From the Department of Cell Biology and Physiology,
Washington University School of Medicine, St. Louis, Missouri 63110 and the ¶ Department of Biochemistry, University of Kentucky
College of Medicine, Chandler Medical Center, Lexington, Kentucky
40536
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ABSTRACT |
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The
N-ethylmaleimide-sensitive
factor (NSF) is required for multiple intracellular vesicle
transport events. In vitro biochemical studies have
demonstrated that NSF, soluble NSF
attachment proteins (SNAPs), and SNAP receptors
form a 20 S particle. This complex is disassembled by the ATPase
activity of NSF. We have studied particle disassembly in a membrane
environment by examining the binding of recombinant SNAPs and NSF to
endosomal membranes. We present evidence that -SNAP is released from
the membranes in a temperature- and time-dependent manner
and that this release is mediated by the ATPase activity of NSF. Our
results indicate that NSF mutants in the first ATP binding domain
completely abrogate
-SNAP release, whereas no inhibitory effect is
observed with a mutant in the second ATP binding domain. Interestingly,
neither
-SNAP nor
-SNAP are released by the ATPase activity of
NSF, indicating that these proteins are retained on the membranes by interactions that differ from those that retain
-SNAP. Although the
small Rab GTPases are known to play a role in SNARE complex assembly,
our results indicate that these GTPases do not regulate the
NSF-dependent release of
-SNAP.
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INTRODUCTION |
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The molecular mechanisms driving movement of proteins between compartments have been unraveled, in part, by the development of cell-free assays that reconstitute various transport and fusion events. Multiple membrane fusion events depend on NSF,1 the N-ethylmaleimide-sensitive factor (for a review see Refs. 1-4) that was originally identified as a protein required for Golgi transport (5). NSF has been shown to be essential for intracellular transport between the endoplasmic reticulum and Golgi (6, 7), from the Golgi to the plasma membrane (8, 9), homotypic vacuole fusion (10), transcytosis (11) and fusion among endosomes (12-14), as well as endocytosis in yeast (15). Recently, NSF also has been found to be involved in exocytosis and neurotransmitter release (2, 16-19).
Membrane association of NSF requires accessory proteins termed -,
-, and
-SNAPs (soluble NSF
attachment proteins) (20, 21).
- and
-SNAPs function synergistically and are widely distributed among
tissues, whereas
-SNAP seems to be a brain specific isoform of
-SNAP. SNAPs bind to SNAREs (SNAP
receptors), specific proteins present in the vesicle and in
the target membrane. A set of SNAREs from bovine brain extracts was
first identified by Rothman and colleagues (22). These receptors are
the neuronal proteins synaptobrevin or VAMP (vesicle
associated membrane protein), syntaxin and SNAP 25 (synaptosomal associated protein of 25 kDa) involved in synaptic transmission (for a review see Ref. 23). In
vitro biochemical studies have demonstrated that NSF, SNAPs, and
SNAREs form a 20 S complex (22, 24) that is proposed to function in
vesicle targeting, docking, or fusion. To form a stable 20 S particle,
a nonhydrolyzable analog of ATP, e.g. ATP
S, is required.
In the presence of ATP, hydrolysis of this nucleotide by the ATPase
activity of NSF results in the dissociation of the 20 S complex into
its component subunits (22). Although the significance of this
disassembly remains controversial, an attractive possibility is that
the energy of ATP hydrolysis drives a conformational change in one or
more of the SNARE proteins. It is important to emphasize that even
though NSF is widely recognized as an essential factor in multiple
transport events this protein's exact function is still unknown.
SNARE complex disassembly has been studied mainly using recombinant
purified components and detergent extracts (22, 25, 26). To gather
further insight into the molecular mechanism of particle disassembly in
a membrane environment, we studied the binding of recombinant SNAPs and
NSF to endosomal membranes. We present evidence that -SNAP is
released from the membranes in a temperature- and
time-dependent manner and that this release is mediated by
the ATPase activity of NSF. Our results indicate that certain NSF
mutants (in the first ATP binding domain) completely abrogate
-SNAP
release, whereas no inhibitory effect is observed with another NSF
mutant (in the second ATP binding domain). Interestingly, neither
-SNAP nor
-SNAP were released by the ATPase activity of NSF,
indicating that these proteins are retained on the membranes by
interactions that are different from those that retain
-SNAP. Our
results also indicate that NSF-dependent release of
-SNAP is not regulated by GTPases.
