©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
SNAP Prevents Mg-ATP-induced Release of N-Ethylmaleimide-sensitive Factor from the Golgi Apparatus in Digitonin-permeabilized PC12 Cells (*)

(Received for publication, September 6, 1995)

Mitsuo Tagaya (§) Akiko Furuno Shoji Mizushima

From the School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(6)-tagged alpha-SNAP exhibited such activity. His(6)-tagged alpha-SNAP also prevented the Mg-ATP-induced release of NSF from isolated Golgi membranes.


INTRODUCTION

The N-ethylmaleimide-sensitive factor (NSF) (^1)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 alpha-, beta-, 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 alpha-SNAP.


EXPERIMENTAL PROCEDURES

Materials

PC12 cells were purchased from the Riken Cell Bank. Chinese hamster ovary Golgi membranes were prepared as described by Balch et al.(19) . Bovine brain cytosol was prepared as described by Malhotra et al.(20) . Monoclonal anti-NSF (2E5) was prepared as described previously(13) . The following plasmid and antibodies were kindly donated (suppliers in parentheses): a plasmid encoding His(6)-tagged alpha-SNAP and polyclonal anti-alpha-SNAP (Drs. M. Brunner and J. E. Rothman), polyclonal anti-mannosidase II (Dr. K. Moremen), and polyclonal anti-VAMP-2 and monoclonal anti-synaptotagmin (Dr. Masami Takahashi).

Cell Culture

PC12 cells were grown on glass or plastic coverslips precoated with collagen (type I) in Dulbecco's modified Eagle's medium containing 50 IU/ml penicillin, 50 µg/ml streptomycin, 7.5% fetal calf serum, and 7.5% horse serum. Neuronal cell cultures were prepared by the treatment of PC12 cells with 40 ng/ml of nerve growth factor for 3-4 days in Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) containing 10 mM HEPES (pH 7.2), 50 IU/ml penicillin, 50 µg/ml streptomycin, and 1 times GMS-G (Life Technologies, Inc.).

Cell Permeabilization

PC12 cells were washed with ice-cold PBS twice and then incubated in EDTA-ATP buffer (25 mM HEPES (pH 7.2) containing 4 mM EDTA, 1 mM ATP, 50 mM KCl, and 0.25 M sucrose) in the presence of 33-40 µM digitonin at 0 °C for 10 min. After washing with ice-cold EDTA-ATP buffer twice, the cells were incubated in EDTA-ATP buffer or Mg-ATP buffer (25 mM HEPES (pH 7.2) containing 5 mM MgCl(2), 1 mM ATP, 50 mM KCl, and 0.25 M sucrose) at 0 °C for 30 min.

Immunofluorescence Staining

Permeabilized cells were fixed in PBS containing 4% paraformaldehyde and 0.15 M sucrose, treated with PBS containing 0.2% Triton X-100, and then incubated in PBS containing 2% bovine serum albumin. After incubation with primary antibodies, the cells were incubated with fluorescein-conjugated goat anti-mouse IgGs (Cappel Organon Teknika) and/or rhodamine-conjugated goat anti-rabbit IgGs (Chemicon). The coverslips were mounted with PBS containing 90% glycerol, 1 mg/ml p-phenylenediamine, and 10 mM sodium azide and then observed under an Olympus BX50 microscope.

NSF Release from Isolated Golgi Membranes

Isolated Golgi membranes were incubated in EDTA-ATP or Mg-ATP buffer (0.4 ml) in the presence or the absence of His(6)-tagged alpha-SNAP on ice. After centrifugation at 100,000 times g for 30 min, the supernatant was concentrated with 6% trichloroacetic acid and 0.02% deoxycholate. The membrane pellet and the concentrated supernatant were subjected to electrophoresis according to the method of Laemmli (21) with 10% gels. Detection of NSF on immunoblots was performed with the ECL system. The amount of NSF on blots was determined densitometrically with a Shimazu CS-9300PC scanning densitometer.


RESULTS

Mg-ATP Induces the Release of NSF from the Golgi Apparatus in Permeabilized PC12 Cells

We first investigated the distribution of NSF in PC12 cells, in which NSF is highly expressed. Significant immunoreactivity for NSF was observed on one side of the perinuclear region (Fig. 1A). Double immunofluorescence analysis revealed that this staining pattern coincides well with that of the mannosidase II, a medial Golgi marker protein (Fig. 1, A and B). The faint immunoreactivity for NSF throughout cells may reflect its presence in the cytosol. When a control antibody was used instead of anti-NSF antibody, no significant staining was observed at any region of cells (Fig. 1C).


