A dileucine motif targets the sulfate anion transporter sat-1 to the basolateral membrane in renal cell lines

Ralf R. Regeer and Daniel Markovich

Department of Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia

Submitted 14 November 2003 ; accepted in final form 2 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The sat-1 transporter mediates sulfate/bicarbonate/oxalate anion exchange in vivo at the basolateral membrane of the kidney proximal tubule. In the present study, we show two renal cell lines [Madin-Darby canine kidney (MDCK) and porcine proximal tubular kidney (LLC-PK1) cells] that similarly target sat-1 exclusively to the basolateral membrane. To identify possible sorting determinants, we generated truncations of the sat-1 cytoplasmic COOH terminus, fused to enhanced green fluorescence protein (EGFP) or the human IL-2 receptor {alpha}-chain (Tac) protein, and both fusion constructs were transiently transfected into MDCK cells. Confocal microscopy revealed that removal of the last three residues on the sat-1 COOH terminus, a putative PDZ domain, had no effect on basolateral sorting in MDCK cells or on sulfate transport in Xenopus oocytes. Removal of the last 30 residues led to an intracellular expression for the GFP fusion protein and an apical expression for the Tac fusion protein, suggesting that a possible sorting motif lies between the last 3 and 30 residues of the sat-1 COOH terminus. Elimination of a dileucine motif at position 677/678 resulted in the loss of basolateral sorting, suggesting that this motif is required for sat-1 targeting to the basolateral membrane. This posttranslational mechanism may be important for the regulation of sulfate reabsorption and oxalate secretion by sat-1 in the kidney proximal tubule.

enhanced green fluorescence protein; Tac; polarized cells; sorting; transport


THE FIRST SULFATE ANION TRANSPORTER (sat-1) was isolated from a rat liver cDNA library by expression cloning using Xenopus oocytes (2). It was found to be strongly expressed in liver and kidney, with weaker expression in skeletal muscle and brain (2). More recently, we isolated two orthologs of rat sat-1 (rsat-1): the mouse sat-1 (msat-1) and human sat-1 (hsat-1) cDNAs and genes (Sat1; SAT1) (21, 39). The three sat-1 proteins share at least 77% amino acid identity (25), consist of 12 putative transmembrane domains with both termini predicted intracellularly (48), and belong to the Human Genome Nomenclature Committee-designated solute-linked carrier (SLC) gene family 26A1. rsat-1 was suggested to function as a sulfate/bicarbonate/oxalate antiporter on the basolateral membrane (BLM) of the kidney proximal tubule (26). Immunolocalization of rsat-1 protein in the kidney confirmed its expression at the BLM of the rat proximal tubule (20), suggesting a role in sulfate reabsorption and oxalate secretion.

The ability of epithelial cells, being morphologically and functionally polarized, to perform transepithelial transport of ions depends on the asymmetrical distribution of cell surface proteins and lipids. To maintain their polarity, newly synthesized proteins have to be properly sorted and targeted to the apical and/or basolateral membrane (3, 16, 32), but the mechanism(s) underlying these processes have not been determined for many proteins. Newly synthesized proteins, with either an apical or basolateral destination, are transported from the endoplasmic reticulum to the trans-Golgi network before reaching the subapical compartment. The subapical compartment is able to deliver proteins to their specific cell surfaces via early endosomes (47).

Protein targeting to the apical membrane of polarized cells has been shown to involve cytoplasmic/transmembrane domain signals (5, 35), via association with rafts, sphingolipids, or oligosaccharides (1, 40, 43), whereas basolateral sorting has been attributed to specific amino acid motifs located in the cytoplasmic tail of proteins (14, 15, 29, 31). Recently, a number of sequence motifs mediating BLM sorting in epithelial cells have been described: 1) A critical tyrosine residue, within either the NPXY or YXXØ sequence (where X represents any amino acid and Ø is an amino acid with a bulky hydrophobic group), has been shown to direct the low-density lipoprotein receptor to the BLM in polarized Madin-Darby canine kidney (MDCK) cells (29). However, the well-characterized basolateral H+-K+-ATPase {beta}-subunit was shown to be sorted to the apical membrane in porcine kidney LLC-PK1 cells (41), suggesting that tyrosine-dependent sequence motifs in some proteins can be interpreted differently in certain polarized epithelial cell lines. 2) A tyrosine-independent dileucine motif has been implicated in BLM sorting in MDCK cells for E-cadherin (31) and the Fc receptor (14). Although the tyrosine-based and dileucine targeting motifs are the most widely documented, other basolateral sorting motifs have been identified for some proteins (13, 22, 42). Furthermore, PDZ (PSD-95/Disc-large/ZO-1)-binding proteins have been suggested to play a role in the localization of proteins to their specific plasma membrane domains, through their ability to mediate protein-protein complexes (8, 9). PDZ proteins have been shown to interact with the actin cytoskeleton, thereby stabilizing proteins at the plasma membrane level (8, 9). One well-studied membrane protein is CFTR, which interacts with the PDZ protein EBP50 (NHERF) via its PDZ domain, and this protein-protein complex is required for the trafficking of CFTR to the apical membrane of MDCK cells (33, 34).

