(Received for publication, September 6, 1995)
From the
The N-ethylmaleimide-sensitive factor (NSF), which is
involved in the multisteps of protein transport, is released from Golgi
membranes on in vitro incubation with
Mg-ATP. However, several lines of evidence suggest
that NSF is associated with membranes in spite of the presence of
Mg
and ATP in vivo. We have used
digitonin-permeabilized PC12 cells to investigate the mechanism
underlying the association of NSF with membranes. In PC12 cells,
immunoreactivity for NSF was observed in the nuclear membranes, the
Golgi apparatus, and neuronal growth cones, where synaptic vesicles are
concentrated. NSF associated with the Golgi apparatus was released on
incubation with Mg
-ATP, whereas NSF in the nuclear
membranes and neuronal growth cones was not released on the same
treatment. The addition of cytosol blocked the
Mg
-ATP-induced release of NSF from the Golgi
apparatus. Chromatographic analyses revealed that the factor(s) that
prevents NSF release from the Golgi apparatus was eluted at the same
position as the soluble NSF attachment proteins (SNAPs). Purified
His
-tagged
-SNAP exhibited such activity.
His
-tagged
-SNAP also prevented the
Mg
-ATP-induced release of NSF from isolated Golgi
membranes.
The N-ethylmaleimide-sensitive factor (NSF) ()was originally characterized as a protein that is
implicated in intra-Golgi vesicle-mediated protein
transport(1, 2) . Several lines of evidence suggest
that NSF most likely mediates the fusion of Golgi-derived transport
vesicles with target membranes(3) . Later studies revealed that
NSF is also involved in protein transport from the endoplasmic
reticulum to the Golgi apparatus(4) , endosome
fusion(5) , and exocytosis of
neurotransmitters(6, 7) . NSF is a soluble protein,
and its attachment to membranes in the absence of
Mg
-ATP is mediated by three peripheral membrane
proteins named
-,
-, and
-SNAPs(8, 9) .
Söllner et al.(10) showed that
syntaxin 1, SNAP-25, and VAMP-2 are membrane-embedded SNAP receptors
(SNAREs). NSF, SNAPs, and SNAREs are associated to form a 20 S complex
in membranes(10, 11) . Incubation of Golgi membranes
with Mg
-ATP induces the disassembly of the 20 S
complex, and thereby results in the release of membrane-bound
NSF(1, 10, 11, 12) . The driving
force disrupting the 20 S complex is probably derived from the
NSF-catalyzed hydrolysis of ATP. NSF possesses N-ethylmaleimide-sensitive ATPase activity (13) , and
the two homologous nucleotide-binding regions of NSF are involved in
ATP hydrolysis(14, 15) .
NSF seems to exist as both
membrane-associated and free forms in vivo, although
intracellular concentrations of Mg and ATP are high
enough to induce the disassembly of the 20 S complex. This is suggested
by the fact that isolated Golgi membranes contain sufficient amounts of
NSF to accomplish intra-Golgi protein
transport(1, 2, 3) . In other cell-free
assays that reconstitute the secretory and endocytotic transport
pathways in which NSF is involved, NSF activity seems to be also
derived from membrane
fractions(4, 5, 6, 16, 17) .
In addition, we recently showed that NSF is associated with isolated
synaptic vesicles (18) . These results raise the possibility
that there is a factor(s) that mediates the association of NSF to
membranes in the presence of Mg
-ATP.
In the
present study we investigated the mechanism underlying the association
of NSF with Golgi apparatus by using digitonin-permeabilized PC12 cells
and isolated Golgi membranes. We found that the
Mg-ATP-induced release of NSF from the Golgi
apparatus is prevented by
-SNAP.
Figure 1: Comparison of the distribution of NSF and Golgi-resident mannosidase II in PC12 cells. Nonneural PC12 cells were double labeled with antibodies against NSF (A) and mannosidase II (B) or labeled with a control mouse IgG (C). The bar corresponds to 20 µm.
