Several groups have now shown a requirement for the N-ethylmaleimide-sensitive fusion protein (NSF/Sec18p) ()in numerous intracellular fusion events of both regulated and constitutive secretion (reviewed in (1) ). Kinetic analysis, using the intra-Golgi transport assay, shows that NSF/Sec18p acts at a late stage in the transport process. N-Ethylmaleimide inhibition of intra-Golgi transport leads to an accumulation of uncoated vesicles that appear to be consumed upon the addition of pure NSF(2) . Studies with the temperature-sensitive mutant alleles, sec18-1 or sec18-2, show that under restrictive conditions there is also a build-up of 50 nm vesicles(3) . These results demonstrate that NSF/Sec18p is required for vesicle consumption and suggest that it is involved at or near the actual vesicle-target membrane fusion step. Recent experiments have pointed to a role for NSF in earlier stages, such as vesicle formation or priming(4, 5) . However, these data conflict with other experiments (6, 7) that show that NSF/Sec18p is not required for production of active transport vesicles in vitro. Although its precise function remains to be elucidated, it is clear that NSF is a general transport factor that plays a central role in many (though perhaps not all; (8) ) of the heterotypic fusion events in the cell.

To explain heterotypic fusion events, Rothman and colleagues proposed the SNAP Receptor (SNARE) hypothesis(9) , in which the specificity of vesicle-target membrane docking is mediated by the matching of a t-SNARE from the target membrane with its cognate v-SNARE in the vesicle membrane, thereby forming a docking or 7 S complex. This complex then provides binding sites for the soluble NSF attachment proteins (SNAPs), which are absolutely required to mediate the correct positioning of NSF(10, 11, 12, 13) . This last step serves to complete the formation of the so-called 20 S fusion particle. At least part of the energy for membrane fusion is thought to be provided by the hydrolysis of ATP by NSF, because mutant forms of NSF that are unable to hydrolyze ATP also fail to complete the vesicular transport process(14) . To describe the molecular basis of vesicle-target membrane fusion, it becomes critical to understand how NSF interacts with the other elements of the fusion machinery (SNAPs, SNAREs, etc.) and how it might use the energy from ATP hydrolysis to facilitate membrane fusion.

The subunit of the homotrimeric NSF can be divided into three domains: an amino-terminal (N, amino acids 1-205) and two ATP-binding domains (D1, amino acids 206-477, and D2, amino acids 478-744)(15, 16) . Initially delineated by sequence analysis, these domains probably represent discrete structural entities because they are released by limited proteolysis of the intact molecule(16) . Mutations in the ATP-binding site of domain D1 (binding mutants Lys to Ala (D1K-A), Gln, or Met or hydrolysis mutant Glu to Gln (D1E-Q)) eliminate intra-Golgi transport activity and cause a 70-80% decrease in ATPase activity relative to wild type NSF(14, 17) . These mutant proteins inhibit intra-Golgi transport in a competitive fashion that as demonstrated in this manuscript (see Fig. 1), is most likely due to their ability to form 20 S fusion particle but not mediate MgATP-dependent particle disassembly. The D2 domain is required for trimerization, but its ability to bind (mutant Lys to Ala (D2K-A), Gln, or Met) or hydrolyze (mutant Asp to Gln (D2D-Q)) ATP does not seem to be specifically required for intra-Golgi transport(14, 17) . The ATP hydrolytic activity of this domain makes only a small contribution to the overall ATPase activity of NSF (30-40%)(14) . The amino-terminal domain has been proposed to exert some control over the ATPase activity of NSF because antibodies directed against it cause a 2-fold increase in hydrolytic activity(17) . A similar increase in ATPase activity was observed when NSF was bound to SNAPs that had been immobilized on a plastic surface(18) . We present data demonstrating that the N domain of NSF is required for interaction with the rest of the 20 S particle components. Deletion of this domain results in a trimeric molecule (D1D2) with ATPase activity but no ability to bind to the SNAPSNARE complex. It has been suggested that each of the three domains of NSF has a distinct contribution to the overall activity of the NSF trimer. In this manuscript we propose a role for the N domain in NSF binding to the SNAPSNARE complex and demonstrate the importance of nucleotide binding and hydrolysis by the D1 domain to 20 S particle dynamics. Further dissection of the role of these domains will undoubtedly shed new light on the cellular function of NSF and may elucidate new aspects of the heterotypic fusion process.

