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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 C-terminal region of the NSF N-D1 linker.(A) SNARE disassembly by wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3A. (B) Binding of wild-type and mutant NSF proteins to the SNARE/α-SNAP complex. Measurements are the same as in Fig. 3B. (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3C.
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pone-0064346-g005: Mutational effects of the C-terminal region of the NSF N-D1 linker.(A) SNARE disassembly by wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3A. (B) Binding of wild-type and mutant NSF proteins to the SNARE/α-SNAP complex. Measurements are the same as in Fig. 3B. (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3C.

Mentions: As shown in Fig. 5A & Table 1, all of the mutations abrogate the ability to disassemble SNARE complex. Similar to the mutations at the middle region of the N-D1 linker, the C-terminal region mutants exhibit an increase in the basal ATPase activity, and marginal impact on the stimulated ATPase activity (Fig. 5C, Table 1). But different from the middle region mutants, these mutants showed severe defect in the SNARE/α-SNAP complex binding activity (Fig. 5B). Three mutants, G219A, I220A, and G221PG222P, fail to bind the substrate, and mutant G221AG222A retains only very weak binding activity compared with wild-type NSF (Fig. 5B), in the ADP-AlFx bound state. This is consistent with our data that the basal ATPase activities of these mutants are similar to their SNARE/α-SNAP stimulated ATPase activities, namely, there are no substrate stimulation effects on these mutants due to their defects in the substrate binding (Fig. 5B&C, 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 C-terminal region of the NSF N-D1 linker.(A) SNARE disassembly by wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3A. (B) Binding of wild-type and mutant NSF proteins to the SNARE/α-SNAP complex. Measurements are the same as in Fig. 3B. (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0064346-g005: Mutational effects of the C-terminal region of the NSF N-D1 linker.(A) SNARE disassembly by wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3A. (B) Binding of wild-type and mutant NSF proteins to the SNARE/α-SNAP complex. Measurements are the same as in Fig. 3B. (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3C.
Mentions: As shown in Fig. 5A & Table 1, all of the mutations abrogate the ability to disassemble SNARE complex. Similar to the mutations at the middle region of the N-D1 linker, the C-terminal region mutants exhibit an increase in the basal ATPase activity, and marginal impact on the stimulated ATPase activity (Fig. 5C, Table 1). But different from the middle region mutants, these mutants showed severe defect in the SNARE/α-SNAP complex binding activity (Fig. 5B). Three mutants, G219A, I220A, and G221PG222P, fail to bind the substrate, and mutant G221AG222A retains only very weak binding activity compared with wild-type NSF (Fig. 5B), in the ADP-AlFx bound state. This is consistent with our data that the basal ATPase activities of these mutants are similar to their SNARE/α-SNAP stimulated ATPase activities, namely, there are no substrate stimulation effects on these mutants due to their defects in the substrate binding (Fig. 5B&C, 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