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作者：Hong C. Li, Emily Y. Li, Lisa Neumeier, Laura Conforti, and Manoocher Soleimani,作者单位：1 Department of Medicine, University of Cincinnati, and 2 Research Services, Veterans Affairs Medical Center, Cincinnati, Ohio 45220 X2 ?: H; T# N6 V8 l$ x
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f% }2 w9 t2 W E* m0 ~( i1 V 【摘要】3 b' N* L7 s) i% w# \
The Na :HCO 3 - cotransporter NBC1 (SLC4A4, variant A, kidney specific) is located exclusively on the basolateral membrane of epithelial cells, implying that this molecule has acquired specific signals for targeting to the basolateral membrane. A motif with the sequence QQPFLS (positions 1010-1015) in the cytoplasmic tail of NBC1 was recently demonstrated to mediate targeting of NBC1 to the basolateral membrane. Here, we demonstrate that mutating the amino acid F (phenylalanine) or L (leucine) at positions 1013 or 1014 to alanine, respectively, resulted in the retargeting of NBC1 to the apical membrane. Furthermore, mutation of the FL motif to FF showed similar properties as the wild-type; however, mutation of the FL motif to LL showed significant intracellular retention of NBC1. Mutating the amino acids Q-Q-P and S (positions 1010-1011-1012 and 1015) to A-A-A and A, respectively, did not affect the membrane targeting of NBC1. Functional studies in oocytes with microelectrode demonstrated that the apically targeted mutants, as well as basolaterally targeted mutants, are all functional. We propose that the FL motif in the COOH-terminal tail of NBC1 is essential for the targeting of NBC1 to the basolateral membrane but is distinct from the membrane-targeting di-leucine motif identified in other membrane proteins. " M( g+ {2 Z7 G# q e
【关键词】 basolateral membrane apical membrane acidbase regulation kidney transmembrane domain
6 P0 l. c" w" D$ K n. ` INTRACELLULAR PH in mammalian tissues is well regulated and maintained through coordinated action of acid-base transporters ( 33 ). Among these acid-base transporters, the Na :HCO 3 - cotransporter (SLC4A4) was first discovered in the kidney proximal tubule ( 3, 5, 11, 16, 31, 32, 41 ). Two well-known variants of NBC1, kidney NBC1 (kNBC1, variant A) and pancreatic NBC1 (pNBC1, variant B), are expressed in various epithelial tissues: kidney proximal tubule, pancreatic duct, gastric parental cells, small intestine, and pulmonary alveolar cells ( 1, 12, 24 - 26, 34, 35 ). In the kidney proximal tubule, the basolateral NBC1 functions in tandem with the apical Na /H exchanger 3 (NHE3) in the kidney proximal tubule and is essential for the reabsorption of the majority of filtered bicarbonate ( 2, 23, 33 ). Mutations in NBC1 result in bicarbonate wasting and proximal renal tubular acidosis in human ( 4, 15, 17 - 19, 21, 37, 38 ), confirming the essential role of this transporter in acid-base regulation.: }/ m) ~0 H2 v4 a9 r2 [1 a" V" F
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In all studies reported to date, kidney NBC1 has been detected in the basolateral membrane ( 9, 20, 21, 27, 28, 36, 40 ), strongly suggesting the presence of specific signal(s) within the NBC1 molecule that is (are) responsible for its targeting to the basolateral membrane. However, little is known about the basolateral targeting signals within this molecule. Recently, the COOH-terminal tail of NBC1 was determined to express signals directing the targeting of NBC1 to the basolateral membrane ( 20 ). Progressive truncation of the COOH-terminal tail identified the motif QQPFLS (positions 1010-1015; full-length 1,035 amino acids), and the amino acid residue F in position 1013, as essential for the targeting of NBC1 to the basolateral membrane ( 20 ).
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$ G6 Y" o, W; Q# B3 Q M% g* [To identify the exact signal that is responsible for the targeting of NBC1 to the basolateral membrane, site-directed mutagenesis was performed on the QQPFLS motif. Thereafter, epitope-tagged or tag-free mutants were expressed in Madin-Darby canine kidney (MDCK) cells and in oocytes and examined for targeting or activity, respectively. Our results identified the FL signal (positions 1013 and 1014) as being essential for the targeting of NBC1 to the basolateral membrane. The FL signal is distinct from the di-leucine (LL) signal that has been identified in other basolaterally targeted membrane proteins, such as nucleotide pyrophosphatase NPP1 and E-cadherin ( 8, 22 ).
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MATERIALS AND METHODS
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# d" s% U( _+ b% C0 Y3 c" tConstruction of tagged full-length NBC1 and mutants. Full-length NBC1 was generated by PCR, using human full-length kidney NBC1 DNA (3,257 bp and 1,035 amino acid residues) as a template (GenBank number AF007216 ). The amplified wild-type NBC1 DNA was fused translationally in-frame to green fluorescent protein (GFP) by cloning into pcDNA3.1/NT-GFP-TOPO vector (Invitrogen, Carlsbad, CA).