Figure 1: A, 20 S Particle formation and disassembly as measured by the SNARE-dependent association of [S]-SNAP with mutant or wild type NSF. Mutant or wild type NSF (15 µg) with the carboxyl-terminal myc epitope were incubated with radiolabeled -SNAP in either the presence or the absence of bovine brain extract (120 µg), which was used as a source of SNARE proteins (SNAREs). Either 0.5 mM ATP (ATP) or ATPS (ATPS) was added to the reactions, and all were maintained in 5 mM MgCl. Anti-myc antibody coupled to protein G-Superose beads was then added, and the immunoprecipitated complexes were collected on glass fiber filters and quantified by scintillation counting. The error bars represent the range of two separate experiments. Abbreviations for the NSF mutants are explained in the text and in Fig. 2. B, MgATP-dependent release of syntaxin from 20 S particle containing either mutant or wild type NSF. Incubations were performed with 0.5 mM ATPS as above except unlabeled -SNAP (54 µg) was added. The complexes were immunoprecipitated and washed in binding buffer. The resulting beads were then incubated with either 5 mM ATP (ATP) or ATPS (ATPS) with 5 mM MgCl, and the proteins released into the supernatant were concentrated and subjected to SDS-polyacrylamide gel electrophoresis and Western blotting using the anti-syntaxin antibody HPC1. Another set of beads was eluted with 0.2 M glycine to determine the total amount of protein bound (Total). The blots presented are representative of two separate experiments.

Figure 2: A, schematic representation of the domain rearrangement mutants. Domain rearrangement mutants were constructed and purified as outlined in Whiteheart et al.(14) . Depicted are the arrangements of the domains in a given mutant subunit together with the abbreviated name of the mutant listed at left. The right column indicates the trimeric state of the recombinant protein. B, point mutations of the D1 and D2 domain ATP-binding sites. The sequences of the D1 and D2 domain ATP-binding sites are depicted with the two point mutations, underlined for both domains. Lysine 266 and 549 were changed to alanine residues as described(14) , and the resulting mutant proteins were denoted D1K-A and D2K-A, respectively. Glutamic acid 329 and aspartic acid 604 were changed to glutamine residues(14) , and the resulting mutant proteins were denoted D1E-Q and D2D-Q respectively. A mutant containing both changes was also used and denoted D1E-Q/D2D-Q.

EXPERIMENTAL PROCEDURES

Materials

Production and Purification of NSF Mutants

20 S Particle Formation and Disassembly Assays

Figure 3: A, SNAP-dependent binding of mutant or wild type NSF to the SNARE complex. Mutant or wild type NSF (15 µg) was incubated with bovine brain extract (200 µg) in the presence or the absence (-SNAP) of -SNAP (5 µg) in the presence of 0.5 mM ATPS (ATPS) or ATP (ATP). The resulting complexes were then precipitated using anti-syntaxin antibody coupled to protein G-Superose. The bound proteins were eluted with 0.2 M glycine and were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with anti-NSF (Bound) or anti-syntaxin (Syntaxin) antibodies. Unbound mutant or wild type NSF in the washes (Unbound) was also concentrated and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. The blots presented are representative of at least two separate experiments. Abbreviations for the NSF mutants are explained in the text and in Fig. 2. B, immunodetection of NSF rearrangement mutants. Equal amounts of wild type and mutant NSF were blotted onto nitrocellulose and immunodecorated with either the monoclonal 6E6 antibody (6E6) or the polyclonal anti-N domain antibody (anti-N). The bands were visualized with the appropriate secondary antibody-horseradish peroxidase conjugates using Enhanced Chemiluminescence. The apparent molecular mass of each full-length protein was: wild type (wt), 82.2 kDa; ND2, 57.4 kDa; D1D2, 63.9 kDa; ND1, 61.6 kDa; N, 25 kDa; and ND2D1, 85.3 kDa.

