Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Mar 20;148(6):1141-49.
doi: 10.1083/jcb.148.6.1141.

Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts

I Martinez  1 , S Chakrabarti, T Hellevik, J Morehead, K Fowler, N W Andrews
Affiliations

Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts

I Martinez et al. J Cell Biol. .

Abstract

Synaptotagmins (Syts) are transmembrane proteins with two Ca(2+)-binding C(2) domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca(2+) sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379-388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290-18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca(2+) concentrations ([Ca(2+)]) below 10 microM, whereas other isoforms require 200-500 microM [Ca(2+)] or show no Ca(2+)-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594-599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1-5 microM [Ca(2+)] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93-104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C(2)A domain of Syt VII inhibit Ca(2+)-triggered secretion of beta-hexosaminidase and surface translocation of Lgp120, whereas the C(2)A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C(2)A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca(2+)-dependent lysosome exocytosis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Syt VII is localized on late endocytic compartments of NRK cells. a, Immunofluorescence with anti-Syt VII antibodies in the presence of scrambled control peptide. b, Same as a, in the presence of Syt VII NH2-terminal peptide. c, Transmission EM of cryosections labeled with anti-Syt VII antibodies. Arrowheads point to 10-nm gold particles at the antibody binding sites; the arrow points to internalized BSA-gold complexes. Bar, 0.5 μm. d, Anti-Syt VII immunofluorescence. e, Dextran-Texas red chased into late endocytic compartments. f, Overlay of d and e. g, Anti-Syt VII immunofluorescence. h, Anti-EEA1 immunofluorescence. i, Overlay of g and h. Arrowheads in d–f point to examples of colocalization between Syt VII and dextran-Texas red. The large spots over the nucleus reactive with anti-Syt VII antibodies in d are artifacts of the methanol fixation.
Figure 2
Figure 2
Syt VII colocalizes with dense lysosomes on subcellular fractionation. a, Western blot of NRK fibroblasts, L6E9 myoblasts, and rat brain extracts (5 μg/lane), and recombinant Syt VII (rSyt VII) with anti-Syt VII antibodies in the presence of the scrambled control peptide (left), or in the presence of the NH2-terminal Syt VII peptide (center). Right, Western blot with antibodies to Syt I (MC17) of the same extracts, and recombinant Syt I (rSyt I). b, Percoll density gradient fractionation of NRK cells. Top, detection of β-hexosaminidase, mannosidase II, and transferrin–HRP on gradient fractions collected from the bottom (activities assayed using 4-methyl-umbellyferyl-N-acetyl-β-d-glucosaminide, 4-methylumbelliferyl a-D-mannopyranoside, and 3,3′, 5,5′-tetramethylbenzidine as substrates, respectively). Bottom, Western blot detection of Syt VII, cathepsin L, rab 7, EEA1, and calnexin on gradient fractions.
Figure 3
Figure 3
Transfected Syt VII-GFP is targeted to mature lysosomes of NRK cells. a, Syt VII-GFP. b, Anti-Lgp120 immunofluorescence. c, Overlay of a and b. d, Syt VII-GFP. e, Transferrin/Texas red-containing early endosomes. f, Overlay of d and e. g, Syt VII-GFP. h, Anti-rab 7 immunofluorescence. i, Overlay of g and h.
Figure 4
Figure 4
Specificity of the antibodies generated against the NH2-terminal and C2A domains of Syt VII. a, Western blot of NRK or L mouse fibroblast extracts (5 μg/lane) with antibodies to Syt VII NH2 terminus peptide (α-Syt VII) or to Syt VII recombinant C2A domain (α-C2A). b, Immunofluorescence of NRK cells with anti-Syt VII C2A antibodies (left) or anti-Lgp120 (right). c, Immunoprecipitation of NRK or rat brain extracts with anti-Syt VII C2A antibodies, Western blotted with antibodies to the NH2 terminus of Syt VII (top) or Syt I (bottom). Syt indicates the position of Syt VII or Syt I, and IgG the position of the heavy chain of rabbit IgG. E, Total extract; PI, immunoprecipitate obtained with preimmune rabbit IgG; α-C2A, immunoprecipitate with anti-Syt VII C2A affinity-purified IgG. d, Western blot of increasing amounts of NRK (10 or 20 μg) or rat brain (5–40 μg) extracts with antibodies against the NH2 terminus of Syt VII (top) or Syt I (bottom).
Figure 5
Figure 5
Antibodies against the Syt VII C2A domain inhibit lysosome exocytosis in NRK cells. a, Cells were permeabilized with SLO, stimulated or not with 1 μM Ca2+ in the presence of the indicated concentrations of preimmune rabbit IgG or anti-Syt VII C2A, and the supernatant was assayed after 10 min for released β-hexosaminidase. b, Surface immunofluorescence of Lgp120 in SLO-permeabilized cells in the absence of Ca2+. c, Same field as b, DAPI stain of cell nuclei. d, Surface immunofluorescence of Lgp120 in cells permeabilized in the presence of 1 μM Ca2+ and 12 μg/ml rabbit preimmune antibodies. e, Same as d, DAPI stain. f, Surface immunofluorescence of Lgp120 in cells permeabilized in the presence of 1 μM Ca2+ and 10 μg/ml anti-Syt VII C2A antibodies. g, Same as f, DAPI stain.
Figure 6
Figure 6
Syt VII C2A domain inhibits lysosome exocytosis in NRK cells. a, SDS-PAGE (10 μg/lane) of recombinant Syt VII C2A (1); Syt VII C2B (2); Syt VII C2A-B (3); Syt I C2A (4); Syt I C2B (5); and Syt I C2A-B (6). b, Permeabilized cells were stimulated with 1 μM Ca2+ in the absence or presence of the indicated concentrations of Syt VII or Syt I C2A domains, and the supernatant was assayed after 10 min for released β-hexosaminidase. c, Permeabilized cells were stimulated with Ca2+ in the absence or presence of 200 μg/ml Syt I fragments, and the supernatant was assayed for released β-hexosaminidase at the indicated time points. d, Same as c, except that 200 μg/ml Syt VII fragments were used. Op-Tc (d) corresponds to an unrelated protein expressed in E. coli and purified under the same conditions. The data is expressed as percentage of the total cellular content of β-hexosaminidase (mean of triplicates ± SD). e–h, Permeabilized cells were stained for surface Lgp120 after incubation in buffer containing: e, no Ca2+; f, 1 μM Ca2+; g, 1 μM Ca2+ plus 200 μg/ml recombinant Syt I C2A fragments; and h, 1 μM Ca2+ plus 200 μg/ml recombinant Syt VII C2A fragments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Black D.L. Splicing in the inner eara common tune, but what are the instruments? Neuron. 1998;20:165–168. - PubMed
    1. Bommert K., Charlton M.P., DeBello W.M., Chin G.J., Betz H., Augustine G.J. Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocytosis. Nature. 1993;363:163–165. - PubMed
    1. Burgoyne R.D., Morgan A. Calcium sensors in regulated exocytosis. Cell Calcium. 1998;24:367–376. - PubMed
    1. Butz S., Fernandez-Chacon R., Schmitz F., Jahn R., Sudhof T.C. The subcellular localizations of atypical synaptotagmins III and VI. Synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles. J. Biol. Chem. 1999;274:18290–18296. - PubMed
    1. Chavez R.A., Miller S.G., Moore H.-P.H.. A biosynthetic regulated secretory pathway in constitutive secretory cells. J. Cell Biol. 1996;133:1177–1191. - PMC - PubMed

Publication types

MeSH terms