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Department of Physiology, University of Wisconsin, Madison, Wisconsin
Received 6 May 2005/ Returned for modification 27 June 2005/ Accepted 8 July 2005
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The apparent Ca2+ affinities of a number of syt isoforms have been explored by measuring the [Ca2+]1/2 for binding to liposomes composed of phosphatidylserine (PS) and phosphatidylcholine (PC). In a number of studies it was shown that all syt isoforms tested exhibited roughly the same apparent affinity for Ca2+. In particular, syt III and VII were shown to have virtually the same [Ca2+]1/2 values as syt I (26, 27, 39). However, more recent studies suggested that syt III and VII may bind PS/PC at much lower [Ca2+] (36). Thus, whether different isoforms of syt have distinct affinities for Ca2+ remains an open issue. Moreover, there are no functional data to address the idea that syt's couple distinct ranges of metals to membrane fusion.
If syt's have in fact diverged to sense different ranges of Ca2+ and if high- and low-affinity isoforms of syt are both targeted to the same secretory organelle, it follows that the ratio of these isoforms might determine the metal (i.e., Ca2+, Ba2+, and Sr2+) requirements for exocytosis. At present, there is little consensus regarding the localization of syt isoforms other than syt I. For example, it has been reported that syt VII is targeted to intracellular structures, including the Golgi (16) and large dense core vesicles (LDCV) in PC12 cells (14). Other studies concluded that syt VII is not targeted to intracellular organelles in PC12 cells but rather is selectively localized to the plasma membrane in this cell type, as well as in neurons, and in non-neuronal cells (19, 35). Finally, Andrews and coworkers reported that syt VII is targeted to lysosomes in non-neuronal cells (28, 32).
Here, we focus on syt VII and PC12 cells to test the hypothesis that syt's have diverged to sense different ranges of [Ca2+] and that more than one isoform of syt is targeted to secretory organelles. Our data indicate that the ratio of syt's with high (e.g., syt VII) and low apparent affinities for Ca2+ (e.g., syt I and IX) can fine-tune the metal requirements for exocytosis. We note that syt IX (15, 22, 37, 48) is sometimes referred to as syt V (8, 21).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oligonucleotides encoding shRNAs were obtained from QIAGEN and were phosphorylated using T4 polynucleotide kinase (Invitrogen). The resulting inserts were ligated into pSHAG-1 vector (provided by G. Hannon, Cold Spring Harbor, NY) via BseRI and BamHI sites. Three constructs were tested for their ability to knockdown exogenous and endogenous syt VII. The construct that exhibited the most effective knockdown of syt VII targets an mRNA sequence that corresponds to the following coding sequence: 1002 to 1020. The oligonucleotide sequence was 5'-TCCAGTTCAGTGTTGGCTAGAAGCTTGTAGCCAACACTGAACTGGATTTTTT-3' (sense) and 5'-GATCAAAAAATCCAGTTCAGTGTTGGCTACAAGCTTCTAGCCAACACTGAACTGGACG-3' (antisense). This construct was designated pSHAG-1-syt VII and was used to knock-down syt VII in the voltammetry assays shown in Fig. 7. All constructs were confirmed by DNA sequencing.
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20 µM [9]). This slight difference is because in the current study the free [Ca2+] was calculated, whereas in previous studies we determined the [Ca2+] by using a Ca2+ electrode (9). Using calculated values simplified comparisons between the relative abilities of Ca2+, Sr2+, and Ba2+ to trigger binding. Binding assays were carried out in triplicate on three independent occasions. The results in all cases were similar; representative data from one experiment are shown in Fig. 1, where we plot the mean ± the standard error of the mean (SEM).
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For immunoblot analysis of RNA interference (RNAi) knockdown cells, transiently transfected cells were harvested and lysed by using lysis buffer (0.5% Triton X-100, 0.05% sodium dodecyl sulfate [SDS], and 5 mM phenylmethylsulfonyl fluoride in phosphate-buffered saline) 48 to 72 h after transfection. Samples were centrifuged at 21,000 x g for 10 min, and the protein concentration of the supernatant was quantified with a BCA assay (Pierce). To detect syt VII expression, 200 µg of supernatant from each sample was subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis. syt VII and IX were detected with polyclonal antibodies described in reference 37; syt I was detected with a monoclonal antibody (41.1; provided by R. Jahn, Gottingen, Germany). For detection of syt I and syt IX, 20 µg of supernatant from each sample was analyzed (37).
