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First published online 25 May 2004
doi: 10.1242/jcs.01141

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Transient calnexin interaction confers long-term stability on folded K⁺ channel protein in the ER

Department of Physiology and Molecular Biology Institute, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095-1751, USA

View larger version (25K): [in a new window]   Fig. 1. Treatment with dNJ prevents association of the core glycosylated Shaker protein with calnexin. (A) Structure of the 14-residue core glycan is shown. Glucose (), mannose () and N- acetylglucosamine (). Arrows indicate sites of trimming by ER glucosidases I and II (ER Glu I and II) and ER mannosidases I and II (ERMI and II). Drugs that inhibit each step are shown in parentheses: dMJ, deoxymannojirimycin; dNJ, deoxynojirimycin; KIF, kifunensine; SWN, swainsonine. (B) Adjacent lanes showing the mobility of the immature, core-glycosylated form of the Shaker protein from cells incubated in the absence (-) or presence (+) of dNJ. When present, dNJ was added before the starvation step and was present throughout the pulse and chase periods. (C) Cells expressing the wild-type Shaker protein or an unglycosylated mutant (N259Q+N263Q) were metabolically labeled for 5 minutes, chased in nonradioactive medium for 0 or 10 minutes, as noted, and lysed with detergent. When present (+), dNJ was added before the starvation step and was present throughout the pulse and chase periods. Proteins were precipitated sequentially with antibodies directed against calnexin and Shaker (Nagaya et al., 1999). The open arrowhead denotes the position of the immature form of the Shaker protein. The open circle indicates the position of a nonspecific background band of variable intensity, which is also seen in untransfected cells upon immunoprecipitation with the Shaker antiserum (data not shown).

View larger version (44K): [in a new window]   Fig. 2. Inhibition of glucose trimming slows ER exit rate of wild-type Shaker protein. (A,B) Wild-type Shaker protein was expressed in HEK293T cells, metabolically labeled for 10 minutes and chased for various times in the absence (-dNJ) or presence of 1 mM dNJ added during the starvation, pulse and chase periods (+dNJ) or during the chase period only (+dNJ*). After detergent solubilization, the Shaker protein was immunoprecipitated and subjected to electrophoresis and fluorography. Representative fluorographs are shown; n=4. Open and closed arrowheads indicate the positions of the immature and mature forms of the Shaker protein, respectively. Open circles indicate the positions of two nonspecific background bands of variable intensity, which are also seen in untransfected cells upon immunoprecipitation with the Shaker antiserum (data not shown). A sharp, unstable band migrating near the mature form of the Shaker protein is also seen in untransfected cells (unmarked). Positions of molecular weight markers are indicated by bars (kD; kilodaltons). (C) The percentage of Shaker protein in the mature form has been plotted as a function of chase time. Data were obtained in the absence of dNJ (; t1/2 for maturation=59 minutes), in the presence of dNJ added during the starvation, pulse and the chase (; t1/2=101 minutes), or in the presence of dNJ added during the chase only (; t1/2=57 minutes) (n=4). Here and in all subsequent figures, the data are presented as mean±s.e.m.

View larger version (37K): [in a new window]   Fig. 3. Inhibition of glucose trimming does not destabilize the wild-type Shaker protein. After a 30 minute pulse, cells were harvested immediately (0 h) or after a 24 hour chase period. The amount of Shaker protein remaining at 24 hours was 86% (-dNJ), 81% (+dNJ) or 81% (+dNJ*). The parenthetical designations have the same meaning as in Fig. 2. Open and closed arrowheads indicate the positions of the immature and mature forms of the Shaker protein, respectively. The open circle indicates the position of a nonspecific background band of variable intensity, which is also seen in untransfected cells upon immunoprecipitation with the Shaker antiserum (data not shown). A representative fluorograph is shown (n=3).

View larger version (41K): [in a new window]   Fig. 4. ER retention does not destabilize wild-type Shaker protein. (A) Shaker-expressing cells were treated with brefeldin A (5 µg/ml) and nocodacole (20 µg/ml) (BFA/NOC) or were untreated. Cells were metabolically labeled for 10 minutes and were harvested immediately (0 h) or after chase periods of 24 or 48 hours, as indicated. Open and closed arrowheads indicate the positions of the immature and mature forms of the Shaker protein, respectively. Compiled data from 5-8 similar experiments indicated that the amount of Shaker protein remaining at 48 hours was 94±12% in untreated cells and 116±22% in BFA/NOC-treated cells. (B,C) Representative differential interference contrast (DIC) (i) and confocal images (ii-iv) of HEK293T cells transiently transfected with wild-type Shaker are shown. At 24 hours post-transfection, cells were incubated in the presence of BFA/NOC (B) or left untreated (C), before permeabilization, labeling with antibodies against calnexin (ii) and Shaker (iii), and visualization with fluorescently conjugated secondary antibodies. Red, calnexin; green, Shaker. The merged images are shown in iv. Bars, 10 µm.

View larger version (28K): [in a new window]   Fig. 5. Wild-type Shaker is destabilized when calnexin interaction and ER exit are blocked. (A) Shaker-expressing cells were treated with BFA/NOC and dNJ (1 mM) added during the starvation, pulse and chase periods; or with BFA/NOC, dNJ and Lac (10 µM). Cells were labeled for 10 minutes and chased for 0, 24 or 48 hours as indicated. Open arrowhead indicates the position of the core-glycosylated, immature form of the Shaker protein. (B) The amount of core glycosylated Shaker protein remaining at 48 hours of chase was quantified by densitometry and expressed relative to the amount present at 0 time of chase (100%). The number of experiments for each condition (n) is shown in parentheses on the appropriate bar.P<0.0001 compared with BFA/NOC; P<0.0001, compared with BFA/NOC+dNJ (Student's t-test).

