Docked Secretory Vesicles Undergo Ca2+-activated Exocytosis in a Cell-free System
1997; Elsevier BV; Volume: 272; Issue: 22 Linguagem: Inglês
10.1074/jbc.272.22.14447
ISSN1083-351X
AutoresThomasF.J. Martin, Judith A. Kowalchyk,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoThe Ca2+-activated fusion of secretory vesicles with the plasma membrane responsible for regulated neurotransmitter and hormone secretion has previously been studied in permeable neuroendocrine cells, where requirements for ATP and cytosolic proteins were identified. As reported here, Ca2+-activated fusion mechanisms are also preserved following cell homogenization. The release of norepinephrine (NE) and other vesicle constituents from a PC12 cell membrane fraction was activated by micromolar Ca2+ (EC50 ∼ 3 μm) and exhibited a dependence upon MgATP and cytosol. Ca2+-dependent NE release was inhibited by botulinum neurotoxins and by CAPS (Ca2+-dependent activator protein for secretion) antibody implying that syntaxin, synaptobrevin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and CAPS are required for regulated exocytosis in this system. The exocytosis-competent membrane fraction consisted of rapidly sedimenting dense core vesicles associated with plasma membrane fragments. Free vesicles did not release NE either in the absence or presence of plasma membranes, indicating that only docked vesicles were competent for exocytosis under the reconstitution conditions used. A cell-free system for Ca2+-activated fusion will facilitate studies on the roles of essential proteins such as syntaxin, synaptobrevin, SNAP-25, and CAPS that act at post-docking steps in the regulated exocytotic pathway. The Ca2+-activated fusion of secretory vesicles with the plasma membrane responsible for regulated neurotransmitter and hormone secretion has previously been studied in permeable neuroendocrine cells, where requirements for ATP and cytosolic proteins were identified. As reported here, Ca2+-activated fusion mechanisms are also preserved following cell homogenization. The release of norepinephrine (NE) and other vesicle constituents from a PC12 cell membrane fraction was activated by micromolar Ca2+ (EC50 ∼ 3 μm) and exhibited a dependence upon MgATP and cytosol. Ca2+-dependent NE release was inhibited by botulinum neurotoxins and by CAPS (Ca2+-dependent activator protein for secretion) antibody implying that syntaxin, synaptobrevin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and CAPS are required for regulated exocytosis in this system. The exocytosis-competent membrane fraction consisted of rapidly sedimenting dense core vesicles associated with plasma membrane fragments. Free vesicles did not release NE either in the absence or presence of plasma membranes, indicating that only docked vesicles were competent for exocytosis under the reconstitution conditions used. A cell-free system for Ca2+-activated fusion will facilitate studies on the roles of essential proteins such as syntaxin, synaptobrevin, SNAP-25, and CAPS that act at post-docking steps in the regulated exocytotic pathway. Cellular compartments within eukaryotic cells maintain discrete identities despite the continuous disassembly and reassembly required for the vectorial transport of lumenal and membrane proteins to specific destinations. This requires a molecular basis for membrane identity and strict regulation of the membrane fusion process (1Rothman J.E. Warren G. Curr. Biol. 1994; 4: 220-233Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). While recent progress toward understanding the molecular basis of vesicle targeting and fusion has been dramatic (2Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar), the biochemical reactions responsible for membrane fusion remain poorly understood. Cell-free systems that reconstitute in vitro aspects of vesicle formation, translocation, targeting, and fusion have provided insights on the mechanisms of vesicle fission and fusion at several stages of the protein trafficking pathway (2Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar).A tightly regulated membrane fusion process underlies the secretion of peptide hormones and neurotransmitters from endocrine and neural cells. Exocytotic fusion of secretory vesicles with the plasma membrane is triggered by intracellular Ca2+ concentration increases into the 1–300 μm range (3Llinas R. Sugimori M. Silver R.B. J. Physiol. 