Miscellaneous Assays of NSF Activity

RESULTS

Role of the ATP-binding Sites of NSF in 20 S Particle Formation and Disassembly

For both assay configurations (Fig. 1, A and B), neither ATP-binding site mutation (Lys-Ala or Asp-Gln; see Fig. 2) in the D2 domain had any effect on 20 S particle formation or the ATP hydrolysis-mediated dissolution of the complex. Binding of radiolabeled -SNAP (Fig. 1A) and syntaxin (Fig. 1B, Total) to the two mutants, (D2K-A and D2D-Q), was essentially identical to the binding by wild type NSF and the addition of MgATP-mediated release of particle components (Fig. 1B, ATP). The D1D2 mutant that lacks the N domain failed to support 20 S particle formation. D1D2 was unable to bind either the radiolabeled SNAP (Fig. 1A) or syntaxin (Fig. 1B, Total; also see Fig. 2A). Despite its trimeric nature and ATPase activity (Table 1), the D1D2 mutant was not active in intra-Golgi transport(14) , probably because it cannot bind to the SNAPSNARE complex.

The ATP-binding mutant D1K-A does not show any 20 S particle formation activity as measured by the [S]-SNAP binding assay (Fig. 1A). When binding was measured in a more sensitive assay under saturating conditions, (Fig. 1B, Total) it appeared that the D1K-A mutant was approximately 10% as efficient at 20 S particle formation as wild type NSF. In the intra-Golgi transport assay, this mutant displayed no transport activity but, interestingly, could inhibit transport when added at high concentrations (IC = 157 nM)(14, 17) . This mutant showed <10% of the inhibitory activity of the ATP hydrolysis mutants D1E-Q (IC = 9.4 nM) or the double mutant D1E-Q/D2D-Q (IC 13.3 nM)(14) . These data suggest that binding of a nucleotide by the D1 domain is an important element of the overall binding of the NSF trimer to the SNAPSNARE complex. The ATP hydrolysis mutants D1E-Q and D1E-Q/D2D-Q participate in 20 S particle formation as does wild type NSF (Fig. 1, A and B, Total). However, when MgATP is added, the particle formed does not disassemble (Fig. 1, A and B, ATP). Because both mutants are inhibitory to intra-Golgi transport (14) but can form 20 S particle, it seems likely that the MgATP-mediated disassembly step by the NSF trimer is a required intermediate in the vesicular transport process and that the ATPase activity of the D1 domain is crucial to that step.

Differential Role of NSF Domains in 20 S Particle Formation

As in Fig. 1, the D1D2 mutant does not exhibit SNAP-dependent binding to the 7 S complex, suggesting that the N domain is a critical element of the interactions of NSF with other components of the 20 S particle (Fig. 3A). Surprisingly, the isolated N domain also failed to show SNAP-dependent binding with the 7 S complex (Fig. 3A). This could simply be due to improper folding of the recombinant domain. However, this His-tagged protein is very stably expressed in E. coli and migrates as a discrete peak of 28 kDa on gel filtration chromatography (data not shown). The fact that the isolated, monomeric N domain does not bind to the SNAPSNARE complex could reflect the need for multiple contact points between NSF and the complex. A single N domain might be incapable of interacting with enough of the requisite binding sites. For the wild type NSF, these contacts could be formed from the cooperative binding of more than one N domain in the context of the trimer or could result from the recognition of more than one NSF domain, i.e. N and D1.

