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• W2078341212 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 Mg2+-ATP. However, several lines of evidence suggest that NSF is associated with membranes in spite of the presence of Mg2+ 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 NSF is associated with membranes-ATP, whereas NSF in the nuclear membranes and neuronal growth cones was not released on the same treatment. The addition of cytosol blocked the Mg2+-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 His6-tagged α-SNAP exhibited such activity. His6-tagged α-SNAP also prevented the Mg2+-ATP-induced release of NSF from isolated Golgi membranes. 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 Mg2+-ATP. However, several lines of evidence suggest that NSF is associated with membranes in spite of the presence of Mg2+ 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 NSF is associated with membranes-ATP, whereas NSF in the nuclear membranes and neuronal growth cones was not released on the same treatment. The addition of cytosol blocked the Mg2+-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 His6-tagged α-SNAP exhibited such activity. His6-tagged α-SNAP also prevented the Mg2+-ATP-induced release of NSF from isolated Golgi membranes. INTRODUCTIONThe N-ethylmaleimide-sensitive factor (NSF) 1The abbreviations used are: NSFN-ethylmaleimide-sensitive factorSNAPsoluble NSF attachment proteinSNARESNAP receptorPBSphosphate-buffered saline. was originally characterized as a protein that is implicated in intra-Golgi vesicle-mediated protein transport(1.Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Crossref PubMed Scopus (208) Google Scholar, 2.Block M.R. Glick B.S. Wilcox C.A. Wieland F.T. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7852-7856Crossref PubMed Scopus (396) Google Scholar). Several lines of evidence suggest that NSF most likely mediates the fusion of Golgi-derived transport vesicles with target membranes(3.Malhotra V. Orci L. Glick B.S. Block M.R. Rothman J.E. Cell. 1988; 54: 221-227Abstract Full Text PDF PubMed Scopus (263) Google Scholar). Later studies revealed that NSF is also involved in protein transport from the endoplasmic reticulum to the Golgi apparatus(4.Beckers C.J.M. Block M.R. Glick B.S. Rothman J.E. Balch W.E. Nature. 1989; 339: 397-398Crossref PubMed Scopus (188) Google Scholar), endosome fusion(5.Diaz R. Mayorga L.S. Weidman P.J. Rothman J.E. Stahl P.D. Nature. 1989; 339: 398-400Crossref PubMed Scopus (157) Google Scholar), and exocytosis of neurotransmitters(6.Morgan A. Burgoyne R.D. EMBO J. 1995; 14: 232-239Crossref PubMed Scopus (95) Google Scholar, 7.DeBello W.M. O'Connor V. Dresbach T. Whiteheart S.W. Wang S.S.-H. Schweizer F.E. Betz H. Rothman J.E. Augustine G.J. Nature. 1995; 373: 626-630Crossref PubMed Scopus (134) Google Scholar). NSF is a soluble protein, and its attachment to membranes in the absence of Mg2+-ATP is mediated by three peripheral membrane proteins named α-, β-, and γ-SNAPs(8.Clary D.O. Rothman J.E. J. Biol. Chem. 1990; 265: 10109-10117Abstract Full Text PDF PubMed Google Scholar, 9.Clary D.O. Griff I.C. Rothman J.E. Cell. 1990; 61: 709-721Abstract Full Text PDF PubMed Scopus (402) Google Scholar). Söllner et al.(10.Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2602) Google Scholar) 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.Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2602) Google Scholar, 11.Wilson D.W. Whiteheart S.W. Wiedmann M. Brunner M. Rothman J.E. J. Cell Biol. 1992; 117: 531-538Crossref PubMed Scopus (210) Google Scholar). Incubation of Golgi membranes with Mg2+-ATP induces the disassembly of the 20 S complex, and thereby results in the release of membrane-bound NSF(1.Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Crossref PubMed Scopus (208) Google Scholar, 10.Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2602) Google Scholar, 11.Wilson D.W. Whiteheart S.W. Wiedmann M. Brunner M. Rothman J.E. J. Cell Biol. 1992; 117: 531-538Crossref PubMed Scopus (210) Google Scholar, 12.Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1573) Google Scholar). 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.Tagaya M. Wilson D.W. Brunner M. Arango N. Rothman J.E. J. Biol. Chem. 1993; 268: 2662-2666Abstract Full Text PDF PubMed Google Scholar), and the two homologous nucleotide-binding regions of NSF are involved in ATP hydrolysis(14.Sumida M. Hong R.-M. Tagaya M. J. Biol. Chem. 1994; 269: 20636-20641Abstract Full Text PDF PubMed Google Scholar, 15.Whiteheart S.W. Rossnagel K. Buhrow S.A. Brunner M. Jaenicke R. Rothman J.E. J. Cell Biol. 1994; 126: 945-954Crossref PubMed Scopus (340) Google Scholar).NSF seems to exist as both membrane-associated and free forms in vivo, although intracellular concentrations of Mg2+ 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.Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Crossref PubMed Scopus (208) Google Scholar, 2.Block M.R. Glick B.S. Wilcox C.A. Wieland F.T. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7852-7856Crossref PubMed Scopus (396) Google Scholar, 3.Malhotra V. Orci L. Glick B.S. Block M.R. Rothman J.E. Cell. 1988; 54: 221-227Abstract Full Text PDF PubMed Scopus (263) Google Scholar). 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.Beckers C.J.M. Block M.R. Glick B.S. Rothman J.E. Balch W.E. Nature. 1989; 339: 397-398Crossref PubMed Scopus (188) Google Scholar, 5.Diaz R. Mayorga L.S. Weidman P.J. Rothman J.E. Stahl P.D. Nature. 1989; 339: 398-400Crossref PubMed Scopus (157) Google Scholar, 6.Morgan A. Burgoyne R.D. EMBO J. 1995; 14: 232-239Crossref PubMed Scopus (95) Google Scholar, 16.Rodriguez L. Stirling C.J. Woodman P.G. Mol. Biol. Cell. 1994; 5: 773-783Crossref PubMed Scopus (78) Google Scholar, 17.Ikonen E. Tagaya M. Ullrich O. Montecucco C. Simons K. Cell. 1995; 81: 571-580Abstract Full Text PDF PubMed Scopus (220) Google Scholar). In addition, we recently showed that NSF is associated with isolated synaptic vesicles (18.Hong R.-M. Mori H. Fukui T. Moriyama Y. Futai M. Yamamoto A. Tashiro Y. Tagaya M. FEBS Lett. 1994; 350: 253-257Crossref PubMed Scopus (59) Google Scholar). These results raise the possibility that there is a factor(s) that mediates the association of NSF to membranes in the presence of Mg2+-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 Mg2+-ATP-induced release of NSF from the Golgi apparatus is prevented by α-SNAP.EXPERIMENTAL PROCEDURESMaterialsPC12 cells were purchased from the Riken Cell Bank. Chinese hamster ovary Golgi membranes were prepared as described by Balch et al.(19.Balch W.E. Dunphy W.G. Braell W.A. Rothman J.E. Cell. 1984; 39: 405-416Abstract Full Text PDF PubMed Scopus (478) Google Scholar). Bovine brain cytosol was prepared as described by Malhotra et al.(20.Malhotra V. Serafini T. Orci L. Shepherd J.C. Rothman J.E. Cell. 1989; 58: 329-336Abstract Full Text PDF PubMed Scopus (319) Google Scholar). Monoclonal anti-NSF (2E5) was prepared as described previously(13.Tagaya M. Wilson D.W. Brunner M. Arango N. Rothman J.E. J. Biol. Chem. 1993; 268: 2662-2666Abstract Full Text PDF PubMed Google Scholar). The following plasmid and antibodies were kindly donated (suppliers in parentheses): a plasmid encoding His6-tagged α-SNAP and polyclonal anti-α-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 CulturePC12 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 × GMS-G (Life Technologies, Inc.).Cell PermeabilizationPC12 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 MgClS2, 1 mM ATP, 50 mM KCl, and 0.25 M sucrose) at 0°C for 30 min.Immunofluorescence StainingPermeabilized 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 MembranesIsolated Golgi membranes were incubated in EDTA-ATP or Mg-ATP buffer (0.4 ml) in the presence or the absence of His6-tagged α-SNAP on ice. After centrifugation at 100,000 × 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.