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) v7 n9 R7 u, y* dSite-directed mutagenesis was performed using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The following mutants were generated: AAA FLS, QQPF A S, QQPFL A, QQPF F S, and QQP L LS. The schemes in Supplemental Diagrams 1 and 2 depict the location and primer sequences for various mutations (all supplemental material is available in the online version of this article). Cycling parameters for the QuikChange site-directed mutagenesis experiments were as follows: segment 1, 95°C, 30 s, 1 cycle; and segment 2, 95°C, 30 s, 55°C, 1 min, 68°C, 10 min, 16 cycles. The sequences of all mutants were confirmed by sequencing at the DNA Core Facility, Cornell University.
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! f) K; E# Q+ r$ nTransient expression of epitope-tagged or tag-free wild-type NBC1 and NBC1 mutants in MDCK cells. The plasmid pcDNA3.1/NT-GFP-NBC1-TOPO and GFP-NBC1 constructs were transformed into TOP 10-competent cells, and single colonies were picked up for growing in 1 ml of TB bacterial culture medium and transfer to 50 ml of TB culture medium. Qiagen EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA) was used to prepare and purify endotoxin-free plasmid. The MDCK cells were maintained in culture medium (DMEM) supplemented with 25 mM NaHCO 3, 10% fetal calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin and incubated at 37°C, 5% CO 2 -95% air. MDCK cells were transiently transfected with the tagged or tag-free NBC1 full-length and mutants and studied 48-72 h later according to established methods ( 20, 21 ). Briefly, cells were seeded on coverslips and transfected at 80% confluence using 1 µg of DNA and 4 µl of Lipofectamine 2000 (Invitrogen). The transfection efficiency was monitored using PCMV-SPORT -gal plasmid as control and. Changes in cell color in response to X-gal addition was used as marker for -gal expression. MDCK cells were grown on cover slides and colabeled with phalloidin-tetramethylrhodamine, as a marker of the actin cytoskeleton, and peanut agglutinin (PNA)-lectin Alexa Fluor 568 conjugate, as a marker of extracellular glycans, which are expressed on the apical membrane. The polarized MDCK cells transiently expressing epitope-tagged wild-type NBC1 or mutant NBC1 (F1013A, F1013L, and L1014A) were labeled with antibodies against E-cadherin, as a marker of lateral membrane, ZO-1, as a marker of tight junction, or PDI (protein-disulfide-isomerase), as a marker of the endoplasmic reticulum (ER). The transient expression experiments were conducted three separate times for each construct.0 X/ d5 }$ M* v0 L! L$ e
5 _' }( E+ K2 \3 S) T1 Q' y* LConfocal microscopy and immunofluorescence labeling of epitope-tagged NBC1 and its mutants. MDCK cells were washed with PBS and fixed in 4% formaldehyde/PBS solution as described ( 20, 21 ). For colabeling studies, MDCK cells were costained with PNA-lectin Alexa Fluor 568 conjugate (Molecular Probes, Eugene, OR) for 30 min. In separate studies, cells were permeabilized with 0.1% Triton X-100 in PBS, washed with PBS, and costained with phalloidin-tetramethylrhodamine or phalloidin-FITC (Sigma, St. Louis, MO) for 30 min. Afterward, cells were washed with PBS and mounted on glass slides in Slow Fade with DAPI (Molecular Probes). Images were taken on a Zeiss LSM510 confocal microscope. Both Z -line ( y-z or x-z projection) and Z -stack ( x-y projection) images were obtained using LSM 5 Image software ( 19, 20 ). Generally, 0.4- to 1.0-µm fixed interval cuts were obtained, and 20-30 images were generated as a gallery. Z -stack images were chosen from the first three to six basal sections, and images ( x-y projections) and corresponding z -lines ( x-z or y-z projections) were obtained.* k8 ^, x( ?2 K& \ |+ j( W
% a g3 ?3 s) vImmunofluorescence labeling of NBC1 in MDCK epithelial cells or oocytes was performed using rabbit IgG anti-NBC1 antibodies(1:200 dilutions) and Alexa Fluor 568-conjugated secondary antibody according to established methods and as reported from our laboratory ( 22 ). Immunofluorescence labeling of GFP-NBC1 in MDCK epithelial cells was also performed using anti E-cadherin and anti ZO-1 antibodies purchased from Zymed (San Francisco, CA). Mouse anti-PDI is a monoclonal antibody purchased from Stressgen Bioreagents (Victoria, BC, Canada).