In this study, we expressed and identified the cellular localization of the rsat-1 protein in renal MDCK and LLC-PK1 cell lines. Furthermore, we investigated the role of the rsat-1 cytoplasmic COOH terminus, including a PDZ domain, as a possible BLM sorting determinant. We have shown that the renal MDCK and LLC-PK1 cell lines exclusively target rsat-1 to the BLM, and we provide the first evidence for a motif in the rsat-1 COOH terminus required for BLM targeting of rsat-1. This information not only furthers our knowledge of the sorting mechanisms regulating the posttranslational expression of sat-1 but also provides a replacement model for studying the in vivo expression of sat-1 in the renal tubule.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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cDNA construction and transport assays in Xenopus oocytes. The open reading frame of rsat-1 wild type (WT) was PCR amplified using specific oligonucleotide primers (5'-CCCAAGCTTGGTGTCCCAGATTAGCCAAAGAG-3' and 5'-ACGCGTCGACGCTAGAGGG-CAGAGTCAGCAG-3') containing artificial endonuclease restriction sites (HindIII and Sal1) and then subcloned for an in-frame fusion at the COOH terminus of the enhanced green fluorescence protein (EGFP) vector pEGFP-C1 (Clontech). The respective construct was designated EGFP/rsat-1 WT and was used as a template for the construction of progressive rsat-1 COOH-terminal truncations lacking the last three residues ({Delta}3), which conform to a PDZ consensus binding motif (SAL), the last 30 residues ({Delta}30), and the last 65 residues ({Delta}65) by the introduction of TGA stop codons using QuickChange (Stratagene) site-directed PCR mutagenesis (Fig. 1A). The same strategy was used for creating a {Delta}3 construct lacking the PDZ protein binding in rsat-1 cloned in pSPORT-1 (Life Technologies) to be used for cRNA synthesis and injection into Xenopus laevis oocytes. Site-directed PCR mutagenesis of the two leucine residues at positions 677 and 678 to alanines was performed using specific oligonucleotide primers (5'-GAACTGCAGAGGAGGCGGCGTTCCCCAGTGTA-CACAGC-3' and 5'-GCTGTGTACACTGGGGAACGCCGCCTCCTCTGCAGTTC-3') (Fig. 1A). The second construct that was prepared consisted of the last 77 amino acids of the cytoplasmic tail of the rsat-1 WT protein attached to the COOH terminus of the human interleukin-2 receptor {alpha}-chain (hIL2R{alpha}, or Tac). The open reading frame of rsat-1 WT was PCR amplified using specific oligonucleotides (5'-CCCCTCGAGAAGCTTGATGTGGCTGGCATGGCCA-3' and 5'-CCGCTCGAGTCTAGACTAGAGGGCAGAGTCAGCAG-3') and was subcloned into the HindIII and XbaI sites of the pCMVIL2R vector. The respective construct was designated Tac/rsat-1 WT and was used as a template for the construction of progressive rsat-1 COOH-terminal truncations as mentioned above (Fig. 1B). All constructs and mutations were confirmed by sequencing using the ABI Prism Big Dye terminator kit (Applied Biosystems) according to the manufacturer's protocol, and gel separation was performed using a ABI 3730xl automatic capillary sequencer at the Australian Genome Research Facility, University of Queensland.



