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The role of the N-D1 linker of the N-ethylmaleimide-sensitive factor in the SNARE disassembly.

Liu CC, Sun S, Sui SF - PLoS ONE (2013)

Bottom Line: Mutating the residues in middle and C-terminal region of the N-D1 linker increases the basal ATPase activity, indicating it may play a role in autoinhibiting NSF activity until it encounters SNARE/α-SNAP complex substrate.Moreover, mutations at the C-terminal sequence GIGG exhibit completely abolished or severely reduced activities of the substrate binding, suggesting that the flexibility of N-D1 linker is critical for the movement of the N domain that is required for the substrate binding.Taken together, these data suggest that the whole N-D1 linker is critical for the biological function of NSF to disassemble SNARE complex substrate with different regions responsible for different roles.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.

ABSTRACT
N-ethylmaleimide-sensitive factor (NSF) is a member of the type II AAA+ (ATPase associated with various cellular activities) family. It plays a critical role in intracellular membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes. Each NSF protomer consists of an N-terminal domain (N domain) followed by two AAA ATPase domains (D1 and D2) in tandem. The N domain is required for SNARE/α-SNAP binding and the D1 domain accounts for the majority of ATP hydrolysis. Little is known about the role of the N-D1 linker in the NSF function. This study presents detailed mutagenesis analyses of NSF N-D1 linker, dissecting its role in the SNARE disassembly, the SNARE/α-SNAP complex binding, the basal ATPase activity and the SNARE/α-SNAP stimulated ATPase activity. Our results show that the N-terminal region of the N-D1 linker associated mutants cause severe defect in SNARE complex disassembly, but little effects on the SNARE/α-SNAP complex binding, the basal and the SNARE/α-SNAP stimulated ATPase activity, suggesting this region may be involved in the motion transmission from D1 to N domain. Mutating the residues in middle and C-terminal region of the N-D1 linker increases the basal ATPase activity, indicating it may play a role in autoinhibiting NSF activity until it encounters SNARE/α-SNAP complex substrate. Moreover, mutations at the C-terminal sequence GIGG exhibit completely abolished or severely reduced activities of the substrate binding, suggesting that the flexibility of N-D1 linker is critical for the movement of the N domain that is required for the substrate binding. Taken together, these data suggest that the whole N-D1 linker is critical for the biological function of NSF to disassemble SNARE complex substrate with different regions responsible for different roles.

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Mutational effects of the N-terminal region of the NSF N-D1 linker.(A) SNARE disassembly by wild-type and mutant NSF. SNARE complexes were incubated with wild-type or mutant NSF, and α-SNAP in the presence of Mg2+-ATP at 37°C for 0 min, 20 min and 60 min, followed by the addition of SDS-PAGE loading buffer and analyzed by SDS-PAGE. The SNARE proteins were quantified by densitometry using ImageJ. The histogram shows the SNARE disassembly activities of wild-type and mutated NSF at 60 min averaged from three independent measurements and calculated as follows: disassembled protein, obtained by subtracting remaining protein (60 min) from total protein (0 min), divided by total protein (0 min). Values have been normalized to that of wild-type (WT) NSF. Error bars indicate the standard deviation. (B) Binding of wild-type and mutant NSF to the SNARE/α-SNAP complex. Wild-type or mutant NSF proteins were incubated with MBP-SNARE complexes in the presence (+) or absence (−) of α-SNAP under the ADP-AlFx state. The bound proteins were collected with amylose magnetic beads, eluted with 10 mM maltose and analyzed by SDS-PAGE. The gels (left panel) presented are representative of at least two separate experiments. The bound proteins were quantified by densitometry using ImageJ (right panel). (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF. Standard ATPase reactions were carried out using 3 μg of wild-type or mutant NSF in the ATPase assay buffer. SNARE/α-SNAP complex was prepared as described in Materials and Methods. Basal and stimulated ATPase activities were measured in the absence (−) or presence (+) of SNARE/α-SNAP complex at 25°C for 1 h, respectively. The histogram shows the rates of ATP hydrolysis averaged from three independent measurements. Error bars indicate standard deviations. The fold increase in ATPase activity (stimulation) was calculated by dividing the stimulated ATPase activity by the basal ATPase activity.
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pone-0064346-g003: Mutational effects of the N-terminal region of the NSF N-D1 linker.(A) SNARE disassembly by wild-type and mutant NSF. SNARE complexes were incubated with wild-type or mutant NSF, and α-SNAP in the presence of Mg2+-ATP at 37°C for 0 min, 20 min and 60 min, followed by the addition of SDS-PAGE loading buffer and analyzed by SDS-PAGE. The SNARE proteins were quantified by densitometry using ImageJ. The histogram shows the SNARE disassembly activities of wild-type and mutated NSF at 60 min averaged from three independent measurements and calculated as follows: disassembled protein, obtained by subtracting remaining protein (60 min) from total protein (0 min), divided by total protein (0 min). Values have been normalized to that of wild-type (WT) NSF. Error bars indicate the standard deviation. (B) Binding of wild-type and mutant NSF to the SNARE/α-SNAP complex. Wild-type or mutant NSF proteins were incubated with MBP-SNARE complexes in the presence (+) or absence (−) of α-SNAP under the ADP-AlFx state. The bound proteins were collected with amylose magnetic beads, eluted with 10 mM maltose and analyzed by SDS-PAGE. The gels (left panel) presented are representative of at least two separate experiments. The bound proteins were quantified by densitometry using ImageJ (right panel). (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF. Standard ATPase reactions were carried out using 3 μg of wild-type or mutant NSF in the ATPase assay buffer. SNARE/α-SNAP complex was prepared as described in Materials and Methods. Basal and stimulated ATPase activities were measured in the absence (−) or presence (+) of SNARE/α-SNAP complex at 25°C for 1 h, respectively. The histogram shows the rates of ATP hydrolysis averaged from three independent measurements. Error bars indicate standard deviations. The fold increase in ATPase activity (stimulation) was calculated by dividing the stimulated ATPase activity by the basal ATPase activity.