To try to address these possibilities, two mutant forms of NSF were employed, N-D1 and N-D2. N-D1 is monomeric (molecular mass = 80 kDa as determined by gel exclusion chromatography) and has some ATPase activity (18% of wild type; Table 1). The low level of ATPase activity is perhaps not surprising because another oligomeric member (p97) of the family of ATPases associated with a variety of cellular activities also has lower ATPase activity when monomeric(27) . N-D2 is trimeric (molecular mass = 186 kDa) but has no measurable ATPase activity (Table 1). The N-D1 truncate mutant binds to the 7 S complex in a SNAP-dependent fashion with a low (10%) binding efficiency compared with wild type NSF. With the addition of MgATP, the bound N-D1 protein releases from the complex in much the same way full-length NSF does (Fig. 3A), but this truncated N-D1 possesses no intra-Golgi transport activity even at very high concentrations (678 nM, data not shown). The N-D2 mutant also binds in a SNAP-dependent fashion, again at much lower affinity, but the bound protein does not release from the complex upon MgATP addition. This same effect is also seen for the ND2D1 mutant (trimeric with ATPase activity(14) ), which binds inefficiently and does not release after MgATP addition (Fig. 3). From these data we conclude that it is not simply the presence of the N domain that is important for interaction with the SNAPSNARE complex but that it is the context in which the N domain are presented. Isolated N domains fail to interact with the SNAPSNARE complex, unless they are either adjacent to a D1 domain, as would be the case for the wild type NSF and the N-D1 mutant, or presented as a multimer, as would be the case for the wild type NSF or the N-D2 and ND2D1 mutants. These two concepts are not mutually exclusive but are most likely synergistic because none of the mutants bind with the same efficiency as the wild type protein.

Inhibition of Golgi Transport by High Concentrations of Domain Rearrangement Mutants

Figure 4: N-D2 and ND2D1 inhibit the Golgi transport activity but only at high concentration. The indicated amounts of each of the proteins were added to a standard (25 µl) intra-Golgi transport assay using wild type Chinese hamster ovary cytosol as a source of soluble transport factors. Intercisternal Golgi transport of marker protein from the mutant Golgi complex (donor) to the wild type Golgi complex (acceptor) was measured by the incorporation of [H]GlcNAc into the vesicular stomatitis virus G glycoprotein. The control 100% value was 5613 dpm with a background of 15 dpm. Abbreviations for the NSF mutants are explained in the text.

Multiple Contact Sites Are Needed for Active NSF Trimers

()

Figure 5: Mixed wild type/D1D2 trimers are active in intra-Golgi transport. His-D1D2 myc and untagged NSF were co-expressed in E. coli, and the resulting mixed trimeric molecules were partially purified by polyethylene glycol precipitation and then subjected to fractionation on NiNTA-agarose using a gradient of imidazole as eluent. The upper panel represents the profile of eluted protein as analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining. In the lower panel, 0.4-µl aliquots were taken from every third 1-ml fraction and assayed for NSF activity using N-ethylmaleimide-treated Golgi membranes under standard reaction conditions as described under ``Experimental Procedures.'' A background of 440 cpm was subtracted from each data point.

DISCUSSION

In this manuscript we attempt to further understand the functional features of NSF by examining which domains of the molecule are required for which protein-protein interactions in the 20 S fusion complex. To this end, we have demonstrated that interactions of SNAPSNARE complex with NSF are primarily through the N domains of the trimer. Consistent with this is the observation that the mutation leading to the sec18-1 temperature-sensitive allele is present in a region (ClaI fragment Ile-Ile) corresponding to the N domain Sec18p(28) . This same mutant allele exhibits synthetic lethality when combined with the sec17-1 mutant, which is the yeast equivalent of -SNAP(3) .

