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Files in this Data Supplement:
Fig. S1. VAMP4-EGFP is incorporated into authentic SNARE complex VAMP4-EGFP-expressing NRK cells were treated with either NEM or DTT-quenched NEM. Cells were lysed and the extracted proteins were used for immunoprecipitation using either rabbit-anti-GFP antibody or control rabbit IgG. The immunoprecipitates were analysed by immunoblotting as indicated. Syntaxin6, syntaxin16 and Vti1a but not VAMP3 were co-immunoprecipitated with VAMP4-EGFP. Since the co-immunoprecipitation is significantly enhanced when SNARE complexes are stabilized by NEM treatment compared with control treatment, the co-immunoprecipitation is probably a reflection of SNARE complex formation in the cell.
Fig. S2. Inhibition of endocytosis of VAMP4-EGFP from the surface by a dominant-negative EPS15 mutant. (A) EPS15 mutants EH29, DIII and D3Δ2 were transiently expressed as FLAG-tagged proteins in HeLa cells. EH29 (panels a-c) or DIII (panels d-f), but not D3Δ2 (panels g-I) inhibited internalization of AF555-conjugated transferrin (Tf-AF555). Green signals were FLAG-fusion proteins; red signals were Tf-AF555. Bar, 10 μm. (B) FLAG-EH29 was transiently expressed in HeLa cells expressing VAMP4-EGFP. Cells were then incubated with anti-GFP antibody and Tf-AF647 for 30 minutes at 37°C. VAMP4-EGFP was showed in green; anti-GFP signal was showed in red and Tf-AF647 was showed in blue. The inhibition of Tf-AF647 was used as a surrogate marker for the expression of exogenous EH29. EH29 potently blocked endocytosis of anti-GPF antibody.
Fig. S3. Knockdown of clathrin reduces endocytosis of VAMP4-EGFP. (A) Knockdown of clathrin heavy chain (CHC). siRNA targeting CHC or scrambled siRNA was transfected into HeLa cells twice with a 24-hour interval. Cells were harvested 48 hours after the second transfection. The lysates (20 μg protein for each sample) were analyzed for the level of CHC by western blot. The result showed that the amount of CHC was noticeably reduced in cells that were transfected with CHC siRNA, compared with non-transfected cells or cells transfected with scrambled siRNA. (B) The effect of CHC depletion on the recycling of VAMP4-EGFP. HeLa cells expressing VAMP4-EGFP were transfected with either scrambled siRNA (panels a-d) or siRNA targeting CHC (panels e-h) twice with a 24-hour interval. 48 hours after the second transfection, cells were incubated with rabbit anti-GFP antibody for 10 minutes at 37°C, followed by a 30-minute chase. The internalization of anti-GFP antibody was performed in the continuous presence of Tf-AF555. The antibody, endocytosed Tf-AF555 and the GFP signal were then revealed by fluorescence microscopy. The inhibition of endocytosis of Tf-AF555 was used as surrogate marker for functional knockdown of CHC. The results showed that in cells where transferrin endocytosis was inhibited (marked by asterisk), the internalization of anti-GFP antibody was also inhibited, whereas in neighboring cells (marked by arrow) where TF-AF555 was endocytosed efficiently, anti-GFP antibody was also efficiently internalized to the perinuclear region (where the Golgi and transferrin-marked peri-Golgi recycling endosome were probably positioned). The results indicate that the transport of VAMP4-EGFP is dependent of clathrin-mediated event. Bar, 10 μm.
Fig. S4. VAMP4-EGFP and Tac-TGN38 are co-transported from the surface to the TGN. CHO-TacTGN38 cells, which express TacTGN38 recycling from the surface to the TGN, were transfected to express VAMP4-EGFP. A stable pool of transfectants was incubated with rabbit anti-GFP antibody and mouse monoclonal anti-Tac at 4°C for 1 hour. After a brief washing step, cells were then incubated at 37°C in the absence of antibodies for the indicated periods of time. EGFP signal is blue, anti-Tac signal is green and anti-GFP signal is red. The merged images are also shown. Bar, 10 μm.
Fig. S5. Various VAMP4-EGFP constructs can be expressed at comparable levels as assessed by flow cytometry. Control NRK cells or pooled transfectants expressing VAMP4-EGFP or various mutants (as indicated) of VAMP4-EGFP were trypsinized and subjected to flow cytometry. The histogram shows the EGFP level in each pool against relative number of events. The percentage and mean intensity of EGFP signal for each cell pool is indicated below each histogram plot.