We next
examined whether Mg-ATP induces the release of NSF
from the Golgi apparatus in digitonin-permeabilized PC12 cells, as
observed in isolated Golgi membranes(1) . Digitonin is a
nonionic detergent that has been used to permeabilize a variety of
cells(22, 23, 24) . During digitonin
permeabilization and subsequent incubation, the cytosol gradually leaks
from digitonin-permeabilized cells(25) . When PC12 cells were
solubilized with digitonin in the presence of EDTA-ATP for 10 min,
washed to remove the detergent, and then incubated with
Mg
-ATP for 30 min, almost all NSF disappeared from
the Golgi area (Fig. 2A). The immunoreactivity for NSF
throughout cells also disappeared. The disappearance of NSF was not due
to distortion or loss of the Golgi apparatus because the mannosidase II
immunostaining pattern did not change with this treatment (Fig. 2B). Interestingly, the presence of NSF in the
nuclear membranes became obvious when NSF was released from the Golgi
apparatus. It should be noted that immunoreactivity for NSF was less
significant but detectable in the nuclear membranes of nontreated PC12
cells (Fig. 1A), suggesting that the presence of NSF in
the nuclear membranes is not a consequence of permeabilization and
incubation with Mg
-ATP. We isolated nuclei from
bovine adrenal medulla and found that NSF is indeed associated with the
nuclear membranes. (
)Previous studies showed that
Mg
is essential for the release of NSF from isolated
Golgi membranes(1, 11) . Consistent with the results,
the release of NSF from the Golgi apparatus in permeabilized cells did
not occur in the absence of Mg
(Fig. 2C). Fig. 2(C and D) shows the co-localization of NSF and mannosidase II more
clearly than Fig. 1(A and B) due to the loss
of cytosolic immunoreactivity.
Figure 2:
NSF
was released from the Golgi apparatus by Mg-ATP but
not EDTA-ATP in digitonin-permeabilized PC12 cells. Permeabilized
nonneural PC12 cells were incubated in Mg-ATP buffer (A and B) or EDTA-ATP buffer (C and D). Double
immunofluorescence for NSF (A and C) and mannosidase
II (B and D). The bar corresponds to 20
µm.
The differentiation of PC12 cells can
be induced by treatment with nerve growth factor(26) . When
neural (nerve growth factor-treated) PC12 cells were used, NSF
associated with the Golgi apparatus behaved in essentially the same
manner as that in the nonneural cells, that is, NSF in the Golgi
apparatus was released by Mg-ATP (Fig. 3A) but not by EDTA-ATP (Fig. 3C). In neural PC12 cells, punctate
immunoreactivity for NSF was observed in neuritic processes including
neuronal growth cones (Fig. 3, A, B, and C). Synaptic vesicles were concentrated at growth cones, as
revealed by the presence of VAMP-2 (Fig. 3D) and
synaptotagmin (Fig. 3E), both of which are synaptic
vesicle-associated
proteins(27, 28, 29, 30) . It is,
therefore, reasonable to assume that the immunoreactivity for NSF in
neuronal growth cones reflects the association of NSF with synaptic
vesicles. It is noteworthy that NSF in neuronal growth cones was not
released on incubation with Mg
-ATP (Fig. 3B). This is consistent with our previous finding
that NSF is not released from rat synaptic vesicles by
Mg
-ATP(18) . When almost all NSF had been
removed from the Golgi area on incubation with
Mg
-ATP, immunoreactivity for VAMP-2 became detectable
in the Golgi area (Fig. 3D). Such a pattern was not
observed when permeabilized PC12 cells were incubated in EDTA-ATP (data
not shown). The co-localization of NSF and VAMP-2 in the Golgi area of
Mg
-ATP-treated cells was recognizable because of the
presence of a trace amount of NSF in the Golgi area (Fig. 3, A and D). This finding can be explained by the idea
that the epitope of VAMP-2 became accessible to an anti-VAMP antibody
after the release of NSF. Because NSF is a large protein, it is
possible that NSF covers the epitope of membrane-embedded VAMP-2. Such
co-localization was not significantly observed in nonneural cells (data
not shown).