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205950) Google Scholar) 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.RESULTSMg2+-ATP Induces the Release of NSF from the Golgi Apparatus in Permeabilized PC12 CellsWe 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).We next examined whether Mg2+-ATP induces the release of NSF from the Golgi apparatus in digitonin-permeabilized PC12 cells, as observed in isolated Golgi membranes(1.Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Crossref PubMed Scopus (208) Google Scholar). Digitonin is a nonionic detergent that has been used to permeabilize a variety of cells(22.Diaz R. Stahl P.D. Methods Cell Biol. 1989; 31: 25-43Crossref PubMed Scopus (24) Google Scholar, 23.Adam S.A. Sterne-Marr R. Gerace L. Methods Cell Biol. 1991; 35: 469-482Crossref PubMed Scopus (46) Google Scholar, 24.Plutner H. Davidson H.W. Saraste J. Balch W.E. J. Cell Biol. 1992; 119: 1097-1116Crossref PubMed Scopus (173) Google Scholar). During digitonin permeabilization and subsequent incubation, the cytosol gradually leaks from digitonin-permeabilized cells(25.Sarafian T. Aunis D. Bader M.-F. J. Biol. Chem. 1987; 262: 16671-16676Abstract Full Text PDF PubMed Google Scholar). 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 Mg2+-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 Mg2+-ATP. We isolated nuclei from bovine adrenal medulla and found that NSF is indeed associated with the nuclear membranes. 2M. Tagaya and S. Mizushima, unpublished data. Previous studies showed that Mg2+ is essential for the release of NSF from isolated Golgi membranes(1.Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Crossref PubMed Scopus (208) Google Scholar, 11.Wilson D.W. Whiteheart S.W. Wiedmann M. Brunner M. Rothman J.E. J. Cell Biol. 1992; 117: 531-538Crossref PubMed Scopus (210) Google Scholar). Consistent with the results, the release of NSF from the Golgi apparatus in permeabilized cells did not occur in the absence of Mg2+ (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 Mg2+-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The differentiation of PC12 cells can be induced by treatment with nerve growth factor(26.Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4816) Google Scholar). 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 Mg2+-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.Jahn R. Schiebler W. Quimet C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4137-4141Crossref PubMed Scopus (727) Google Scholar, 28.Wiedenmann B. Franke W.W. Cell. 1985; 41: 1017-1028Abstract Full Text PDF PubMed Scopus (1214) Google Scholar, 29.Trimble W.S. Cowan D.M. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4538-4542Crossref PubMed Scopus (449) Google Scholar, 30.Baumert M. Maycox P.R. Navone F. De Camilli P. Jahn R. EMBO J. 1989; 8: 379-384Crossref PubMed Scopus (396) Google Scholar). 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 Mg2+-ATP (Fig. 3B). This is consistent with our previous finding that NSF is not released from rat synaptic vesicles by Mg2+ -ATP(18.Hong R.-M. Mori H. Fukui T. Moriyama Y. Futai M. Yamamoto A. Tashiro Y. Tagaya M. FEBS Lett. 1994; 350: 253-257Crossref PubMed Scopus (59) Google Scholar). When almost all NSF had been removed from the Golgi area on incubation with Mg2+-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 Mg2+-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). Mg2+-ATP-treated cells were double labeled for NSF (A) and VAMP-2 (D). A neuronal growth cone in a Mg2+-ATP-treated cell was labeled for NSF (B).EDTA-ATP-treated cells were labeled for NSF (C). Mg2+-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 Mg2+-ATP treatment. The bars correspond to 20 μm (A, C, D, and E) or 10 μm (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)SNAP Prevents the Mg2+-ATP-induced Release of NSF from the Golgi ApparatusWhen a