For stable transfections, tetracycline-off PC12 cells were cotransfected with 20 µg of pTRE-syt VII and 20 µg of pTK-hygromycin. Hygromycin was added after 48 h to a final concentration of 0.2 mg/ml. Medium was replaced with fresh hygromycin-containing medium every 4 days. Individual cell colonies were picked after 3 to 4 weeks of selection and placed in 96-well plates. Control cell lines were generated in parallel by cotransfection of 20 µg of pTRE lacking an insert and 20 µg of pTK-hygromycin. To compare expression levels of syt VII in wild-type PC12 cells, syt VII+ cell lines, and control cell lines, cells were harvested and lysed to obtain a crude total membrane fraction (37). Quantitative immunoblot assays were carried out as described previously (37) by using a fragment of syt VII (residues 1 to 260) fused to GST as a standard (20 µg of membrane preparation was assayed for each cell line). Signals were quantified by densitometry, and the abundance of syt VII in the membrane preparation was calculated from the standard curve made from recombinant protein. These data are plotted in Fig. 2 (lower panel); error bars represent the SEM from triplicate determinations. As controls, we also blotted for synaptobrevin II (monoclonal antibody 69.1), syt I, and syt IX. We assayed for tetracycline induction of syt VII expression but did not observe consistent increases in expression. Thus, all experiments were carried out without inducing the cells.
Immunocytochemistry.
syt VII+ PC12 cells were plated on 0.01% poly-L-lysine-coated coverslips at approximately the same density for each antibody labeling condition. Cells were washed once in phosphate-buffered saline (PBS) without Ca2+ or Mg2+ and fixed for 15 min in 4% paraformaldehyde with 4% sucrose in PBS. After one quick wash in PBS, residual paraformaldehyde was quenched by a 15-min incubation in 0.15 M glycine in PBS. Cells were simultaneously permeabilized and blocked overnight with 0.25 mg of saponin/ml in blocking buffer consisting of 10% goat serum, 6% bovine serum albumin (BSA), 0.1 M NH4Cl, 1 mM MgCl2, and 1 mM EGTA in 0.1 M morpholineethanesulfonic acid (pH 6.8). Primary and secondary antibodies were diluted in blocking buffer containing saponin. Cells were incubated for 1 h with a combination of the following primary antibodies: polyclonal rabbit anti-syt VII antibody (1:350) (37), monoclonal anti-syt I antibody (1:500; antibody 41.1), monoclonal anti-chromogranin B antibody (Cg; 1:500; provided by W. Huttner, Dresden, Germany), monoclonal anti-Lamp1 antibody (1:500; provided by N. Andrews, New Haven, CT), polyclonal rabbit anti-secretogranin II (Sg; 1:500; QED Biosciences, San Diego, CA), or monoclonal anti-syntaxin 1A antibody (1:500; provided by R. Jahn, Gottingen, Germany). The cells were then washed four times in PBS plus saponin and incubated for 1 h with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies (Molecular Probes, Eugene, OR) diluted 1:800. After four 10-min washes in PBS with saponin, coverslips were washed once in distilled H2O and mounted on slides with Prolong mounting reagent (Molecular Probes, Eugene, OR). Cells were imaged on a Nikon TE300 inverted microscope using a 100x, 1.4-numerical-aperture objective lens. Images were acquired by using a Princeton Instruments Micromax cooled charge-coupled device camera (Roper Scientific, Princeton, NJ) controlled by Metamorph software (Universal Imaging Corp, West Chester, PA). Z-series images were obtained at x100 magnification with 200-nm sectioning. The resulting Z-stacks were deconvolved (100 to 200 iterations) by using Autodeblur/autovisualize software (AutoQuant Imaging Inc, Watervliet, NY). Colocalization was quantified by thresholding the green and red images and measuring the overlap between the two (red to green and green to red) for each optical section by using ImageJ (National Institutes of Health) software. Thresholds were set to include the top 33% of the total integrated fluorescence of the cell, which corresponds to 8% Lamp1, 15% syt VII, 11% chromogranin and secretogranin, and 14% syt I of the total cell volume. As such, if the distributions of, for example, syt VII and chromogranin, were random (uncorrelated), one would predict that they would overlap 1.2% of the time. In each case, we corrected for this factor. For each pair of antibodies six cells were analyzed, taken from at least two coverslips. For each cell, all voxels were analyzed for colocalization (50 optical sections per cell) to ensure z position within the cell did not influence the analysis. Colocalization was assessed by overlap of thresholds for the two labels. Pooled data are expressed as mean ± the SEM.
