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Stimulation of NSF ATPase activity by alpha-SNAP is required for SNARE complex disassembly and exocytosis.

Barnard RJ, Morgan A, Burgoyne RD - J. Cell Biol. (1997)

Bottom Line: Deletion of up to 160 NH2-terminal amino acids had little effect on the ability of alpha-SNAP to stimulate the ATPase activity of NSF.However, deletion of as few as 10 COOH-terminal amino acids resulted in a marked decrease.Mutation of leucine 294 to alanine (alpha-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. alpha-SNAP (1-285) and alpha-SNAP (L294A) were unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells.

View Article: PubMed Central - PubMed

Affiliation: The Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, UK.

ABSTRACT
N-ethylmaleimide-sensitive fusion protein (NSF) and alpha-SNAP play key roles in vesicular traffic through the secretory pathway. In this study, NH2- and COOH-terminal truncation mutants of alpha-SNAP were assayed for ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH2-terminal amino acids had little effect on the ability of alpha-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids resulted in a marked decrease. Both NH2-terminal (1-160) and COOH-terminal (160-295) fragments of alpha-SNAP were able to bind to NSF, suggesting that alpha-SNAP contains distinct NH2- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs revealed only leucine 294 to be conserved in the final 10 amino acids of alpha-SNAP. Mutation of leucine 294 to alanine (alpha-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. alpha-SNAP (1-285) and alpha-SNAP (L294A) were unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells. In addition, alpha-SNAP (1-285), and alpha-SNAP (L294A) were able to inhibit the stimulation of exocytosis by exogenous alpha-SNAP. alpha-SNAP, alpha-SNAP (1-285), and alpha-SNAP (L294A) were all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, alpha-SNAP (1-285) and alpha-SNAP (L294A) were unable to fully disassemble the 20S complex and did not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that alpha-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the alpha-SNAP stimulation of exocytosis.

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α-SNAP (1–285) and α-SNAP (L294A) associate with but are unable to support dissociation of the 20S complex. (A) A detergent extract of rat brain membrane proteins was incubated with 15 μg of NSF, 30 μg α-SNAPs for 30 min with 0.5 mM MgATP or  MgATPγS as indicated at 4°C. Proteins were immunoprecipitated with an antisyntaxin antibody conjugated to protein G–Sepharose,  and bound proteins were solubilized with SDS sample buffer and separated on a 12.5% polyacrylamide gel. Proteins were detected using specific antisera to NSF, α-SNAP, syntaxin, and VAMP. (B) Extracts were incubated without (control) or with added α-SNAP and  with 15 μg NSF in the presence of 0.5 mM MgATP or MgATPγS as indicated. Endogenous α-SNAP in control incubations was sufficient to recruit exogenous NSF. (C) Extracts were incubated with NSF with no added α-SNAP (control) or with added α-SNAPs as indicated. The presence of VAMP in syntaxin immunoprecipitates was determined by immunoblotting, and the amount of VAMP dissociated in the presence of MgATP was calculated as a percentage of the amount of bound VAMP in MgATPγS incubations.The data  shown are the mean values from two experiments.
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Figure 6: α-SNAP (1–285) and α-SNAP (L294A) associate with but are unable to support dissociation of the 20S complex. (A) A detergent extract of rat brain membrane proteins was incubated with 15 μg of NSF, 30 μg α-SNAPs for 30 min with 0.5 mM MgATP or MgATPγS as indicated at 4°C. Proteins were immunoprecipitated with an antisyntaxin antibody conjugated to protein G–Sepharose, and bound proteins were solubilized with SDS sample buffer and separated on a 12.5% polyacrylamide gel. Proteins were detected using specific antisera to NSF, α-SNAP, syntaxin, and VAMP. (B) Extracts were incubated without (control) or with added α-SNAP and with 15 μg NSF in the presence of 0.5 mM MgATP or MgATPγS as indicated. Endogenous α-SNAP in control incubations was sufficient to recruit exogenous NSF. (C) Extracts were incubated with NSF with no added α-SNAP (control) or with added α-SNAPs as indicated. The presence of VAMP in syntaxin immunoprecipitates was determined by immunoblotting, and the amount of VAMP dissociated in the presence of MgATP was calculated as a percentage of the amount of bound VAMP in MgATPγS incubations.The data shown are the mean values from two experiments.

