Chaperone stress 70 protein (STCH) binds and regulates two acid/base transporters NBCe1-B and NHE1 - PubMed (original) (raw)

Chaperone stress 70 protein (STCH) binds and regulates two acid/base transporters NBCe1-B and NHE1

Jun-Seok Bae et al. J Biol Chem. 2013.

Abstract

Regulation of intracellular pH is critical for the maintenance of cell homeostasis in response to stress. We used yeast two-hybrid screening to identify novel interacting partners of the pH-regulating transporter NBCe1-B. We identified Hsp70-like stress 70 protein chaperone (STCH) as interacting with NBCe1-B at the N-terminal (amino acids 96-440) region. Co-injection of STCH and NBCe1-B cRNA into Xenopus oocytes significantly increased surface expression of NBCe1-B and enhanced bicarbonate conductance compared with NBCe1-B cRNA alone. STCH siRNA decreased the rate of Na(+)-dependent pHi recovery from NH4(+) pulse-induced acidification in an HSG (human submandibular gland ductal) cell line. We observed that in addition to NBCe1-B, Na(+)/H(+) exchanger (NHE)-dependent pHi recovery was also impaired by STCH siRNA and further confirmed the interaction of STCH with NHE1 but not plasma membrane Ca(2+) ATPase. Both NBCe1-B and NHE1 interactions were dependent on a specific 45-amino acid region of STCH. In conclusion, we identify a novel role of STCH in the regulation of pHi through site-specific interactions with NBCe1-B and NHE1 and subsequent modulation of membrane transporter expression. We propose STCH may play a role in pHi regulation at times of cellular stress by enhancing the recovery from intracellular acidification.

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Figures

FIGURE 1.

The interaction of NBCe1-B with STCH. A, shown is a schematic diagram of NBCe1-B indicating the position of the bait region (amino acids 96–440) for yeast two-hybrid screening. B, shown is an α-galactosidase assay. The positive control (PC, n = 3) is comprised of the well characterized interaction between p53 (bait) and SV40 large T-antigen (target). The negative control (NC) is a co-transformation of human lamin C (bait) and SV40 large T-antigen (target), which is known to have no interaction. (n = 3, ***, p < 0.0001). NBCe1-B + STCH is a co-transformation of bait region (amino acids 96–440) and STCH (target). (n = 3, *** p < 0.0001). C, NBCe1-B transcripts (left panel, n = 2) and STCH transcripts (right panel, n = 2) in primary hSMG ducts are shown. D, hSMG tissues were subjected to immunoprecipitation (IP) with normal rabbit IgG (Control), rabbit anti-STCH, and rabbit anti-NBC1 antibodies. Immunoprecipitates were subjected to Western blotting (IB) with rabbit anti-NBC1 antibody (n = 2). E, shown is the current-voltage (I-V) relationship of NBCe1-B current in oocytes injected with cRNA of NBCe1-B or NBCe1-B + STCH. Reversal potentials were −96.54 mV (NBCe1-B, n = 4) and −114.4 mV (NBCe1-B +STCH, n = 6). F, shown is immunolabeling of cultured HSG cell using polyclonal anti-STCH (green, left, n = 3); monoclonal anti-protein disulfide isomerase (PDI) antibody (red, middle, n = 3) was used for detection of endoplasmic reticulum. A merged image of double staining is shown on the right. Scale bars, 10 μm.

FIGURE 2.

The functional effect of STCH on NBCe1-B activity in Xenopus oocytes. A, shown is a diagram of wild type and NBCe1-B deletion mutants. AID, autoinhibitory domain. B, shown is the current-voltage (I-V) relationship of NBCe1-B current in Xenopus oocytes injected with cRNA for NBCe1-B wild type, Δ95aa, and Δ345aa. Reversal potentials were −96.54 mV (NBCe1-B, n = 4) and −111.9 mV (Δ95aa, n = 5). C and D, shown are membrane potential (Vm) changes by exposure to HCO3−/Na+ or Na+-free solution mediated by NBCe1-B wild type (n = 6) and Δ95aa mutant (n = 7) alone or co-expressed with STCH (wild type, n = 7; Δ95aa mutant, n = 9. *, p < 0.05) in oocytes.

