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First published online 8 December 2005
doi: 10.1242/jcs.02706

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Deglycosylation of Na⁺/K⁺-ATPase causes the basolateral protein to undergo apical targeting in polarized hepatic cells

¹ Institute of Microbiology and Immunology, National Yang-Ming University, 155 Sec. 2 Linong Street, Taipei 112, Taiwan
² Department of Life Science, National Yang-Ming University, 155 Sec. 2 Linong Street, Taipei 112, Taiwan
³ Institute of Biophotonics Engineering, National Yang-Ming University, 155 Sec. 2 Linong Street, Taipei 112, Taiwan
⁴ Department of Surgery, Veteran General Hospital, 201 Sec. 2 Shih-Pai Road, Taipei 112, Taiwan

View larger version (90K): [in a new window]   Fig. 1. Deglycosylation by pharmacological treatments causes aberrant apical targeting of the basolateral Na+/K+-ATPase. (A) Polarized Hep G2 cells (CTL) were subjected to immunofluorescence (IF) staining for Na+/K+-ATPase, E-cadherin, CD147 and DPPIV (green in the colour-merged channel), and co-labelled with Rhodamine-phalloidin (red) to identify F-actin-enriched bile canaliculi (arrows). Na+/K+-ATPase, E-cadherin and CD147 were present mainly on the basolateral membrane (arrowheads), whereas DPPIV was an apical marker (arrows). (B) Hep G2 cells were treated with 20 µM tunicamycin (TM) for 24 hours before the IF experiments. Note that the Na+/K+-ATPase was aberrantly targeted to the apical domain (double arrowheads, stained yellow in the colour panel). Tunicamycin treatment had little effect on the localization of basolateral E-cadherin, CD 147 or apical DPPIV. (C) Statistical analysis of the bile canaliculi that contained the membrane proteins indicated, before (open bars) and after (filled bars) tunicamycin treatment. More than 500 bile canaliculi were counted in each experiment; data are the means ± s.d. of three experiments. (D) Hep G2 cells were subjected to double-IF staining to reveal Na+/K+-ATPase (green) and ER (by anti-calreticulin antibody, red) before and after tunicamycin treatment. Na+/K+-ATPase was present mainly along the basolateral membrane (arrowheads) in control cells. After tunicamycin treatment, the presence of Na+/K+-ATPase was greatly reduced from the basolateral membrane, and gradually increased in the cytoplasmic vesicles (arrows), with no obvious accumulation in the ER. (E) Western blot analysis using Na+/K+-ATPase ß-subunit-specific antibodies on Hep G2 cells treated with mock solution (CTL) or various glycosylation inhibitors: tunicamycin (TM), 1-deoxy-mannojirimycin (DMJ), 1-deoxynojirimycin (DNJ) and Kifunensine (KIF). The fully glycosylated (***ß), intermediately glycosylated (**ß) and the core (*ß) proteins of Na+/K+-ATPase ß-subunit are indicated. Note that the KIF treatment resulted in significant degradation of the ß-subunit protein (arrow). (F) After adding deglycosylating drugs for 24 hours, the percentage of bile canaliculi that contained mistargeted Na+/K+-ATPase was calculated. The bile canaliculi were recognized by F-actin staining; those positively stained for Na+/K+-ATPase ß-subunit were identified. At least 500 bile canaliculi obtained from approximately 25 microscopic images were included in the calculation. Data are the means ± s.d. of three experiments. Bars, 20 µm (A,B); 2 µm (D).

View larger version (77K): [in a new window]   Fig. 2. Deglycosylation by site-directed mutagenesis also caused apical targeting of Na+/K+-ATPase ß-subunit. (A) Hep G2 cells transfected with control vector (Mock), the wild type (WT) or one of the three deglycosylated and FLAG-tagged ß-subunit genes (N124Q, N240Q, N124/240Q) were treated with tunicamycin (TM) or not (-) before western blot analyses using anti-FLAG antibody. The fully glycosylated (***ß), intermediately glycosylated (**ß), and the core (*ß) proteins of the Na+/K+-ATPase ß-subunit are indicated. (B) Hep G2 cells transfected with control vector (CTL), the wild-type (WT), or one of the three deglycosylated and FLAG-tagged ß-subunit genes (N124Q, N240Q, N124/240Q) were subjected to IF staining using anti-FLAG antibody (green in the merged panel); F-actin staining (red) was used to localize the apical domain (arrow). Note that the wild-type ß-subunit proteins were found only along the basolateral membrane (arrowheads), whereas all three mutants exhibited apical presence (double arrowheads). Bar, 5 µm.

View larger version (116K): [in a new window]   Fig. 3. Deglycosylated Na+/K+-ATPase ß-subunits can still interact with the catalytic -subunits. (A) Hep G2 cells treated with tunicamycin (TM) or untreated were subjected to immunoprecipitation (IP) experiments using either anti--subunit antibody (anti-) or anti-ß-subunit antibody (anti-ß). The resulting immunoprecipitates were analyzed by western blotting. The positions of the -subunit and the fully glycosylated (***ß), intermediately glycosylated (**ß), and the core (*ß) proteins of the ß-subunit are indicated. (B) The interactions between the exogenous FLAG-tagged ß-subunit (the wild-type and three deglycosylated mutant constructs) and the endogenous protein were tested by IP-western analyses; co-transfected EYFP--tubulin were used as a control. (C) The localization of - and ß-subunits before and after tunicamycin treatment was visualized by IF using antibodies specific to either - or ß-subunit (green), together with F-actin staining (red). Note the basolateral only (arrowheads) and the apical presence (double arrowheads) of both subunits in the control (CTL) and tunicamycin-treated (TM) cells, respectively. (D) Hep G2 cells transfected with EGFP-labelled ß-subunit (wild-type or N124Q mutant, green), were stained for endogenous -subunit (blue) and F-actin (red). Note both wild-type ß-subunit and -subunit were found only along the basolateral membrane (arrowheads) in the wild-type transfectants, whereas a portion of the N124Q ß-subunit and the endogenous -subunit was aberrantly translocated to the apical domain (double arrowheads) in Hep G2 cell transfected with N124Q ß-subunit. Bars, 10 µm.