Figure 3:
Distribution of NSF and synaptic
vesicle-associated proteins in digitonin-permeabilized neural PC12
cells. Permeabilized neural PC12 cells were incubated in Mg-ATP buffer (A, B, D, and E) or EDTA-ATP (C). Mg-ATP-treated cells were double
labeled for NSF (A) and VAMP-2 (D). A neuronal growth
cone in a Mg
-ATP-treated cell was labeled for NSF (B). EDTA-ATP-treated cells were labeled for NSF (C).
Mg
-ATP-treated cells were labeled for synaptotagmin (E). In C, the Golgi apparatus is located in punctate
structures surrounding the nucleus. The arrow in A indicates NSF in the Golgi area that slightly remained after
Mg
-ATP treatment. The bars correspond to 20
µm (A, C, D, and E) or 10
µm (B).
Figure 4: A cytosolic factor(s) prevents the release of NSF from the Golgi apparatus in permeabilized PC12 cells. Permeabilized PC12 cells were incubated in Mg-ATP buffer containing the indicated concentrations of bovine brain cytosol or heat-treated cytosol. Heat treatment was carried out at 100 °C for 5 min. The bar corresponds to 20 µm.
When bovine brain cytosol was fractionated
with Superose 12, the factor(s) was eluted in fractions 12 to 14 (Fig. 5). This elution position corresponds to that of
30-40-kDa proteins. Because the relative molecular masses of
-,
-, and
-SNAPs are 35, 36, and 39 kDa,
respectively(8) , the co-elution of SNAPs in these fractions
was examined. Immunoblotting analysis with an anti-SNAP antibody that
recognizes
- and
-SNAPs revealed that these SNAPs were eluted
in these fractions. When bovine brain cytosol was fractionated with a
Q-cartridge column, the factor(s) was also co-eluted with SNAPs (data
not shown).
Figure 5:
The activity that prevents the
Mg-ATP-induced release of NSF from the Golgi
apparatus was co-fractionated with SNAPs. Elution profile on Superose
12 chromatography. A bovine brain cytosolic fraction (0.6 ml) was
applied to a Superose 12 column that had been equilibrated with 25
mM Tris-HCl (pH 7.5) containing 50 mM KCl and 0.5
mM dithiothreitol. The column was developed with the same
buffer at the flow rate of 0.25 ml/min, and fractions of 1 ml each were
collected. Portions of the fractions were used for measurement of the
activity preventing the release of NSF from the Golgi apparatus in
permeabilized neural PC12 cells and for immunoblotting for SNAPs.
- and
-SNAPs were not resolved with this electrophoretic
system. The bar corresponds to 20
µm.
- and
-SNAPs are expressed in a wide range of
tissues(31) , and
-SNAP shows about 6-fold higher activity
than
-SNAP in an intra-Golgi protein transport
assay(8, 9, 31) . We therefore examined the
effect of
-SNAP on the Mg
-ATP-induced release of
NSF from the Golgi apparatus. When His
-tagged
-SNAP
purified from Escherichia coli was added on incubation with
Mg
-ATP after permeabilization, it prevented the
Mg
-ATP-induced release of NSF from the Golgi
apparatus (Fig. 6). The inhibition of NSF release was detectable
at 0.6 µg/ml
-SNAP, and almost complete inhibition was
observed at concentrations of 1-2 µg/ml.
His
-tagged
-SNAP lost this inhibitory activity on heat
treatment, as observed in bovine brain cytosol.
Figure 6:
-SNAP prevents the
Mg
-ATP-induced NSF release from the Golgi apparatus
in permeabilized PC12 cells. Permeabilized nonneural PC12 cells were
incubated in Mg-ATP buffer containing the indicated concentrations of
His
-tagged
-SNAP or heat-treated
His
-tagged
-SNAP. Essentially the same results were
obtained for neural PC12 cells. The bar corresponds to 20
µm.
