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First published online 3 June 2008
doi: 10.1242/jcs.022905

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Fast degradation of the auxiliary subunit of Na⁺/K⁺-ATPase in the plasma membrane of HeLa cells

¹ Graduate School of Biostudies, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto, 606-8502, Japan
² Max-Planck-Institute for Biophysics, Max-von-Laue-Str. 3, 60438, Frankfurt, Germany

View larger version (55K): [in this window] [in a new window]   Fig. 1. Characterization of the cells and antibodies used in this study. (A) Detection of isoform-specific mRNA in HeLa S3 cells by RT-PCR. The total RNA isolated from HeLa S3 cells used in this study was subjected to RT-PCR to examine the expression of four -isoforms (1-4) and four β-isoforms (β1-β4) of Na+/K+-ATPase. Primers used were designed to amplify an approximately 300 bp DNA fragment of each isoform cDNA (for the primer sequences, see supplementary material Table S1). The amplified fragment was subcloned into the cloning vector pT7blue (Takara) and the nucleotide sequences were confirmed by nucleotide sequencing. The expression patterns of these isoforms in human whole brain, human skeletal muscle and human testis were also examined. (B) Immunoblotting of HeLa S3 cell lysate using isoform-specific antibodies. The HeLa cell lysate was subjected to SDS-PAGE analyses followed by immunoblotting using isoform specific antibodies (anti-1, -β1 and -β3 antibodies). For the analyses of the N-linked glycosylation states, the lysate was pretreated with (+) or without (–) N-glycosidase F before the SDS-PAGE analysis. The positions of the molecular size markers (in kDa) are indicated on the right. (C) Characterization of the sensitivities of the isoform-specific antibodies in immunodetection. HAx2-tagged 1-, β1- and β3-subunits of human Na+/K+-ATPase were transiently expressed in HeLa cells and immunoprecipitated with anti-HA polyclonal antibody. These purified HA-tagged proteins were then subjected to SDS-PAGE and immunoblot analyses using anti-1, anti-β1 or anti-β3 isoform-specific antibodies. The exposure time was adjusted to avoid signal saturation. To normalize the number of protein molecules in each sample, the same samples were subjected to immunoblot analyses using anti-HA monoclonal antibody. The sensitivity ratio of the antibodies was obtained by dividing the signal intensity from the specific antibody by that from the anti-HA antibody (see Materials and Methods for the detailed procedure). Because the anti-β3-antibody is a rabbit polyclonal antibody, the signal from the immunoglobulin (rabbit anti-HA polyclonal antibody) used in the immunoprecipitation was detected on the immunoblot slightly overlapping with the bands from the β3-subunit. To obtain an accurate signal from the β3-subunit, the immunoglobulin signal was obtained from the control immunoprecipitation, in which the cell lysate was replaced with buffer and was subtracted from the total signal (immunoglobulin + β3-subunit).

