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Files in this Data Supplement:
Fig. S1. Intracellular localization of Yip1A in HeLa cells and the organelle structures in Yip1A-knockdown cells. (A) HeLa cells were fixed and immunostained using the anti-Yip1A antibody and antibodies against GM130, p230, and Sec31A. Scale bar: 10 µm. (B) After transfection of the scrambled siRNA (cont RNAi) or siRNA against Yip1A (Yip1A RNAi) for 72 hours, the cells were immunostained with ER tracker or antibodies against p115, βCOP, and Sec31A.
Fig. S2. Biochemical analysis of VSVG or Stx transport in Yip1A-knockdown cells. (A) 48 hours after transfection with the Yip1A or scrambled siRNA, the plasmid that encoded GFP-tagged VSVGts045 was transfected into the cells. After 24 hours at 39°C, the cells were incubated at 32°C for 0, 15, 30, 60 and 90 minutes. The cells were lysed with lysis buffer (25 mM HEPES-KOH pH 7.4, 100 mM KCl, 2 mM EDTA, 1% Triton X-100, 5 µg/ml leupeptin, antipain, chymostatin, and pepstatin A). The lysate was passed through a 27G needle 10 times, rotated at 4°C for 30 minutes, and centrifuged at 17,800 g at 4°C for 10 minutes. The supernatant was collected, combined with 10× denaturing buffer (5 % SDS, 10% β-mercaptoethanol), and incubated at 100°C for 10 minutes. The supernatant was then further combined with 10× G5 buffer (0.5 M sodium citrate pH 5.5) and 1.5 µl endoglycosidase H (New England Biolabs) and incubated at 37°C for 3 hours. The lysate was added to 2× SDS-sample buffer and boiled at 100°C for 5 minutes, and subjected to western blotting with an anti-GFP antibody (MBL). The kinetics of the change in sensitivity to endo-H in the Yip1A-knockdown cells were indistinguishable from those in the control cells. (B) 72 hours after transfection with the Yip1A (open circle) or scrambled siRNA (closed circle), the cells were incubated with 1 mg/ml of Cy2-Stx at 37°C for 0, 3, 5, 8, and 10 hours as described in Materials and Methods. We quantified the number and the percentage of live cells using Cell Counting Kit-8 (Dojindo) according to the manufacturer’s instructions. Cell viability decreased more slowly in Yip1A-knockdown cells compared with that in control cells.
Fig. S3. Morphometric analysis of GFP-tagged cation-independent mannose-6-phosphate receptor (GFP-CI-MPR) transport in Yip1A-knockdown cells. (A) 48 hours after transfection with the Yip1A or control siRNA, the plasmid that encoded GFP-CI-MPR was transfected into the cells. After 24 hours, the cells were treated with 10 µg/ml cycloheximide (CHX; Sigma) at 37°C for 30 minutes to inhibit protein synthesis. GFP-CI-MPR that had accumulated near the nucleus was photobleached by high-intensity laser illumination, and then and the recovery of fluorescence in this region was monitored for 1, 3, 5, and 10 minutes. Scale bar: 10 µm. (B) Kinetics of recovery of fluorescence intensity in the perinuclear region in control or Yip1A-knockdown cells. We arbitrarily assigned the average intensity of fluorescence in a selected region in the prebleached cells as 100% and in the bleached cells as 0%. For all experiments, three independent transport assays were performed, and the means and standard deviations were plotted. (C) Fluorescence recovery in the perinuclear region at 10 minutes after bleaching in control cells (control RNAi), Yip1A-knockdown cells (Yip1A RNAi), or cells that had been treated with 20 mM 3-methyladenine for 3 hours (3-MA). For a positive control, we examined the effect of 3-methyladenine (3-MA) on the transport. 3-MA, which is a phosphoinositide 3 kinase inhibitor, is known to inhibit retrograde transport from the endosomes to the Golgi (Hirosako et al., 2004). We incubated HeLa cells that had been transfected with GFP-CI-MPR with 20 mM 3-MA for 3 hours and then performed FRAP. We found that treatment with 3-MA decreased the fluorescence recovery in the Golgi region to 28%. n=300.
Fig. S4. No reduction in total amount of Rab6, Rab1b, and Rab33b in Yip1A-knockdown cells. Control (RNAi −) or Yip1A (RNAi +) siRNA was transfected into HeLa cells. After 72 hours, the amount of Rab6, Rab1b, Rab33b, and GAPDH was quantified by western blotting.
