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First published online 22 March 2005
doi: 10.1242/jcs.02294

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Protein synthesis inhibitors and the chemical chaperone TMAO reverse endoplasmic reticulum perturbation induced by overexpression of the iodide transporter pendrin

¹ Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
² Johns Hopkins Bay View Medical Center, Johns Hopkins University, Baltimore, MD 21224, USA
³ Department of Internal Medicine E, Meir Hospital Sapir Medical Center, Kfar-Sava, Israel

View larger version (58K): [in a new window]   Fig. 1. Intracellular distribution of GFP-PDS and effect on ER function in COS7 cells. (A) Confocal images of fixed cells expressing untagged PDS, and living cells expressing GFP-PDS and GFP-CFTR, 16 hours after transfection. Cells expressing untagged PDS were fixed, permeabilized and probed with anti-PDS antibody and secondary Cy3 anti-rabbit antibody as described in Materials and Methods. GFP and Cy3 were detected with Ar 488 nm and HeNe 543 nm laser lines, respectively. Arrows indicate the plasma membrane (PM), endoplasmic reticulum (ER) and perinuclear aggregate (AGG). Bars, 10 µm. (B) Perinuclear GFP-PDS forms aggregates as demonstrated by photobleaching. A rectangle in the middle of a perinuclear aggregate was photobleached in a cell expressing GFP-PDS using a high-power 488 nm laser. Bars, 10 µm. (C) Detection of surface GFP-PDS and GFP-L236P by biotinylation. Cells were biotinylated using membrane-impermeable sulfo-NHS-biotin. Surface-labeled proteins were affinity purified using avidin-agarose and probed by western blotting using anti-GFP mAb. Results of quantitative densitometry are presented, on the right, as percentage of the sum of total lysate and low speed (14K) pellet. (D) Western blot analysis of the effect of GFP-PDS, GFP-L236P, GFP-CFTR and GFP-508 expression on proteasomal degradation. Cells were transfected with equal amounts of plasmids as described in Materials and Methods and harvested after 16 hours. Western blot analysis showing the accumulation of ubiquitilated proteins as a result of overexpression of GFP-tagged proteins probed with anti-GFP (top panel) or anti-ubiquitin (bottom panel) antibodies. (E) Effect of co-expressed GFP-PDS on GFP-VSVG surface-biotinylation labeling. Cells expressing GFP-VSVG in the presence or absence of GFP-PDS were preincubated overnight at 40°C prior to 2 hours incubation at 32°C. Cells were biotinylated using membrane-impermeable sulfo-NHS-biotin. Surface-labeled proteins were affinity purified using avidin-agarose and probed by western blotting using anti-GFP mAb. Biotinylated GFP-VSVG bands are indicated with an asterisk.

View larger version (54K): [in a new window]   Fig. 2. Effect of GFP-PDS overexpression on ER morphology in COS7 cells. (A) Confocal images of cells co-expressing YFP-PDS and free cytosolic CFP. The ER is vesiculated and CFP (red) is excluded from the lumen of the vesicular membranes (white arrows). (B) Confocal images of cells co-expressing YFP-PDS and ER-luminal helss-CFP. The lumen of the vesicular membrane structures is labeled with helss-CFP, confirming that they are ER membranes. The relative topology and distributions of YFP-PDS (green) and CFP or helss-CFP (red) in each panel are summarized in the scheme on the right side.

View larger version (53K): [in a new window]   Fig. 3. Effect of inhibition of protein synthesis on solubility and intracellular distribution of GFP-PDS. (A) Live-cell analysis of CHX-mediated recovery of GFP-PDS-expressing cells. Representative images of a time-lapse sequence of cells expressing GFP-PDS incubated in the presence of 25 µg/ml CHX for 5.3 hours. Images were captured at 45-second intervals. Arrows indicate cells with recovering ER. Right: regions of interest of total cellular fluorescence are plotted against time for the three cells outlined in the 0 minute panel. Bars, 20 µm. See also Movie 1 in supplementary material. (B) Live-cell microscopy of puromycin-mediated redistribution of the ER-retained GFP-PDS. Confocal images of cells expressing GFP-PDS incubated for 6 hours with or without 160 µg/ml puromycin. Bars, 10 µm. (C) Western blot analysis of the effect of CHX on GFP-PDS solubilization and degradation. Cells expressing GFP-PDS were incubated with increasing concentrations of CHX for 6 hours. Cell lysates (15 µg protein, top panel) and the 14K pellet (total pellet loaded, bottom panel) were probed with anti-PDS antibodies. (D) CHX-mediated degradation of GFP-PDS and GFP-L236P by western blot. Cells expressing GFP-PDS or GFP-L236P were incubated for the designated times with 25 µg/ml CHX. Cell lysates (15 µg protein) were probed with anti-PDS antibodies.

