Synaptotagmin IX, a possible linker between the perinuclear endocytic recycling compartment and the microtubules.

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.

Synaptotagmin III is a critical factor for the formation of the perinuclear endocytic recycling compartment and determination of secretory granules size.

Early endosomes and a perinuclear, Rab-11-positive compartment have been implicated in the recycling of internalized receptors. In this study, we show that synaptotagmin III (Syt III), a member of the Syt family of proteins, is required for the formation and delivery of cargo to the perinuclear endocytic recycling compartment (ERC). We demonstrate that rat basophilic leukemia (RBL-2H3) mast cells endogenously express Syt III, and >70% of this protein colocalizes with early endosomal markers, such as EEA1, annexin II and syntaxin 7, and the remaining protein colocalizes with secretory granule (SG) markers such as beta-hexosaminidase, histamine and serotonin. To study the functional role of Syt III, we stably transfected RBL cells with Syt III antisense cDNA and monitored the route of transferrin (Tfn) internalization in cells that displayed substantially reduced (<90%) levels of Syt III (RBL-Syt III(-)). In these cells, Tfn binding and internalization into early endosomes were unaltered. However, whereas in the mock-transfected cells Tfn was subsequently delivered to the ERC, in the RBL-Syt III(-) cells, Tfn remained associated with dispersed peripheral vesicles and Rab 11 remained cytosolic. Nevertheless, the rates of Tfn internalization and recycling were not affected. RBL-Syt III(-) cells also displayed enlarged SGs, reminiscent of the SGs present in Chediak-Higashi (beige) mice. However, morphometric analyses suggested that granule formation was unaltered and that the calculated unit granule volume is the same in both cell lines. Therefore, our results implicate Syt III as a critical factor for the generation and delivery of internalized cargo to the perinuclear endocytic recycling compartment and suggest a possible link between ERC and recycling from immature SGs during the process of SG maturation.

Suppression of Synaptotagmin II restrains phorbolester-induced downregulation of protein kinase Calpha by diverting the kinase from a degradative pathway to the recycling endocytic compartment.

Downregulation of protein kinase Calpha (PKCalpha) following long-term exposure to phorbol esters such as TPA is traffic dependent and involves delivery of the active, membrane-associated PKCalpha to endosomes. In this study, we show that synaptotagmin II (Syt II), a member of the Syt family of proteins, is required for TPA-induced degradation of PKCalpha. Thus, whereas the kinase half-life in TPA-treated cultured mast cells (the mast cell line rat basophilic leukemia RBL-2H3) is 2 hours, it is doubled in RBL-Syt II(-) cells, in which the cellular level of Syt II is reduced by >95% by transfection with Syt II antisense cDNA. We demonstrate that in TPA-treated RBL cells, PKCalpha travels from the cytosol to the plasma membrane, where it is delivered to early endosomes on its route to degradation. By contrast, in TPA-treated RBL-Syt II(-) cells, PKCalpha is diverted to recycling endosomes and remains distributed between the plasma membrane and the perinuclear recycling endocytic compartment. Notably, in both RBL and RBL-Syt II(-) cells, a fraction of PKCalpha is delivered and maintained in the secretory granules (SG). These results implicate Syt II as a critical factor for the delivery of internalized cargo for degradation. As shown here, one consequence of Syt II suppression is a delay in PKCalpha downregulation, resulting in its prolonged signaling.