For the line scan data plotted in Fig. 3C, images of syntaxin 1A and syt VII were obtained and deconvolved as described above (using syt VII+ clone G10). Two 0.5-µm-thick lines were drawn across individual cells at roughly the angles shown in Fig. 3B. To better compare overlap, we normalized the syt VII and syntaxin fluorescence signals to the same arbitrary value. We then determined the degree of overlap between signals. This was carried out by using data from 10 cells, two line scans per cell. The overall degree of overlap was 12% (SEM = 1.95). A representative example of the normalized line scan data is shown in Fig. 3C.
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Release of catecholamine was monitored in real time with a rotating disk electrode voltammetry setup as described previously (12, 37). Release curves were fitted with single-exponential functions by using Axograph software, yielding time constants (
). The rate (i.e., the reciprocal of the time constant, 1/), was plotted against [divalent cation]. The amplitude of response 80 s after addition of metal was also determined. These values were normalized to the maximum amplitude and plotted versus [divalent cation]. The data are shown as the mean ± the SEM (from triplicate determinations) in all plots and tables.
For the Sr2+ and Ba2+ titrations, data were fit to sigmoidal dose-response curves as described above for liposome binding assays. Since the Ca2+ dose-response data were not well fitted by sigmoidal dose-response equations, we connected the datum points and determined the effective concentration for 50% effect (EC50) values by drawing a line through the 50% Ymax and reading the X value at the point where this line intersects the data curve. This analysis was carried out using data from three independent Ca2+ titrations for the experiments shown in Fig. 5C and D and Fig. 7B. The mean ± the SEM for these studies are provided in Tables 1 and 3.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in Fig. 1, the EC50 values for Ca2+-, Sr2+-, and Ba2+-triggered binding of syt VII to PS/PC liposomes were markedly lower than for syt I (16-, 3-, and 9-fold lower, respectively). Thus, syt VII exhibits a higher apparent affinity than syt I for all divalent cations capable of triggering exocytosis (i.e., Ca2+, Sr2+, and Ba2+) (24) and is a bona fide high-affinity divalent cation sensor.
Upregulation and subcellular localization of syt VII in PC12 cells. If syt's function as metal sensors in vivo, syt VII would be expected to trigger release at lower [Ca2+], [Ba2+], and [Sr2+] than syt I. We addressed this question using PC12 cells as a model system. In this cell line, the most abundant isoforms are syt I and IX (37), both of which are relatively low affinity divalent cation sensors (Fig. 1; see also reference 22, where it is reported that the [Ca2+]1/2 for syt IX-liposome interactions is 14 µM). The only high-affinity isoform expressed in PC12 cells, syt VII, is at least 30 times less abundant than syt I (37).
In order to change the ratio between syt VII and syt I/IX (i.e., between high- and low-affinity sensors), we increased the expression level of syt VII so that it approached the abundance of syt I and IX. To this end, we transfected PC12 cells with an expression vector that encodes untagged syt VII. Three independent stable cell lines that overexpressed syt VII (designated as syt VII+) were characterized, as were three control cell lines transfected with the empty vector. Quantitative immunoblot analysis revealed that syt VII was upregulated to different extents in each of the VII+ clones (Fig. 2; syt VII+ clones A1, G10 and G3 exhibited 17-, 10-, and-6-fold increases in the level of syt VII expression, respectively). In clones A1, G10, and G3, syt VII had 50, 30, and 18% of the abundance of syt IX, and 30, 18, and 10% of the levels of syt I, respectively (note that the levels of syt I and IX were first determined in reference 37; similar values were obtained with the cells described in the present study [data not shown]). syt VII was not upregulated in any of the control lines.
We then explored the subcellular localization of untagged syt VII in the syt VII+ cell lines. It is currently the subject of debate as to whether syt VII-green fluorescent protein (GFP) fusion proteins are targeted to the plasma membrane (19, 35) or to intracellular organelles, including LDCVs (14, 16), in PC12 cells. To address this question, we first determined whether our syt VII antibody (used for the immunoblot analysis shown in Fig. 2) could be used for immunocytochemistry. As shown in Fig. 3A, the low levels of endogenous syt VII in control PC12 cells fell just below the limits of detection at the camera/microscope settings used. Under these conditions, strong syt VII staining was observed in syt VII+ clone G10, establishing that the antibody stains the upregulated protein. The same camera/microscope settings were used for the following syt VII localization experiments.