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EXPERIMENTAL PROCEDURES |
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Cells and Materials--
J774 E-clone (mannose receptor
positive), a macrophage cell line, was grown to confluence in minimum
essential medium containing Earle's salts and supplemented with 10%
fetal calf serum. Cytosol from J774 was the high speed supernatant of a
cell homogenate obtained as described (27, 28) and stored at
80 °C. Cytosol samples (200 µl) were gel filtered through 1-ml
G-25 Sephadex spin columns just before use in the fusion assay. Protein
concentration after filtration was 3-5 mg/ml. Recombinant NSF wild
type and mutants were prepared and purified essentially as described
(29, 30). Recombinant
-,
-, and
-SNAPs were prepared and
purified as described previously (31). The monoclonal antibody, CI 77.1 anti
/
SNAP, was a generous gift from Dr. Reinhard Jahn (Yale University, New Haven, CT). All other chemicals were obtained from
Sigma.
Preparation of Endosomal Membranes--
J774 macrophages (1 × 108 cells) were washed sequentially with 150 mM NaCl, 5 mM EDTA, 10 mM phosphate
buffer, pH 7.0, and with 250 mM sucrose, 0.5 mM
EGTA, 20 mM Hepes-KOH, pH 7.0 (homogenization buffer) and
homogenized in the latter buffer (2 ml) using a cell homogenizer (28).
Homogenates were centrifuged at 800 × g for 5 min to
eliminate nuclei and intact cells. Postnuclear fractions were pelleted
for 1 min at 37,000 × g in a Beckman L 100 centrifuge. The supernatants were centrifuged for an additional 5 min at
50,000 × g. The pellets of this second centrifugation
were enriched with 5 min endosomes. Endosomal fractions were washed
with homogenization buffer to remove cytosolic proteins, pelleted again
and resuspended in the same buffer. The samples (200-µl aliquots)
were frozen quickly in liquid nitrogen and stored at 80 °C.
NSF/SNAP Binding to Endosomes--
Endosomal fractions (200-µl
aliquot) were thawed quickly, diluted with homogenization buffer
containing 1 M KCl, and supplemented with trypsin inhibitor
and p-amidinophenylmethylsulfonyl fluoride hydrochloride as
protease inhibitors. Samples were incubated for 10 min at 4 °C
followed by a 5-10 min incubation period at 37 °C to remove
endogenous NSF and SNAP. The stripped endosomal fractions were
collected by centrifugation at 45,000 rpm for 15 min using a TL 100 rotor (Beckman). The membranes (15 µg/tube) were incubated in the
same buffer (250 mM sucrose, 0.5 mM EGTA, 20 mM Hepes-KOH, pH 7.0, 1 mM dithiothreitol, 1.5 mM MgCl2, 50 mM KCl, 1 mM ATP, 8 mM creatine phosphate, 31 units/ml
creatine phosphokinase) used in a standard endosome fusion assay (27,
28) containing a mixture of protease inhibitors. Binding assays were
carried out in fusion buffer in a final volume of 30 µl supplemented
with 100 nM recombinant NSF and/or SNAPs. In some assays,
the ATP-regenerating system was replaced by an ATP-depleting system (5 mM mannose, 25 units/ml hexokinase). Proteins were bound to
the membranes for 5-10 min at 4 °C. Subsequently, membranes were
incubated for 20 min at either 4 or 37 °C to study the effect of
temperature on the release of bound proteins. After incubation, the
endosomal membranes were washed with 1 ml of homogenization buffer and
sedimented by centrifugation. In some experiments, the samples were
centrifuged to separate bound proteins (pellet) from unbound proteins
(supernatant). Membrane pellets were solubilized in SDS-polyacrylamide
gel electrophoresis sample buffer. Proteins were separated by
SDS-polyacrylamide gel electrophoresis and transferred onto
nitrocellulose membranes using standard techniques. NSF was detected by
Western blot analysis with the mouse monoclonal antibody 6E6 (32) at a
1:500 dilution. NSF-truncated mutants were detected with both the
monoclonal antibody 6E6 and an additional rabbit polyclonal anti-NSF.
/
-SNAPs were detected using the monoclonal antibody CI 77.1 (26)
at a dilution of 1:10,000, whereas
-SNAP was detected using a rabbit
polyclonal antibody generated against the whole recombinant protein.
Visualization was performed with a horseradish peroxidase rabbit
anti-mouse (1:5,000) or horseradish peroxidase-coupled goat anti-rabbit
antibody (1:2,000) with the Lumiglo chemiluminescence reagents (Pierce) according to the manufacturer's instructions.
