Limits...
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.

Show MeSH

Related in: MedlinePlus

Protein purification and the oligomerization analysis of the NSF mutants.(A) Purity of the NSF proteins. The final purified proteins (3 μg of each proteins) were resolved by 13.5% SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. The molecular size standards are shown on the left. (B) Oligomeric state analysis by size-exclusion chromatography in the buffer containing ADP-AlFx. The typical profiles of NSF (WT), E203A (a representative mutant of N-terminal region), P211A (a representative mutant of middle region), G221PG222P (a representative mutant of C-terminal region), N210A and E216A (the mutants that show the strongest effect in SNARE disassembly assay) from a Superose-6 column were present.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646813&req=5

pone-0064346-g002: Protein purification and the oligomerization analysis of the NSF mutants.(A) Purity of the NSF proteins. The final purified proteins (3 μg of each proteins) were resolved by 13.5% SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. The molecular size standards are shown on the left. (B) Oligomeric state analysis by size-exclusion chromatography in the buffer containing ADP-AlFx. The typical profiles of NSF (WT), E203A (a representative mutant of N-terminal region), P211A (a representative mutant of middle region), G221PG222P (a representative mutant of C-terminal region), N210A and E216A (the mutants that show the strongest effect in SNARE disassembly assay) from a Superose-6 column were present.

Mentions: Plasmids expressing wild-type NSF and N-D1 linker mutants were introduced into E. coli BL21 (DE3). The proteins were initially purified by Ni-NTA superflow affinity chromatography, then further purified by gel filtration chromatography using a Superdex-200 column. The final purity was analyzed by the SDS-PAGE gel, showing the high purity of each protein (Fig. 2A).


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)

Protein purification and the oligomerization analysis of the NSF mutants.(A) Purity of the NSF proteins. The final purified proteins (3 μg of each proteins) were resolved by 13.5% SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. The molecular size standards are shown on the left. (B) Oligomeric state analysis by size-exclusion chromatography in the buffer containing ADP-AlFx. The typical profiles of NSF (WT), E203A (a representative mutant of N-terminal region), P211A (a representative mutant of middle region), G221PG222P (a representative mutant of C-terminal region), N210A and E216A (the mutants that show the strongest effect in SNARE disassembly assay) from a Superose-6 column were present.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0064346-g002: Protein purification and the oligomerization analysis of the NSF mutants.(A) Purity of the NSF proteins. The final purified proteins (3 μg of each proteins) were resolved by 13.5% SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. The molecular size standards are shown on the left. (B) Oligomeric state analysis by size-exclusion chromatography in the buffer containing ADP-AlFx. The typical profiles of NSF (WT), E203A (a representative mutant of N-terminal region), P211A (a representative mutant of middle region), G221PG222P (a representative mutant of C-terminal region), N210A and E216A (the mutants that show the strongest effect in SNARE disassembly assay) from a Superose-6 column were present.
Mentions: Plasmids expressing wild-type NSF and N-D1 linker mutants were introduced into E. coli BL21 (DE3). The proteins were initially purified by Ni-NTA superflow affinity chromatography, then further purified by gel filtration chromatography using a Superdex-200 column. The final purity was analyzed by the SDS-PAGE gel, showing the high purity of each protein (Fig. 2A).

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