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Article |
Address correspondence to William T. Wickner, Dept. of Biochemistry, 7200 Vail Bldg., Dartmouth Medical School, Hanover, NH 03755-3844. Tel.: (603) 650-1701. Fax: (603) 650-1353. email: william.wickner{at}dartmouth.edu
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Abstract |
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Key Words: membrane; docking; fusion; Rab GTPase; SM-protein
Abbreviations used in this paper: GDI, GDP dissociation inhibitor; RDI, Rho GDI; rVam7p, recombinant Vam7p; SM, Sec1p/Munc18.
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Introduction |
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Cytosolic Ca2+ is required for many membrane fusion events. In some cases, ambient [Ca2+] is sufficient for fusion (Beckers and Balch, 1989; Baker et al., 1990); in other cases, transient increases in [Ca2+] are needed (Sullivan et al., 1993; Peters and Mayer, 1998; Chen et al., 1999; Pryor et al., 2000; Reddy et al., 2001; Rettig and Neher, 2002). In neurons, depolarization opens voltage-gated Ca2+ channels, triggering exocytosis (Rettig and Neher, 2002; Jahn et al., 2003). However, little is known about how Ca2+ regulates intracellular fusion events such as endosomeendosome fusion. Even less is known about how Ca2+ signals that regulate intracellular membrane fusion are themselves regulated. Ca2+ channels in the plasma membranes of mammalian cells physically and functionally interact with the Q-SNAREs syntaxin and SNAP-25 (Bennett et al., 1992; Yoshida et al., 1992; Sheng et al., 1994; Mochida et al., 1996; Wiser et al., 1996; Rettig et al., 1997). Ca2+ channels also interact with the exocytic machinery via the Sec6/8 complex and Rab3-interacting molecule binding proteins (Shin et al., 2000; Hibino et al., 2002). On the yeast vacuole, the R-SNARE Nyv1p inhibits the vacuolar Ca2+ ATPase Pmc1p (Takita et al., 2001). It is unclear how such interactions are integrated into cycles of membrane docking and fusion.
Trans interactions between SNARES on opposite membranes have been proposed to facilitate or trigger Ca2+ signals in response to docking (Bezprozvanny et al., 1995; Wiser et al., 1996; Schekman, 1998), but technical obstacles have precluded direct tests of this hypothesis. The yeast vacuole offers powerful genetic and biochemical tools that allow trans-SNARE interactions to be manipulated in a physiologically relevant in vitro docking and fusion reaction. Vacuole fusion begins with priming. In this step, Sec17/18p (yeast -SNAP/NSF) consumes ATP to disassemble cis-SNARE complexes (Mayer et al., 1996; Ungermann et al., 1998a). In the next stage, docking, the Rab GTPase Ypt7p promotes membrane tethering (Mayer and Wickner, 1997; Ungermann et al., 1998b), and specialized subdomains called vertex sites assemble at the docking junction (Wang et al., 2002, 2003). During Ypt7p-mediated docking, Ca2+ is released from the vacuole lumen (Peters and Mayer, 1998; Eitzen et al., 2000). This Ca2+ signal is necessary for fusion because depletion of lumenal Ca2+ makes fusion dependent on added Ca2+, and chelation of extralumenal Ca2+ with BAPTA prevents fusion (Peters and Mayer, 1998). We now show that Ca2+ release during docking occurs in response to trans-SNARE interactions.
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Results |
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Docking factors and SNAREs promote Ca2+ release
To see if Vam7p influences Ca2+ efflux from the vacuole lumen, we used the photoprotein aequorin to monitor the extralumenal Ca2+ concentration [Ca2+]E during in vitro docking and fusion (Peters and Mayer, 1998). We began by evaluating the importance of proteins that function during docking.
Vacuoles actively sequester extralumenal Ca2+ during the first minutes of incubation with ATP (Fig. 2 A, standard). Experiments with the slow Ca2+ chelator EGTA indicate that the relatively high initial [Ca2+]E is due to ambient Ca2+ present in buffer solutions, and is not necessary for docking or fusion (Peters and Mayer, 1998; unpublished data; Margolis, N., personal communication). During docking, Ca2+ is released from the vacuole lumen into the extralumenal space, with [Ca2+]E typically peaking at 3040 min (Fig. 2 A). As reported previously (Peters and Mayer, 1998), this Ca2+ release requires both Sec17/18p-mediated priming and Ypt7p-mediated docking. Ca2+ was sequestered in the vacuole lumen but not released in the presence of anti-Sec17p antibody (Fig. 1 A). Recombinant Gdi1p (GDI; GDP dissociation inhibitor [GDI]) or affinity-purified anti-Ypt7p antibody, both inhibitors of Ypt7p function, also prevented Ca2+ release (Fig. 2 A).