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RESULTS |
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NSF-dependent Release of -SNAP Requires ATP
Hydrolysis--
It has been shown previously that ATP hydrolysis by
NSF disassembles the SNARE complex (22, 25). However, the formation and
disruption of this complex has been studied mainly with recombinant purified components and detergent extracts (22, 25, 26, 44, 45). To
gain further insight into the molecular mechanism involved in particle
disassembly in a membrane environment, we studied the binding of
recombinant SNAPs and NSF to endosomal membranes. For this purpose, an
endosomal fraction was prepared as described under "Experimental
Procedures." The membranes were incubated with 1 M KCl in
homogenization buffer to strip off the endogenous peripheral
membrane-associated proteins. Membranes then were incubated in fusion
buffer supplemented with recombinant NSF and/or
-SNAP for 5-10 min
at 4 °C to allow for binding of the proteins. Subsequently, the
samples were incubated for an additional 20 min at either 4 or
37 °C. The endosomal membranes were then sedimented by
centrifugation, and bound proteins were detected by Western blotting.
Because
-SNAP binds to plastic tubes (35), we precoated all of the
tubes used in the binding experiments with 10% ovalbumin in
phosphate-buffered saline. Very little binding of
-SNAP and NSF was
detected when ovalbumin-coated tubes were incubated with these proteins
in the absence of endosomal membranes (data not shown).
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NSF Regulates -SNAP but not
- or
-SNAP Release--
Three
different forms of SNAPs,
-,
- and
-SNAP (35, 36 and 39 KDa,
respectively), have been purified from bovine brain (21).
- and
-SNAP are widely distributed among tissues, whereas
-SNAP seems
to be a brain specific isoform of
-SNAP (
- and
-SNAP are 83%
identical to each other). Therefore, we were interested in determining
whether
- and
-SNAP were released from the membranes in a manner
similar to that of
-SNAP. As shown in Fig.
4, panel A, neither
-SNAP
nor
-SNAP were released from the membranes by the ATPase activity of
NSF, suggesting that they are retained by the membranes through
interactions that are different from those that retain
-SNAP.
Interestingly, when
-SNAP and
-SNAP were added together,
-SNAP
was released by wild-type NSF while
-SNAP remained bound (Fig. 4,
panel B). This result suggests that, in contrast to what is
observed in the in vitro particle disassembly reaction (35),
-SNAP is not released by the ATPase activity of NSF when monitored
in a membrane environment.
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NSF-dependent Release of -SNAP Is Not Regulated by
Rab GTPases--
Genetic interactions and biochemical studies have
demonstrated that Rab proteins, a class of small GTPases, play a role
in the assembly of specific SNARE complexes (38, 39). We were interested in determining the role of GTPases in the NSF-mediated release of
-SNAP. For this purpose, salt-washed endosomal membranes were preincubated for 30 min at 25 °C in the absence (control) or
presence of 100 µM GDP
S or GTP
S. Samples then were
incubated for 20 min at either 4 or 37 °C in fusion buffer
containing 100 nM
-SNAP with or without recombinant NSF.
Fig. 5, panel A shows that
neither GDP
S nor GTP
S blocked the NSF-mediated release of
-SNAP.
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DISCUSSION |
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An important conceptual highlight regarding the molecular machinery involved in intracellular transport and neurotransmitter release came from the discovery of the SNAREs (for a review see Ref. 42), membrane receptors for SNAP (22). NSF binds through SNAPs to a complex of neuronal proteins comprised of synaptobrevin, syntaxin, and SNAP 25. Disassembly of this complex by NSF occurs in the presence of ATP and is thought to be critical for membrane fusion not only at the synaptic level but also in intracellular transport events (reviewed in Refs. 1, 3, 4, and 42).
In the present report, we present evidence that -SNAP is released
from the membranes in a temperature- and time-dependent manner. This release is absolutely dependent on the ATPase activity of
NSF, as mutations in NSF that render the protein defective in either
ATP binding or hydrolysis result in almost complete inhibition of
-SNAP release. Our data indicate that the release of
-SNAP takes
place after a few minutes of incubation at 37 °C and is almost
complete by 15-20 min. Our results agree with those of Wickner and
collaborators (43) showing that Sec18p (yeast NSF homologue) drives the
release of Sec17p (yeast
-SNAP homologue) after a short incubation
at 37 °C. Interestingly, we have observed previously that endosome
fusion becomes insensitive to NSF mutants after 15 min (14). However,
at that point only 20% of the total measurable fusion has occurred.
Therefore,
-SNAP is released long before the fusion reaction is
complete. Both results are consistent with the idea of an early action
for NSF. This hypothesis receives independent support from studies in
regulated exocytosis that show NSF acting at a prefusion
ATP-dependent step but not at the Ca2+
triggered fusion step (16, 18). The recent finding that Sec18p/Sec17p are required in a priming step before the actual docking event (44)
provides additional support for the idea that NSF is required for early
steps.
NSF·SNAP·SNARE complex formation requires the presence of ATP, and
disassembly is driven by conditions that favor ATP hydrolysis (22). It
has been shown that in the presence of ATP NSF bound to syntaxin
through -SNAP catalyzes a conformational change in the syntaxin
molecule that induces the release of both NSF and
-SNAP (26).