bovine brain cytosolic fraction was added on incubation with Mg2+-ATP, NSF was not released from the Golgi apparatus by Mg2+-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 Mg2+-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 α-, β-, and γ-SNAPs are 35, 36, and 39 kDa, respectively(8.Clary D.O. Rothman J.E. J. Biol. Chem. 1990; 265: 10109-10117Abstract Full Text PDF PubMed Google Scholar), the co-elution of SNAPs in these fractions was examined. Immunoblotting analysis with an anti-SNAP antibody that recognizes α- and β-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 Mg2+-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. α- and β-SNAPs were not resolved with this electrophoretic system. The bar corresponds to 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)α- and γ-SNAPs are expressed in a wide range of tissues(31.Whiteheart S.W. Griff I.C. Brunner M. Clary D.O. Mayer T. Buhrow S.A. Rothman J.E. Nature. 1993; 362: 353-355Crossref PubMed Scopus (223) Google Scholar), and α-SNAP shows about 6-fold higher activity than γ-SNAP in an intra-Golgi protein transport assay(8.Clary D.O. Rothman J.E. J. Biol. Chem. 1990; 265: 10109-10117Abstract Full Text PDF PubMed Google Scholar, 9.Clary D.O. Griff I.C. Rothman J.E. Cell. 1990; 61: 709-721Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 31.Whiteheart S.W. Griff I.C. Brunner M. Clary D.O. Mayer T. Buhrow S.A. Rothman J.E. Nature. 1993; 362: 353-355Crossref PubMed Scopus (223) Google Scholar). We therefore examined the effect of α-SNAP on the Mg2+-ATP-induced release of NSF from the Golgi apparatus. When His6-tagged α-SNAP purified from Escherichia coli was added on incubation with Mg2+-ATP after permeabilization, it prevented the Mg2+-ATP-induced release of NSF from the Golgi apparatus (Fig. 6). The inhibition of NSF release was detectable at 0.6 μg/ml α-SNAP, and almost complete inhibition was observed at concentrations of 1-2 μg/ml. His6-tagged α-SNAP lost this inhibitory activity on heat treatment, as observed in bovine brain cytosol.Figure 6:α-SNAP prevents the Mg2+-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 His6-tagged α-SNAP or heat-treated His6-tagged α-SNAP. Essentially the same results were obtained for neural PC12 cells. The bar corresponds to 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 α- and β-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.Clary D.O. Rothman J.E. J. Biol. Chem. 1990; 265: 10109-10117Abstract Full Text PDF PubMed Google Scholar). 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 Mg2+-ATP-induced NSF release from the Golgi apparatus in bovine brain cytosol is due to α- and β-SNAPs. Of course, it is possible that γ-SNAP synergistically prevents the release of NSF from the Golgi apparatus, as observed in the binding of NSF to isolated Golgi membranes(11.Wilson D.W. Whiteheart S.W. Wiedmann M. Brunner M. Rothman J.E. J. Cell Biol. 1992; 117: 531-538Crossref PubMed Scopus (210) Google Scholar).α-SNAP Prevents the Mg2+-ATP-induced Release of NSF from Isolated Golgi MembranesWe next examined whether or not α-SNAP prevents the Mg2+-ATP-induced release of NSF from isolated Golgi membranes. For this purpose, isolated Golgi membranes were incubated with EDTA-ATP or Mg2+-ATP in the presence or the absence of α-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. 7 and Table 1). On the other hand, on incubation with Mg2+-ATP, the amount of NSF recovered in the supernatant increased and that in the pellet decreased. The amount of total NSF in the the Mg2+-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 Mg2+-ATP-dependent manner. When 0.5 μg/ml or higher concentrations of His6-tagged α-SNAP were added during incubation with Mg2+-ATP, the amount of NSF recovered in the supernatant decreased to a level found on incubation with EDTA-ATP, suggesting that α-SNAP also prevents the Mg2+-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 Mg2+-ATP. As demonstrated by the previous (18.Hong R.