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When rVam7p was added to a standard fusion assay, it caused an increase in docking-dependent Ca2+ efflux (Fig. 3 A). Ca2+ release was blocked by affinity-purified anti-Vam7p antibody or PX domain (Fig. 3, A and B). rVam7p reversed the PX and anti-Vam7p blocks (Fig. 3, A and B). Ca2+ release was also prevented by antibodies against the Q-SNAREs Vam3p (Fig. 3 C) or Vti1p (Fig. 3 D), or against the R-SNARE Nyv1p (Fig. 3, C and D). Control IgG, heat-denatured antibodies, and antibodies against the vacuolar SNARE Ykt6p had no detectable effect on Ca2+ release under these conditions (unpublished data). Thus the SNAREs Vam3p, Vti1p, Vam7p, and Nyv1p regulate docking-dependent Ca2+ signaling.
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Cycles of trans-SNARE complex assembly promote Ca2+ release
Sec17/18p disassemble complexed SNARE proteins. Recombinant Sec17p and Sec18p, and antibodies against these proteins, have provided useful tools for the manipulation of SNARE assembly dynamics. To further test whether the oligomeric state of SNARE proteins regulates Ca2+ release, we tested the effect of recombinant Sec18p (Fig. 5 A). Sec18p stimulated Ca2+ release at up to 300 nM; at higher concentrations this stimulation declined (see Discussion). The stimulatory effect of rVam7p and Sec18p (Fig. 5 A) combined was greater than with either reagent alone.
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To see if Sec17/18p action and Ca2+ release could be uncoupled, we used an antipeptide antibody that permits reversible inhibition of the Ypt7p GTPase. Ypt7p block-reversal can be used to allow Sec17/18p-mediated priming while accumulating the vacuoles at an early, Ypt7p-dependent stage of docking. Relief of the anti-Ypt7p block by adding the antigenic peptide allows the vacuoles to proceed through docking and fusion in a relatively synchronous manner (Eitzen et al., 2001, 2002). Reactions were initiated either without or with the Ypt7p antibody (Fig. 6). In reactions blocked with anti-Ypt7p for 35 min and then reversed (Fig. 6), anti-SNARE antibodies added 5 min before reversal were still inhibitory, indicating that SNARE function is still needed after priming, and during or after Ypt7p function. In marked contrast, anti-Sec17p antibodies did not prevent Ca2+ release upon peptide rescue. In a control reaction, anti-Sec17p, when added from the start of a reaction without anti-Ypt7p, prevented Ca2+ release. Thus, Sec17/18p function is not required during Ca2+ release step itself.
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Second, we took advantage of recent observations that, under appropriate conditions, rVam7p can bypass the requirement for Sec17/18p-mediated priming (unpublished data; Thorngren, N., personal communication). It appears that the only essential event mediated by Sec17/18p in vitro is the liberation of Vam7p from cis-SNARE complexes; sufficient quantities of unpaired integral-membrane SNAREs are present on unprimed vacuoles to catalyze fusion when the reaction is supplemented with rVam7p. When rVam7p was added to an anti-Sec17p-blocked reaction (Fig. 7 A, trace b), a fast spike of Ca2+ release was observed and this spike rapidly decayed. Similar results were obtained when rVam7p was used to bypass an anti-Sec18p block (unpublished data). We also asked if rVam7p could interact with Vam3p and Nyv1p located in trans to one another to promote efflux. For this purpose, a complementation experiment similar to the one shown in Fig. 3 was used. We found that rVam7p elicited transient Ca2+ efflux from anti-Sec17p-blocked mixtures of vacuoles derived from vam3 and nyv1
cells (Fig. 7 B), but it did not elicit Ca2+ efflux from either population alone. Anti-Vam3p antibody completely suppressed rVam7p rescue (Fig. 7 B). rVam7p bypass of an anti-Sec17p block thus requires Vam3p and Nyv1p, even when these SNAREs are available only in trans and not in cis. These results suggest that on pretethered vacuoles, rVam7 promotes near-synchronous formation of many trans-SNARE complexes and rapid Ca2+ efflux. In the absence of Sec17/18p activity, rVam7p-elicited efflux is short-lived relative to the sustained release promoted by Sec17/18p (Fig. 7 A, compare trace a with trace b).
In the third block-reversal protocol, PX domain was used to block Vam7p reassociation, and rVam7p was used to reverse the PX block as in Fig. 1 F. Under a PX block, priming and Ypt7p-dependent docking occur, but vertex assembly is incomplete and trans-SNARE pairing is prevented (Boeddinghaus et al., 2002; Wang et al., 2003). When the PX block was reversed, an immediate Ca2+ spike was observed (Fig. 7 A, trace c), and this spike was followed by prolonged Ca2+ release. When we plot the average values of the immediate spike from the anti-Sec17p/rVam7p reversal (Fig. 7 A, trace b) and the sustained but delayed spike from the anti-Sec18p/Sec18p reversal (Fig. 7 A, trace a), we obtain a curve (Fig. 7 A, dashed line) that closely matches the rescue curve for PX/rVam7p reversal. In addition, when the PX block was reversed by rVam7p in the presence of anti-Sec17p, the sustained component of the Ca2+ flux was absent and only the spike was observed as in trace b (unpublished data). Together, these experiments show that the reversal of inhibitor blocks by rVam7p can be used to synchronize Ca2+ release, and confirm that Sec17/18p promotes sustained cycles of Ca2+ release but need not operate during a single Ca2+ release event. We conclude that Sec17/18p disassembles SNARE complexes, producing unpaired SNAREs, which in turn enter new trans complexes that elicit Ca2+ release.