Mentions: It has been shown previously that ATP hydrolysis by NSF is required to disassemble the 20S complex of NSF, α-SNAP and the SNAREs syntaxin, SNAP-25, and VAMP (Söllner et al., 1993b). As α-SNAP (1–285) and α-SNAP (L294A) are unable to stimulate either the ATPase activity of NSF or exocytosis in adrenal chromaffin cells, they were analyzed for their ability to support assembly and disassembly of the 20S complex. A detergent extract of rat brain membrane proteins was incubated with NSF and with α-SNAP, α-SNAP (1–285), or α-SNAP (L294A) in the presence of MgATP or the nonhydrolyzable analogue MgATPγS. The 20S complex was immunoprecipitated with an antisyntaxin antibody, and immunoprecipitated syntaxin and proteins bound to syntaxin were visualized by immunoblotting. In the presence of ATP-γ-S, added α-SNAP, α-SNAP (1–285), and α-SNAP (L294A) were able to bind to the SNARE complex and recruit NSF, forming the 20S complex (Fig. 6 A). In the presence of MgATP, α-SNAP was able to disassemble the 20S complex, and the levels of VAMP, α-SNAP, and NSF in the syntaxin immunoprecipitate were significantly reduced (Fig. 6 A, second lane). The level of NSF dissociation from the 20S complex was similar with both wild-type and mutant α-SNAPs (Fig. 6 A). However, in the presence of MgATP, both α-SNAP (1–285) and α-SNAP (L294A) were still significantly associated with syntaxin, and in addition, VAMP did not fully disassemble from the 20S complex formed with these mutants (Fig. 6 A, fourth and sixth lanes). In five experiments under varying conditions, no consistent differences were seen in the extent of VAMP dissociation with α-SNAP (1–285) or α-SNAP (L284A), which was always less than with α-SNAP. In the absence of added exogenous α-SNAP, low levels of endogenous α-SNAP, sufficient to recruit added NSF, could be detected by immunoblotting (Fig. 6 B). Since some VAMP dissociation was seen with the mutants, additional experiments were carried out in which the extent of VAMP dissociation was compared to incubations in which NSF but no α-SNAP was added. Quantification was based on densitometric analysis of blots within the linear range of the assay, and to account for variations between individual samples, the data shown is based on two separate experiments. Partial VAMP dissociation occurred in an ATP- dependent manner in the control incubation and to the same extent as with the mutants (Fig. 6 C), indicating that α-SNAP (1–285) and α-SNAP (L294A) are unable to support any VAMP dissociation.


Stimulation of NSF ATPase activity by alpha-SNAP is required for SNARE complex disassembly and exocytosis.

Barnard RJ, Morgan A, Burgoyne RD - J. Cell Biol. (1997)