However, most of these studies have been carried out with recombinant
proteins and/or detergent extracts (22, 25, 26, 33, 34). Our binding
studies with membranes indicate that upon incubation with
Mg2+-ATP most
-SNAP is released, whereas NSF is released
only partially from the membranes. These data suggest that although
part of NSF is bound to membranes in an
-SNAP-dependent
manner there is a fraction of NSF that is retained by the membranes
through an
-SNAP-independent mechanism. However, this NSF binding in
the absence of SNAP was sensitive to proteinase K and heat treatment,
indicating that NSF associates with a protein factor perhaps distinct
from the classical NSF/SNAP receptors. Our results are consistent with the observations of Mayer et al. (43). They showed that in
yeast, although the release of Sec17p is rapid and complete, little
Sec18p is released from the membranes. This finding indicates that
Sec18p must be bound to the membranes via a protein distinct from
Sec17p. Moreover, in a recent publication, it has been shown that
Sec18p (yeast NSF) binds to GST-Pep12p, a putative yeast SNARE, in a Sec17p (yeast SNAP) independent manner (45). Interestingly, Woodman and
co-workers have shown that treatment of coated vesicles with
Mg2+-ATP did not result in release of NSF (46). Therefore,
it appears that NSF is retained on coated vesicles by interactions that
are different from the classical NSF·SNAP·SNARE complexes. In
addition, it has been shown previously that NSF associated with
synaptic vesicles (47) and with growth cones (48) was not released upon
incubation with Mg2+-ATP. Considered together, these
results indicate that in the presence of Mg2+-ATP there is
a pool of NSF that remains associated with membranes perhaps in an
-SNAP independent manner.
In contrast to -SNAP, neither
-SNAP nor
-SNAP were released
from the membranes by the ATPase activity of NSF, suggesting that they
are retained by interactions that are different from those that retain
-SNAP. Indeed, it has been shown that
-SNAP unlike
-SNAP binds
synaptotagmin and recruits NSF (49). In agreement with our data, this
complex is unaffected by Mg2+-ATP. Interestingly, when
-
and
-SNAP were added together,
-SNAP was released by NSF whereas
-SNAP was not released. Although cross-linking experiments indicate
that
- and
-SNAP interact directly when bound to membranes (31),
it is possible that this interaction is transient and occurs only under
certain experimental conditions (e.g. in the absence of
Mg2+-ATP). Moreover, it has been shown that
-SNAP binds
to a different membrane receptor protein or at a site that is distinct
from the
/
-SNAP-binding site (34, 35). This differential
interaction is consistent with our observation that the nearly complete
release of
-SNAP from membranes is not accompanied by
-SNAP
release.
Among the proteins that have been implicated in the regulation of
intracellular membrane transport are the members of the Rab family of
monomeric GTPases (for review see Refs. 50-53). Although Rab proteins
do not seem to be core components of the SNARE complex, genetic
evidence supports a role for the Rab proteins in the assembly of this
complex (38, 39). In addition, evidence for a functional link between
Rab3 and the SNARE complex has been presented recently (54). Our data
indicate that NSF-driven release of -SNAP is not regulated by
GTPases. Therefore, although complex assembly relies on Rab function,
it is likely that the NSF-mediated disassembly does not. Our results
are consistent with previous results showing that the release of sec17p
in yeast was not influenced by GTP
S or by antibodies against the Rab
protein homologue Ypt7p (43).
We believe that binding studies using a membrane environment, such as those described in this report, should prove to be useful models for further biochemical analyses and for the identification of other molecules that regulate the assembly and disassembly of the docking/fusion machinery.
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ACKNOWLEDGEMENTS |
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We thank Drs. Phyllis Hanson and Carmen Alvarez-Dominguez for critical reading of this manuscript. We thank Dr. Chun Zhi Yang for kindly providing purified GDI and for advice regarding the proper use of this protein. We also are grateful to Moges Taddese and Aysel Monniae for technical assistance.
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FOOTNOTES |
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* This work was supported in part by a National Institutes of Health grant (to P. D. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendoza, Argentina. Tel.: 54-61-205-115 (ext. 2670); Fax: 54-61-494-117.
1
The abbreviations used are: NSF,
N-ethylmaleimide-sensitive factor; SNAP, soluble NSF
attachment protein; SNARE, SNAP receptor; GTPS, guanosine
5
-3-O-(thio)triphosphate; GDP
S, guanosine
5'-O-2-(thio)diphosphate; GDI, GTP dissociation inhibitor;
ATP
S, adenosine 5
-O-(thiotriphosphate).
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REFERENCES |
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