View larger version (32K): [in a new window]   Fig. 4. The rate of protein degradation was similar between the wild-type and the deglycosylated mutants of ß-subunits. (A) Hep G2 cells transfected with control vector (Mock), the wild type (WT) or one of the three deglycosylated and FLAG-tagged ß-subunit genes (N124Q, N240Q, N124/240Q) were labelled with [35S]methionine for 15 minutes, followed by incubation in unlabelled culture medium for the time periods indicated. Whole-cell lysates were extracted and examined by anti-FLAG IP and autoradiography. (B) Quantitative analysis of the autoradiograph. The co-transfected EYFP tagged -tubulin was used as a control for transfection efficiency. The 35S signals of the ß-subunit were first normalized by the signals of the co-transfected EYFP--tubulin. The data recorded at 1, 3 or 6 hours were normalized against that recorded immediately after the washout of [35S]methionine label (0 hour).

View larger version (123K): [in a new window]   Fig. 5. Apical targeting by the deglycosylated Na+/K+-ATPase ß-subunit was mediated via the indirect transcytosis pathway. (A) Polarized Hep G2 cells were treated with monoclonal anti-Na+/K+-ATPase ß-subunit antibody at 4°C for 15 minutes. After washout of the primary antibody, the cells were warmed to 37°C for the time periods indicated, then fixed and stained with fluorescently labelled secondary antibody. Within the 60 minute observation period, no fluorescent signal appeared at the bile canaliculi (arrows). (B) The same experiments were applied to the Hep G2 cells treated with tunicamycin. The fluorescent signals were originally found only along the basolateral membrane (arrowheads); over time, there was a progressive increase in fluorescence at the apical canalicular domain (double arrowheads). (C) The percentage of bile canaliculi (BC) that contained the fluorescence generated from the mistargeted Na+/K+-ATPase ß-subunit was calculated in the control () and tunicamycin-treated cells (). The bile canaliculi were recognized by F-actin staining; those also positively stained for Na+/K+-ATPase ß-subunit were identified. At least 500 bile canaliculi obtained from approximately 20 microscopic images were included in the calculation. Results shown are the means ± s.d. of three independent experiments. Bars, 10 µm.

View larger version (24K): [in a new window]   Fig. 6. Cells containing apical Na+/K+-ATPase were defective in maintaining sodium homeostasis. (A) Polarized Hep G2 cells were loaded with non-ratio sodium indicator, Sodium Green and then treated with mock solution (CTL), tunicamycin (TM) for 12 hours, ouabain for 10 minutes, or both. The changes in fluorescence intensity before, during and after washout of the high Na+ solution (Bar, 10 seconds) were monitored under a fluorescence microscope, and plotted as a function of time. The mean (line) ± s.d. (dashed line) curves are shown. (B) The same experiment as in A, but the cells were loaded with the ratio sodium indicator, SBFI. Ratio imaging by the intensity of emission fluorescence excited by 340 nm and 380 nm was plotted as a function of time. The pharmacological and mutagenesis treatments are as indicated. (C) The quantification of intracellular sodium concentrations for the data set shown in B, in vivo calibration for SBFI loading was performed. Cells treated with tunicamycin and ouabain (*), or transfected with three deglycosylated ß-subunit mutant constructs (+), all exhibited a higher [Na+]i than the control cells before the high-Na+ challenge (P<0.05 by Student's t-test). Means ± s.d. are shown.

View larger version (85K): [in a new window]   Fig. 7. Hep G2 cells containing apical Na+/K+-ATPase are defective in canalicular excretion. (A) Polarized Hep G2 cells were treated with 20 µg/ml tunicamycin for 24 hours (TM) or untreated (CTL). Fluorescein diacetate (FDA, green) was added to the culture media for 30 minutes; the locations of bile canaliculi were visualized by F-actin staining (red). The bile canaliculi capable of excreting FDA via transcytotic transport were stained yellow (arrows), or stained red otherwise (arrowheads). (B) Fluorescently labelled 3 kDa dextrans (green) were added to the culture media for 1 hour; the locations of bile canaliculi were visualized by F-actin staining (red). The fluorescent dextran could enter the canalicular lumen via permeable tight junctions between the neighbouring cells (the paracellular pathway). Bile canaliculi active in paracellular transport were stained yellow (arrows), and stained red if inactive (arrowheads). (C) Quantification of the bile canaliculi (BC) apical domains active in transcytotic (grey bars) or paracellular transport (open bars) in the presence (TM) or absence (-) of tunicamycin treatments. More than 500 apical domains were counted for each experiment and results show the means ± s.d. *P<0.05 by Student's t-test. Bars, 20 µm.