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Fig. 1. Schematic representation of wild type (WT) and truncations of the rat sulfate anion transporter (rsat-1) protein fused to the COOH terminus of enhanced green fluorescent protein (EGFP; A) or to human IL-2 receptor {alpha}-chain (Tac; B). A: transmembrane domains (TMD), including connecting loops, are represented by filled rectangles; lines represent the NH2 (NT) and COOH termini (CT) of the rsat-1 protein. B: chimeras consisting of the cytoplasmic tail (CT) of rsat-1 (open rectangle) fused to the extracellular and transmembrane domain of Tac (filled ovals and rectangles, respectively). LL677/678AA, dileucine (LL) motif at amino acid position 677/678.

 
Methods for the handling of Xenopus laevis oocytes, in vitro transcription, and transport assays have been described previously (26, 28). Briefly, stage V-VI oocytes were injected with either 50 nl of water (control) or 5 ng of either rsat-1 WT or rsat-1 {Delta}3 cRNA with the use of a Nanojet automatic injector (Drummond Scientific).

Cell culture and transfections. The MDCK and LLC-PK1 cell lines were grown in Dulbecco's modified Eagle's medium, each supplemented with 10% cosmic calf serum (Progen), 2 mM glutamine, and 100 µg/ml penicillin-streptomycin at 37°C with 5% CO2. All reagents, except serum, were obtained from Life Technologies. For transient transfections, cells were grown without antibiotics on millicell-CM transwell filters (Millipore), and subconfluent monolayers were transiently transfected with 1 µg of plasmid DNA with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. The cells were processed 2 days after transfection for use in confocal microscopy analysis. All data were obtained in at least three independent experiments with cells of different passages (between passages 15 and 35). Endeavors to acquire a MDCK cell line stably expressing EGFP/rsat-1 WT were not successful. For the brefeldin A (Sigma) experiments, transiently transfected MDCK cells were incubated with 5 µg/ml brefeldin A for 1–3 h at 37°C with 5% CO2 before being processed as described above.

Confocal microscopy. Confluent layers of cells grown on transwell filters were fixed for 30 min at room temperature by using 4% paraformaldehyde in PBS, followed by quenching using 50 mM NH4Cl in PBS for 10 min. The cell membranes were permeabilized using 0.1% saponin-0.1% Triton X-100, and cells were incubated in either the actin filament stain phalloidin-rhodamine (0.1 µM; Calbiochem, San Diego, CA) or in solution blocking nonspecific antibodies [0.2% BSA-0.2% fish skin gelatin (Sigma) in PBS for 10 min]. The cells were incubated with a polyclonal antibody directed against rsat-1 (27) in block solution for 1 h at room temperature and washed extensively with PBS, followed by an incubation of 45 min at room temperature with goat anti-rabbit rhodamine-conjugated IgG (Calbiochem). For the Tac studies, after the cells were fixed and permeabilized, a 1:50 dilution of a mouse monoclonal Tac antibody (B-B10; Biosource International, Camarillo, CA) in 0.5% BSA-PBS blocking solution was added to the transwell filters for 60 min. Filters were washed three times with blocking solution before the addition of a 1:400 dilution of secondary Cy3-conjugated sheep anti-mouse antibody (Jackson ImmunoResearch Labs, West Grove, PA). All transwell filters were washed extensively with PBS before mounting using Mowiol (Calbiochem), and confocal immunofluorescence images were obtained using a Nikon Eclipse E600 upright microscope with a Bio-Rad Radiance 2000 confocal scanning system with a x100 oil-immersion Nikon objective. XY and XZ sections were generated using Bio-Rad software (Lasersharp 2000). EGFP fluorescence is shown in green, and actin staining is shown in red. In cross sections (XZ scatter), apical membranes are on top and basolateral membranes are on the bottom.

Data presentation and statistics. All experiments were performed at least three times, and statistical significance was tested using the unpaired Student's t-test, with P < 0.05 considered significant.