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5 u8 w! d3 W" g, F6 SFunctional expression of full-length and NBC1 mutants in oocytes. Stage IV-V oocytes were isolated as previously described and used for expression studies according to established methods ( 20, 21, 39 ). The capped tag-free (no GFP) or tagged NBC1 (GFP-NBC1 full-length, GFP-NBC1 point-mutations, and GFP only) cRNAs were generated using an mMESSAGE mMACHINE T7 Kit (Ambion, Austin, TX) according to the manufacturer's instruction. Fifty nanoliters cRNA (0.5 µg/µl) were injected with a Drummond 510 microdispenser via a sterile glass pipette with a tip of 20-30 µm. After injection, the oocytes were maintained in a solution of the following composition (in mM): 96 NaCl, 2.0 KCl, 1.0 MgCl 2, 1.8 CaCl 2, 5 HEPES, 2.5 Na pyruvate, and 0.5 theophylline as well as 100 U/ml penicillin and 100 µg/ml streptomycin, pH 7.5. Injected oocytes were stored in an incubator at 17°C and were used for electrophysiological experiments after 2-4 days.
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For membrane potential recording, tagged or tag-free constructs were used. Briefly, oocytes were placed on nylon mesh in a perfusion chamber and continuously perfused (3 ml/min perfusion rate). The perfusion solution had the following composition (in mM): 96 NaCl, 2 KCl, 1 MgCl 2, 1.8 CaCl 2, and 15 HEPES, pH 7.5. After a stabilization period when the membrane potential ( V m ) was constant, the perfusion solution was switched to a CO 2 /bicarbonate-containing solution of the following composition (in mM: 30 NaHCO 3, 33 TMACl, 66 NaCl, 2 KCl, 1 MgCl 2, 1.8 CaCl 2 and 15 HEPES, pH 7.5) and gassed with 5% CO 2. Experiments were performed at room temperature (22-25°C). Membrane potentials were recorded with conventional microelectrode techniques by glass microelectrodes (resistance 3-5 mµ) filled with 3 M KCl and connected to an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA) ( 20, 38 ). The digitized signals were stored and analyzed on a personal computer using Axotape (Axon Instruments).
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Materials. The High-fidelity PCR Amplification Kit, GFP Fusion TOPO TA Expression Kit, and Lipofectamine 2000 were purchased from Invitrogen. The mMESSAGE mMACHINE T7 Kit was purchased from Ambion. EndoFree Plasmid Maxi Kit was purchased from Qiagen. DMEM was purchased from Invitrogen. All other chemicals were purchased from Sigma.2 R) K4 T! X8 s9 A8 c. E
/ x* t8 X5 y' A, `8 B. `: LStatistics. Results are given as means ± SE. Student's t -test and ANOVA were performed among and between the groups. The data were considered significant if P ! D* U/ p! ~, S8 b/ F
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RESULTS
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Both the epitope-tagged and tag-free NBC1 full lengths are targeted exclusively to the basolateral membrane in MDCK epithelial cells. In the first series of experiments, we examined the expression of GFP vector without the NBC1 insert in MDCK cells. Figure 1A ( x-y projection) shows that transfection of cultured cells with the GFP vector alone (no NBC1 insert) results in the accumulation of GFP in the cytoplasm, when merged images with the plasma membrane F-actin marker phalloidin-tetramethylrhodamine is obtained, with no localization on the membrane. However, transfection with the GFP-NBC1 full-length cDNA shows exclusive localization in the membrane, as visualized by x-y projection, front view ( Fig. 1 B, top, colabeled with phalloidin-tetramethylrhodamine). Z -line image ( x-z projection) analysis indicates that NBC1 is exclusively detected on the basolateral membrane ( Fig. 1 B, middle, stained with phalloidin-tetramethylrhodamine; bottom, stained with PNA-lectin). The images in Fig. 1 C are immunofluorescent labeling of tag-free wild-type NBC1 (no GFP) in MDCK cells, with or without phalloidin-FITC costaining. As shown, wild-type NBC1 is detected on the basolateral membrane ( top images: x-y projections or front views; bottom : Z -line images; x-z projections or side views; from both above and down images). No labeling was detected in mock-transfected cells (data not shown), confirming the specificity of the staining. Figure 1 D demonstrated NBC1 immunofluorescence labeling in MDCK cells transfected with the GFP-NBC1 cDNA. As shown, epitope-tagged NBC1 (green) and tag-free NBC1, detected by immunofluorescence labeling, colocalize to the same membrane domain. These results demonstrate that GFP does not interfere with the targeting and trafficking of NBC1. The results in Fig. 1 are consistent with published reports on the basolateral membrane localization of NBC1 in epithelial cells ( 9, 20, 21, 27, 28, 36, 37, 40 )., Q; G/ v1 u f0 |3 Z