We then addressed the overall localization of syt VII (in clone G10) using higher-magnification images of cells in which the plasma membrane was demarcated by staining with an anti-syntaxin antibody (Fig. 3B). From this image, it is clear that the majority of syt VII is intracellular. To quantify these data, we carried out line scan (e.g., Fig. 3B) analysis of the syntaxin 1A and syt VII signals, and these data are plotted in Fig. 3C. Only 12% of the syt VII signal was colocalized with syntaxin 1A; hence, 
88% of the syt VII signal is intracellular. We conclude that syt VII is not a plasma membrane protein (note that additional images, showing strong punctate staining of syt VII within cells are shown in Videos S1 and S2 in the supplemental material).
Next, we carried out quantitative immunolocalization experiments, again using syt VII+ clone G10, to determine which intracellular organelles harbor syt VII. As a negative control for our colocalization approach, we measured the overlap of the LDCV marker secretogranin II (Sg) (23), with the lysosomal marker Lamp1 (25). As expected, little overlap was observed (Fig. 4A, top row; Fig. 4C, 3 to 6% of the Lamp1 and Sg signals colocalized with each other). As a positive control, we costained for two well-documented LDCV markers, Sg and syt I (23, 29, 41), and extensive colocalization was observed (Fig. 4A, bottom row; Fig. 4C, 55 to 60% of the Sg and syt I signals colocalized with each other).
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33% of the total syt VII signal was colocalized with Cg (Fig. 4C). In the reciprocal analysis, 49% of the Cg signal (i.e., the LDCV signal) was colocalized with syt VII (Fig. 4C). Thus, a significant fraction of LDCVs harbor upregulated syt VII. This finding was confirmed by colabeling syt VII and syt I (Fig. 4B, middle row); 45% of the syt VII colocalized with syt I, and 53% of syt I colocalized with syt VII (Fig. 4C). These data strongly indicate that LDCVs harbor both syt I and upregulated syt VII. These findings allow us to further address the impact of syt VII on the metal sensitivity of LDCV exocytosis, as detailed below. To complete our syt VII localization experiments, we also determined whether syt VII is targeted to lysosomes by costaining with antibodies against the lysosomal marker Lamp1 (Fig. 4B, bottom row). Based on these experiments, 20% of the syt VII signal colocalized with Lamp1, and 35% of the Lamp1 signal colocalized with syt VII. Thus, a significant fraction of upregulated syt VII is targeted to lysosomes, a finding consistent with previous reports studying the localization of syt VII in nonneuronal cells (32).
Upregulation of syt VII increases the metal sensitivity of LDCV exocytosis.
Since syt VII is a high-affinity metal sensor (Fig. 1) that is targeted, at least in part, to LDCVs in PC12 cells (Fig. 4B and C), we sought to determine whether syt VII could increase the metal sensitivity of exocytosis. We addressed this question using RDE voltammetry (12). Cells were mechanically permeabilized so that the release machinery could be directly exposed to different concentrations of Ca2+. The amplitudes of the voltammetry signals (voltage) were determined at 80 s. Also, curves were fitted with single exponential functions to yield the time constants () of release. Representative release profiles are shown for control (Fig. 5A) and syt VII+ clone A1 (Fig. 5B), where it is apparent that syt VII increased the Ca2+ sensitivity of release. To quantify this effect, normalized amplitudes (amp) and rate constants (1/) were plotted against [Ca2+]. As shown in Fig. 5C and D, both types of dose-response curves revealed that the [Ca2+]1/2 for release from syt VII+ clone A1 was shifted to lower values compared to wild-type PC12 cells (summarized in Table 1; e.g., from the amplitude plot, the [Ca2+]1/2 = 0.7 ± 0.1 µM for wild-type PC12 cells and [Ca2+]1/2 = 0.4 ± 0.1 µM for clone A1 [P < 0.05, unpaired t test]). All three control PC12 cell lines (H1, B2, and E1 [data not shown]), as well as the parental cell line (Fig. 5C,D), yielded similar [Ca2+]1/2 values that are consistent with data from a previous study (42). We also assayed for shifts in the [Ca2+]1/2 in syt VII+ clones G10 and G3 and observed slight shifts to the left, but these changes were within the error of the assay (data not shown). These findings indicate that syt VII expression must reach 30% the levels of syt I to give rise to significant changes in the Ca2+ responsiveness of the secretory machinery.
