(Received for publication, August 1, 1995; and in revised form, August 30, 1995)
From the
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 SNAPSNARE
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.
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
SNAP
SNARE 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 SNAP
SNARE 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 ATP
S (ATP
S) 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 ATP
S 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
ATP
S (ATP
S) 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.
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 ATP
S (ATP
S) 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.
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 SNAP
SNARE 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 SNAP
SNARE
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.
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 SNAP
SNARE 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 SNAP
SNARE complex but that it is the context
in which the N domain are presented. Isolated N domains fail to
interact with the SNAP
SNARE 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.
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.
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.
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 SNAP
SNARE 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
SNAP
SNARE 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.