View larger version (62K): [in a new window]   Fig. 4. GFP-PDS is ubiquitilated. (A) Immunofluorescence co-localization of GFP-PDS with ubiquitin. Confocal images of cells expressing GFP-PDS were treated with ALLN for 6 hours. Cells were fixed, permeabilized and probed with anti-ubiquitin mAb and secondary anti-mouse Cy3-tagged mAb. GFP and Cy3 were detected with Ar 488 nm and HeNe 543 nm laser lines, respectively. Magnified views on the left show aggregates of ubiquitilated (CK SP) GFP-PDS. Arrowheads in the left panels point to a non-GFP-PDS-expressing cell. Arrows indicate the nucleus. Boxed regions in the center panels are magnified fivefold to show perinuclear aggregates. N, nucleus; AG, aggregate. Bar, 20 µm. (B) Western blot analysis showing ubiquitilation of GFP-PDS by immunoprecipitation. Cells expressing GFP-PDS were incubated for 1 hour with ALLN. Anti-GFP mAb-GFP-PDS complexes were immunoisolated (IP) from lysates using protein G agarose and probed with anti-ubiquitin mAb. (C) Live-cell microscopy of CHX-mediated recovery of GFP-PDS-expressing cells in the presence of ALLN. Representative images from a time-lapse series of cells expressing GFP-PDS, incubated in the presence of 25 µg/ml CHX and 20 µg/ml ALLN for 3.5 hours. Images were captured at 45-second intervals. Arrows indicate GFP-PDS in aggregates and in collapsed ER. Bar, 20 µm. See also Movie 2 in supplementary material.

View larger version (60K): [in a new window]   Fig. 5. Effect of inhibition of protein synthesis on folding and export of GFP-PDS. (A) Detection of CHX- and puromycin-mediated surface expression of GFP-PDS by biotinylation. Cells expressing GFP-PDS were incubated for 1 hour in culture medium containing 450 mM sucrose and 25 µg/ml CHX or 160 µg/ml puromycin. Cells were biotinylated using membrane-impermeable sulfo-NHS-biotin. Surface-labeled proteins were affinity-purified using avidin-agarose and probed by western blotting using anti-GFP and anti-actin mAbs as a loading control. (B) Confocal image analysis of CHX- or puromycin-mediated accumulation of YFP-PDS in the Golgi apparatus using a 20°C temperature block. Cells co-expressing YFP-PDS and the Golgi marker GalT-CFP were fixed after being incubated for 4 hours at 20°C in the presence of CHX or puromycin. Enlarged inserts show the perinuclear Golgi region. CFP- and YFP-tagged proteins were detected with Ar 458 nm and 514 nm laser lines, respectively. Bars, 10 µm.

View larger version (81K): [in a new window]   Fig. 6. Effect of TMAO on GFP-PDS aggregates and on ER morphology. (A) Western blot analysis of the effect of TMAO on GFP-PDS and GFP-L236P turnover. Cells expressing GFP-PDS were incubated with CHX or TMAO for 1 hour. Cell lysates (15 µg) were probed with anti-PDS antibodies and anti-actin mAbs as a loading control. (B) Protease protection assay of TMAO- and CHX-mediated GFP-PDS aggregate dissociation. Cells expressing GFP-PDS were incubated with CHX or TMAO for 1 hour. Cell lysates were incubated with protease for 5 and 15 minutes and probed with anti-GFP mAb by western blotting. (C) Analysis of TMAO-mediated ER recovery using live-cell microscopy. Confocal images of cells expressing GFP-PDS were captured at 45-second intervals for 3 hours after the addition of 150 mM TMAO. Bar, 10 µm. See also Movie 3 in supplementary material.