FIGURE 3.

STCH binds to specific regions of NBCe1-B. A, Xenopus oocytes (n = 30 per transcript) injected with indicated cRNAs were extracted, and membrane fractions were subjected to Western blotting (IB) with indicated antibodies. Observations were repeated in at least two independent experiments for each construct: WT (n = 3), Δ95aa (n = 3), Δ345aa (n = 2). B, shown is densitometry quantification of bands illustrating wild type and mutant NBCe1 membrane expression in the presence of STCH as a percentage of control (without STCH) (*, p < 0.05). C, shown is immunolabeling of NBCe1-B wild type and deletion mutants in Xenopus oocytes transfected with NBCe1 alone (upper panels) or co-transfected with STCH (lower panels). Scale bars, 200 μm. Images are representative of two to three independent experiments for each transfection. D, shown is fluorescence intensity of membrane region corresponding to white boxed area in C. AU, absorbance units. E, HSG cells were transfected with FLAG-NBCe1-B, deletion mutants (Δ95aa, Δ345aa), and GFP-STCH plasmid. Cell lysates were subjected to immunoprecipitation (IP) with mouse anti-FLAG antibody, and immunoprecipitates were subjected to Western blotting with mouse anti-GFP antibody (n = 2).

FIGURE 4.

STCH interaction with NBCe1-B occurs independently of IRBIT. A, NBCe1 currents measured in oocytes co-expressed with STCH and/or IRBIT were evoked by a single depolarizing step pulse from −120 to +20 mV in a HCO3− solution. B, shown is a summary of mean current amplitude at +20 mV in oocytes transfected with NBCe1-B (n = 5), IRBIT (n = 4; *, p < 0.05), and STCH (n = 6; *, p < 0.05) either alone or as co-transfection (n = 4; *, p < 0.05). C, HSG cells were subjected to immunoprecipitation (IP) with normal mouse IgG, mouse anti-IRBIT, and anti-NBC1 antibodies. Immunoprecipitates were subjected to Western blotting (IB) with anti-NBC1 (n = 2) and anti-IRBIT (n = 2) antibodies. The filled triangle indicates the migration of endogenous IRBIT. D, HSG cells were subjected to immunoprecipitation with anti-STCH and anti-IRBIT antibodies. Immunoprecipitates were subjected to Western blotting with anti-STCH (n = 2) and anti-IRBIT (n = 2) antibodies. Filled and empty triangles indicate the migration of endogenous IRBIT and STCH, respectively.

FIGURE 5.

Knockdown of STCH inhibits pH_i_ recovery from acidification. A, shown are NBCe1-B (left panel, n = 2) and STCH transcripts (right panel, n = 2) in HSG cells. Note PCR products for NBCe1-A were not observed (left panel). B, HSG cells were transfected with one of two siRNA sequences targeting STCH or scrambled siRNA. IB, immunoblot. C, densitometry quantification of bands illustrating STCH expression in the presence of STCH siRNA 1 (n = 3) and 2 (n = 3) as a percentage of control (scrambled siRNA) (n = 3; *, p < 0.05). D, Na+-dependent pH_i_ recovery from NH4+ pulse (black bars) in HSG cells in the presence of STCH (the resting pH_i_ of cells was ∼6.92, n = 5; *, p < 0.05) or scrambled siRNA (the resting pH_i_ of cells was ∼7.45, n = 5) recorded in a HCO3− solution. EIPA (25 μ

) and DIDS (500 μ

) applications are indicated by the horizontal bars. E, shown is a summary quantification of the EIPA (n = 5; *, p < 0.05)- and DIDS-sensitive (n = 5; *, p < 0.05) pH_i_ recovery rate in the presence of scrambled or STCH siRNA. F, HSG cells were subjected to immunoprecipitation with mouse IgG, anti-STCH, anti-NHE1, and mouse anti-PMCA antibodies. Immunoprecipitates (IP) were subjected to Western blotting (IB) with anti-NHE1 (n = 2), anti-STCH (n = 2), and anti-PMCA (n = 2) antibodies. In the case of Western blotting with anti-STCH (second panel), ReliaBLOT reagents were used to remove contaminating heavy chain IgG (see “Experimental Procedures”).