View larger version (38K): [in this window] [in a new window]   Fig. 2. Quantification of the Na+/K+-ATPase subunits in the plasma membrane and the whole cell fraction of HeLa cells. The amount of the 1-, β1- and β3-subunits in the whole cell lysate and in the plasma membrane of HeLa cells at 80% confluence was quantified by immunoblot analysis using isoform-specific antibodies. The detailed procedures of cell-surface protein labeling and purification are described in supplementary material Fig. S1. (A) The total HeLa cell lysate (total) and the purified cell-surface proteins (surface) were subjected to SDS-PAGE and immunoblot analysis using the isoform-specific antibodies (1, β1 and β3) described in Fig. 1C. Note that the cell-surface fraction contains only matured forms of the β-subunits, whereas the total cell lysate contains the molecules with various sugar modifications. The positions of the molecular size markers (in kDa) are indicated on the right. Three different amounts of the samples (1, 5 and 10 µl) were subjected to the analyses to obtain a linear relationship between the amount of protein and the intensity of the immunoreactive signal on the immunoblot. The surface and total samples were blotted to the same membrane filter and subjected to exactly the same procedure to avoid experimental error. (B) A summary of the quantitative analyses. The immunoreactive bands in A were quantified, avoiding signal saturation. To compare the ratio of three subunits in each fraction, the band intensity was multiplied by the mean value of the antibody `sensitivity ratio' obtained in Fig. 1C (1:9.6:7.2 for 1:β1:β3). To compare the surface and total samples, the difference in the sample volume (50 µl surface and 500 µl total) was considered. The intracellular fraction of each subunit was obtained by subtracting the surface amount from the total amount. The surface and the intracellular fractions of 1-, β1- and β3-subunits are represented with the total amount of 1 as 1. The values in the figure were the mean values obtained from 4-5 independent experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 3. The differential degradation of the Na+/K+-ATPase subunits in the plasma membrane. HeLa cells at 80% confluence were pulse-labeled with membrane-impermeable biotinylation reagent (sulfo-NHS-SS-biotin) at 4°C for 1 hour, placed back in DMEM and incubated at 37°C for 0, 1, 3 or 5 hours in the absence or presence of inhibitors for degradation pathways. At the end of the chase period, the cells were harvested before (–GSH) and after (+GSH) treatment with glutathione to remove biotin moieties remaining on the cell surface. The labeled proteins were purified with streptavidin-agarose and analyzed by immunoblotting using antibodies against 1-, β1- and β3-subunits. (A) A typical result of the immunoblot analyses. (B) Quantitative analyses of the immunoblot. The results from at least three independent experiments are summarized.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Ubiquitylation of the Na+/K+-ATPase subunits. The HeLa cells in DMEM were incubated with or without the proteasome inhibitor lactacystin for 5 hours. The ubiquitylated proteins were immunoprecipitated from the cell lysate with anti-ubiquitin antibody (polyclonal antibody for 1, β1, ubiquitin and IgG immunoblots and monoclonal antibody for β3 immunoblot) in the presence of the inhibitor and then subjected to immunoblotting using the antibodies indicated. Mouse IgG was used in the immunoblotting as a negative control (IgG). The positions of the molecular size markers (in kDa) are indicated on the right.

View larger version (22K): [in this window] [in a new window]   Fig. 5. The effect of specific β-isoform knockdown by RNAi on the amount of other isoforms. siRNA for β1 or β3 or both was introduced into HeLa cells. (A) After 24 hours, the cells were either directly harvested as the total cell lysate or harvested after the surface labeling with sulfo-NHS-SS-biotin as described in Fig. 2. The total cell lysate (whole cell) and the affinity-purified biotinylated proteins (surface) were analyzed by immunoblotting using 1-, β1- or β3-specific antibodies. A typical result of the immunoblot is shown. The positions of the molecular size markers (in kDa) are indicated on the right. (B) The band intensity in A was quantified and represented as the ratio to that of the nontransfected cells. More than three independent experiments were performed to obtain error bars (s.d). (C) The amount of Na+/K+-ATPase subunits in the plasma membrane in β1/β3-double-knockdown cells. The siRNAs for both β1- and β3-subunits were introduced into the HeLa cells. After the indicated time, the cell surface proteins were labeled with biotin and purified. These samples were subjected to immunoblot analysis as in A and the amount of the 1-, β1- and β3-subunits were quantified and represented relative to the amount at the beginning of the chase. The loading volumes were adjusted based on the total protein amount in the sample. The results from three independent experiments are summarized (mean ± s.d.).

View larger version (18K): [in this window] [in a new window]   Fig. 6. The number of β-subunits but not -subunits in the plasma membrane is dependent on the cell density. HeLa cells with different confluencies (20, 40, 60, 80 and 100%) were prepared. The amount of individual isoforms in the whole cell lysate (A), as well as in the plasma membrane (B), were examined by quantitative immunoblotting as described in Fig. 2. The loading volumes were adjusted based on the total protein amount. The immunoreactive bands were quantified and plotted as described in Fig. 2, with the amount of the 1-subunit at 20% confluence adjusted to 1. Three independent experiments were performed to obtain mean ± s.d. The immunoblotting results are also shown as insets.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Model of the life cycle of the Na+/K+-ATPase subunits in HeLa cells. The results obtained in this study, combined with those of other previous studies, are summarized. The - and β-subunits first co-translationally assemble in the ER. The assembled enzymes leave the ER for the Golgi network, where they are subjected to sugar modifications for maturation. Once they reach the plasma membrane, the subunits dissociate and the unassembled β-subunit undergoes ubiquitylation, endocytosis and fast degradation, whereas the -subunit remains in the plasma membrane and is subjected to slow degradation. As a result, the plasma membrane contains more -subunit than β-subunit. Reassembly of the subunits may occur in the plasma membrane, although it is not clear how this assembly-disassembly is regulated.

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