1995; 89: 77-81Google Scholar). Recent studies have identified some of the proteins required for regulated fusion (see "Discussion"), but an understanding of the events in which these proteins participate and their relationship to membrane bilayer fusion remains limited due to the lack of functional cell-free in vitro assays for Ca2+-regulated fusion.Studies of regulated fusion have instead utilized permeable cell preparations, most of which exhibit limited permeability or possess limited stability. Exceptions where exocytotic fusion reactions have been studied in vitro in cell-free systems are reconstituted sea urchin egg cortices (4Crabb J.H. Jackson R.C. J. Cell Biol. 1985; 101: 2263-2273Crossref PubMed Scopus (65) Google Scholar) and acinar pancreatic zymogen granule-plasma membrane preparations (5MacLean C.M. Edwardson J.M. Biochem. J. 1992; 286: 747-753Crossref PubMed Scopus (22) Google Scholar); however, these systems exhibit a poorly understood partial or complete loss of Ca2+ regulation. An extension of previous studies (6Walent J.H. Porter B.W. Martin T.F.J. Cell. 1992; 70: 765-775Abstract Full Text PDF PubMed Scopus (222) Google Scholar, 7Hay J.C. Martin T.F.J. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (244) Google Scholar) of Ca2+-activated exocytosis in mechanically permeabilized PC12 neuroendocrine cells led to the development of a simple purified membrane preparation consisting of large dense core vesicles (LDCVs) 1The abbreviations used areLDCVlarge dense core vesicle;NEnorepinephrine;FITCfluorescein isothiocyanate;WGAwheat germ agglutinin;CAPSCa2+-dependent activator protein for secretion;NSFN-ethylmaleimide-sensitive factor;SNAPsoluble NSF attachment protein;SNARESNAP receptor;SNAP-25synaptosome-associated protein of 25 kDa;PIphosphatidylinositol;PI(4,5)P2phosphatidylinositol 4,5-bisphosphate;GMPPNPguanylyl imidotriphosphate. associated with plasma membrane fragments. This preparation may be enriched for a very late stage intermediate of the regulated exocytotic pathway in which LDCVs are stably docked at the plasma membrane and in which the final steps of Ca2+-regulated fusion can be studied in vitro.MATERIALS AND METHODSCell Culture and LabelingPC12 cells were cultured in 10-cm dishes as described previously (8Lomneth R.L. Martin T.F.J. DasGupta B.R. J. Neurochem. 1991; 57: 1413-1421Crossref PubMed Scopus (57) Google Scholar) and incubated with 0.5 μC/ml [3H]norepinephrine (NE; 45 Ci/mmol; Amersham Corp.) for 16 h in the presence of 0.5 mm ascorbic acid. Labeled cells were washed extensively with and incubated in Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin to remove unincorporated [3H]NE, and the cells were detached from culture dishes by pipetting in cold Kglu buffer (0.02m HEPES, pH 7.2, 0.12 m potassium glutamate, 0.02 m potassium acetate, 0.002 m EGTA, 0.1% bovine serum albumin). For plasma membrane labeling, cells were incubated with 0.5 mg/ml fluorescein isothiocyanate (FITC) in 0.1m sodium borate pH 8.0, 0.15 m NaCl or with 20 μg/ml FITC-wheat germ agglutinin (FITC-WGA) in Dulbecco's modified Eagle's medium for 60 min at 0 °C.Cell Homogenization and Sucrose GradientsCell homogenates were prepared by passing cell suspensions 15 times through a narrow clearance (10 μm) stainless steel ball homogenizer (9Martin T.F.J. Methods Enzymol. 