FIGURE 6.

Effect of STCH on surface expression of NBCe1-B. A, HSG cells were transfected with empty vector or STCH. Surface-biotinylated proteins (1 mg) were subjected to Western blotting (IB) with rabbit anti-NBC1 and anti-PMCA, and whole cell lysates were subjected to Western blotting with anti-GFP and anti-β-actin antibodies. Densitometry quantification of bands illustrate NBCe1-B surface expression (n = 3; *, p < 0.05; B) and PMCA surface expression (n = 3; C) in the presence of STCH as a percentage of control (without STCH). D, HSG cells were transfected with scrambled or STCH siRNA. Surface-biotinylated proteins (1 mg) were subjected to Western blotting with rabbit anti-NBC1 and anti-PMCA, and whole cell lysates were subjected to Western blotting with anti-STCH and anti-β-actin antibodies. Densitometry quantification of bands illustrating NBCe1 surface expression (n = 3; *, p < 0.05; E) and PMCA surface expression (n = 3; F) in the presence of STCH siRNA as a percentage of control (scrambled siRNA).

FIGURE 7.

Effect of STCH on the functional activity of NHE. A, shown is Na+-dependent pH_i_ recovery from NH4+ pulse (black bars) in HSG cells transfected with STCH (the resting pH_i_ of cells was ∼7. 63) or empty vector (the resting pH_i_ of cells was ∼7.40, control) in a HCO3−-free solution and the effect of EIPA (25 μ

) application (white bar). B, summary quantification of the EIPA-sensitive pH_i_ recovery rate with (n = 4; *, p < 0.05) or without STCH (n = 3) transfection is shown. C, HSG cells were transfected with empty vector or STCH. Surface-biotinylated proteins (1 mg) were subjected to Western blotting with rabbit anti-NHE1 and anti-PMCA, and whole cell lysates were subjected to Western blotting (IB) with anti-GFP and anti-β-actin antibodies. D, densitometry quantification of bands illustrating NHE1 surface expression (n = 3; *, p < 0.05) is shown in the presence of STCH as a percentage of control (without STCH). E, shown is Na+-dependent pH_i_ recovery from NH4+ pulse (black bars) in HSG cells in the presence of STCH (the resting pH_i_ of cells was ∼7.11) or scrambled siRNA (the resting pH_i_ of cells was ∼7.10) recorded in a HCO3−-free solution and the effect of EIPA (25 μ

) application (white bar). F, shown is summary quantification of the EIPA-sensitive pH_i_ recovery rate in the presence of scrambled (n = 5) or STCH siRNA (n = 5; *, p < 0.05). G, HSG cells were transfected with scrambled or STCH siRNA. Surface-biotinylated proteins (1 mg) were subjected to Western blotting with rabbit anti-NHE1 and anti-PMCA, and whole cell lysates were subjected to Western blotting with anti-STCH and anti-β-actin antibodies. H, shown is densitometry quantification of bands illustrating NHE1 surface expression (n = 3; *, p < 0.05) in the presence of STCH siRNA as a percentage of control (scrambled siRNA).

FIGURE 8.

Specific region of STCH required for interaction with NBCe1-B and NHE1. A, shown are diagrams of wild type STCH and two STCH deletion mutants. B and C, HSG cells were transfected with GFP-tagged wild type, Δ314aa, and Δ359aa STCH. Cell lysates were subjected to immunoprecipitation with anti-GFP antibody, and immunoprecipitates (IP) were subjected to Western blotting (IB) with anti-NBC1 (n = 3) (B) or anti-NHE1 antibodies (n = 3) (C) (upper panels). To confirm transfection efficiency of GFP-tagged constructs, whole lysates of HSG cells were subjected to Western blotting with anti-GFP (middle panels). Endogenous expression of NBC1 and NHE1 was confirmed with anti-NBC1 and anti-NHE1 antibodies, respectively (lower panels).

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Chaperone stress 70 protein (STCH) binds and regulates two acid/base transporters NBCe1-B and NHE1 - PubMed (original) (raw)