Under standard in vitro conditions (Conradt et al., 1994; Wang et al., 2002), two to three rounds of fusion occur in 6090 min (unpublished data). Because of the prompt onset and decay of Ca2+ release when rVam7p was used to reverse an anti-Sec17p block, we asked if fusion was accelerated under these conditions. Three approaches were used. First, we used anti-Vam3p antibody to block docking at various times after reversal (Fig. 8 A). Within 2 min after reversal, the shortest time tested, fusion had become completely resistant to anti-Vam3p. In contrast, when anti-Vam3p was added immediately before rVam7p, no fusion was observed. Thus, Vam3p is required at the time of rVam7p addition, but it is needed (or its functional determinants are antibody accessible) for <2 min after rVam7p addition. To examine fusion more directly, we measured vacuole size (surface area) under four conditions: at 14 min after Vam7p reversal of anti-Sec17p; at 2025 min after reversal; in reactions that were blocked but not reversed; and in reactions not blocked with anti-Sec17p (Fig. 8 B). We found that the geometric mean surface area of the vacuoles increased by 54% within 4 min after rVam7p reversal of the anti-Sec17 block, but no additional size increase was detected at later times. This methodology underestimates the actual amount of fusion because some limiting membrane is lost into the lumen in most fusion events (Wang et al., 2002), and because vacuoles too small to resolve (<250-nm diam) likely fuse to form small vacuoles that are then resolved, lowering the population's measured size. Thus, at least 0.5 round of fusion occurred in the experiment shown, a result consistent with the amount of alkaline phosphatase maturation observed in our standard fusion assay (unpublished data). In the third approach, we observed fusion in real time during the 14 min interval (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200310105/DC1). During this interval, we observed fusion among 15% of the vacuoles. This measurement also is likely to underestimate fusion because recording was initiated
1 min after reversal of the block by rVam7p, and because events out of the focal plane could not be clearly resolved. Thus, docking (assayed by sensitivity to anti-Vam3p), Ca2+ efflux, and fusion occur promptly upon rVam7p-mediated bypass of the priming block. Together, our data show that the forward pathway of trans-SNARE complex assembly leads to Ca2+ release and fusion. Sec17/18p are not needed during Ca2+ efflux but promote sustained Ca2+ efflux by catalyzing SNARE complex turnover and making unpaired SNAREs available for additional cycles of trans-complex assembly.
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Discussion |
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Trans-SNARE interactions can occur only during docking. If Ca2+ release is triggered by trans-SNARE interactions, then docking-independent SNARE complex assembly and disassembly should not cause Ca2+ release. Consistent with this prediction, staging experiments show that SNAREs are needed for Ca2+ release at a post-priming step of the reaction (Figs. 6 and 7). Moreover, the addition of excess recombinant Sec17p, which inhibits fusion by promoting the reformation of cis-SNARE complexes from unpaired SNAREs, attenuates Ca2+ release (Fig. 4 C), indicating that cis-SNARE complex reformation does not trigger Ca2+ release. Together, these results indicate that neither cis-SNARE complex disassembly nor cis-complex reformation trigger Ca2+ release.
Ypt7p is required for tethering, vertex assembly, and trans-SNARE pairing (Mayer and Wickner, 1997; Ungermann et al., 1998b; Wang et al., 2002, 2003). As shown in Fig. 5, SNAREs are needed for Ca2+ release at a stage during or after Ypt7p function, for example, during docking. In addition, Rho GTPases and the SM-protein Vps33p act at docking, regulate SNARE complex formation (Sato et al., 2000; Eitzen et al., 2001; Muller et al., 2001), and are needed for Ca2+ release.
The most direct evidence that trans-SNARE interactions trigger Ca2+ release is shown in Fig. 4: in vitro complementation between populations of vacuoles lacking either Vam3p or Nyv1p indicates that these SNAREs can act from opposite membranes to trigger docking-dependent Ca2+ release. Moreover, rVam7p stimulation of Ca2+ release requires both Vam3p and Nyv1p, even when these SNAREs are located in trans to one another (Fig. 7 B).
Role of Sec17/18p
Two types of staging experiments, Ypt7p block release and Vam7p bypass of Sec17/18p function, indicate that Sec17/18p need not function during the Ca2+ release event itself. Instead, Sec17/18p are required for sustained cycles of Ca2+ release, as shown in Fig. 5 B, Fig. 6 B, and Fig. 7 A. Sec17/18p catalyze the disassembly of SNARE complexes, liberating unpaired SNAREs which can participate in new trans interactions. Recombinant Sec18p promotes Ca2+ release with maximal stimulation at 300 nM (Fig. 5 A), close to the in vivo level of
200 nM (Ghaemmaghami et al., 2003). At higher levels of Sec18p (>400 nM), the stimulation of Ca2+ release is attenuated, possibly due to the disassembly of trans-SNARE complexes (Ungermann et al., 1998b) before they can complete their stimulation of Ca2+ efflux. When Sec17/18p function is prevented, Ca2+ release decays rapidly. These observations suggest that each trans-SNARE interaction event triggers only a short-lived transient of Ca2+ release, and that repeated cycles of trans-SNARE interactions are needed for sustained Ca2+ release.