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. (^2)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).



SNAP Prevents the Mg-ATP-induced Release of NSF from the Golgi Apparatus

When a bovine brain cytosolic fraction was added on incubation with Mg-ATP, NSF was not released from the Golgi apparatus by Mg-ATP (Fig. 4). This effect was dose-dependent, and almost complete inhibition of the release of NSF was observed at cytosol concentrations of 200-700 µg/ml. The cytosol lost this inhibitory activity on heat treatment. These results suggest the presence of a protein factor(s) in bovine brain cytosol that prevents Mg-induced NSF release from the Golgi apparatus.


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 alpha-, beta-, 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 alpha- and beta-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. alpha- and beta-SNAPs were not resolved with this electrophoretic system. The bar corresponds to 20 µm.



alpha- and -SNAPs are expressed in a wide range of tissues(31) , and alpha-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 alpha-SNAP on the Mg-ATP-induced release of NSF from the Golgi apparatus. When His(6)-tagged alpha-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 alpha-SNAP, and almost complete inhibition was observed at concentrations of 1-2 µg/ml. His(6)-tagged alpha-SNAP lost this inhibitory activity on heat treatment, as observed in bovine brain cytosol.


Figure 6: alpha-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(6)-tagged alpha-SNAP or heat-treated His(6)-tagged alpha-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 alpha- and beta-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 alpha- and beta-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) .

alpha-SNAP Prevents the Mg-ATP-induced Release of NSF from Isolated Golgi Membranes

We next examined whether or not alpha-SNAP prevents the Mg-ATP-induced release of NSF from isolated Golgi membranes. For this purpose, isolated Golgi membranes were incubated with EDTA-ATP or Mg-ATP in the presence or the absence of alpha-SNAP, and the amounts of membrane-associated NSF and released NSF were estimated as described under ``Experimental Procedures.'' When Golgi membranes were incubated with EDTA-ATP, only 17% of total NSF was released from membranes, whereas 83% was not ( Fig. 7and Table 1). On the other hand, on incubation with Mg-ATP, the amount of NSF recovered in the supernatant increased and that in the pellet decreased. The amount of total NSF in the the Mg-ATP-treated sample was 1.4 times larger than that in the EDTA-ATP-treated sample. This was probably due to the slight underestimation of the amount of NSF in the pellet. The efficiency of immunostaining seemed to be low when samples contained membranes. Although the results were semiquantitative, it was obvious that a major fraction of NSF associated with membranes is released in a Mg-ATP-dependent manner. When 0.5 µg/ml or higher concentrations of His(6)-tagged alpha-SNAP were added during incubation with Mg-ATP, the amount of NSF recovered in the supernatant decreased to a level found on incubation with EDTA-ATP, suggesting that alpha-SNAP also prevents the Mg-ATP-induced release of NSF from isolated Golgi membranes. We do not know why 30-35% of the membrane-associated NSF was not released on incubation with Mg-ATP. One possibility is that our Golgi membrane preparations contain membranes in which NSF is not released by Mg-ATP. As demonstrated by the previous (18) and present studies, NSF associated with synaptic vesicles and the nuclear membranes are not released by Mg-ATP. Likewise, NSF associated with other membranes such as endosome vesicles, which are present in Golgi membrane preparations, may not be released by Mg-ATP.


Figure 7: alpha-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(6)-tagged alpha-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 alpha-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 alpha-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, alpha-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 alpha-SNAP after a 15-min incubation resulted in the reassociation of the released NSF with Golgi membranes, suggesting that alpha-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 alpha-SNAP was added at the start of the incubation. It is possible that alpha-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: alpha-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(6)-tagged alpha-SNAP (lanes 4 and 8). NSF in the pellets (lanes 1-4) and the supernatants (lanes 5-8) was visualized by immunoblotting.






DISCUSSION

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(6)-tagged alpha-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 alpha- and beta-SNAPs. alpha-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 alpha-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 alpha-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 alpha-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) .


FOOTNOTES

*
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and by the Ciba-Geigy Foundation (Japan) for the Promotion of Science. 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: School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan. Fax: 81-426-76-8866.

(^1)
The abbreviations used are: NSF, N-ethylmaleimide-sensitive factor; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; PBS, phosphate-buffered saline.

(^2)
M. Tagaya and S. Mizushima, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Masami Takahashi (Mitsubishi Kasei Institute of Life Science), James E. Rothman and Michael Brunner (Memorial Sloan-Kettering Cancer Center), and Kelly Moremen (University of Georgia) for providing plasmid and antibodies.


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