Fig. S5. Biochemical requirements for the retrograde transport of GT-GFP. (A) After CHX treatment to inhibit protein synthesis, GT-GFP within the ER region was photobleached by laser illumination (bleach). Semi-intact cells were then incubated with cytosol/ATP at 32°C for the indicated times (sec). PI represents propidium iodide. Scale bar: 10 µm. (B) Semi-intact CHO-GT8 cells that had been treated with CHX were incubated with cytosol/ATP (cytosol+ATP), an ATP regenerating system only (ATP), cytosol plus 1 mM AMP-PNP (AMP-PNP), or cytosol/ATP plus 1 mM GTPγS (GTPγS), and then subjected to the retrograde (GolgirER) transport assay. In addition, intact CHO-GT8 cells were treated with nocodazole (nocodazole), and then subjected to the retrograde transport assay. (C) CHX-treated, semi-intact CHO-GT8 cells were incubated with or without 5 µg of His-tagged Sar1T39N protein (Sar1T39N) or 5 mg of GST-tagged RabT27N protein in the presence of cytosol/ATP, and subjected to the assay for the retrograde transport of GT-GFP. To synthesize GST-tagged Rab6T27N recombinant protein, the coding sequence for human Rab6T27N was amplified from Rab6T27N cDNA in a modified pcDNA3 vector (a gift from Drs Arai and Aoki at the University of Tokyo) and subcloned into the BamHI-EcoRI site of pGEX-5X-1. We found that Rab6T27N, but not Sar1T39N, inhibited retrograde transport.
Fig. S6. Reconstitution of anterograde transport of GT-GFP. (A) CHO-GT8 cells were pretreated with CHX. GT-GFP in the Golgi region of a single semi-intact CHO-GT8 cell was photobleached by laser illumination (bleach). The cell was incubated in the presence of cytosol/ATP for the indicated times (sec). PI, propidium iodide. Scale bar: 10 µm. (B) Kinetics of fluorescence recovery after photobleaching in the Golgi region in the presence of cytosol/ATP (cytosol+ATP), an ATP regenerating system only (ATP), or cytosol/ATP plus 1 mM GTPγS (GTPγS). (C) After CHX treatment, GT-GFP in the Golgi region was photobleached, and the semi-intact cells were incubated at 32°C for 10 minutes in the presence of cytosol/ATP. Cells were then treated with 10 µg/ml brefeldin A (BFA) for 30 minutes to relocate the Golgi-localized GT-GFP to the ER. This indicated that the recovered fluorescent structure was the Golgi apparatus. Scale bar: 10 µm. (D) Semi-intact CHO-GT8 cells that had been treated with CHX were incubated with cytosol/ATP (cytosol+ATP), an ATP regenerating system only (ATP), cytosol plus 1 mM AMP-PNP (AMP-PNP), or cytosol/ATP plus 1 mM GTPγS (GTPγS), and then subjected to the anterograde (ERrGolgi) transport assay. In addition, intact CHO-GT8 cells were treated with nocodazole (nocodazole), and then subjected to the anterograde transport assay. (E) CHX-treated, semi-intact CHO-GT8 cells were incubated with or without 5 µg His-tagged Sar1T39N protein (Sar1T39N) or 5 µg GST-tagged RabT27N protein in the presence of cytosol/ATP, and subjected to the assay for the anterograde transport of GT-GFP. Sar1T39N inhibited the anterograde transport, but Rab6T27N did not. And the amount of anterograde transport in the presence of Rab6T27N did not change when compared to that in the presence of rat liver cytosol alone. This result indicated that, in our anterograde transport assay, the fluorescence recovery in the Golgi was attributable to the anterograde transport of GT-GFP alone, and was not affected by retrograde transport from the Golgi.
Fig. S7. Kinetics of anterograde transport of GT-GFP in the presence of Yip1A peptides. CHX-treated, semi-intact CHO-GT cells were incubated with Yip1A peptides that corresponded to amino acids 1-56, 1-109, or 57-109 fused to GST in the presence of rat liver cytosol and an ATP regenerating system, and subjected to the anterograde transport assay. Fluorescence recovery in the ER was monitored every minute.
Fig. S8. The anti-EAGE2 antibody inhibited VSVG transport from the ER in semi-intact cells. The plasmid that encoded GFP-tagged VSVFts045 was transfected into HeLa cells. After 24 hours at 39°C, the cells were permeabilized with SLO and incubated with rat liver cytosol and 5 µg of normal rabbit IgG or anti-EAGE2 antibody on ice for 30 minutes. The cells were further incubated at 32°C for 45 minutes and observed by confocal microscopy. The cells were incubated at 39°C for 24 hours to allow the accumulation of the VSVG protein within the ER, and subsequently incubated at 32°C for the indicated times to induce the transport of VSVG from the ER. The cells were divided into three classes upon examination under the microscope (A). We counted the number of cells that showed localization of VSVG to the Golgi or the ER. The percentages of cells in which VSVG reached the Golgi in the presence of the control and anti-EAGE2 antibodies were 88.2±5.0 % and 46.1±7.9 %, respectively (B).
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