1989; 168: 225-233Crossref PubMed Scopus (30) Google Scholar). Homogenates were clarified by centrifugation at 800 × g for 10 min twice in succession to rigorously remove broken cells (monitored microscopically), and the postnuclear supernatant was centrifuged at 5000 × g for 10 min to recover rapidly sedimenting LDCVs. The 5000 × g membrane pellet was washed by resuspension in Kglu buffer and centrifugation at 10,000 ×g for 10 min. Approximately 28% of the [3H]NE in post-nuclear supernatants was rapidly sedimenting and recovered in 5000 × g pellets. The remaining free LDCVs were quantitatively isolated from a 10,000 × g supernatant by centrifugation at 40,000 × g for 15 min. Whereas the majority of synaptic vesicle-like microvesicles containing synaptophysin was present in the supernatant of the 40,000 ×g centrifugation, LDCVs in PC12 cells also contain synaptophysin (see Fig. 2). For sucrose gradient purification, 5000 × g pellet fractions were collected on a pad of 2m sucrose in 0.02 m Tris, pH 7.8, 0.15m NaCl, 0.002 m EGTA (TNE buffer), diluted 5-fold, and loaded onto either continuous or discontinuous sucrose gradients. Continuous gradients consisting of 9 ml of a linear gradient of 0.25–2 m sucrose and discontinuous gradients consisting of 2 ml each of 0.25, 0.5, 1.0, 1.5, and 2 m sucrose in TNE were centrifuged in a Beckman SW 41 rotor at 20,000 ×g for 30 min and collected from the bottom. For Western blotting, membranes were recovered from gradient fractions by 8-fold dilution in TNE buffer and centrifugation at 150,000 ×g for 16 h. Pellets were solubilized in sample buffer for electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose for immunoblotting with antibodies to chromogranin B (Pel-Freez, Rogers, AR), synaptophysin (Boehringer Mannheim), SV2 (R.H. Scheller, Stanford), syntaxin (Sigma), and SNAP-25 (Sternberger Monoclonals Inc., Baltimore, MD). Rabbit polyclonal antibodies to synaptotagmin I were generated using a glutathioneS-transferase-cytoplasmic domain fusion protein of synaptotagmin I. Rabbit polyclonal antibodies to synaptobrevin-2 were generated using an N-terminal peptide (SATAATVPPAAPAGEGGPPC) conjugated to bovine serum albumin. Western blots were processed with125I-protein A or 125I-labeled goat anti-mouse antibody (DuPont NEN) and subjected to autoradiography.Exocytosis AssaysSecretion assays to monitor the release of [3H]NE were conducted in 0.2-ml reactions that contained Kglu buffer adjusted with CaCl2 to the indicated [Ca2+]f, 0.002 m MgCl2, 0.002 m ATP, and the resuspended 5000 × g pellet. Incubations at 30 °C were terminated by chilling on ice, followed by centrifugation at 100,000 × g for 90 min. [3H]NE in supernatant and in 1% Triton X-100-solubilized pellet fractions were determined by liquid scintillation counting and used to calculate the percentage of NE release as [dpm supernatant]/[dpm total] × 100%. Supernatant values were corrected by subtraction of zero time incubation values determined for each experiment. Antibody to rat CAPS was generated by immunizing rabbits with a glutathione S-transferase fusion protein representing amino acids 264–364 (accession no. U16802) and an IgG fraction was purified by chromatography on protein A-Sepharose (Pharmacia Biotech). The recombinant botulinum neurotoxin E light chain was kindly provided by A. Zdanovsky (Promega Corp., Madison, WI).RESULTSRegulated Exocytosis in PC12 Cell HomogenatesThe Ca2+-regulated secretion of NE is faithfully reconstituted in PC12 cells permeabilized by single passage through a narrow clearance ball homogenizer (6Walent J.H. Porter B.W. Martin T.F.J. Cell. 1992; 70: 765-775Abstract Full Text PDF PubMed Scopus (222) Google Scholar, 7Hay J.C. Martin T.F.J. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (244) Google Scholar). The Ca2+-dependent release of NE and other vesicle constituents by exocytotic fusion of LDCVs with the plasma membrane is ATP- and cytosol protein-dependent and inhibited by clostridial neurotoxins that specifically proteolyze synaptobrevin, SNAP-25, and syntaxin (6Walent J.H. Porter B.W. Martin T.F.J. Cell. 1992; 70: 765-775Abstract Full Text PDF PubMed Scopus (222) Google Scholar, 7Hay J.C. Martin T.F.J. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (244) Google Scholar, 8Lomneth R.L. Martin T.F.J. DasGupta B.R. J. Neurochem. 1991; 57: 1413-1421Crossref PubMed Scopus (57) Google Scholar, 9Martin T.F.J. Methods Enzymol. 