Mentions: We first assessed the effect of the mutations at the N-terminal region of the N-D1 linker on the biological function of NSF to disassemble SNARE complex. To assay for this disassembly activity, SNARE complexes were preincubated with wild-type NSF or the mutants and α-SNAP on ice, following the addition of 2 mM Mg2+-ATP. The reactions were performed at 37°C for the indicated times, then immediately stopped with the addition of the SDS-PAGE loading buffer and analyzed by SDS-PAGE. As expected, the amount of SNARE complexes progressively decreased with the increased incubation time (Fig. S1). But all of the mutants, E203A, N204A, Q206A, S207A, I208A, I209A and N210A, had severe defect. None of them were able to disassemble the SNARE complex to the extent of wild-type NSF (Fig. 3A&S1, Table 1). Among them, the N210A mutant shows the most seriously defective in disassembly efficiency, only 6% of that of wild-type NSF (Fig. 3A, Table 1).


The role of the N-D1 linker of the N-ethylmaleimide-sensitive factor in the SNARE disassembly.

Liu CC, Sun S, Sui SF - PLoS ONE (2013)

Mutational effects of the N-terminal region of the NSF N-D1 linker.(A) SNARE disassembly by wild-type and mutant NSF. SNARE complexes were incubated with wild-type or mutant NSF, and α-SNAP in the presence of Mg2+-ATP at 37°C for 0 min, 20 min and 60 min, followed by the addition of SDS-PAGE loading buffer and analyzed by SDS-PAGE. The SNARE proteins were quantified by densitometry using ImageJ. The histogram shows the SNARE disassembly activities of wild-type and mutated NSF at 60 min averaged from three independent measurements and calculated as follows: disassembled protein, obtained by subtracting remaining protein (60 min) from total protein (0 min), divided by total protein (0 min). Values have been normalized to that of wild-type (WT) NSF. Error bars indicate the standard deviation. (B) Binding of wild-type and mutant NSF to the SNARE/α-SNAP complex. Wild-type or mutant NSF proteins were incubated with MBP-SNARE complexes in the presence (+) or absence (−) of α-SNAP under the ADP-AlFx state. The bound proteins were collected with amylose magnetic beads, eluted with 10 mM maltose and analyzed by SDS-PAGE. The gels (left panel) presented are representative of at least two separate experiments. The bound proteins were quantified by densitometry using ImageJ (right panel). (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF. Standard ATPase reactions were carried out using 3 μg of wild-type or mutant NSF in the ATPase assay buffer. SNARE/α-SNAP complex was prepared as described in Materials and Methods. Basal and stimulated ATPase activities were measured in the absence (−) or presence (+) of SNARE/α-SNAP complex at 25°C for 1 h, respectively. The histogram shows the rates of ATP hydrolysis averaged from three independent measurements. Error bars indicate standard deviations. The fold increase in ATPase activity (stimulation) was calculated by dividing the stimulated ATPase activity by the basal ATPase activity.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646813&req=5