! S. K s2 L7 w! cFig. 1. Transfection of Madin-Darby canine kidney (MDCK) cells with green fluorescent protein (GFP) vector only (no NBC1 insert) or tagged or tag-free wild-type NBC1. A : expression of GFP vector only (no NBC1 insert). Z -stack analysis ( x-y projection, front view). Transfection with GFP vector alone, with no NBC1 insert, results in the accumulation of GFP in the cytoplasm and does not reach plasma membrane. Green, NBC1-GFP; red, phalloidin-tetramethylrhodamine. B : expression of epitope-tagged wild-type NBC1. Top : Z -stack ( x-y projection, front view) image analysis. As demonstrated, full-length NBC1 is targeted to the plasma membrane. Green, NBC1-GFP; red, phalloidin-tetramethylrhodamine. Middle : Z -line ( x-z projection, side view) image analysis. Full-length NBC1 is exclusively targeted to the basolateral membrane. Green, GFP-NBC1; red, phalloidin-tetramethylrhodamine. Bottom : Z -line ( x-z projection, side view) image analysis. GFP-NBC1 full-length colabeled with peanut agglutinin (PNA)-lectin, which exclusively binds to the apical membrane, demonstrates the localization of full-length NBC1 to the basolateral membrane. Green, GFP-NBC1; red, PNA-lectin. C : NBC1 immunofluorescence labeling in MDCK cells transfected with the epitope-tagged free NBC1 (no GFP). Top : Z -stack ( x-y projection, front view) image analysis. Bottom : Z -line ( x-z projection, side view) image analysis. As shown, wild-type NBC1 displays similar membrane localization ( top ) and basolateral localization ( bottom ) as epitope tagged construct, detected by immunofluorescence labeling. Rabbit IgG anti-NBC1 antibodies (1:200) and Alexa-Fluor 568-conjugated secondary antibody (1:200) were used. Bottom : Z-stack ( x-y projection, front view) image analysis. Bottom : Z -line ( x-z projection, side view) image analysis. As shown, costained with phalloidin-FITC, wild-type NBC1 displays similar membrane localization ( top ) and basolateral localization ( bottom ) as epitope-tagged construct, detected by immunofluorescence labeling. D : NBC1 immunofluorescence labeling in MDCK cells transfected with epitope-tagged NBC1. Top : Z -stack ( x-y projection, front view) image analysis. Bottom : Z -line ( x-z projection, side view) image analysis. Green, GFP-NBC1; red, Alexa Fluor 568. As shown, full-length GFP-NBC1 (green) displays a merged image with membrane localization as epitope-tagged free construct, detected by immunofluorescence labeling. Rabbit IgG anti-NBC1 antibodies (1:500) and Alexa Fluor 568-conjugated secondary antibody (1:200) were used.: v' G5 T; y+ y. I6 {- ?
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Mutations in Q-Q-P and S amino acid residues in the COOH-terminal QQPFLS region do not affect the targeting of NBC1 to the basolateral membrane. In the next series of experiments, we examined the expression of two mutants generated by site-directed mutagenesis. In the first series of experiments, the AAA FLS mutant, where Q(1010), Q(1011), and P(1012) residues, was simultaneously mutagenized to Alanine(Q-Q-Q to A-A-A; see MATERIALS AND METHODS ), was transiently expressed, and examined. As demonstrated in Fig. 2 A ( top and bottom ), the AAA FLS NBC1 mutant was targeted to the membrane, with exclusive localization on the basolateral membrane domain. Similar to the AAA FLS mutant, the NBC1 mutant (S1015A) with the sequence QQPFL A was exclusively targeted to the basolateral membrane Fig. 2 B ( top and bottom ).
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Fig. 2. Transfection of MDCK cells with epitope-tagged NBC1 mutants AAA FLS and QQPFL A. A : expression of epitope-tagged NBC1 mutant (Q1010A-Q1011A-P1012A) with COOH-terminal AAA FLS region. Top : Z -stack ( x-y projection, front view) image analysis. As demonstrated, the NBC1 mutant (Q1010A-Q1011A-P1012A) AAA FLS is targeted to the plasma membrane. Green, GFP-NBC; red, phalloidin-tetramethylrhodamine. Bottom : Z -line ( x-z projection, side view) image analysis. The NBC1 mutant (Q1010A-Q1011A-P1012A) AAA FLS is targeted to the basolateral membrane. Green, GFP-NBC1; red, phalloidin-tetramethylrhodamine. B : expression of epitope-tagged NBC1 mutant (S1015A) with COOH-terminal QQPFL A region. Top : Z -stack image ( x-y projection, front view) analysis. As demonstrated, the NBC1 mutant (S1015A) QQPFL A is detected in the plasma membrane. Green, GFP-NBC1; red, phalloidin-tetramethylrhodamine. Bottom : Z -line ( x-z projection, side view) image analysis. The NBC1 mutant (S1015A) QQPFL A is targeted to the basolateral membrane. Green, GFP-NBC1; red, phalloidin-tetramethylrhodamine.
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Mutations in F or L amino acid residues in the COOH-terminal QQPFLS (1010-1015) region result in the retargeting of NBC1 to the apical membrane. We next examined the expression of tagged-NBC1 mutants QQP A LS (F1013A) and QQPF A S (L1014A) in MDCK cells (see MATERIALS AND METHODS ). Figure 3 A, top, demonstrates Z -stack images (front view, x-y projection) of NBC1 point-mutation QQP A LS (F1013A) colabeled with phalloidin-tetramethylrhodamine in MDCK cells and indicates the retargeting of this mutant to the membrane. Figure 3 A, middle and bottom, shows x-z projections (side views) of the QQP A LS (F1013A) mutant colabeled with phalloidin-tetramethylrhodamine or PNA-lectin, respectively. As shown, the NBC1 mutant QQP A LS (F1013A) is localized predominantly on the apical membrane.