1989; 168: 225-233Crossref PubMed Scopus (30) Google Scholar, 10Banerjee A. Kowalchyk J.A. DasGupta B.R. Martin T.F.J. J. Biol. Chem. 1996; 271: 20227-20230Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Multiple passes of PC12 cells through a wider clearance ball homogenizer generated a cell homogenate consisting of small membrane fragments and intact LDCVs. Surprisingly, ATP- and cytosol-dependent, Ca2+-activated release of NE was preserved in membranes recovered from the homogenate (Fig. 1 A). The Ca2+-dependent release of NE was not affected by dilution of the membranes, suggesting that the responsive subcellular fraction contained all of the membranes that participate in exocytotic fusion. The responsive fraction was recovered by centrifugation at 5000 × g and was found to contain ∼28% (12–44%, n = 8) of the cellular LDCVs present in post-nuclear supernatants based on the recovery of [3H]NE or vesicle-specific antigens (see below). Free LDCVs in the homogenate fail to sediment at 5000 × g but are fully recovered by centrifugation at 40,000 ×g consistent with the size (∼75 nm) of LDCVs in PC12 cells (11Wagner J.A. J. Neurochem. 1985; 45: 1244-1253Crossref PubMed Scopus (33) Google Scholar). Comparison of the membranes sedimented at 5000 ×g and 40,000 × g showed that only the former exhibited Ca2+-activated NE release (Fig.1 B), indicating that free LDCVs do not release their contents under standard incubation conditions. Because the LDCVs in the 5000 × g pellet fraction exhibited Ca2+-dependent NE release and sedimented more rapidly than free LDCVs, it was likely that these LDCVs were associated with plasma membranes.Figure 1Ca2+-activated NE release from membrane fractions of a PC12 cell homogenate.[3H]NE-labeled PC12 cells were homogenized by 15 passes through a ball homogenizer. A, membranes collected at 15,000 × g and 40,000 × g were resuspended in Kglu buffer, pooled and incubated at 30 °C for 10 min in the absence (○) or presence (•) of 10 μmCa2+, 2 mm MgATP, and 0.5 mg/ml cytosol. A fixed aliquot of membranes was incubated in the indicated volumes that varied 8-fold. B, membranes from post-nuclear supernatants were collected by sequential centrifugation at 5000 ×g and 40,000 × g. Pellets were resuspended separately and incubated for the indicated times at 30 °C in Kglu buffer (○), buffer supplemented with 3 μm MgGMPPNP (▴), or Ca2+, MgATP, and cytosol (•).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Regulated Exocytosis in a LDCV-Plasma Membrane ComplexTo characterize the membranes present in the 5000 × g pellet fraction, analysis by velocity gradient centrifugation was conducted (Fig. 2). LDCVs in the 5000 ×g pellet fraction sedimented rapidly into the gradient (Fig.2 A, arrow) as indicated by the co-sedimentation of LDCV contents markers [3H]NE, chromogranin B (Fig.2 A), and dopamine β-hydroxylase (data not shown). The vesicle membrane proteins synaptotagmin I, synaptophysin (Fig. 2,A and B), SV2, and synaptobrevin (data not shown) co-sedimented with the contents markers. Rapidly sedimenting LDCVs also exhibited reserpine-sensitive amine transporter activity characteristic of NE-containing LDCVs (Fig. 2 C). Variable amounts of LDCVs were dislodged during preparation and sedimented nearer the top of the gradients at a position characteristic of free LDCVs (e.g. fraction 12 in Fig. 2 A). Incubation of the 5000 ×g pellet fraction under exocytosis conditions prior to gradient analysis revealed that the release of NE occurred from the rapidly sedimenting but not free LDCVs (Fig. 2 D).Plasma membranes were monitored by surface labeling of cells and by immunoblotting with SNAP-25 and syntaxin antibodies. The majority of plasma membrane in the PC12 cell homogenate sedimented at 800 ×g. About 23% of the plasma membrane was recovered in the 5000 × g pellet fraction and the majority of this co-sedimented with LDCVs in sucrose gradients (Fig. 2, B andE, arrow). Co-sedimentation of LDCVs and plasma membranes was also observed in isopycnic gradient centrifugation in sucrose, Percoll and Ficoll (data not shown). Incubation of 5000 × g pellet fractions under exocytosis conditions prior to gradient analysis revealed a redistribution of plasma membranes following membrane fusion (Fig. 2 E). Collectively, the results indicated that