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First published online 22 March 2005
doi: 10.1242/jcs.02276

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Classical protein kinase C(s) regulates targeting of synaptotagmin IX to the endocytic recycling compartment

Yael Haberman^1,*, Idit Ziv¹, Yaara Gorzalczany¹, Mitsunori Fukuda² and Ronit Sagi-Eisenberg^1,

¹ Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
² Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

View larger version (56K): [in a new window]   Fig. 1. Distribution of Syt IX in RBL cells stably transfected with T7-Syt IX or Flag-Syt IX-GFP. (A) RBL cells, stably transfected with T7-Syt IX or Flag-Syt IX-GFP cDNA, were grown on glass coverslips and labeled with polyclonal anti-Syt IX C2A antibodies followed by Cy3-conjugated donkey anti-rabbit IgG (a). Bars, 3 µm. (B) Cell extracts (60 µg) derived from T7-Syt IX- (1) or Flag-Syt IX-GFP (2)-expressing cells were resolved by SDS-PAGE and subjected to immunoblotting with anti-Syt IX C2A antibodies.

View larger version (78K): [in a new window]   Fig. 2. Biosynthetic trafficking of transiently transfected T7-Syt IX and Flag-Syt IX-GFP in RBL cells. RBL cells, transiently transfected with Flag-Syt IX-GFP (a-f) or T7-Syt IX (g-l) cDNA were grown on glass coverslips. Two hours post transfection, cells were placed for 2 hours at 19° and subsequently shifted to 37°C (time zero) for the indicated time periods, in the absence or presence of BFA (5 µg/ml) for the last 30 minutes of incubation at 37°C. Cells were subsequently labeled with polyclonal anti-Syt IX C2A antibodies followed by Cy3-conjugated donkey anti-rabbit IgG (g-l). Bars, 3 µm.

View larger version (18K): [in a new window]   Fig. 3. Binding of AP-2 by Syt IX. Twenty microgram of GST, GST-Syt IX-C2A or GST-Syt IX-C2B were immobilized on glutathione-Sepharose beads and incubated in the absence or the presence of Ca2+ (3 mM) as indicated, for 4 hours at 4°C with RBL cells lysates (500 µg). Bound proteins were eluted by sample buffer, resolved on SDS-PAGE and analyzed by western blot using anti- adaptin (AP-2) antibodies.

View larger version (64K): [in a new window]   Fig. 4. Antibody-induced endocytosis is not sufficient for targeting of Syt IX to the ERC. RBL cells stably transfected with Flag-Syt IX-GFP cDNA were grown on glass coverslips. Cells were then incubated with monoclonal anti-Flag antibodies (10 µg/ml) for 30 minutes at 4°C, washed and subsequently shifted to 37°C for the indicated time periods (A) or for 2 hours (B). The reaction was stopped by placing the cells on ice. Cells were either labeled with Cy3-conjugated donkey anti-mouse IgG (A) or were allowed to internalize Texas Red-conjugated human Tfn for the last 10 minutes of incubation (B). Bars, 3 µm. Arrows mark the vesicular localization of Flag-Syt IX-GFP and Tfn.