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6 M- M" _6 E) H! @Fig. 3. Transfection of MDCK cells with the epitope-tagged NBC1 mutants QQP A LS and QQPF A S. A : expression of epitope-tagged NBC1 mutant (F1013A) with COOH-terminal QQP A LS region. Top : Z -stack ( x-y projection, front view) image analysis. As demonstrated, the NBC1 mutant (F1013A) QQP A LS is detected in the plasma membrane. Green, GFP-NBC1; red, phalloidin-tetramethylrhodamine. Middle : Z -line ( x-z projection, side view) image analysis. As demonstrated, the NBC1 mutant (F1013A) QQP A LS is retargeted to the apical membrane. Green, GFP-NBC1; red, phalloidin-tetramethylrhodamine. Bottom : Z -line ( x-z projection, side view) image analysis (colabeling with PNA-lectin). As demonstrated, the NBC1 mutant (F1013A) QQP A LS is retargeted to the apical membrane. Green, NBC1-GFP; red, PNA-lectin. B : expression of epitope-tagged NBC1 mutant (L1014A) with COOH-terminal QQPF A S region. Top : Z -stack ( x-y projection, front view) image analysis. As demonstrated, the NBC1 mutant (L1014A) QQPF A S is detected in the plasma membrane. Green, GFP-NBC1; red, phalloidin-tetramethylrhodamine. Middle : Z -line ( x-z projection, side view) image analysis. The NBC1 mutant (L1014A) QQPF A S is retargeted to the apical membrane. Green, GFP-NBC1; red, phalloidin-tetramethylrhodamine. Bottom : Z -line ( x-z projection, side view) image analysis (colabeling with PNA-lectin). As demonstrated, the NBC1 mutant (L1014A) QQPF A S is retargeted to the apical membrane. Green, GFP-NBC1; red, PNA-lectin., U) `+ {/ a7 o
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Figure 3 B, top, demonstrates Z -stack (front view, x-y projection) images of NBC1 point-mutation QQPF A S (L1014A), colabeled with phalloidin-tetramethylrhodamine in MDCK cells, and indicates the targeting of this mutant to the membrane. Figure 3 B, middle and bottom, shows x-z projections (side views) of the QQPF A S (L1014A) mutant colabeled with phalloidin-tetramethylrhodamine or PNA-lectin, respectively, in MDCK cells and shows the retargeting of NBC1 mutant QQPF A S (L1014A) to the apical membrane. Taken together, these results demonstrate that the dihydrophobic FL motif in the NBC1 cytoplasmic tail plays a vital role in NBC1 targeting to the basolateral membrane, which is essential for normal functioning of the cotransporter. Y: [$ L y7 S9 E
4 o+ F: w1 P* z" l# CNBC1 COOH-terminal dihydrophobic FL motif is distinct from the di-leucine motif. The purpose of the next series of experiments was to further characterize the specificity of dihydrophobic FL motif to NBC1 basolateral membrane targeting by mutating the FL motif to either FF or LL (di-leucine). Accordingly, the following two mutants FF (QQPF F S, L1014F) and LL (QQP L LS, F1013L) were generated and expressed in MDCK cells. As demonstrated in Fig. 4 A ( top and bottom ), the NBC1 mutant FF (QQPF F S, L1014F) was targeted to the basolateral membrane, suggesting that the leucine (L) residue in the cytoplasmic tail FL motif can be replaced with another hydrophobic amino acid such as phenylalanine (F), without affecting its trafficking. However, the NBC1 mutant LL (QQP L LS, F1013L), where phenylalanine 1013 is mutagenized to leucine ( MATERIALS AND METHODS ), was significantly retained in the cytoplasm ( Fig. 4 B, top and bottom ). Studies with the tag-free mutants displayed similar trafficking pattern as the epitope-tagged construct, indicating that the splicing of GFP to NBC1 does not affect its trafficking or functional activity (data not shown).
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( k- x& {4 W* \2 v; C$ |* QFig. 4. Transfection of MDCK cells with epitope-tagged NBC1 mutants QQPF F S and QQPL L S. A : expression of epitope-tagged NBC1 mutant (L1014F) with COOH-terminal QQPF F S region. Top : Z -stack ( x-y projection, front view) image analysis. As demonstrated, the NBC1 mutant (L1014F) QQPF F S is detected in the plasma membrane. Green, GFP-NBC1; red, phalloidin-tetramethylrhodamine. Bottom : Z -line ( x-z projection, side view) image analysis. The NBC1 mutant (L1014F) QQPF F S is targeted to the basolateral membrane. Green, FP-NBC1; red, phalloidin-tetramethylrhodamine. B : expression of epitope-tagged NBC1 mutant (F1013L) with COOH-terminal QQP L LS region. Top : Z -stack ( x-y projection, front view) image analysis. As demonstrated, the NBC1 mutant (F1013L) QQP L LS is predominantly retained in the cytoplasm. Green, GFP-NBC1; red, phalloidin-tetramethylrhodamine. Bottom : Z -line ( x-z projection, side view) image analysis. The NBC1 mutant (F1013L) QQP L LS is accumulated in the cytoplasm. Green, NBC1-GFP; red, phalloidin-tetramethylrhodamine.