the rapidly sedimenting, exocytosis-competent subcellular fraction consisted of LDCVs associated with plasma membrane fragments.Electron microscopy of the membrane complex purified on sucrose gradients was consistent with this conclusion revealing the presence of LDCVs on membrane fragments (Fig. 3, left panel). LDCVs were not evident in membrane fractions that were incubated with Ca2+ in the presence of ATP and cytosol (Fig. 3, right panel) as anticipated if exocytotic fusion and the release of LDCV constituents had occurred.Figure 3Electron micrographs of rapidly sedimenting LDCVs. Resuspended 5000 × g pellet fractions were incubated for 10 min at 30 °C in Kglu buffer without additions (left panel) or with Ca2+, MgATP, and cytosol (right panel). Material corresponding to fractions 6 and 7 of Fig. 2 A was collected from continuous sucrose gradients and membranes were recovered by centrifugation for fixation in glutaraldehyde and analysis by transmission electron microscopy. Several LDCVs in left panel are indicated byarrowheads. Scale bar corresponds to 1 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The majority of LDCVs in PC12 cells are morphologically docked at the plasma membrane (12Banerjee A. Barry V.A. DasGupta B.R. Martin T.F.J. J. Biol. Chem. 1996; 271: 20223-20226Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), but only about one-third of the LDCVs in a post-nuclear supernatant fraction were isolated in a homogenization-resistant complex with plasma membranes. To evaluate the possibility that LDCV-plasma membrane complexes were disrupted during homogenization, the conditions of homogenization were systematically varied. The results (data not shown) indicated that an optimal yield of rapidly sedimenting LDCVs required 5–10 passes through the ball homogenizer, whereas 15–20 passes did not further increase or decrease the number of LDCVs isolated in rapidly sedimenting complexes. These results suggest that a fraction of the LDCVs in PC12 cells exist in an unusually stable docked state, which could represent a very late stage intermediate in the LDCV exocytotic pathway.Characterization of Ca2+-activated Exocytosis from LDCV-Plasma Membrane ComplexThe release of NE from LDCV-plasma membrane preparations exhibited characteristics similar to those described for permeable PC12 cells prepared by a single pass through the ball homogenizer (6Walent J.H. Porter B.W. Martin T.F.J. Cell. 1992; 70: 765-775Abstract Full Text PDF PubMed Scopus (222) Google Scholar, 7Hay J.C. Martin T.F.J. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (244) Google Scholar, 8Lomneth R.L. Martin T.F.J. DasGupta B.R. J. Neurochem. 1991; 57: 1413-1421Crossref PubMed Scopus (57) Google Scholar). NE release was entirely dependent on Ca2+, largely dependent on cytosol, and partially dependent on MgATP (Fig. 4 A). Ca2+activation was half-maximal at 2–5 μm and optimal at 10 μm (Fig. 4 B). Ca2+ stimulated the concomitant release of several LDCV constituents in addition to NE including preloaded quinacrine (Fig. 4 C) and chromogranin B (Fig. 4 D). The Ca2+-activated release of LDCV constituents was entirely abolished by treatment with type B, type C (data not shown), and type E (Fig. 4 E) botulinum neurotoxins, indicating a requirement for functional synaptobrevin, syntaxin, and SNAP-25 as anticipated for Ca2+-dependent exocytotic fusion (13Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar). Botulinum neurotoxin-sensitive release of NE from LDCV-plasma membrane preparations was alternatively activated by nonhydrolyzable GTP analogs such as GMPPNP in the absence of Ca2+ (Fig. 1 B), although the rate of stimulated secretion was lower than with Ca2+ even at maximally effective concentrations (3 μm) of MgGMPPNP.Figure 4Ca2+-activated LDCV fusion in LDCV-plasma membrane complexes requires MgATP and cytosol. A, resuspended 5000 × g pellets from [3H]NE-labeled PC12 cells were incubated at 30 °C for 10 min in the presence of 10 μm Ca2+, 2 mm MgATP, 0.5 mg/ml cytosol, 2 mg/ml rabbit IgG (IgG), or 2 mg/ml IgG purified from CAPS antiserum (αCAPS) as indicated. B, incubations