A secondary interaction with the D1 domain of NSF also appears to play a role in 20 S particle formation, but only when D1 is adjacent to the N domain. ATP binding by the D1 may be an important element for NSF to attain the conformation needed for binding to 20 S particle, although this is not completely clear because other replacements of lysine 266 do not exhibit exactly the same inhibitory behavior(17) . One might expect that the D1K-A mutant would have similar particle binding properties to the wild type protein because both elements for binding, trimerization and adjacency to the D1 domain, are present in the molecule. The fact that D1K-A mutant can bind only weakly (like N-D2 and ND2D1), suggests that there may be a nucleotide-induced conformational change in D1 that affects the N domain. We speculate that binding of NSF to the SNAPSNARE complex may be promoted when an appropriate nucleotide (ATP) is bound to the D1 domain. The mutant proteins (especially the N-D1) described in this manuscript should aid in dissecting the role of nucleotide binding to the D1 domain and its relationship to NSF binding to the SNAPSNARE complex.

The amino acid sequences around the ATP-binding sites in the D1 and D2 domains are characteristic of the family of ATPases associated with a variety of cellular activities(29) . Like NSF, these proteins use ATP hydrolysis to carry out their distinctive cellular functions, and mutations of their ATP-binding sites (especially in the domains most homologous to D1) appear to affect the function of the proteins in ways similar to that shown for NSF (discussed in (14) ). These similarities between the various ATPases associated with a variety of cellular activities suggest that a common mechanism might be used by all of these proteins to carry out their varied cellular functions. In this manuscript, we demonstrate that the ATPase activity of the D1 domain is directly required for fusion complex disassembly and that the N domain is required for NSF localization. This concept that each domain has a distinct contribution to the overall function of NSF may prove to be a useful paradigm for the study of other ATPases associated with a variety of cellular activities.

The major role of the D2 domain is trimer formation, which appears to be essential for NSF activity(26) . The isolated domain, expressed as a recombinant protein in E. coli, is trimeric. ()Earlier studies showed that ATP binding and hydrolysis by this domain are not required for intra-Golgi transport (14, 17) and, as shown in this manuscript, these properties are also not required for 20 S particle dynamics. The N-D2 and ND2D1 mutants bind to the SNAPSNARE complex because the N domains are presented as a trimer, but they cannot release once bound because they lack the adjacent D1 domain. These data suggest that even when the D2 domain is placed adjacent to the N domain in a chimeric molecule, it cannot mimic the conformational effects that D1 exerts on the N domain.

In summary, the data presented here suggest roles for each of the three domains of NSF. The N domain is required for SNAPSNARE complex binding but must be either adjacent to the D1 domain or in a trimeric configuration. These two structural features are most likely synergistic, because neither alone is sufficient to promote the binding efficiency seen for wild type NSF. The D1 domain must be able to hydrolyze ATP to disassemble the 20 S particle, and therefore this step is a required intermediate in the transport process. Consistent with this observation is the recent discovery that the temperature-sensitive, paralytic, mutant comatose is caused by a point mutation (equivalent to Gly to Glu) in the Drosophila NSF gene near the D1 ATP-binding site(30) . From the data presented in this manuscript, we speculate that the binding of nucleotide (ATP) by D1 induces conformational changes in the D1 and N domains that are critical to the interaction of NSF with the other elements of the 20 S fusion particle. In this way, only ATP-charged NSF would be capable of participating in 20 S particle formation. By pairing biochemical and structural analyses, it should be possible to unravel the mechanism of NSF in vesicular transport.

FOOTNOTES

*

This work was supported by Grant CB-153 from the American Cancer Society (to S. W. W.) and by a grant from the University of Kentucky Research Foundation (to S. W. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Biochemistry, University of Kentucky College of Medicine, Chandler Medical Center, 800 Rose St., Lexington, KY 40536-0084. Tel.: 606-323-1065; Fax: 606-323-1037.

()

The abbreviations used are: NSF, N-ethylmaleimide-sensitive fusion protein; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; NiNTA, nickel nitrotriacetic acid; ATPS, adenosine 5`-O-(3-thiotriphosphate).

()

S. W. Whiteheart, unpublished observation.

()

S. W. Whiteheart and B. Boggess, unpublished observation.

ACKNOWLEDGEMENTS

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