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4 g- [- Z4 S, q, o+ N0 |To define the localization of those NBC1 mutants that are targeted to the apical membrane (Fl013A and L1014A) or retained in the cytoplasm (F1013L) in more detail, they were transiently expressed in polarized MDCK cells and colabeled with E-cadherin (marker of lateral membrane), ZO-1 (marker of cell junction), or PDI (marker of ER; MATERIALS AND METHODS ). The expression of wild-type NBC1 was examined as the control. Figure 5 A displays the colabeling of wild-type NBC1 and ZO-1, and Fig. 5, B and C, demonstrates the colabeling of the apically targeted NBC1 mutants (F1013A and L1014A) with ZO-1 in MDCK cells. Comparison of the images in Fig. 5, A - C, clearly demonstrates the localization of wild-type NBC1 on the lateral plasma membrane on the x-y projection and close to the basal membrane ( Fig. 5 A ), while NBC1 mutant proteins are densely expressed on the surface region on the x-y projection close to the apical membrane ( Fig. 5, B and C ).& c) z+ [7 z6 S% M
0 `+ Q* M9 h, @; wFig. 5. Transfection of MDCK cells with GFP-kNBC1 wild-type, F1013A and L1014A mutants colabeled with ZO-1 monoclonal antibody. A : transfection of MDCK cells with GFP-kNBC1 wild-type. The result shows that GFP-kNBC1 is exclusively localized in the basolateral membrane as ZO-1 is a cellular junction marker. B : transfection of MDCK cells with GFP-kNBC1 mutant F1013A. The results demonstrate that GFP-kNBC1 F1013A mutation is preferentially localized in the apical membrane. C : transfection of MDCK cells with GFP-kNBC1 mutant L1014A. The results demonstrate that GFP-kNBC1 L1014A mutation is preferentially localized in the apical membrane.; F% t% i0 g; W7 A
' Q! Q) z; O" P! R# bWe next examined the colabeling of E-cadherin with the wild-type and the cytoplasmically retained NBC1 mutants (F1013L) in MDCK cells. Comparison of the images ( Fig. 6 A ) demonstrates the expression of wild-type NBC1 on the lateral membrane in the polarized MDCK cells, whereas GFP-NBC1 mutant F1013 L (mutant LL) was mistargeted to the cytoplasm. The cytoplasmically retained NBC1 mutant (F1013L; also referred to as mutant LL) was colocalized with PDI, an ER marker ( Fig. 6 B ). The unstained oval regions represent cellular nuclei in both wild-type and mutant (mutant LL) NBC1.
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. _4 Q8 o! K6 IFig. 6. Transfection of MDCK cells with GFP-kNBC1 wild-type, F1013L (mutant LL) colabeled with E-cadherin, and PDI monoclonal antibodies. A : transfection of MDCK cells with GFP-kNBC1 wild type, F1013L (mutant LL) colabeled with E-cadherin monoclonal antibody. Colabeled with monoclonal antibody E-cadherin, a lateral marker in MDCK cells, GFP-NBC1 wild-type was expressed in the plasma membrane, whereas GFP-NBC1 mutant F1013L (mutant LL) mistargeted on the cytoplasm. B : transfection of MDCK cells with GFP-kNBC1 wild-type, F1013L (mutant LL) colabeled with PDI monoclonal antibody. The results show that GFP-kNBC1 F1013L mutation (mutant LL) mistargeted in the cytoplasm, colocalized with PDI, an ER marker.+ g3 x0 a% a3 C+ O* Q
9 A/ z$ T0 K3 E& b& T h% s7 pFunctional properties of NBC1 mutants. In the last series of experiments, we examined the functional characteristics of NBC1 mutants in oocytes by membrane potential measurement, as recorded by intracellular microelectrodes 2-4 days after injection with respective cRNAs. For the experiments, following an equilibrating period, the perfusion solution was switched to a solution containing 30 mM HCO 3 - and gassed with 5% CO 2 -95% O 2 at pH 7.5. Exposure to the HCO 3 - /CO -containing solution activates NBC1 and results in membrane hyperpolarization due to the inward movement of NBC1 down the Na and bicarbonate gradients. As the data indicated, the resting V m was not significantly different among full-length GFP-NBC1 (-55.8 ± 3.3 mV, n = 17), mutants [F1013A (QQP A LS), -53.0 ± 2.0 mV, n = 4; L1014A (QQPF A S) -52.0 ± 2.0 mV, n = 11; FF (L1014F or QQPF F S) -58.0 ± 3.0 mV, n = 7; LL (F1013L, or QQP L LS) -53.0 ± 3.1 mV, n = 6], and GFP only (-46.9 ± 4.9 mV, n = 4) ( P 0.05 among all groups). As shown in Fig. 7, A and B, the tagged wild-type NBC1 showed significant hyperpolarization (-38.2 ± 2.5 mV, n = 17 ) compared with GFP only (14.4 ± 4.8 mV, n = 4), which showed no activity ( P