The Ca2+ release pathway
Our data show that docking-dependent trans-SNARE interactions promote Ca2+ release. However, we emphasize that the functional trans-SNARE interactions defined in this paper might not be identical to SNARE core complexes or trans-SNARE complexes isolated from vacuole detergent extracts (Ungermann et al., 1998b); additional work is needed to understand SNARE association cycles and dynamics. Docking-dependent Ca2+ release may be triggered directly by SNARE interactions with a Ca2+-releasing channel, or release may result from sequential interactions of SNAREs on apposed membranes with unidentified intermediary molecules.
It is unlikely that the observed Ca2+ release occurs as an indirect consequence of fusion events. Mayer's group recently reported that vacuoles isolated from vph1 cells dock and release Ca2+ in a Ypt7p-dependent manner, but do not go on to fuse (Bayer et al., 2003). In addition, manipulation of Sec17/18p function after priming can dramatically raise or lower the amplitude of Ca2+ release with only modest effects on fusion. These observations, together with the Ca2+-dependence of fusion itself (Peters and Mayer, 1998), strongly suggest that the Ca2+ release events studied here occur during docking, not after fusion.
A channel or transporter required for docking-dependent Ca2+ release from the vacuole has not yet been identified, despite our efforts (Fig. 8) and studies of vacuolar Ca2+ homeostasis in other laboratories (Takita et al., 2001; Bayer et al., 2003; Zhou et al., 2003). A number of uncharacterized ORFs in S. cerevisiae appear to encode ion transporters, and one of these could be responsible for docking-dependent Ca2+ release. However, redundancy among more than one transporter might frustrate efforts to identify the relevant proteins through analyses of single knockouts.
Docking-dependent Ca2+ release might occur through less conventional mechanisms. In most models of fusion, lipids at the fusion site transiently assume nonbilayer morphologies. Simulations (Muller et al., 2003) indicate that these rearrangements may form transient pores between cytoplasmic and noncytoplasmic compartments, and careful measurements of fusion events mediated by the influenza hemagglutinin protein confirm that transient leakage currents can accompany fusion (Frolov et al., 2003). Trans-SNARE complex formation might directly promote ion flux by perturbing bilayer structure.
Implications of SNARE-dependent Ca2+ release during docking
The physical proximity of Ca2+ channels to the fusion machinery is suggested by experiments in which fusion is prevented by fast, but not slow, Ca2+ chelators (Sullivan et al., 1993; Neher, 1998; Peters and Mayer, 1998; Pryor et al., 2000), and by the brief interval (200 µs) between channel gating and exocytosis in neurons (Llinas et al., 1981). Furthermore, many reports document physical and regulatory interactions between Ca2+ channels and SNARE proteins in animal cells (Bennett et al., 1992; Yoshida et al., 1992; Sheng et al., 1994; Mochida et al., 1996; Wiser et al., 1996; Rettig et al., 1997). Ca2+ channels associate with other fusion factors, including Rab3-interactor binding proteins (Hibino et al., 2002) and the synaptic Ca2+ sensor synaptotagmin (Sudhof, 2002). SNAREs are also implicated in store-operated Ca2+ entry, which may require membrane docking (Yao et al., 1999). Interactions between Ca2+ signaling proteins and docking and fusion factors could have two functions: to allow channels to monitor the functional status of docking over time, and to ensure that the fusion machinery and regions of peak Ca2+ flux coincide in space (Neher, 1998). For intracellular fusion events, these interactions may trigger Ca2+ flux in response to successful docking. In synapses, where voltage-gated Ca2+ channels respond to membrane depolarization, similar mechanisms might bias Ca2+ flux toward channels associated with primed and docked vesicles. Our experiments with vacuoles suggest that trans-SNARE complex formation is a checkpoint that controls progression to fusion. In this view, trans-SNARE interactions signify that docked membranes reside within a certain minimum distance and verify that specific biochemical events have transpired (e.g., priming and vertex subdomain assembly), triggering Ca2+ release and downstream events leading to fusion.