α-SNAP (1–285) and α-SNAP (L294A) associate with but are unable to support dissociation of the 20S complex. (A) A detergent extract of rat brain membrane proteins was incubated with 15 μg of NSF, 30 μg α-SNAPs for 30 min with 0.5 mM MgATP or  MgATPγS as indicated at 4°C. Proteins were immunoprecipitated with an antisyntaxin antibody conjugated to protein G–Sepharose,  and bound proteins were solubilized with SDS sample buffer and separated on a 12.5% polyacrylamide gel. Proteins were detected using specific antisera to NSF, α-SNAP, syntaxin, and VAMP. (B) Extracts were incubated without (control) or with added α-SNAP and  with 15 μg NSF in the presence of 0.5 mM MgATP or MgATPγS as indicated. Endogenous α-SNAP in control incubations was sufficient to recruit exogenous NSF. (C) Extracts were incubated with NSF with no added α-SNAP (control) or with added α-SNAPs as indicated. The presence of VAMP in syntaxin immunoprecipitates was determined by immunoblotting, and the amount of VAMP dissociated in the presence of MgATP was calculated as a percentage of the amount of bound VAMP in MgATPγS incubations.The data  shown are the mean values from two experiments.
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Figure 6: α-SNAP (1–285) and α-SNAP (L294A) associate with but are unable to support dissociation of the 20S complex. (A) A detergent extract of rat brain membrane proteins was incubated with 15 μg of NSF, 30 μg α-SNAPs for 30 min with 0.5 mM MgATP or MgATPγS as indicated at 4°C. Proteins were immunoprecipitated with an antisyntaxin antibody conjugated to protein G–Sepharose, and bound proteins were solubilized with SDS sample buffer and separated on a 12.5% polyacrylamide gel. Proteins were detected using specific antisera to NSF, α-SNAP, syntaxin, and VAMP. (B) Extracts were incubated without (control) or with added α-SNAP and with 15 μg NSF in the presence of 0.5 mM MgATP or MgATPγS as indicated. Endogenous α-SNAP in control incubations was sufficient to recruit exogenous NSF. (C) Extracts were incubated with NSF with no added α-SNAP (control) or with added α-SNAPs as indicated. The presence of VAMP in syntaxin immunoprecipitates was determined by immunoblotting, and the amount of VAMP dissociated in the presence of MgATP was calculated as a percentage of the amount of bound VAMP in MgATPγS incubations.The data shown are the mean values from two experiments.
Mentions: It has been shown previously that ATP hydrolysis by NSF is required to disassemble the 20S complex of NSF, α-SNAP and the SNAREs syntaxin, SNAP-25, and VAMP (Söllner et al., 1993b). As α-SNAP (1–285) and α-SNAP (L294A) are unable to stimulate either the ATPase activity of NSF or exocytosis in adrenal chromaffin cells, they were analyzed for their ability to support assembly and disassembly of the 20S complex. A detergent extract of rat brain membrane proteins was incubated with NSF and with α-SNAP, α-SNAP (1–285), or α-SNAP (L294A) in the presence of MgATP or the nonhydrolyzable analogue MgATPγS. The 20S complex was immunoprecipitated with an antisyntaxin antibody, and immunoprecipitated syntaxin and proteins bound to syntaxin were visualized by immunoblotting. In the presence of ATP-γ-S, added α-SNAP, α-SNAP (1–285), and α-SNAP (L294A) were able to bind to the SNARE complex and recruit NSF, forming the 20S complex (Fig. 6 A). In the presence of MgATP, α-SNAP was able to disassemble the 20S complex, and the levels of VAMP, α-SNAP, and NSF in the syntaxin immunoprecipitate were significantly reduced (Fig. 6 A, second lane). The level of NSF dissociation from the 20S complex was similar with both wild-type and mutant α-SNAPs (Fig. 6 A). However, in the presence of MgATP, both α-SNAP (1–285) and α-SNAP (L294A) were still significantly associated with syntaxin, and in addition, VAMP did not fully disassemble from the 20S complex formed with these mutants (Fig. 6 A, fourth and sixth lanes). In five experiments under varying conditions, no consistent differences were seen in the extent of VAMP dissociation with α-SNAP (1–285) or α-SNAP (L284A), which was always less than with α-SNAP. In the absence of added exogenous α-SNAP, low levels of endogenous α-SNAP, sufficient to recruit added NSF, could be detected by immunoblotting (Fig. 6 B). Since some VAMP dissociation was seen with the mutants, additional experiments were carried out in which the extent of VAMP dissociation was compared to incubations in which NSF but no α-SNAP was added. Quantification was based on densitometric analysis of blots within the linear range of the assay, and to account for variations between individual samples, the data shown is based on two separate experiments. Partial VAMP dissociation occurred in an ATP- dependent manner in the control incubation and to the same extent as with the mutants (Fig. 6 C), indicating that α-SNAP (1–285) and α-SNAP (L294A) are unable to support any VAMP dissociation.

Bottom Line: Deletion of up to 160 NH2-terminal amino acids had little effect on the ability of alpha-SNAP to stimulate the ATPase activity of NSF.However, deletion of as few as 10 COOH-terminal amino acids resulted in a marked decrease.Mutation of leucine 294 to alanine (alpha-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. alpha-SNAP (1-285) and alpha-SNAP (L294A) were unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells.

View Article: PubMed Central - PubMed

Affiliation: The Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, UK.

ABSTRACT
N-ethylmaleimide-sensitive fusion protein (NSF) and alpha-SNAP play key roles in vesicular traffic through the secretory pathway. In this study, NH2- and COOH-terminal truncation mutants of alpha-SNAP were assayed for ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH2-terminal amino acids had little effect on the ability of alpha-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids resulted in a marked decrease. Both NH2-terminal (1-160) and COOH-terminal (160-295) fragments of alpha-SNAP were able to bind to NSF, suggesting that alpha-SNAP contains distinct NH2- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs revealed only leucine 294 to be conserved in the final 10 amino acids of alpha-SNAP. Mutation of leucine 294 to alanine (alpha-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. alpha-SNAP (1-285) and alpha-SNAP (L294A) were unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells. In addition, alpha-SNAP (1-285), and alpha-SNAP (L294A) were able to inhibit the stimulation of exocytosis by exogenous alpha-SNAP. alpha-SNAP, alpha-SNAP (1-285), and alpha-SNAP (L294A) were all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, alpha-SNAP (1-285) and alpha-SNAP (L294A) were unable to fully disassemble the 20S complex and did not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that alpha-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the alpha-SNAP stimulation of exocytosis.

Show MeSH
Related in: MedlinePlus