View larger version (75K): [in a new window]   Fig. 6. Shaker wild-type protein remains competent for ER-to-Golgi transport despite treatment with BFA/NOC and dNJ. (A) Cells expressing the Shaker wild-type protein were pre-incubated with BFA/NOC and dNJ for 2 hours; the drugs were then maintained during a 30 minute starvation period, 30 minute pulse and 2 hour chase. After 2 hours of chase, cells were washed with PBS and the medium was replaced. The chase was then continued in the presence of dNJ with or without BFA/NOC. At various times, cells were harvested and solubilized with detergent, and the Shaker protein was immunoprecipitated and subjected to electrophoresis and fluorography. Lanes show Shaker protein from cells: (1) harvested immediately after pulse; (2) harvested after 2 hours of chase; (3) harvested after 9 hours of chase in the presence of dNJ and BFA/NOC; (4) harvested after 9 hours of chase in the presence of dNJ but no BFA/NOC after the first 2 hours of chase; (5) untreated with dNJ and BFA/NOC and harvested after 9 hours of chase. Open arrowhead indicates the position of the immature form of the Shaker protein. Closed arrowhead indicates the position of the mature form of the Shaker protein from untreated cells (lane 5). Filled circle indicates the position of a diffuse band seen upon removal of BFA/NOC (lane 4). This putative mature band was first detected approximately 2-3 hours after the removal of BFA/NOC. Representative results are shown from a total of six experiments. (B) Cells expressing wild-type Shaker were preincubated, starved, pulsed and chased for 2 hours in the presence of dNJ and BFA/NOC. At 2 hours of chase, cells were washed with PBS and the medium was replaced. The chase was then continued for 3 more hours in the presence of dNJ with (lane 1) or without (lanes 2-4) BFA/NOC. Lane 3: immunoprecipitated protein was treated with endo H. Lane 4: immunoprecipitated protein was treated with PNGase F. Filled circle indicates the position of the diffuse band seen upon removal of BFA/NOC. Open arrowhead indicates the position of the immature form of the Shaker protein. Open square indicates the deglycosylated Shaker protein. Representative results are shown from a total of two experiments. (C) Cells expressing the misfolded Shaker mutant D316K were preincubated, starved, pulsed and chased for 2 hours in the presence of dNJ and BFA/NOC. The chase was then continued in the presence of dNJ with or without BFA/NOC. Lanes show D316K protein from cells: (1) harvested after 2 hours of chase; (2) harvested after 6 hours of chase in presence of dNJ and BFA/NOC; (3) harvested after 6 hours of chase in presence of dNJ but no BFA/NOC after the first 2 hours of chase; (4) untreated with dNJ and BFA/NOC and harvested after 6 hours of chase. Open arrowhead indicates the position of the immature form of the Shaker protein. Representative results are shown from a total of two experiments.

View larger version (24K): [in a new window]   Fig. 7. Transient interaction with calnexin provides long-term protection from ERAD. (A) Shaker-expressing cells were treated with BFA/NOC or were untreated, as indicated. Cells were metabolically labeled for 2 minutes, chased for the indicated times and lysed. Proteins were precipitated sequentially with antibodies directed against calnexin and Shaker (Nagaya et al., 1999). A representative experiment is shown; n=3. Open arrowhead indicates the position of the core-glycosylated, immature form of the Shaker protein. (B) The amount of Shaker protein obtained after sequential immunoprecipitation with calnexin and Shaker antibodies in the presence () or absence () of BFA/NOC treatment was quantified by densitometry, normalized to the maximum value obtained during the experiment and plotted versus chase time. A representative experiment is shown; n=3. (C) Comparison of the time course of calnexin interaction and ERAD for the wild-type Shaker protein. The percentage of Shaker protein remaining at 0, 24 and 48 hours of chase after treatment with BFA/NOC () or with BFA/NOC and dNJ () was quantified by densitometry and plotted versus time of chase. The values of the percentage of Shaker protein remaining in BFA/NOC-treated samples were 160±44% (24 hours) and 83±4.9% (48 hours) and in BFA/NOC+dNJ-treated samples were 83±8% (24 hours) and 31±5% (48 hours). P<0.001 for BFA/NOC/dNJ versus BFA/NOC at the 48 hour chase time; Student's t-test. The data from B have been replotted on the same axes using the same symbols as in B (, ).

View larger version (37K): [in a new window]   Fig. 8. ER mannosidase I inhibitors block ERAD of wild-type Shaker protein. (A) Shaker-expressing cells were pulsed for 10 minutes and either harvested immediately or chased for 48 hours. Cells were treated with BFA/NOC and no additional drug; BFA/NOC and dNJ (1 mM); BFA/NOC and dMJ (1 mM); BFA/NOC, dNJ and dMJ; BFA/NOC, dNJ and KIF (10 µM); or BFA/NOC, dNJ and SWN (1 mM). A representative fluorograph is shown. Open arrowhead indicates the position of the core glycosylated, immature form of the Shaker protein. (B) The amount of core glycosylated Shaker protein remaining at 48 hours of chase was quantified by densitometry and expressed relative to the amount present at 0 time of chase (100%). The number of experiments for each condition (n) is shown in parentheses on the appropriate bar. P<0.001 compared with all other treatments except dNJ+SWN; Student's t-test. Data are presented as mean±s.e.m. except for the dMJ sample, where n=2. For the dMJ sample, the mean value is shown.

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