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Materials and methods |
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Reagents
rVam7p (residues 2316) and Vam7p PX domain (residues 2123) were expressed as GST fusions from the pGEX-KT vector (Hakes and Dixon, 1992) in BL21-pRP cells (Stratagene). VAM7 sequences were amplified from BJ3505 DNA using a forward primer with an engineered BamH1 site (5'-cgcGGATCCGCAgctaattctgtaggg-3') and reverse primers with engineered EcoR1 sites (5'-cgGAATTCTCAagcactgttgttaaaatgtctagc-3' for rVam7p, and 5'-cgGAATTCACTTtgacaactgcaggaagac-3' for PX). Cells were grown in TB medium (Maniatis et al., 2001), 200 mg/l ampicillin, and 34 mg/l chloramphenicol to OD600 = 2.3, and expression was induced with 0.5 mM IPTG for 4 h at 26°C. Cell pellets (10,000 g, 20°C, 5 min) were resuspended in two pellet volumes of PBS with 2 mM EGTA, 1 mM EDTA, 1x protease inhibitor cocktail (Haas, 1995), 1 mM PMSF, and 0.01% 2-mercaptoethanol. The cell suspension was frozen dropwise in liquid N2 and stored at 80°C. Cells were thawed, lysed in a French press, mixed with Triton X-100 (0.5% final), and rocked for 20 min at 4°C before clearing (20,000 g, 4°C, 30 min). Glutathione-agarose (1 ml of slurry per 10 ml of lysate; Amersham Biosciences) was added to cleared lysate and rocked overnight at 4°C. The beads were washed with PBS supplemented as above (20 bed volumes), then with PBS alone (20 bed volumes), and finally with cleavage buffer (50 mM TrisCl, pH 8.0, 100 mM NaCl, 2 mM CaCl2, 0.1% 2-mercaptoethanol; 10 bed volumes). Proteins were cleaved from the support with thrombin (10 U/mg fusion protein, in two to three bed volumes of cleavage buffer; Sigma-Aldrich) for 34 h at 23°C. Thrombin was removed by flowing the eluted cleavage products directly over a second column containing 1 ml p-aminobenzamidineagarose (Sigma-Aldrich). Proteins were exchanged into reaction buffer (20 mM PIPES/KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2, 200 mM sorbitol) on G-25 resin (Amersham Biosciences). Aliquots were frozen in liquid N2, stored at 80°C, and thawed just before use.
Antibodies were raised against peptides from Ypt7p (Eitzen et al., 2001), Vps33p (for Vps33p, NH2-CLEDTEQWQKDGFDLNSKKT, and NH2-CIEDEHAADKITNENDDFSEA); against rVam7p and Sec17p (Haas and Wickner, 1996); and against recombinant cytoplasmic domains of Vam3p (Nichols et al., 1997), Nyv1p (Ungermann et al., 1998a), and Vti1p (Ungermann et al., 1999). IgG or affinity-purified antibodies were prepared as described previously (Harlow and Lane, 1999). For anti-Ypt7p reversal (Eitzen et al., 2001) peptide was used at 20 µg/ml final. Antibodies were stored in PS buffer (20 mM Pipes/KOH, 200 mM sorbitol, pH 6.8) at 4°C or 80°C after buffer exchange on Sepharose G-25 resin (Amersham Biosciences) or dialysis. Some antibody preparations were concentrated in Microcon-30 ultrafiltration devices (Millipore).
Fusion
30 µl of standard fusion reactions contained a 1:1 mixture of vacuoles (Haas, 1995) from strains BJ3505 and DKY6281 (6 µg total protein by Bradford assay; premixed in PS buffer) added to a mixture containing: 20 mM Pipes/KOH, pH 6.8, 200 mM sorbitol, 125 mM KCl, 5 mM MgCl2, 10 µM coenzyme A, 4.5 µM recombinant Pbi2p (IB2), 10 nM recombinant His6-Sec18p (except as noted), 1% (wt/vol) defatted BSA (Sigma-Aldrich), and 0.03x protease inhibitor cocktail (Haas, 1995). ATP-regenerating system (Haas, 1995) was added from a 10x stock to 1x final (0.67x for Ca2+ efflux assays). Fusion assays were incubated for 80 min at 27°C or as noted in figure legends, and fusion was measured by determining the amount of active alkaline phosphatase as described previously (Haas, 1995) except that phosphatase assay buffer was supplemented with 10 mM CaCl2. Ca2+ efflux assays were performed with 10 µg rather than 6 µg of vacuoles per 30 µl vol. For experiments using vacuoles from mutant cells, highly pure oxalyticase (Enzogenetics) was used to prepare spheroplasts for vacuole isolation.
Ca2+ assay
Aequorin luminescence assays were performed as described previously (Peters and Mayer, 1998; Muller et al., 2002) with modifications. Samples were analyzed in 96-well, low protein binding, conical bottom plates (Nunc) in a luminometer (Molecular Devices). IGOR Pro 4 (WaveMetrics) and JMP 5 (SAS Institute) were used for data analysis. Calibration was done using buffered Ca2+ EGTA standard solutions containing reaction salts including the ATP regenerating system, prepared as described previously (Tsien and Pozzan, 1989), and checked with MAX Chelator (Bers et al., 1994) version WEBMAXC 10.14.2002 (http://www.stanford.edu/~cpatton). Commercial Ca2+ standards gave similar results. A curve was fit to the luminance for each Ca2+ standard divided by peak luminance at saturating Ca2+ (L/Lmax), using a two-state model with three Ca2+-binding sites (Allen et al., 1977): L/Lmax = [(1 + KR [Ca2+])/(1 + KTR + KR [Ca2+])]3.