were conducted at 2 mm MgATP in the indicated Ca2+concentrations in the absence (○) or presence (•) of cytosol.C, PC12 cells were incubated with 10 μmquinacrine for 60 min prior to preparation of 5000 × g pellet fractions. Incubations similar to those in B were conducted with 2 mm MgATP in the absence or presence of 10 μm Ca2+ and cytosol as indicated. Membranes were pelleted, and the fluorescence of supernatants was measured (Ex = 424 nm; Em = 505 nm). D, incubations were conducted as in C with 5000 × g pellet fractions from unlabeled PC12 cells, and the supernatants following centrifugation were analyzed by immunoblotting with chromogranin B antibodies. E, incubations were conducted as in A with Ca2+, MgATP, and cytosol following a 3-min incubation at 30 °C with indicated concentrations of the botulinum neurotoxin E light chain (BoNT).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Previous studies with permeable PC12 cells had shown that the requirement for MgATP in regulated exocytosis was for priming steps that precede the final step of Ca2+-triggered fusion (7Hay J.C. Martin T.F.J. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (244) Google Scholar). The MgATP requirement for NE release from LDCV-plasma membrane preparations was similarly found to be largely met in preincubations with MgATP lacking Ca2+ (Fig. 5), indicating that a priming process was required for optimal exocytosis of docked LDCVs. In permeable cells, the Ca2+-triggered fusion step that follows priming was shown to require a 145-kDa cytosolic protein CAPS (6Walent J.H. Porter B.W. Martin T.F.J. Cell. 1992; 70: 765-775Abstract Full Text PDF PubMed Scopus (222) Google Scholar, 7Hay J.C. Martin T.F.J. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (244) Google Scholar). 2K. Ann, J. A. Kowalchyk, and T. F. J. Martin, submitted for publication. CAPS was also found to be necessary for Ca2+-triggered fusion in the LDCV-plasma membrane preparation, as indicated by the specific inhibition observed with IgGs from CAPS immune serum but not control IgGs (Fig.4 A). A nearly complete (90%) inhibition of cytosol-dependent NE release was observed in incubations with a different CAPS antibody (data not shown), indicating that the incomplete inhibition shown in Fig. 4 A was attributable to properties of the antibody.Figure 5The MgATP requirement for Ca2+-activated LDCV fusion in LDCV-plasma membrane complexes is met in a priming preincubation. Two-stage incubations were conducted with additions indicated. First stage 30-min incubations contained 1 mg/ml cytosol in the absence or presence 2 mmMgATP. Membranes were washed by centrifugation and incubated for 10 min in second stage incubations containing 10 μmCa2+ and 0.5 mg/ml cytosol with or without 2 mmMgATP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Docked but Not Undocked LDCVs Undergo Regulated ExocytosisThe results of Figs. 1 B and 2 D indicated that only LDCVs associated with plasma membranes were triggered to exocytose by Ca2+. It was of interest to determine whether free LDCVs could undergo exocytosis in the presence of plasma membranes, and whether LDCV-plasma membrane complexes were competent to dock and fuse additional LDCVs. Mixing experiments were undertaken in which free LDCVs were incubated with LDCV-plasma membrane preparations under exocytosis conditions. Incubating unlabeled LDCV-plasma membrane preparations with [3H]NE-loaded free LDCVs in various proportions under exocytosis conditions failed to elicit release of [3H]NE (data not shown). Similarly, co-incubating [3H]NE-loaded LDCV-plasma membrane preparations with [3H]NE-loaded free LDCVs in the presence of Ca2+, MgATP and cytosol failed to elicit more [3H]NE release than that observed for the LDCV-plasma membrane complexes alone (Fig. 6). Similar studies with GMPPNP to trigger fusion at low Ca2+ concentrations also failed to provide evidence for fusion of free LDCVs with the plasma membrane (data not shown). The results indicated that exocytosis was restricted to LDCVs already docked at the plasma membrane and that LDCV recruitment to and functional docking at the plasma membrane was not reconstituted in the standard exocytosis assay.Figure 6Free LDCVs fail to dock and fuse with LDCV-plasma membrane complexes. Free LDCVs and LDCV-plasma membrane complexes were prepared from [3H]NE-labeled PC12 cells as described under "Materials and Methods." Exocytosis incubations were conducted in the presence of Ca2+, MgATP, and cytosol with LDCV-plasma membrane complexes alone, LDCVs alone, or LDCV-plasma membrane complexes plus LDCVs where indicated. Co-incubations contained free LDCVs at 1.5- or 3-fold the number of complexed LDCVs based on [3H]NE content.