To determine whether
or not SNAPs are major factors in bovine brain cytosol that prevent the
release of NSF from the Golgi apparatus, we determined the content of
SNAPs in our cytosol preparation. Immunoblotting analysis revealed that
the content of SNAPs (the sum of - and
-SNAPs) comprised
approximately 0.3-0.4% of the total cytosolic proteins (data not
shown). This value is in good agreement with that reported by Clary and
Rothman(8) . Based on this estimation, the concentration of
SNAPs was calculated to be 0.6-2.8 µg/ml in 200-700
µg/ml cytosol. Because 1-2 µg/ml SNAPs is required for
almost complete inhibition, we concluded that the majority of the
activity that prevents Mg
-ATP-induced NSF release
from the Golgi apparatus in bovine brain cytosol is due to
- and
-SNAPs. Of course, it is possible that
-SNAP synergistically
prevents the release of NSF from the Golgi apparatus, as observed in
the binding of NSF to isolated Golgi membranes(11) .
Figure 7:
-SNAP prevents the
Mg
-ATP-induced NSF release from isolated Golgi
membranes. Isolated Golgi membranes were incubated at 0 °C for 30
min in EDTA-ATP buffer (lanes 1 and 6) or Mg-ATP
buffer in the absence (lanes 2 and 7) or the presence
of 0.5 µg/ml (lanes 3 and 8), 2.0 µg/ml (lanes 4 and 9), or 10 µg/ml
His
-tagged
-SNAP (lanes 5 and 10).
NSF in the pellets (lanes 1-5) and the supernatants (lanes 6-10) was visualized by
immunoblotting.
There are several possible explanations for the inhibitory effect of
-SNAP on the release of NSF from the Golgi apparatus. Because the
release of NSF occurs under conditions favoring ATP hydrolysis, one
possibility is that
-SNAP inhibits the ATPase activity of NSF.
However, this possibility is unlikely because a recent study
demonstrated that SNAPs stimulate the ATPase activity of
NSF(32) . Another possibility is that SNAPs bind to released
NSF and return it to the Golgi apparatus. To examine the latter
possibility,
-SNAP was added after a 15-min incubation of isolated
Golgi membranes with Mg
-ATP, and then the incubation
was continued for another 15 min. The amount of NSF released by 15 min
was comparable to that released by 30 min, indicating that the release
of NSF by Mg
-ATP is almost completed by 15 min ( Fig. 8and Table 2). The addition of
-SNAP after a
15-min incubation resulted in the reassociation of the released NSF
with Golgi membranes, suggesting that
-SNAP has the ability to
mediate the reassociation of NSF with Golgi membranes. It was not
certain whether NSF was not released from Golgi membranes or released
and then returned to Golgi membranes when
-SNAP was added at the
start of the incubation. It is possible that
-SNAP binds to
disassembled NSF before its release from Golgi membranes and makes NSF
become attached to the membranes if it is present at the start of the
incubation with Mg
-ATP.
Figure 8:
-SNAP mediates reassociation of NSF
with isolated Golgi membranes. Isolated Golgi membranes were incubated
at 0 °C in EDTA-ATP buffer for 30 min (lanes 1 and 5) or Mg-ATP buffer for 30 (lanes 2 and 6)
or 15 min (lanes 3 and 7). Alternatively, Golgi
membranes were incubated at 0 °C in Mg-ATP buffer for 15 min and
then further incubated for 15 min in the presence of 10 µg/ml
His
-tagged
-SNAP (lanes 4 and 8).
NSF in the pellets (lanes 1-4) and the supernatants (lanes 5-8) was visualized by
immunoblotting.