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Fig. 7. Functional activity of wild-type and NBC 1 mutants F1013A, L1014A, FF (L1014F), and LL (F1013L) in oocytes as measured by membrane potential recording. A : representative tracings. Membrane potential was measured by conventional intracellular microelectrodes in Xenopus laevis oocytes 2-4 days after injection with epitope-tagged wild-type NBC1 or various mutants. After an equilibrating period, the perfusion solution was switched to a solution containing 30 mM HCO 3 - and gassed with 5% CO 2 -95% O 2 at pH 7.5. A solid horizontal line indicates the time of exposure to CO 2 /HCO 3 -. Oocytes that were injected with GFP only (no NBC insert, top ), GFP-NBC1 full-length (second panel), mutants F1013A (QQP A LS), L1014A (QQPF A S), FF (L1014F or QQPF F S), LL (F1013L, or QQP L LS; in the subsequent order shown) were exposed to CO 2 /HCO 3 -. As shown, there was an immediate and sustained hyperpolarization in the full-length (second panel) and all NBC1 mutants except the LL (F1013L, QQP L LS) mutant ( bottom ) and GFP only ( top ). The hyperpolarization was reversible on removal of CO 2 /HCO 3 - from the perfusion medium. Exposure of control (water-injected) oocytes to CO 2 /HCO 3 - also did not alter the membrane potential (data not shown). All cRNAs were injected at 0.5 µg/µl. B : results summation. In the cRNA-injected oocytes, full-length NBC1 (**) showed significant hyperpolarization activity vs. GFP only (*), which showed no activity ( P
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The targeting of NBC1 to the basolateral membrane was examined using a series of mutants generated by site-directed mutagenesis. The results demonstrated that full-length NBC1 is targeted to the basolateral membrane ( Fig. 1 ), consistent with published reports. Site-directed mutagenesis in the QQPFLS motif (positions 1010-1015) in the COOH-terminal tail of NBC1 resulted in several distinct trafficking patterns. AAA FLS and QQPFL A mutants were targeted to the basolateral membrane, whereas QQP A LS and QQPF A S mutants were retargeted to the apical membrane ( Figs. 2 and 3 ). Mutation of the conserved residue L (leucine) at position 1014 to F (phenylalanine), which resulted in the QQPF F S mutant, showed a pattern similar to the wild-type and detected exclusively in the basolateral membrane ( Fig. 4 ). However, and unexpectedly, mutation of the conserved residue F (phenylalanine) at position 1013 to L (leucine), which resulted in QQP L LS mutant, was predominantly retained in the ER ( Figs. 4 and 6 ). Membrane potential measurements in oocytes demonstrated that all mutants that are located on the membrane (irrespective of whether they are targeted to the apical or basolateral membrane) are functional and comparable to wild-type NBC1 ( Fig. 7 ).; Z0 U/ y7 ~! S# X5 y# L
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The most salient feature of the current study is the identification of the dihydrophobic FL motif in the COOH-terminal tail, which is essential for the targeting of NBC1 to the basolateral membrane. It is noteworthy that mutating the amino acid residue leucine (L) at position 1014 to phenylalanine (F), with the resultant FF mutant (QQP FF S), had no discernible impact on NBC1 targeting and functional activity compared with the wild-type ( Figs. 4 and 7 ). However, mutating the amino acid residue phenylalanine (F) at position 1013 to leucine (L), with the resultant LL mutant (QQP L LS), caused significant cytoplasmic accumulation of NBC1 by retaining in the ER, along with reduced functional activity ( Figs. 4, 6, and 7 ). These results indicate that the substitution of leucine with another hydrophobic amino acid, such as phenylalanine, in the form of FF mutant, has no impact on the targeting and activity of NBC1; however, the opposite is not the case, as shown by the cytoplasmic accumulation and ER retention of LL mutant. Taken together, these results indicate that the amino acid residue phenylalanine on position 1013 is essential for the basolateral targeting of NBC1 and cannot be substituted with another hydrophobic residue such as leucine.