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONElucidation of the process of Ca2+-regulated membrane fusion will require identification of proteins that mediate secretory vesicle transport to, docking at, and fusion with the plasma membrane as well as those that mediate the Ca2+ regulation of steps in this sequence. Some of the proteins required for regulated exocytosis have recently been identified, but their precise roles are poorly understood. The SNARE proteins (for soluble NSF attachment protein receptors) synaptobrevin, syntaxin, and SNAP-25, which are the targets of clostridial neurotoxin proteolytic activity (14Niemann H. Blasi J. Jahn R. Trends Cell Biol. 1994; 4: 179-185Abstract Full Text PDF PubMed Scopus (291) Google Scholar) and the receptors for the NSF/SNAP proteins required for constitutive fusion (2Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar, 15Sollner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2603) Google Scholar), form oligomeric complexes that are proposed to function in vesicle docking and/or fusion (2Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar, 16Sollner 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). Studies on the Ca2+-binding vesicle protein synaptotagmin suggest its possible role as a Ca2+-dependent regulator of exocytosis (13Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar). Many additional proteins potentially involved in regulated secretion have been identified genetically or biochemically but remain poorly understood (13Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar, 17Martin T.F.J. Trends Cell Biol., in press. 1997; Google Scholar). Physiological studies in neuroendocrine cells indicate that secretory granules are staged at numerous points in the exocytotic pathway in transit to the plasma membrane for fusion (18Henkel A.W. Almers W. Curr. Opin. Neurobiol. 1996; 6: 350-357Crossref PubMed Scopus (142) Google Scholar) and that Ca2+ accelerates granule transit through several stages that include recruitment to the plasma membrane for docking, post-docking priming steps, fusion pore formation, and fusion pore expansion (17Martin T.F.J. Trends Cell Biol., in press. 1997; Google Scholar). It will be essential to reconstitute these stages along the exocytotic route in vitro as distinct biochemical steps in which the role of specific proteins can be assessed and their mechanisms determined.Some progress toward elucidating partial reactions in the exocytotic pathway has been achieved with permeable neuroendocrine cells. In permeable PC12 cells where the majority of LDCVs are morphologically docked, ATP-dependent priming steps precede and are required for Ca2+-activated fusion steps (7Hay J.C. Martin T.F.J. J. Cell Biol. 1992; 119: 139-151Crossref PubMed Scopus (244) Google Scholar, 12Banerjee A. Barry V.A. DasGupta B.R. Martin T.F.J. J. Biol. Chem. 1996; 271: 20223-20226Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Ca2+-activated secretion in LDCV-plasma membrane preparations similarly exhibited a requirement for ATP. Because the LDCVs are stably docked at the plasma membrane, the results indicate that priming is distinct from vesicle recruitment or docking and that it occurs in vitro following docking and prior to fusion.Two distinct ATP-dependent priming reactions have been characterized in permeable PC12 cells. In the first of these, PI transfer protein and phosphoinositide kinases catalyze synthesis of PI(4,5)P2, which is req
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