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Neuronal and non-neuronal tissues show distinctly different intracellular localization of synaptotagmin (Syt) homologues. Therefore, cell type-specific mechanisms are likely to direct Syt homologues to their final cellular destinations. Syt IX localizes to dense core vesicles in PC12 cells. However, in the rat basophilic leukemia (RBL-2H3) mast cell line, as well as in CHO cells, Syt IX is localized at the endocytic recycling compartment (ERC). We show that targeting of Syt IX to the ERC involves constitutive trafficking to the plasma membrane followed by internalization and transport to the ERC. We further show that internalization from the plasma membrane and delivery to the ERC are dependent on phosphorylation by Ca2+-dependent protein kinase Cα or β. As such, correct targeting of Syt IX is facilitated by the phorbol ester TPA but prevented by the cPKC inhibitor Go 6976.

The pericentriolar endocytic recycling compartment (ERC) is involved in receptor and lipid recycling as well as in the delivery of internalized cargo from early endosomes to the trans Golgi network (TGN). We show that synaptotagmin (Syt) IX, a member of the Syt family of proteins, localizes to the ERC and is required for export from the ERC to the cell surface. We demonstrate that rat basophilic leukemia (RBL-2H3) mast cells endogenously express Syt IX mRNA and protein. Localization studies employing fractionation on linear sucrose gradients combined with confocal microscopy by indirect immunofluorescence or stable expression of a Syt IX-green fluorescent fusion protein demonstrate that Syt IX colocalizes with internalized transferrin (Tfn) and with Rab 11 at the perinuclear ERC. Syt IX also colocalizes with tubulin at the microtubules organizing center (MTOC) and remains associated with tubulin clusters formed in taxol-treated cells. Moreover, Syt IX coimmunoprecipitates with tubulin from intact RBL cells, and chimeric fusion proteins comprising either the C2A or the C2B domain of Syt IX are able to pull down tubulin from RBL cell lysates. To study the functional role of Syt IX, we have stably transfected RBL cells with Syt IX sense or antisense cDNA and monitored the routes of Tfn internalization and recycling in cells that overexpress (RBL-Syt IX+) or display substantially reduced (<90%) levels of Syt IX (RBL-Syt IX–). In these cells, Tfn binding and internalization into early endosomes and the ERC are unaltered. However, recycling from the ERC to the cell surface is significantly slowed down in the RBL-Syt IX– cells. These results therefore indicate that Syt IX is involved in regulating transport from the ERC to the cell surface, and suggest that it may play a role in linking vesicles that exit the ERC with the microtubules network.

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