    RESULTS
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 MATERIALS AND METHODS
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Analysis of the rsat-1 amino acid sequence using the TopPred2 program (48) suggests a protein comprising 12 transmembrane-spanning domains, with both NH2 and COOH termini located intracellularly (25). To characterize the expression and targeting of rsat-1 protein in renal epithelial cell lines, we transiently transfected EGFP/rsat-1 constructs (Fig. 1) into two renal cell lines: proximal tubular LLC-PK1 cells and distal tubular MDCK cells. Confocal microscopy of confluent monolayers of pEGFP-transfected LLC-PK1 (Fig. 2A) and MDCK cells (Fig. 3A) revealed diffuse intracellular staining with faint fluorescence in the cytoplasm and strong expression in the perinuclear compartment (Figs. 2E and 3D), as demonstrated previously (12, 18). In contrast, EGFP/rsat-1 WT-transfected LLC-PK1 (Fig. 2, B and F) and MDCK cells (Fig. 3, B and F) showed exclusive BLM expression. To confirm that the BLM fluorescence corresponded to the rsat-1 protein, we immunostained EGFP/rsat-1 WT-transfected MDCK cells with a rsat-1-specific antibody (27), which showed exclusive BLM staining (Fig. 3, C and F), resembling its in vivo expression in the renal proximal tubule (20). To study in more detail the sorting pathway for rsat-1 from the endoplasmic reticulum to the plasma membrane, we tested brefeldin A (BFA) on EGFP/rsat-1-expressing MDCK cells. BFA, a fungal metabolite that causes disassembly of the Golgi apparatus and disrupts vesicular transport from the Golgi to the plasma membrane, led to random plasma membrane sorting of rsat-1 in MDCK cells to both apical (Fig. 4, A and C) and basolateral membranes (Fig. 4, B and C), suggesting that rsat-1 most likely undergoes the Golgi trafficking pathway via the Golgi complex, which is disrupted by BFA.



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Fig. 2. Expression of EGFP and EGFP/rsat-1 WT in proximal tubule porcine kidney LLC-PK1 cells. LLC-PK1 cells were transiently transfected with either EGFP (A, C, E) or EGFP/rsat-1 WT (B, D, F). Images in A and B represent expression in the focal plane (XY scatter), and images in CF show a confocal cross section (XZ scatter), through the focal plane positioned at the white line (A and B), of either actin (C and D) or EGFP signal (E and F).

 


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Fig. 3. Expression of EGFP and EGFP/rsat-1 WT in Madin-Darby canine kidney (MDCK) cells. MDCK cells were transiently expressed with either EGFP (A, D, G) or EGFP/rsat-1 (corresponding images in B and C, E and F, and H and I). EGFP fluorescence is stained green and actin is stained red (A and B; G and H). rsat-1 localization (red) was detected using a rsat-1-specific antibody (C, I). Images in A–C represent the focal plane (XY scatter), and images in D–I represent a confocal cross section along planes (XZ scatter) depicted by white lines.

 


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Fig. 4. Effect of brefeldin A on expression of EGFP/rsat-1 in MDCK cells. Transiently transfected MDCK cells were incubated with 5 µg/ml brefeldin A for 1–3 h at 37°C with 5% CO2. EGFP fluorescence is stained green and actin is stained red. A: EGFP fluorescence at the apical membrane of MDCK cells (XY scatter). B: EGFP fluorescence at the basolateral membrane of MDCK cells (XY scatter). Images in C and D show a confocal cross section (XZ scatter) through the focal plane positioned at the white line.

 
Several proteins that are targeted to the BLM in MDCK cells (i.e., IgG Fc receptor, LDL receptor, E-cadherin) were shown to contain sorting motifs in their cytoplasmic COOH termini (14, 29, 31). To identify possible BLM sorting motif(s), we made progressive truncations of the rsat-1 intracellular COOH terminus (Fig. 1A). Removal of the last three residues (SAL; rsat-1{Delta}3), a consensus PDZ-binding motif (8), still led to rsat-1 BLM targeting (Fig. 5, B and F). To test whether this protein was functional, we expressed rsat-1{Delta}3 protein in Xenopus oocytes and tested them for radiotracer sulfate uptake: rsat-1{Delta}3 led to strong induction in sulfate uptake with no significant difference in transport capacity compared with rsat-1 WT-expressing oocytes (data not shown). These data together suggest that in both mammalian (MDCK) and amphibian (Xenopus) cells, the removal of the putative COOH-terminal PDZ domain in the last three residues of rsat-1 does not affect its plasma membrane expression and that the rsat-1 sorting motif(s) may be located further upstream in its COOH terminus. To narrow down the region containing potential rsat-1 BLM sorting motifs, we generated two further rsat-1 COOH-terminal truncations, lacking either the last 30 (EGFP/rsat-1{Delta}30) or the last 65 (EGFP/rsat-1{Delta}65) residues (Fig. 1A). Removal of either the last 30 (Fig. 5, C and G) or 65 amino acids of rsat-1 (Fig. 5, D and H) abolished BLM expression, showing intracellular staining, suggesting that the BLM targeting motif(s) for rsat-1 may lie between the last 3 and 30 residues in its COOH terminus. Sequence analysis of the last 30 amino acids revealed the presence of a putative targeting motif, a dileucine at position 677/678 (26/27 residues from its COOH-terminal tail). Alanine substitution of this dileucine motif (Fig. 1A) led to intracellular staining in both MDCK (Fig. 6, B and E) and LLC-PK1 cells (Fig. 6, C and F), comparable to COOH-terminal {Delta}65 and {Delta}30 truncations (Fig. 5, C, D, G, and H), suggesting that the 677/678 dileucine motif may be required for rsat-1 basolateral targeting.