pone-0064346-g003: Mutational effects of the N-terminal region of the NSF N-D1 linker.(A) SNARE disassembly by wild-type and mutant NSF. SNARE complexes were incubated with wild-type or mutant NSF, and α-SNAP in the presence of Mg2+-ATP at 37°C for 0 min, 20 min and 60 min, followed by the addition of SDS-PAGE loading buffer and analyzed by SDS-PAGE. The SNARE proteins were quantified by densitometry using ImageJ. The histogram shows the SNARE disassembly activities of wild-type and mutated NSF at 60 min averaged from three independent measurements and calculated as follows: disassembled protein, obtained by subtracting remaining protein (60 min) from total protein (0 min), divided by total protein (0 min). Values have been normalized to that of wild-type (WT) NSF. Error bars indicate the standard deviation. (B) Binding of wild-type and mutant NSF to the SNARE/α-SNAP complex. Wild-type or mutant NSF proteins were incubated with MBP-SNARE complexes in the presence (+) or absence (−) of α-SNAP under the ADP-AlFx state. The bound proteins were collected with amylose magnetic beads, eluted with 10 mM maltose and analyzed by SDS-PAGE. The gels (left panel) presented are representative of at least two separate experiments. The bound proteins were quantified by densitometry using ImageJ (right panel). (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF. Standard ATPase reactions were carried out using 3 μg of wild-type or mutant NSF in the ATPase assay buffer. SNARE/α-SNAP complex was prepared as described in Materials and Methods. Basal and stimulated ATPase activities were measured in the absence (−) or presence (+) of SNARE/α-SNAP complex at 25°C for 1 h, respectively. The histogram shows the rates of ATP hydrolysis averaged from three independent measurements. Error bars indicate standard deviations. The fold increase in ATPase activity (stimulation) was calculated by dividing the stimulated ATPase activity by the basal ATPase activity.
Mentions: We first assessed the effect of the mutations at the N-terminal region of the N-D1 linker on the biological function of NSF to disassemble SNARE complex. To assay for this disassembly activity, SNARE complexes were preincubated with wild-type NSF or the mutants and α-SNAP on ice, following the addition of 2 mM Mg2+-ATP. The reactions were performed at 37°C for the indicated times, then immediately stopped with the addition of the SDS-PAGE loading buffer and analyzed by SDS-PAGE. As expected, the amount of SNARE complexes progressively decreased with the increased incubation time (Fig. S1). But all of the mutants, E203A, N204A, Q206A, S207A, I208A, I209A and N210A, had severe defect. None of them were able to disassemble the SNARE complex to the extent of wild-type NSF (Fig. 3A&S1, Table 1). Among them, the N210A mutant shows the most seriously defective in disassembly efficiency, only 6% of that of wild-type NSF (Fig. 3A, Table 1).

Bottom Line: Mutating the residues in middle and C-terminal region of the N-D1 linker increases the basal ATPase activity, indicating it may play a role in autoinhibiting NSF activity until it encounters SNARE/α-SNAP complex substrate.Moreover, mutations at the C-terminal sequence GIGG exhibit completely abolished or severely reduced activities of the substrate binding, suggesting that the flexibility of N-D1 linker is critical for the movement of the N domain that is required for the substrate binding.Taken together, these data suggest that the whole N-D1 linker is critical for the biological function of NSF to disassemble SNARE complex substrate with different regions responsible for different roles.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.

ABSTRACT
N-ethylmaleimide-sensitive factor (NSF) is a member of the type II AAA+ (ATPase associated with various cellular activities) family. It plays a critical role in intracellular membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes. Each NSF protomer consists of an N-terminal domain (N domain) followed by two AAA ATPase domains (D1 and D2) in tandem. The N domain is required for SNARE/α-SNAP binding and the D1 domain accounts for the majority of ATP hydrolysis. Little is known about the role of the N-D1 linker in the NSF function. This study presents detailed mutagenesis analyses of NSF N-D1 linker, dissecting its role in the SNARE disassembly, the SNARE/α-SNAP complex binding, the basal ATPase activity and the SNARE/α-SNAP stimulated ATPase activity. Our results show that the N-terminal region of the N-D1 linker associated mutants cause severe defect in SNARE complex disassembly, but little effects on the SNARE/α-SNAP complex binding, the basal and the SNARE/α-SNAP stimulated ATPase activity, suggesting this region may be involved in the motion transmission from D1 to N domain. Mutating the residues in middle and C-terminal region of the N-D1 linker increases the basal ATPase activity, indicating it may play a role in autoinhibiting NSF activity until it encounters SNARE/α-SNAP complex substrate. Moreover, mutations at the C-terminal sequence GIGG exhibit completely abolished or severely reduced activities of the substrate binding, suggesting that the flexibility of N-D1 linker is critical for the movement of the N domain that is required for the substrate binding. Taken together, these data suggest that the whole N-D1 linker is critical for the biological function of NSF to disassemble SNARE complex substrate with different regions responsible for different roles.

Show MeSH
Related in: MedlinePlus