-M. Mori H. Fukui T. Moriyama Y. Futai M. Yamamoto A. Tashiro Y. Tagaya M. FEBS Lett. 1994; 350: 253-257Crossref PubMed Scopus (59) Google Scholar) and present studies, NSF associated with synaptic vesicles and the nuclear membranes are not released by Mg2+-ATP. Likewise, NSF associated with other membranes such as endosome vesicles, which are present in Golgi membrane preparations, may not be released by Mg2+-ATP.Figure 7:α-SNAP prevents the Mg2+-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 His6-tagged α-SNAP (lanes 5 and 10). NSF in the pellets (lanes 1-5) and the supernatants (lanes 6-10) was visualized by immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tabled 1 Open table in a new tab There are several possible explanations for the inhibitory effect of α-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 α-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.Morgan A. Dimaline R. Burgoyne R.D. J. Biol. Chem. 1994; 269: 29347-29350Abstract Full Text PDF PubMed Google Scholar). Another possibility is that SNAPs bind to released NSF and return it to the Golgi apparatus. To examine the latter possibility, α-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 Mg2+-ATP is almost completed by 15 min (Fig. 8 and Table 2). The addition of α-SNAP after a 15-min incubation resulted in the reassociation of the released NSF with Golgi membranes, suggesting that α-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 α-SNAP was added at the start of the incubation. It is possible that α-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 Mg2+-ATP.Figure 8:α-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 His6-tagged α-SNAP (lanes 4 and 8). NSF in the pellets (lanes 1-4) and the supernatants (lanes 5-8) was visualized by immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tabled 1 Open table in a new tab DISCUSSIONIn 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 Mg2+-ATP, as observed with isolated Golgi membranes(1.Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Crossref PubMed Scopus (208) Google Scholar), whereas NSF located in the nuclear membranes and neuronal growth cones was not released by Mg2+-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 His6-tagged α-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 α- and β-SNAPs. α-SNAP also prevented the Mg2+-ATP-induced release of NSF from isolated Golgi membranes.SNAPs were identified as components that mediate the attachment of NSF to Golgi membranes(33.Weidman P.J. Melançon P. Block M.R. Rothman J.E. J. Cell Biol. 1989; 108: 1589-1596Crossref PubMed Scopus (113) Google Scholar). Because NSF is released from Golgi membranes by Mg2+-ATP(1.Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Crossref PubMed Scopus (208) Google Scholar), SNAP activity had only been measured in the presence of EDTA-ATP(8.Clary D.O. Rothman J.E. J. Biol. Chem. 1990; 265: 10109-10117Abstract Full Text PDF PubMed Google Scholar, 9.Clary D.O. Griff I.C. Rothman J.E. Cell. 1990; 61: 709-721Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 33.Weidman P.J. Melançon P. Block M.R. Rothman J.E. J. Cell Biol. 1989; 108: 1589-1596Crossref PubMed Scopus (113) Google Scholar). 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 Mg2+-ATP. The present results clearly show that α-SNAP prevents the Mg2+-ATP-induced release of NSF from Golgi membranes by mediating the reassociation of disassembled NSF with membranes. Söllner et al.(10.Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2602) Google Scholar, 12.Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1573) Google Scholar) demonstrated that a 20 S complex comprising NSF, SNAPs, and SNAREs is completely disassembled by Mg2+-ATP. In their experiments, reconstitution of the 20 S complex was performed by incubation of NSF and α-SNAP in an equimolar ratio. This may be the reason why the formation of the complex does not occur in the presence of Mg2+-ATP. In the present study, an excess amount of α-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.Morgan A. Burgoyne R.D. EMBO J. 1995; 14: 232-239Crossref PubMed Scopus (95) Google Scholar) and the transport of vesicular stomatitis-virus encoded glycoprotein to the basolateral membrane(17.Ikonen E. Tagaya M. Ullrich O. Montecucco C. Simons K. Cell. 1995; 81: 571-580Abstract Full Text PDF PubMed Scopus (220) Google Scholar). Paradoxically, NSF does not stimulate regulated exocytosis(6.Morgan A. Burgoyne R.D. EMBO J. 1995; 14: 232-239Crossref PubMed Scopus (95) Google Scholar). Morgan and Burgoyne (6.Morgan A. Burgoyne R.D. EMBO J. 1995; 14: 232-239Crossref PubMed Scopus (95) Google Scholar) assumed that NSF is expressed at supramaximal levels in chromaffin cells and that SNAPs expression is limiting. Because Mg2+-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 Mg2+-ATP and therefore stimulates protein transport.According to the model proposed by Rothman and his colleagues(10.Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2602) Google Scholar, 12.Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1573) Google Scholar, 34.Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar), 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 Mg2+-ATP (18.Hong R.-M. Mori H. Fukui T. Moriyama Y. Futai M. Yamamoto A. Tashiro Y. Tagaya M. FEBS Lett. 1994; 350: 253-257Crossref PubMed Scopus (59) Google Scholar). Based on these findings and the results of kinetic studies involving an intra-Golgi protein transport assay(14.Sumida M. Hong R.-M. Tagaya M. J. Biol. Chem. 1994; 269: 20636-20641Abstract Full Text PDF PubMed Google Scholar, 35.Wattenberg B.W. Raub T.J. Hiebsch R.R. Weidman P.J. J. Cell Biol. 1992; 118: 1321-1332Crossref PubMed Scopus (37) Google Scholar), 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 Mg2+-ATP. The present results combined with the recent finding that syntaxin 1 is associated with vesicles as well as target membranes (36.Walch-Solimena C. Blasi J. Edelmann L. Chapman E.R. Fischer von Mollard G. Jahn R. J. Cell Biol. 1995; 128: 637-645Crossref PubMed Scopus (312) Google Scholar, 37.Tagaya M. Toyonaga S. Takahashi M. Yamamoto A. Fujiwara T. Akagawa K. Moriyama Y. Mizushima S. J. Biol. Chem. 1995; 270: 15930-15933Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) may raise serious questions regarding Rothman's hypothesis(34.Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). Similar questions were also recently raised by Morgan and Burgoyne(38.Morgan A. Burgoyne R.D. Trends Cell Biol. 1995; 5: 335-339Abstract Full Text PDF PubMed Scopus (95) Google Scholar). INTRODUCTIONThe N-ethylmaleimide-sensitive factor (NSF) 1The abbreviations used are: NSFN-ethylmaleimide-sensitive factorSNAPsoluble NSF attachment proteinSNARESNAP receptorPBSphosphate-buffered saline. was originally characterized as a protein that is implicated in intra-Golgi vesicle-mediated protein transport(1.Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Crossref PubMed Scopus (208) Google Scholar, 2.Block M.R. Glick B.S. Wilcox C.A. Wieland F.T. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7852-7856Crossref PubMed Scopus (396) Google Scholar). Several lines of evidence suggest that NSF most likely mediates the fusion of Golgi-derived transport vesicles with target membranes(3.Malhotra V. Orci L. Glick B.S. Block M.R. Rothman J.E. Cell. 1988; 54: 221-227Abstract Full Text PDF PubMed Scopus (263) Google Scholar). Later studies revealed that NSF is also involved in protein transport from the endoplasmic reticulum to the Golgi apparatus(4.Beckers C.J.M. Block M.R. Glick B.S. Rothman J.E. Balch W.E. Nature. 1989; 339: 397-398Crossref PubMed Scopus (188) Google Scholar), endosome fusion(5.Diaz R. Mayorga L.S. Weidman P.J. Rothman J.E. Stahl P.D. Nature. 