We extended this analysis by carrying out RDE assays with Sr2+ and Ba2+ to trigger release. Although Sr2+ and Ba2+ can substitute to some extent for Ca2+, the abilities of these divalent cations to trigger release from intact cells differ (24). This may be due to a number of complicating factors, including differences in flux through voltage activated channels, differences in buffering, extrusion, and/or sequestration or differences in the relative abilities of Sr2+ and Ba2+ to activate Ca2+ sensors to trigger release (46, 47). These complicating factors were mitigated by the use of permeabilized cells, which bypass Ca2+ channels and make it possible to wash out mobile divalent cation buffers.
Again, three independent syt VII+ (A1, G10, and G3) and three independent control cell lines (H1, B2, and E1), as well as the wild-type parental cell line, were analyzed. Release profiles for control and syt VII+ PC12 cells are shown in Fig. 6A. Normalized amplitudes (amp) (Fig. 6B and C) and the rate constants (1/) (see Fig. S1A and B in the supplemental material) were plotted against [divalent cation]. The data were fitted with sigmoidal dose-response curves, and the EC50 values and Hill coefficients were determined. Hill coefficients for all cell lines were similar (ca. 1.1 to 1.5). However, in both amplitude (Table 2) and 1/ plots (see Table S1 in the supplemental material), syt VII+ cell lines exhibited significantly lower EC50 values compared to wild-type PC12 cells. The tightly clustered [divalent cation]1/2 values for the control PC12 cell lines and the parental PC12 cell line (Fig. 6B) indicate that there is little clonal variation, supporting the idea that the increase in syt VII expression mediates the shift in the Sr2+ and Ba2+ dose-response curves observed for all three syt VII+ clones.
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To achieve this, we used an RNAi approach (11). We screened three short hairpin RNA (shRNA)-encoding constructs for their abilities to knock down the level of endogenous syt VII. The most effective shRNA is shown diagrammatically in Fig. 7A (left panel). After transient transfection of this construct, the level of syt VII expression was reduced by 60% to 70% (Fig. 7A, right panel). Knockdown was specific, since the shRNA had no effect on syt I or syt IX expression levels (Fig. 7A, right panel). Secretion from the knockdown cells (designated syt VII–) was analyzed as a function of [divalent cation] using RDE voltammetry. As a control, cells transfected with the empty pSHAG-1 vector were tested in parallel. As shown in Fig. 7B to D, EC50 values were increased by 1.4- to 2.6-fold, depending on the divalent cation used, in syt VII– cells but not in the control cells (in control cells, the EC50 values were within error of these values obtained with wild-type untransfected cells [Tables 1 and 3]). These results provide further support for the idea that the metal sensitivity of secretion in PC12 cells is dictated by the relative abundance of syt isoforms expressed in these cells, in conjunction with the affinity of each isoform for Ca2+, Ba2+, or Sr2+.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are 16 isoforms of syt, but whether different isoforms have distinct apparent affinities for Ca2+ is unresolved (e.g., see references 26 and 27 versus reference 36). Using liposomes that contain PS as a binding partner, we demonstrate here that syt VII exhibits a higher apparent affinity for Ca2+, Ba2+, and Sr2+ than syt I (Fig. 1). We then upregulated syt VII in PC12 cells in order to test the idea that the ratio of high- and low-affinity isoforms of syt dictates the metal requirements for exocytosis. We chose PC12 cells because they express relatively high levels of the low-affinity isoforms syt I and IX (22) and express relatively low levels of the high-affinity sensor, syt VII (Fig. 2) (37).
The metal requirements of exocytosis from LDCVs were determined by using RDE voltammetry, which reports the release of catecholamines. syt VII can affect release only if it is targeted to membranes that participate in the LDCV fusion reaction. However, the localization of syt VII remained unclear, with different reports concluding that this isoform is targeted to lysosomes in nonneuronal cells (28, 32), to the plasma membrane (35) or to intracellular compartments—including the Golgi and LDCVs (14, 16)—in PC12 cells, or to secretory granules in pancreatic beta cells (17). Moreover, the idea that multiple syt isoforms are targeted to a common compartment such as a secretory vesicle (e.g., references 13, 15, and 30) has been questioned in a recent study focused on syt localization in Drosophila (1).