Fig. S6. Detection of VAMP4-EGFP on the cell surface is enhanced by mutation of double-Leu motif as assessed by flow cytometry. (A) Stable pooled transfectants expressing either VAMP4-EGFP or V4-LL-EGFP were trypsinized and incubated with anti-GFP antibody on ice for 1 hour. After cold washing, cells were then incubated with PE-conjugated secondary antibody for 1 hour on ice before being subjected to flow cytometry analysis. The intensities of PE signals in the two pools were shown in the right panels, respectively. As a control, cells were incubated with PE-conjugated secondary antibody without prior incubation with the anti-GFP antibody (thus representing the non-specific signal) for 1 hour on ice before being analyzed by flow cytometry. The results for these controls were shown in the left panels. The mean of intensity was indicated below each panel. (B) Analysis of the other VAMP4-EGFP mutants by flow cytometry. Stable pooled transfectants expressing V4-EDD-EGFP, V4-5A-EGFP or V4-FF-EGFP were subjected to flow cytometry analysis as described in A. The intensities of PE signals were shown in the lower panels. The background signals derived from analysis omitting the anti-GFP antibody were shown in the upper panels. The mean of intensity is indicated below each panel.
Fig. S7. Low but detectable amounts of VAMP4-EGFP and V4nV5-EGFP are accessible to surface biotinylation. The cell surface biotinylation assay was performed as reported previously (Zeng et al., 2003). NRK cells or stable pooled NRK expressing VAMP4-EGFP, VAMP5-EGFP or V4nV5-EGFP were biotinylated twice (15-20 minutes each) on ice with sulfo-NHS-biotin (0.5 mg/ml). The reaction was stopped by washing the cells four times (10 minutes each) with 50 mM NH4Cl at 4°C. After rinsing twice (10 minutes each) with cold PBSCM, cells were lysed in lysis buffer (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% BSA, 10% FBS, and 1 mM PMSF). The cell lysate was then incubated with streptavidin-agarose at 4°C for 2 hours. Bound proteins on the beads, along with 20% of cell lysates were analyzed by western blot. The result shown in the upper panel was from a short (5 seconds) exposure which shows that VAMP5-EGFP (indicated by red #) is accessible to surface biotinylation. The lower panel is a longer (60-second) exposure of the same western blot which shows that low but detectable amounts of VAMP4-EGFP and V4nV5-EGFP (indicated by red asterisk) were accessible to surface biotinylation.
Fig. S8. Knockdown of syntaxin 16 does not affect endocytosis and recycling to the Golgi of VAMP4-EGFP. siRNA targeting Syn16 (Wang et al., 2004) or scrambled siRNA was transfected into HeLa cells twice with a 24-hour interval. Cells were harvested 48 hours after the second transfection. The lysates (20 μg protein for each sample) were analysed for the level of Syn16 by western blot (A). The result showed that the amount of Syn16 was significantly reduced in cells that were transfected with Syn16 siRNA, compared with non-transfected cells or cells transfected with scrambled siRNA. We then examined the effect of Syn16 depletion on the endocytosis and recycling of monoclonal antibodies against EGFP to assess the trafficking of VAMP4-EGFP (B). HeLa cells expressing VAMP4-EGFP were transfected with either scrambled siRNA (panels a-d) or siRNA against Syn16 (panels e-h) twice with a 24-hour interval. 48 hours after the second transfection, cells were incubated with mouse anti-GFP antibody for 10 minutes at 37°C, followed by a 30-minute chase. The mouse anti-GFP antibody, endogenous Syn16 and the GFP signal were revealed by fluorescence microscopy. The antibody was endocytosed and recycled to the Golgi when Syn16 was effectively knocked down (panels e-h), suggesting that the recycling pathway of VAMP4-EGFP is probably independent of Syn16. Bar, 10 μm.
Fig. S9. Knockdown of GS15 does not affect endocytosis and recycling to the Golgi of VAMP4-EGFP. GS15 depletion was achieved using siRNA targeting GS15 as described previously (Tai et al., 2004). HeLa cells expressing VAMP4-EGFP were transfected with either scrambled siRNA (panels a-d) or siRNA targeting GS15 (panels e-h) twice with a 24-hour interval. 48 hours after the second transfection, cells were incubated with rabbit anti-GFP antibody for 10 minutes at 37°C, followed by a 30-minute chase. The antibody, endogenous GS15 and the GFP signal were revealed by fluorescence microscopy. The antibody was endocytosed and recycled to the Golgi when GS15 was effectively knocked down (panels e-h), suggesting that the recycling pathway of VAMP4-EGFP is independent of GS15. Bar, 10 μm.
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