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Functional and biochemical analysis of the C2 domains of synaptotagmin IV - PubMed

Functional and biochemical analysis of the C2 domains of synaptotagmin IV

D M Thomas et al. Mol Biol Cell. 1999 Jul.

Free PMC article

Abstract

Synaptotagmins (Syts) are a family of vesicle proteins that have been implicated in both regulated neurosecretion and general membrane trafficking. Calcium-dependent interactions mediated through their C2 domains are proposed to contribute to the mechanism by which Syts trigger calcium-dependent neurotransmitter release. Syt IV is a novel member of the Syt family that is induced by cell depolarization and has a rapid rate of synthesis and a short half-life. Moreover, the C2A domain of Syt IV does not bind calcium. We have examined the biochemical and functional properties of the C2 domains of Syt IV. Consistent with its non-calcium binding properties, the C2A domain of Syt IV binds syntaxin isoforms in a calcium-independent manner. In neuroendocrine pheochromocytoma (PC12) cells, Syt IV colocalizes with Syt I in the tips of the neurites. Microinjection of the C2A domain reveals that calcium-independent interactions mediated through this domain of Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of Syt isoforms, suggests that the secretory machinery contains a vast repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly.

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Figures

Figure 1
Figure 1

Distinct Syt–Stx interactions exist for Syt I and Syt IV isoforms. (A) The indicated recombinant GST fusion proteins were immobilized on glutathione–agarose and incubated with 800 μg of rat brain synaptosomes in the absence (−) or presence (+) of 3 mM calcium. Protein complexes were isolated, fractionated by SDS-PAGE, and examined by Western analysis. Stx 1a binding was detected with HPC-1, followed by enhanced chemiluminescence. One hundred nanograms of soluble Stx 1a (Con) were used as a Western control. (B) Ten micrograms of immobilized recombinant Syt I and IV C2A domains were incubated with 100 nM soluble recombinant Stx 1a (amino acids 4–266) or HA-tagged Stxs 2 (amino acids 4–264), 3 (amino acids 4–264), or 4 (amino acids 1–268), and the protein complexes were examined by Western analysis. Stx 1a binding was detected with the anti-Stx 1a antibody HPC-1. Binding of HA-tagged Stxs 2–4 was detected with the anti-HA antibody 12CA5. No binding was detected with GST alone (Con).

Figure 2
Figure 2

Calcium-dependent, C2B domain–mediated homo- and hetero-oligomerization of Syt I and Syt IV. (A) Five microliters of in vitro–translated, [35S]methionine-labeled cytoplasmic domain of Syt I and Syt IV were incubated with 10 μg of immobilized GST-Syt IV C2A, GST-Syt IV C2B, or GST alone in the presence of calcium. Protein complexes were isolated and analyzed by SDS-PAGE. Gels were dried and exposed to autoradiographic film overnight. In vitro–translated, [35S]methionine-labeled Syt I and Syt IV products are shown in the left two lanes (Input). (B) Five microliters of in vitro–translated, [35S]methionine-labeled cytoplasmic domain of Syt I and Syt IV were incubated with 10 μg of immobilized GST-Syt I C2B or GST-Syt IV C2B in the absence (−) or presence (+) of calcium. Protein complexes were analyzed as described above.

Figure 3
Figure 3

Affinity-purified anti-Syt IV antibody does not react with other Syt isoforms. (A) Five hundred nanograms of each soluble Syt I–VIII-GST fusion protein were subjected to SDS-PAGE and analyzed by Western blotting. Filters were probed with the affinity-purified Syt IV antibody in the absence (A, top panel) or presence (A, middle panel) of 500 μg of soluble Syt IV. The presence of GST-Syt fusion proteins was confirmed by probing a filter with an anti-GST mAb (A, bottom panel). (B) Cell lysates from COS-7 cells transiently transfected with full-length Syt IV cDNA cloned into the eukaryotic expression vector pcDNA3.1 zeo+, in the forward (+) and reverse (−) orientations, were examined for expression of Syt IV by Western analysis. Filters were probed in the absence (B, top panel) or presence (B, bottom panel) of 500 μg of soluble Syt IV. (C) PC12 cells treated as indicated were examined for Syt IV expression by Western analysis. Filters were probed with anti-Syt IV antibody in the absence (C, top panel) or presence (C, bottom panel) of 500 μg of soluble Syt IV.

Figure 4
Figure 4

Syt I and Syt IV colocalize to the tips of neurites in PC12 cells in vivo. NGF-differentiated PC12 cells were K+ depolarized and 2 h after depolarization analyzed by double immunofluorescence confocal microscopy. A phase-contrast image of representative cells is shown in A. Cells were costained for endogenous Syt I (B) and Syt IV (C) and detected using secondary antibodies coupled to RITC and FITC, respectively. Representative areas of overlap between Syt I and Syt IV in these panels and the merged image (D) are indicated by the arrows in the tips of PC12 cells. Bar, 20 μm.

Figure 5
Figure 5

The polybasic motif in the C2A domain of Syt IV functions in calcium-regulated secretion. (A) The indicated recombinant GST fusion proteins were immobilized on glutathione–agarose and incubated with 800 μg of rat brain synaptosomes in the presence (+) or absence (−) of 3 mM calcium. Protein complexes were isolated, fractionated by SDS-PAGE, and examined by Western analysis. Stx 1a binding was detected with HPC-1, followed by enhanced chemiluminescence. One hundred nanograms of soluble Stx 1a (Con) were used as a Western control. (B) NGF-differentiated PC12 cells were coinjected with Texas Red–conjugated dextran and the indicated soluble recombinant Syt IV or Nedd4 fragments. Control cells were injected with Texas Red–conjugated dextran only (Texas Red) or coinjected with a control GST extract (GST). One hour after microinjection, the cells were K+ depolarized in the presence of calcium, and DβH surface immunoreactivity was detected with a fluorescein-labeled secondary antibody. The numbers of individual fluorescent particles were counted, regardless of their size, and are presented as a percent of the total number of cells injected. Data are shown as the mean of two independent experiments ± SD with the total number of injected cells (n). Significant differences (p ≤ 0.01, χ2 analysis) between experimental and control treatments are indicated with an asterisk.

Figure 6
Figure 6

α-Latrotoxin reverses the inhibitory effect of the Syt IV C2A domain on calcium-regulated secretion from PC12 cells. NGF-differentiated PC12 cells were injected with Texas Red–conjugated dextran alone (Texas Red) or a soluble recombinant fragment encompassing the C2A domain of Syt IV (Syt IV C2A). One hour after injection, cells were K+ depolarized in a calcium-free buffer supplemented with 2.2 mM MgCl2, 5 mM EGTA, and α-latrotoxin at a final concentration of 9.6 μM. DβH surface immunoreactivity was quantified and is presented as described in the legend to Figure 5. Data are shown as the mean of two independent experiments ± SD with the total number of injected cells (n). Significant differences (p ≤ 0.01, χ2 analysis) between experimental and control treatments are indicated with an asterisk.

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