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The apically targeted NBC1 mutants are functionally active in the Xenopus laevis frog oocytes ( Fig. 7 ). This is in agreement with published reports, which show that the deletion of the last 26 amino acids on the COOH-terminal end, which encompasses residues 1009-1035, does not affect NBC1 functional activity ( 19 ). While these results suggest that possible mutations or deletions in the NBC1 FL motif do not impair its functional activity in the X. laevis frog oocytes ( Fig. 7 ), such mutations or deletions may cause significant impairment of bicarbonate reabsorption in vivo due to the mistargeting of NBC1 to the apical membrane, which will impair the vectorial transport of bicarbonate and lead to proximal tubular acidosis due to the absence of an exit pathway for bicarbonate transport across the basolateral membrane.& U% z A# C: Z f
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Kidney NBC1, which encodes 1,035 amino acid residues, consists of 11 transmembrane domains and a COOH-terminal tail that is distributed intracellularly and is 93 amino acids long. It is plausible that the retargeting of NBC1 to the apical membrane in the QQP A LS or QQPF A S mutant occurs by default, consistent with the possibility that this cotransporter physically binds to a basolateral scaffolding protein through the FL motif. A similar targeting pattern is also detected in the bicarbonate transporter AE1. A COOH-terminal cytoplasmic YXXØ motif was shown to mediate the targeting of AE1 to the basolateral membrane, through binding with subunits of adaptor-protein complexes ( 10, 14 ). Furthermore, the AE1 Y904A mutant and the truncated AE1 11 mutant show significant localization on the apical membrane in cultured epithelial cells ( 14 ). Although the FL motif in NBC1 differs from the above AE1 motifs with regard to its primary amino acid sequence, these studies demonstrate an important role for the cytoplasmic tail in the membrane targeting of these two bicarbonate transporters which belong to the same superfamily.% W& p! ^; |' d: R5 C
+ i( Y9 m6 P T! f0 `A dihydrophobic motif, in the form of leucine-valine, di-leucine, or valine-tryptophan (LL, LV, or VW), has been shown to play a vital role in the targeting of several membrane proteins, with either single or multiple transmembrane domains, to the basolateral membrane ( 7, 8, 10, 22, 30 ). Among these proteins, the E-cadherin that encodes a single-pass transmembrane protein (1-728 amino acid residues) is noteworthy for a di-leucine motif on its NH 2 -terminal end, which is responsible for basolateral targeting, with subsequent maintenance of cell-cell adhesion and cell polarity in the epithelial cells ( 22 ). While our current studies identify a dihydrophobic motif that is essential for the targeting of NBC1 to the basolateral membrane, this motif is distinct from other leucine-based dihydrophobic motifs in that its mutation to di-leucine (LL) actually disrupts the membrane trafficking of NBC1 and causes retention in the ER ( Figs. 4, 6, 7 ).! d+ t# Q9 Q9 w8 I
7 p! r/ M) i6 e* H0 U3 [$ {: N! S3 B5 E3 NA GenBank search of other members of the NBC/AE superfamily demonstrated that the QQPFLS motif is highly conserved in NBC2, NBC4, NCBE, AE1, and AE2, all known bicarbonate transporters located on the basolateral membrane of various epithelia. An analysis of other known chloride/bicarbonate or anion exchangers from the SLC26 family, which share little homology with NBC/AE superfamily, demonstrates that those anion exchangers that are targeted to the basolateral membrane (SLC26A1 and SLC26A7) contain motifs in their cytoplasmic tail with high homology to the current motif (GenBank NM_022042 for SLC26A1, from residue 675 to 678, with amino acid sequence QLFL, with the full-length of 701 amino acid.; GenBank NM_134266 for SLC26A7, from residue 343 to 346, with amino acid sequence QEFL, with the full-length of 663 amino acids). However, SLC26 exchangers that are targeted to the apical membrane (SLC26A3 and SLC26A4) do not express signals with significant homology to the current motif.
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B! z' ~% J2 b6 p7 e @+ X3 HIn conclusion, the dihydrophobic FL motif in positions 1013 and 1014, located in the COOH tail, is essential for the targeting of NBC1 to the basolateral membrane. Mutating F or L to alanine resulted in the retargeting of NBC1 to the apical membrane, without affecting its functional activity. However, mutating the FL to LL disrupts the membrane trafficking of NBC1 and causes retention in the ER. It is speculated that the FL motif binds with other membrane protein or proteins that anchor or retain NBC1 in the basolateral membrane.. R0 |/ m, E- |4 q8 H, h
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In the Articles in Press version of this manuscript, Fig. 3 A was incorrect in that it contained panels that were erroneously derived from the same images as those in Fig. 3 B. The authors sincerely apologize for this mistake. Here, in this final-published version, Fig. 3 A is replaced with the correct version.7 b i7 P2 b7 f p) z, s1 L
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GRANTS. A2 l* o- X u7 y' M
c. @& [9 Z$ [1 MThese studies were supported by National Institute of Health Grants DK-62809 (M. Soleimani) and CA-95286 (L. Conforti), a Merit Review Award, a Cystic Fibrosis Foundation grant, and grants from Dialysis Clinic, Incorporated (to M. Soleimani). R! s. D* Z3 x
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发表于 2009-4-22 09:41