To address these issues, we carried out quantitative immunolocalization studies of untagged syt VII that was stably overexpressed in PC12 cells. One potential problem with previous localization experiments was the reliance on GFP fusion proteins that in many cases were transiently overexpressed in PC12 cells. The localization data reported here overcome the problems associated with tagged proteins, since we used untagged syt VII. In addition, our experiments made use of stable cell lines in which syt VII was unlikely to spill over into the wrong compartment because the upregulated syt VII levels (in the syt VII clone G10 used for immunolocalization) were only 18% the level of endogenous syt I. Our results revealed that a significant fraction of syt VII was colocalized with syt I on LDCVs (Fig. 4B and C), further establishing that a single type of secretory vesicle can harbor multiple isoforms of syt (13, 15, 30). Costaining with antibodies against syt VII and Lamp1 also suggested that a portion of the total pool of syt VII was targeted to lysosomes (Fig. 4B, bottom row; Fig. 4C) (28, 32). Some cortical syt VII immunoreactivity was observed, and this has been interpreted to reflect targeting to the plasma membrane (19, 35). However, only 12% of the total syt VII signal overlaps with syntaxin 1A, a bona fide plasma membrane protein (Fig. 3B and C). Whether this small fraction is indeed in the plasma membrane will require ultrastructural studies, but clearly this is a minor pool. We note that qualitative electron microscopy data indicate that endogenous syt VII is present in presynaptic nerve terminals (35). Immunogold labeling suggested that overexpressed syt VII fusion proteins were at least partially localized to synaptic vesicles (40). In fruit flies, a putative syt VII ortholog was not detected on synaptic vesicles (1).
Having established that syt VII is targeted, at least in part, to LDCVs, we determined the impact that this protein has on the metal requirements for release. The key finding in our study is that secretion from cells that overexpress syt VII is more sensitive to Ca2+, Ba2+, and Sr2+ than from control cells that express only low levels of syt VII (Fig. 5 and 6; see also Fig. S1 in the supplemental material). Thus, upregulation of the sole high-affinity isoform expressed in PC12 cells, syt VII (37, 48), decreases the metal requirements for LDCV exocytosis. We note that upregulation of syt VII resulted in a greater increase in the apparent sensitivity of the release machinery to Sr2+ and Ba2+ compared to Ca2+. Based on the liposome-binding studies in Fig. 1, one might have expected that upregulation of syt VII would have the strongest effect on Ca2+-stimulated release compared to Ba2+- and Sr2+-stimulated release. However, syt's are likely to regulate exocytosis via a series of effector interactions, in addition to binding PS (5), and these other interactions could be differentially regulated by Ba2+ and Sr2+ compared to Ca2+.
Our model predicts that downregulation of syt VII would increase the metal requirements for release, since less high-affinity sensor would be available to regulate exocytosis. Indeed, knockdown of syt VII expression shifted the Ca2+, Sr2+, and Ba2+ dose responses to the right (Fig. 7), presumably because a larger fraction of release was mediated by the low-affinity sensors, syt I and IX. These data further support the idea that the affinity of syt for metals, and the complement of syt's expressed in a given cell, determine the metal requirements for exocytosis. Consistent with this model, overexpression of syt IV, which is thought to bind Ca2+ weakly, increases the Ca2+ requirements for secretion from PC12 cells (42).
The C2A domain of syt VII is a potent inhibitor of exocytosis in PC12 cells (35, 37). This was interpreted as demonstrating that syt VII functions as a high-affinity Ca2+-sensor for exocytosis in this cell type (35). However, it is clear that the ability of a C2 domain to block secretion is unrelated to the expression profile of syt's in PC12 cells. For example, the C2A domain of syt III is a potent inhibitor of release but is not expressed in PC12 cells (37). C2 domains block release by binding to molecules that are essential for exocytosis but cannot provide information regarding syt expression patterns. In the case of syt C2 domains, inhibition appears to be mediated by binding to t-SNAREs and PIP2 (37). The RNAi experiments shown in Fig. 7 provide the first evidence that endogenous syt VII functions as a high-affinity Ca2+ sensor in PC12 cells.
The data reported here begin to explain why there are so many isoforms of syt. Namely, the distinct Ca2+ requirements for release that have been observed in a number of different cell types might be mediated by different isoforms of syt. Within a given cell type, isoforms of syt might also be targeted to distinct compartments, endowing the fusion machinery in various compartments with different sensitivities to Ca2+. Finally, as shown here, multiple syt isoforms can be targeted to the same compartment, where the precise ratio of isoforms can fine-tune the metal requirements for exocytosis.
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ACKNOWLEDGMENTS |
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We thank members of the Chapman lab and M. Jackson for discussions and comments.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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