View larger version (66K): [in a new window]   Fig. 5. Phosphorylation by a Ca2+-dependent PKC isozyme (s) is required for the delivery of Flag-Syt IX-GFP to the ERC. (A) RBL cells stably transfected with Flag-Syt IX-GFP cDNA and grown on glass coverslips, were incubated with or without TPA (50 nM) for 30 minutes at 37°C as indicated, prior to fixation and monitoring. (B) RBL cells stably transfected with Flag-Syt IX-GFP cDNA and grown on glass coverslips, were incubated with TPA (50 nM) for 30 minutes at 37°C and allowed to internalize Texas Red-conjugated human Tfn for the last 15 minutes of incubation as indicated. Arrows indicate the vesicular localization of Flag-Syt IX-GFP and Tfn. (C) RBL cells stably transfected with Flag-Syt IX-GFP cDNA and grown on glass coverslips, were either incubated with hypertonic sucrose (0.45 M) for 40 minutes or preincubated with hypertonic sucrose (0.45 M) for 10 minutes, followed by incubation with both sucrose and TPA (50 nM) for an additional 30 minutes as indicated. (D) Quantitative analysis of the cellular distribution of Flag-Syt IX-GFP in stably transfected RBL cells treated as indicated. Analysis was performed on 200 cells for each treatment. White bars: cells in which Flag-Syt IX-GFP resides at the plasma membrane only; gray bars: cells in which Flag-Syt IX-GFP is distributed between the plasma membrane and the ERC; black bars: cells in which Flag-Syt IX-GFP resides at the ERC. (E) RBL cells stably transfected with Flag-Syt IX-GFP cDNA and grown on glass coverslips, were preincubated with GF109203X (100 nM) or Go 6976 (100 nM) for 15 minutes. Buffer or TPA (50 nM) was subsequently added and the cells incubated for further 30 minutes. (F) RBL cells stably transfected with Flag-Syt IX-GFP cDNA and grown on glass coverslips were incubated with monoclonal anti-Flag antibodies (10 µg/ml) for 30 minutes at 4°C, washed and delivered to 37°C for 1 hour. TPA (50 nM) was added for the last 30 minutes of incubation at 37°C. Cells were labeled with Cy3-conjugated donkey anti-mouse IgG. Bars, 3 µm.

View larger version (70K): [in a new window]   Fig. 6. PKC-mediated phosphorylation is crucial for ERC localization of Syt IX. (A) RBL cells stably transfected with T7-Syt IX cDNA were grown on glass coverslips. Cells were left untreated or incubated with TPA (50 nM) for 30 minutes at 37°C prior to labeling with polyclonal anti-Syt IX C2A antibodies followed by Cy3-conjugated donkey anti-rabbit IgG. Bars, 3 µm. (B) RBL cells, transiently transfected with T7-Syt IX or Flag-Syt IX-GFP cDNA were grown on glass coverslips for 3 hours. Cells were then either left untreated or incubated with Go 6976 (100 nM) for additional 15 hours. Cells were subsequently labeled with polyclonal anti-Syt IX C2A antibodies followed by Cy3-conjugated donkey anti-rabbit IgG. Bars, 3 µm.

View larger version (45K): [in a new window]   Fig. 7. The slower-migrating form of Syt IX resides exclusively at the ERC. (A) Cell extracts (80 µg) derived from untreated or TPA (50 nM, 30 minutes)-treated Flag-Syt IX-GFP-expressing cells were resolved by SDS-PAGE and subjected to immunoblotting with anti-Syt IX C2A antibodies. (B) Cell homogenates derived from RBL cells stably transfected with T7-Syt IX or Flag-Syt IX-GFP cDNA were fractionated on continuous sucrose gradients as described under Materials and Methods. Fractions were collected from the top, subjected to SDS-PAGE and immunoblotted with anti-Syt IX C2A (a) or anti-GFP (b) antibodies.

View larger version (50K): [in a new window]   Fig. 8. Localization of Syt IX in CHO cells. CHO cells stably transfected with Syt IX (a) or Flag-Syt IX-GFP (b,c) cDNA were grown on glass coverslips in the absence or presence of TPA (50 nM) as indicated. Cells were subsequently labeled with polyclonal anti-Syt IX C2A antibodies followed by Cy3-conjugated donkey anti-rabbit IgG (a). Bars, 3 µm.

View larger version (21K): [in a new window]   Fig. 9. The route of trafficking of Syt IX. A cartoon illustrating the proposed route of trafficking of Syt IX, from the TGN to the plasma membrane (PM), early endosomes (EE) and the ERC. According to this model, Syt IX travels from the TGN to the plasma membrane by the constitutive secretory pathway. At the plasma membrane, Syt IX is phosphorylated by a classical PKC resulting in its internalization and delivery to the ERC. The rate-limiting factor in this step is the phosphorylation by PKC, then the phosphorylated protein is rapidly internalized and delivered to the ERC, where it undergoes dephosphorylation.