaxin mutation (syxΔ229) [23]. In syx3–69/syxΔ229 mutants, the mini frequency was 22.3 Hz (n = 9), which is significantly higher than that in the wild-type larvae (3.2 Hz, n = 8; p < 0.0001) (Figure 10A). At 0.8 mM Ca2+, the evoked EPSP amplitude was also significantly increased to 37.9 mV (n = 9) from 29.5 mV (n = 8) in the wild-type larvae (p < 0.01) (Figure 10B). The resting potential of the mutant animal (−72.4 mV) was similar to that (−73.8 mV) in the wild-type larvae. These results are highly similar to those found in the syx3–69/syx3–69 homozygote. Along with the molecular evidence presented earlier, these results provide further genetic and electrophysiological evidence that the effects we have observed in the syx3–69 mutant is specifically caused by the T254I mutation in the syntaxin gene.

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