We obtained KR = 3.88 ± 0.88 x 106 M-1 and KTR = 1.45 ± 0.29 x 102 (mean ± SD) similar to values reported by Allen et al. (1977). KR and KTR were used to calculate [Ca2+] in experimental samples. In addition to Ca2+-dependent luminance, aequorin undergoes irreversible, Ca2+-dependent inactivation. At constant-free [Ca2+] (i.e., in buffered standard solutions), the apparent [Ca2+] gradually decays exponentially over time. At 640 nM of free [Ca2+], the decay had a time constant = 95 ± 4 min (mean r2 > 0.97). The traces shown are not adjusted for aequorin inactivation.
Microscopy
Images were obtained on a microscope (model BX51/61; Olympus) with a Hg arc lamp, a 60x 1.4 NA Plan Apochromat objective, and a camera (model Sensicam QE; Cooke). The camera and microscope are controlled by IP Lab (Scanalytics). Vacuole sizes were measured by photographing wet mounts of reactions containing 2 µM MDY-64, a fluorescent lipid probe (Molecular Probes). Vacuoles were measured using Image/J (http://rsb.info.nih.gov/ij/) by manually circumscribing vacuoles with best-fit ellipses. The mean of the major and minor diameters of each ellipse was used to estimate vacuole surface area, by approximating vacuoles as spheres of radius diam/2. Surface areas were distributed approximately log normally, so these data were log transformed before statistical analysis (in JMP 5; SAS Institute) and geometric means were reported.
Online supplemental material
Video 1. Video microscopy of fusion occurring after rVam7p rescue of an anti-Sec17p block. Video microscopy images were acquired with a 60x 1.4 NA Plan Apochromat objective and a camera (model Sensicam QE; Cooke) mounted on a hybrid BX51/61 platform (Olympus). The camera and microscope were controlled by IP Lab (Scanalytics) running under Mac OS X. Illumination was provided by an Hg arc lamp (Olympus). Time-lapse observations were made under illumination attenuated by >98% using neutral density filters (Olympus) and an infrared-blocking filter (Schott), and data was acquired with 2 x 2 pixel binning. Images were acquired at ambient temperature, measured at 2325°C with a thermocouple on the microscope stage. For time-lapse sequences a sharpening filter (5 x 5 hat) was applied to each frame. Online supplemental material is available at htttp://www.jcb.org/cgi/content/full/jcb.200310105/DC1.
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Acknowledgments |
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Submitted: 22 October 2003
Accepted: 4 December 2003
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References |
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Allen, D.G., J.R. Blinks, and F.G. Prendergast. 1977. Aequorin luminescence: relation of light emission to calcium concentrationa calcium-independent component. Science. 195:996998.[Medline]
Baker, D., L. Wuestehube, R. Schekman, D. Botstein, and N. Segev. 1990. GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction. Proc. Natl. Acad. Sci. USA. 87:355359.[Abstract]
Bayer, M.J., C. Reese, S. Buhler, C. Peters, and A. Mayer. 2003. Vacuole membrane fusion: V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel. J. Cell Biol. 162:211222.
Beckers, C.J., and W.E. Balch. 1989. Calcium and GTP: essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus. J. Cell Biol. 108:12451256.[Abstract]
Bennett, M.K., N. Calakos, and R.H. Scheller. 1992. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science. 257:255259.[Medline]
Bers, D.M., C.W. Patton, and R. Nuccitelli. 1994. A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol. 40:329.[Medline]
Bezprozvanny, I., R.H. Scheller, and R.W. Tsien. 1995. Functional impact of syntaxin on gating of N-type and Q-type calcium channels. Nature. 378:623626.[CrossRef][Medline]
Boeddinghaus, C., A.J. Merz, R. Laage, and C. Ungermann. 2002. A cycle of Vam7p release from and PtdIns 3-Pdependent rebinding to the yeast vacuole is required for homotypic vacuole fusion. J. Cell Biol. 157:7989.
Brachmann, C.B., A. Davies, G.J. Cost, E. Caputo, J. Li, P. Hieter, and J.D. Boeke. 1998. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 14:115132.[CrossRef][Medline]
Cheever, M.L., T.K. Sato, T. de Beer, T.G. Kutateladze, S.D. Emr, and M. Overduin. 2001. Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes. Nat. Cell Biol. 3:613618.[CrossRef][Medline]
Chen, Y.A., S.J. Scales, S.M. Patel, Y.C. Doung, and R.H. Scheller. 1999. SNARE complex formation is triggered by Ca2+ and drives membrane fusion. Cell. 97:165174.[Medline]
Conradt, B., A. Haas, and W. Wickner. 1994. Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro. J. Cell Biol. 126:99110.[Abstract]
Cunningham, K.W., and G.R. Fink. 1994. Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases. J. Cell Biol. 124:351363.[Abstract]
Eitzen, G., E. Will, D. Gallwitz, A. Haas, and W. Wickner. 2000. Sequential action of two GTPases to promote vacuole docking and fusion. EMBO J. 19:67136720.
