[29] Expression and purification of recombinant N-ethylmaleimide-sensitive fusion protein from Escherichia coli

Wilson, Duncan W.; Rothman, James E.

doi:10.1016/0076-6879(92)19031-z

Cited by 13 publications

(8 citation statements)

References 8 publications

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“…LY294002 was kindly provided by Dr. P. Shepherd (Department of Biochemistry, University College, London, UK), aliquoted, and kept at −20°C as a 10 mg/ml stock in DMSO. Recombinant Myc-tagged NSF was purified from cultures of Escherichia coli (strain from Dr. J. Rothman provided with permission by Dr. P. Woodman, Department of Biochemistry and Molecular Biology, University of Manchester, UK) by the procedure of Wilson and Rothman (1992) . Recombinant His-tagged α- and γ-SNAPs were obtained from the same source and purified according to Whiteheart et al (1993) .…”

Section: Methodsmentioning

confidence: 99%

Fusion of Lysosomes with Late Endosomes Produces a Hybrid Organelle of Intermediate Density and Is NSF Dependent

Mullock

Bright

Fearon

et al. 1998

The Journal of Cell Biology

207

183

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Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37°C, late endosome–lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide–sensitive factor– dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.

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Section: Methodsmentioning

confidence: 99%

Fusion of Lysosomes with Late Endosomes Produces a Hybrid Organelle of Intermediate Density and Is NSF Dependent

Mullock

Bright

Fearon

et al. 1998

The Journal of Cell Biology

207

183

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“…1, all mutants were constructed with a carboxyl-terminal myc epitope that allows immunoprecipitation of the recombinant protein by the 9E10 antibody (19). This epitope has been shown to have no effect on the activity of the wild type NSF molecule (24). For purification of the recombinant proteins, E. coli cells were disrupted using a French press, insoluble material was removed by centrifugation, and the His 6 -tagged proteins were purified by NiNTA-agarose (QIAGEN) affinity chromatography as described (22).…”

Section: Methodsmentioning

confidence: 99%

Each Domain of the N-Ethylmaleimide-sensitive Fusion Protein Contributes to Its Transport Activity

Nagiec

Bernstein

Whiteheart

1995

Journal of Biological Chemistry

127

173

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N-Ethylmaleimide-sensitive fusion protein (NSF) has been shown to be involved in numerous intracellular transport events. In an effort to understand the basic mechanism of NSF in vesicle-target membrane fusion events, we have examined the role that each of its three domains play in how NSF interacts with the SNAP.SNARE complex. Mutagenesis of the first ATP-binding domain (D1, amino acids 206-477) demonstrates that nucleotide binding by this domain is required for 20 S particle assembly. A second mutation, which permits ATP binding but not hydrolysis, yields a protein that can form 20 S particle but fails to mediate its disassembly. Similar mutations of the second ATP-binding domain (D2, amino acids 478-744) result in trimeric molecules that behave like wild type NSF. Domain rearrangement mutants were used to further probe the functional role of each domain. The amino-terminal domain (N, amino acids 1-205) is absolutely required for binding of NSF to the SNAP.SNARE complex, because the truncated mutant, D1D2, is unable to form 20 S particle. When tested as an isolated recombinant protein, the N domain is not sufficient for binding to the SNAP.SNARE complex, but when adjacent to the D1 domain or in a trimeric molecule, the N domain does mediate binding to the SNAP.SNARE complex. Monomeric N-D1 and trimeric N-D2 could both participate in particle formation. Only the N-D1 mutant was able to facilitate MgATP-dependent release from the SNAP.SNARE complex. These data demonstrate that NSF binding to the SNAP.SNARE complex is mediated by the N domain and that both ATP binding and hydrolysis by the D1 domain are essential for 20 S particle dynamics. The intramolecular interactions outlined suggest a mechanism by which NSF may use ATP hydrolysis to facilitate the vesicle fusion process.

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“…To address this question, mixed NSF trimers were produced in E. coli by coexpressing a K266A mutant with a His~ at the amino terminus and a myc epitope tag at the carboxy terminus together with a wild-type NSE The mixed NSF trimer could then be separated by Ni2+NTA agarose chromatography into pools that contained no K266A subunits (trimers that did not adhere to the resin) and those that contain at least one K266A mutant subunit. Using monoclonal antibodies that recognized the myc epitope (56), it is possible to show that in the initial pool of NSF (runthrough) does not contain any of the mutant subunit (Fig. 6, A and B).…”

Section: Oligomeric Nature Of Nsf and Its Functional Relevancementioning

confidence: 99%

N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion.

Whiteheart

Rossnagel

Buhrow

et al. 1994

The Journal of Cell Biology

384

404

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Abstract. The NEM-sensitive fusion protein, NSF, together with SNAPs (soluble NSF attachment proteins) and the SNAREs (SNAP receptors), is thought to be generally used for the fusion of transport vesicles to their target membranes. NSF is a homotrimer whose polypeptide subunits are made up of three distinct domains: an amino-terminal domain (N) and two homologous ATP-binding domains (D1 and D2). Mutants of NSF were produced in which either the order or composition of the three domains were altered. These mutants could not support intra-Golgi transport, but they indicated that the D2 domain was required for trimerization of the NSF subunits. Mutations of the first ATP-binding site that affected either the binding (K266A) or hydrolysis (E329Q) of ATP completely eliminated NSF activity. The hydrolysis mutant was an effective, reversible inhibitor of Golgi transport with an IC50 of 125 ng/50 #1 assay. Mutants in the second ATP-binding site (binding, K549A; hydrolysis, D604Q) had either 14 or 42 % the specific activity of the wild-type protein, respectively. Using coexpression of an inactive mutant with wild-type subunits, it was possible to produce a recombinant form of trimeric NSF that contained a mixture of subunits. The mixed NSF trimers were inactive, even when only one mutant subunit was present, suggesting that NSF action requires each of the three subunits in a concerted mechanism. These studies demonstrate that the ability of the D1 domain to hydrolyze ATP is required for NSF activity and, therefore is required for membrane fusion. The D2 domain is required for trimerization, but its ability to hydrolyze ATP is not absolutely required for NSF function.

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[29] Expression and purification of recombinant N-ethylmaleimide-sensitive fusion protein from Escherichia coli

Cited by 13 publications

References 8 publications

Fusion of Lysosomes with Late Endosomes Produces a Hybrid Organelle of Intermediate Density and Is NSF Dependent

Fusion of Lysosomes with Late Endosomes Produces a Hybrid Organelle of Intermediate Density and Is NSF Dependent

Each Domain of the N-Ethylmaleimide-sensitive Fusion Protein Contributes to Its Transport Activity

N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion.

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