In the present study, we used digitonin-permeabilized PC12
cells to investigate the mechanism underlying the association of NSF
with membranes. NSF associated with the Golgi apparatus was released by
Mg-ATP, as observed with isolated Golgi
membranes(1) , whereas NSF located in the nuclear membranes and
neuronal growth cones was not released by Mg
-ATP. The
addition of bovine brain cytosol prevented the release of NSF from the
Golgi apparatus in a concentration-dependent manner. This effect was
prevented by heat treatment. These results suggest the presence of a
protein factor(s) that inhibits the release of NSF. Gel filtration and
ion exchange chromatography of bovine brain cytosol revealed that the
factor(s) was co-eluted with SNAPs. Furthermore, purified
His
-tagged
-SNAP exhibited such activity. Estimation
of the SNAP content in bovine brain cytosol suggested that the majority
of the activity that prevents the release of NSF from the Golgi
apparatus is due to
- and
-SNAPs.
-SNAP also prevented
the Mg
-ATP-induced release of NSF from isolated Golgi
membranes.
SNAPs were identified as components that mediate the
attachment of NSF to Golgi membranes(33) . Because NSF is
released from Golgi membranes by Mg-ATP(1) ,
SNAP activity had only been measured in the presence of
EDTA-ATP(8, 9, 33) . No one has so far
investigated whether or not SNAPs have the ability to mediate the
attachment of NSF to Golgi membranes in the presence of
Mg
-ATP. The present results clearly show that
-SNAP prevents the Mg
-ATP-induced release of NSF
from Golgi membranes by mediating the reassociation of disassembled NSF
with membranes. Söllner et
al.(10, 12) demonstrated that a 20 S complex
comprising NSF, SNAPs, and SNAREs is completely disassembled by
Mg
-ATP. In their experiments, reconstitution of the
20 S complex was performed by incubation of NSF and
-SNAP in an
equimolar ratio. This may be the reason why the formation of the
complex does not occur in the presence of Mg
-ATP. In
the present study, an excess amount of
-SNAP was added over NSF
that is present in permeabilized cells and isolated Golgi membranes.
Under this condition, SNAP may bind to the disassembled NSF and thereby
cause reassociation of the 20 S complex.
It was recently found that
SNAPs stimulate the regulated exocytosis of catecholamine in chromaffin
cells (6) and the transport of vesicular stomatitis-virus
encoded glycoprotein to the basolateral membrane(17) .
Paradoxically, NSF does not stimulate regulated exocytosis(6) .
Morgan and Burgoyne (6) assumed that NSF is expressed at
supramaximal levels in chromaffin cells and that SNAPs expression is
limiting. Because Mg-ATP is indispensable for protein
transport and therefore always included in cell-free protein transport
assays, some fraction of NSF must be released from membranes because of
the limited amount of SNAPs in the assay mixture. If so, it is expected
that the addition of SNAPs increases the number of NSF molecules
associated with membranes in the presence of Mg
-ATP
and therefore stimulates protein transport.
According to the model
proposed by Rothman and his
colleagues(10, 12, 34) , synaptotagmin, a
vesicle-SNARE (VAMP-2), and target SNAREs (syntaxin and SNAP-25) form a
complex, and then NSF and SNAPs bind to this complex in the presence of
calcium. Subsequent ATP hydrolysis by NSF causes the disassembly of the
complex, which in turn promotes membrane fusion. This model predicts
the transient binding of NSF from the cytosol to the SNAREs complex on
the plasma membrane. However, there is so far no direct evidence that
NSF and SNAP derived from the cytosolic pool mediate membrane fusion.
We previously showed that NSF is associated with synaptic vesicles in
the absence of calcium influx and not released on incubation with
Mg-ATP (18) . Based on these findings and the
results of kinetic studies involving an intra-Golgi protein transport
assay(14, 35) , we suggested that NSF is a
constitutive component of transport vesicles. Consistent with this
idea, NSF is present in punctate structures in the neuronal growth
cones of neural PC12 cells, where synaptic vesicles are concentrated.
As in the case of rat brain synaptic vesicles, NSF in the cone area is
not released on incubation with Mg
-ATP. The present
results combined with the recent finding that syntaxin 1 is associated
with vesicles as well as target membranes (36, 37) may
raise serious questions regarding Rothman's
hypothesis(34) . Similar questions were also recently raised by
Morgan and Burgoyne(38) .