Eitzen, G., N. Thorngren, and W. Wickner. 2001. Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking. EMBO J. 20:56505656.
Eitzen, G., L. Wang, N. Thorngren, and W. Wickner. 2002. Remodeling of organelle-bound actin is required for yeast vacuole fusion. J. Cell Biol. 158:669679.
Fasshauer, D., R.B. Sutton, A.T. Brunger, and R. Jahn. 1998. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc. Natl. Acad. Sci. USA. 95:1578115786.
Frolov, V.A., A.Y. Dunina-Barkovskaya, A.V. Samsonov, and J. Zimmerberg. 2003. Membrane permeability changes at early stages of influenza hemagglutinin-mediated fusion. Biophys. J. 85:17251733.
Ghaemmaghami, S., W.K. Huh, K. Bower, R.W. Howson, A. Belle, N. Dephoure, E.K. O'Shea, and J.S. Weissman. 2003. Global analysis of protein expression in yeast. Nature. 425:737741.[CrossRef][Medline]
Haas, A. 1995. A quantitative assay to measure homotypic vacuole fusion in vitro. Meth. Cell Sci. 17:283294.
Haas, A., and W. Wickner. 1996. Homotypic vacuole fusion requires Sec17p (yeast -SNAP) and Sec18p (yeast NSF). EMBO J. 15:32963305.[Abstract]
Haas, A., B. Conradt, and W. Wickner. 1994. G-protein ligands inhibit in vitro reactions of vacuole inheritance. J. Cell Biol. 126:8797.[Abstract]
Hakes, D.J., and J.E. Dixon. 1992. New vectors for high level expression of recombinant proteins in bacteria. Anal. Biochem. 202:293298.[Medline]
Harlow, E., and D. Lane. 1999. Purification of antibodies on an antigen column. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 7780.
Hibino, H., R. Pironkova, O. Onwumere, M. Vologodskaia, A.J. Hudspeth, and F. Lesage. 2002. RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca2+ channels. Neuron. 34:411423.[Medline]
Jahn, R., T. Lang, and T.C. Sudhof. 2003. Membrane fusion. Cell. 112:519533.[Medline]
Jones, E.W. 2002. Vacuolar proteases and proteolytic artifacts in Saccharomyces cerevisiae. Methods Enzymol. 351:127150.[Medline]
Llinas, R., I.Z. Steinberg, and K. Walton. 1981. Relationship between presynaptic calcium current and postsynaptic potential in squid giant synapse. Biophys. J. 33:323351.[Abstract]
Maniatis, T., D.W. Russell, and J. Sambrook. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2.4.
Mayer, A., and W. Wickner. 1997. Docking of yeast vacuoles is catalyzed by the Ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF). J. Cell Biol. 136:307317.
Mayer, A., W. Wickner, and A. Haas. 1996. Sec18p (NSF)-driven release of Sec17p (-SNAP) can precede docking and fusion of yeast vacuoles. Cell. 85:8394.[Medline]
Mochida, S., Z.H. Sheng, C. Baker, H. Kobayashi, and W.A. Catterall. 1996. Inhibition of neurotransmission by peptides containing the synaptic protein interaction site of N-type Ca2+ channels. Neuron. 17:781788.[Medline]
Muller, O., D.I. Johnson, and A. Mayer. 2001. Cdc42p functions at the docking stage of yeast vacuole membrane fusion. EMBO J. 20:56575665.
Muller, O., M.J. Bayer, C. Peters, J.S. Andersen, M. Mann, and A. Mayer. 2002. The Vtc proteins in vacuole fusion: coupling NSF activity to V(0) trans-complex formation. EMBO J. 21:259269.
Muller, M., K. Katsov, and M. Schick. 2003. A new mechanism of model membrane fusion determined from monte carlo simulation. Biophys. J. 85:16111623.
Neher, E. 1998. Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron. 20:389399.[Medline]
Nichols, B.J., C. Ungermann, H.R. Pelham, W.T. Wickner, and A. Haas. 1997. Homotypic vacuolar fusion mediated by t- and v-SNAREs. Nature. 387:199202.[CrossRef][Medline]
Peters, C., and A. Mayer. 1998. Ca2+/calmodulin signals the completion of docking and triggers a late step of vacuole fusion. Nature. 396:575580.[CrossRef][Medline]
Price, A., D. Seals, W. Wickner, and C. Ungermann. 2000. The docking stage of yeast vacuole fusion requires the transfer of proteins from a cis-SNARE complex to a Rab/Ypt protein.PG. J. Cell Biol. 148:12311238.
Pryor, P.R., B.M. Mullock, N.A. Bright, S.R. Gray, and J.P. Luzio. 2000. The role of intraorganellar Ca2+ in late endosomelysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles. J. Cell Biol. 149:10531062.