1989; 339: 398-400Crossref PubMed Scopus (157) Google Scholar), and exocytosis of neurotransmitters(6.Morgan A. Burgoyne R.D. EMBO J. 1995; 14: 232-239Crossref PubMed Scopus (95) Google Scholar, 7.DeBello W.M. O'Connor V. Dresbach T. Whiteheart S.W. Wang S.S.-H. Schweizer F.E. Betz H. Rothman J.E. Augustine G.J. Nature. 1995; 373: 626-630Crossref PubMed Scopus (134) Google Scholar). NSF is a soluble protein, and its attachment to membranes in the absence of Mg2+-ATP is mediated by three peripheral membrane proteins named α-, β-, and γ-SNAPs(8.Clary D.O. Rothman J.E. J. Biol. Chem. 1990; 265: 10109-10117Abstract Full Text PDF PubMed Google Scholar, 9.Clary D.O. Griff I.C. Rothman J.E. Cell. 1990; 61: 709-721Abstract Full Text PDF PubMed Scopus (402) Google Scholar). Söllner et al.(10.Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2602) Google Scholar) 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.Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2602) Google Scholar, 11.Wilson D.W. Whiteheart S.W. Wiedmann M. Brunner M. Rothman J.E. J. Cell Biol. 1992; 117: 531-538Crossref PubMed Scopus (210) Google Scholar). Incubation of Golgi membranes with Mg2+-ATP induces the disassembly of the 20 S complex, and thereby results in the release of membrane-bound NSF(1.Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Crossref PubMed Scopus (208) Google Scholar, 10.Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2602) Google Scholar, 11.Wilson D.W. Whiteheart S.W. Wiedmann M. Brunner M. Rothman J.E. J. Cell Biol. 1992; 117: 531-538Crossref PubMed Scopus (210) Google Scholar, 12.Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1573) Google Scholar). 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.Tagaya M. Wilson D.W. Brunner M. Arango N. Rothman J.E. J. Biol. Chem. 1993; 268: 2662-2666Abstract Full Text PDF PubMed Google Scholar), and the two homologous nucleotide-binding regions of NSF are involved in ATP hydrolysis(14.Sumida M. Hong R.-M. Tagaya M. J. Biol. Chem. 1994; 269: 20636-20641Abstract Full Text PDF PubMed Google Scholar, 15.Whiteheart S.W. Rossnagel K. Buhrow S.A. Brunner M. Jaenicke R. Rothman J.E. J. Cell Biol. 1994; 126: 945-954Crossref PubMed Scopus (340) Google Scholar).NSF seems to exist as both membrane-associated and free forms in vivo, although intracellular concentrations of Mg2+ 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.Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Crossref PubMed Scopus (208) Google Scholar, 2.Block M.R. Glick B.S. Wilcox C.A. Wieland F.T. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7852-7856Crossref PubMed Scopus (396) Google Scholar, 3.Malhotra V. Orci L. Glick B.S. Block M.R. Rothman J.E. Cell. 1988; 54: 221-227Abstract Full Text PDF PubMed Scopus (263) Google Scholar). 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.Beckers C.J.M. Block M.R. Glick B.S. Rothman J.E. Balch W.E. Nature. 1989; 339: 397-398Crossref PubMed Scopus (188) Google Scholar, 5.Diaz R. Mayorga L.S. Weidman P.J. Rothman J.E. Stahl P.D. Nature. 1989; 339: 398-400Crossref PubMed Scopus (157) Google Scholar, 6.Morgan A. Burgoyne R.D. EMBO J. 1995; 14: 232-239Crossref PubMed Scopus (95) Google Scholar, 16.Rodriguez L. Stirling C.J. Woodman P.G. Mol. Biol. Cell. 1994; 5: 773-783Crossref PubMed Scopus (78) Google Scholar, 17.Ikonen E. Tagaya M. Ullrich O. Montecucco C. Simons K. Cell. 1995; 81: 571-580Abstract Full Text PDF PubMed Scopus (220) Google Scholar). In addition, we recently showed that NSF is associated with isolated synaptic vesicles (18.Hong R.-M. Mori H. Fukui T. Moriyama Y. Futai M. Yamamoto A. Tashiro Y. Tagaya M. FEBS Lett. 1994; 350: 253-257Crossref PubMed Scopus (59) Google Scholar). These results raise the possibility that there is a factor(s) that mediates the association of NSF to membranes in the presence of Mg2+-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 Mg2+-ATP-induced release of NSF from the Golgi apparatus is prevented by α-SNAP." @default.
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