Reddy, A., E.V. Caler, and N.W. Andrews. 2001. Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes. Cell. 106:157169.[Medline]
Rettig, J., and E. Neher. 2002. Emerging roles of presynaptic proteins in Ca2+-triggered exocytosis. Science. 298:781785.
Rettig, J., C. Heinemann, U. Ashery, Z.H. Sheng, C.T. Yokoyama, W.A. Catterall, and E. Neher. 1997. Alteration of Ca2+ dependence of neurotransmitter release by disruption of Ca2+ channel/syntaxin interaction. J. Neurosci. 17:66476656.
Sato, T.K., P. Rehling, M.R. Peterson, and S.D. Emr. 2000. Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion. Mol. Cell. 6:661671.[Medline]
Schekman, R. 1998. Membrane fusion. Ready...aim...fire! Nature. 396:514515.[CrossRef][Medline]
Seals, D.F., G. Eitzen, N. Margolis, W.T. Wickner, and A. Price. 2000. A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion. Proc. Natl. Acad. Sci. USA. 97:94029407.
Sheng, Z.H., J. Rettig, M. Takahashi, and W.A. Catterall. 1994. Identification of a syntaxin-binding site on N-type calcium channels. Neuron. 13:13031313.[Medline]
Shin, D.M., X.S. Zhao, W. Zeng, M. Mozhayeva, and S. Muallem. 2000. The mammalian Sec6/8 complex interacts with Ca2+ signaling complexes and regulates their activity. J. Cell Biol. 150:11011112.
Sudhof, T.C. 2002. Synaptotagmins: why so many? J. Biol. Chem. 277:76297632.
Sullivan, K.M., W.B. Busa, and K.L. Wilson. 1993. Calcium mobilization is required for nuclear vesicle fusion in vitro: implications for membrane traffic and IP3 receptor function. Cell. 73:14111422.[Medline]
Sutton, R.B., D. Fasshauer, R. Jahn, and A.T. Brunger. 1998. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature. 395:347353.[CrossRef][Medline]
Takita, Y., L. Engstrom, C. Ungermann, and K.W. Cunningham. 2001. Inhibition of the Ca2+-ATPase Pmc1p by the v-SNARE protein Nyv1p. J. Biol. Chem. 276:62006206.
Tsien, R., and T. Pozzan. 1989. Measurement of cytosolic free Ca2+ with quin2. Methods Enzymol. 172:230262.[Medline]
Ungermann, C., and W. Wickner. 1998. Vam7p, a vacuolar SNAP-25 homolog, is required for SNARE complex integrity and vacuole docking and fusion. EMBO J. 17:32693276.
Ungermann, C., B.J. Nichols, H.R. Pelham, and W. Wickner. 1998a. A vacuolar vt-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion. J. Cell Biol. 140:6169.
Ungermann, C., K. Sato, and W. Wickner. 1998b. Defining the functions of trans-SNARE pairs. Nature. 396:543548.[CrossRef][Medline]
Ungermann, C., G.F. von Mollard, O.N. Jensen, N. Margolis, T.H. Stevens, and W. Wickner. 1999. Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion. J. Cell Biol. 145:14351442.
Vollmer, P., E. Will, D. Scheglmann, M. Strom, and D. Gallwitz. 1999. Primary structure and biochemical characterization of yeast GTPase-activating proteins with substrate preference for the transport GTPase Ypt7p. Eur. J. Biochem. 260:284290.
Wang, L., C. Ungermann, and W. Wickner. 2000. The docking of primed vacuoles can be reversibly arrested by excess Sec17p (-SNAP). J. Biol. Chem. 275:2286222867.
Wang, L., E.S. Seeley, W. Wickner, and A.J. Merz. 2002. Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle. Cell. 108:357369.[Medline]
Wang, L., A.J. Merz, K.M. Collins, and W.T. Wickner. 2003. A hierarchy of protein interactions organizes assembly of the vacuolar vertex membrane docking complex. J. Cell Biol. 160:365374.
Weber, T., B.V. Zemelman, J.A. McNew, B. Westermann, M. Gmachl, F. Parlati, T.H. Sollner, and J.E. Rothman. 1998. SNAREpins: minimal machinery for membrane fusion. Cell. 92:759772.[Medline]
Wiser, O., M.K. Bennett, and D. Atlas. 1996. Functional interaction of syntaxin and SNAP-25 with voltage-sensitive L- and N-type Ca2+ channels. EMBO J. 15:41004110.[Abstract]
Yao, Y., A.V. Ferrer-Montiel, M. Montal, and R.Y. Tsien. 1999. Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger. Cell. 98:475485.[Medline]
Yoshida, A., C. Oho, A. Omori, R. Kuwahara, T. Ito, and M. Takahashi. 1992. HPC-1 is associated with synaptotagmin and omega-conotoxin receptor. J. Biol. Chem. 267:2492524928.
Zhou, X.L., A.F. Batiza, S.H. Loukin, C.P. Palmer, C. Kung, and Y. Saimi. 2003. The transient receptor potential channel on the yeast vacuole is mechanosensitive. Proc. Natl. Acad. Sci. USA. 100:71057110.
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