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Fig. 5. Expression of EGFP/rsat-1 WT and several truncations in MDCK cells: EGFP/rsat-1 WT (A), EGFP/rsat-1 {Delta}3 (–PDZ; B), EGFP/rsat-1 {Delta}30 (C), and EGFP/rsat-1 {Delta}65 (D), as described in Fig. 1A. Images in A–D represent the expression of EGFP and actin as seen in the focal plane, and images in E–L show a confocal cross section (XZ scatter) through the focal plane positioned at the white line.

 


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Fig. 6. Expression of the dileucine mutant in MDCK and LLC-PK1 cells. A: EGFP/rsat-1 WT in MDCK cells. B: EGFP/rsat-1 LL677/678AA in MDCK cells. C: EGFP/rsat-1 LL677/678AA in LLC-PK1 cells. Images in A–C represent expression of EGFP and actin as seen in the focal plane, and images in D–I show a confocal cross section (XZ scatter) through the focal plane positioned at the white line.

 
To provide further evidence that the rsat-1 COOH terminus has a functional BLM sorting determinant, we fused the complete rsat-1 cytoplasmic tail (the last 77 amino acids) to the COOH terminus of hIL2R{alpha} (Tac) protein (Fig. 1B) and expressed this fusion protein in MDCK cells. Tac, a 273-amino acid single-transmembrane domain protein, sorts to the apical membrane of MDCK (Fig. 7, A and E), as shown previously (23, 38), whereas the Tac/rsat-1 WT chimera largely sorts to the BLM of MDCK cells (Fig. 7, D and F), with minor staining at the apical membrane (Fig. 7B), suggesting that rsat-1 COOH terminus does contain a BLM sorting determinant. Removal of the rsat-1 COOH-terminal PDZ domain ({Delta}3) resulted in BLM sorting (Fig. 8, D and G), similar to Tac/rsat-1 WT staining (Fig. 7, D and F), suggesting that the PDZ domain is not required for rsat-1 plasma membrane trafficking. Deletion of the last 30 amino acids ({Delta}30) of rsat-1 (Fig. 1B) resulted in a return to apical expression (Fig. 8, B and H), similar to Tac staining (Fig. 7, A and E), suggesting that a sorting motif is present between the last 3 and 30 residues of the rsat-1 COOH terminus. Alanine substitution of the dileucine motif at position 677/678 (Fig. 1B) also led to apical expression (Fig. 8, C and I), confirming that this dileucine motif, located 26/27 residues from its COOH-terminal end, is essential for basolateral trafficking of rsat-1.



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Fig. 7. Expression of human interleukin-2 receptor {alpha}-chain (Tac) and Tac/rsat-1 fusion protein in MDCK cells. MDCK cells transiently transfected with Tac (A, C, E) or Tac/rsat-1 (B, D, F) (see Fig. 1B) were fixed, permeabilized, stained with Tac antibody and Cy3, and visualized by confocal fluorescence microscopy. Images in A and B represent XY scans at the apical membrane level, and images in C and D represent scans at the basolateral membrane level. Images in E and F represent a confocal cross section (XZ scatter) through the focal plane.

 


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Fig. 8. Expression of Tac/rsat-1 truncations in MDCK cells. MDCK cells transiently transfected with Tac/rsat-1 {Delta}3 (A, D, G), Tac/rsat-1 {Delta}30 (B, E, H), or Tac/sat-1 LL677/678AA (C, F, I) (see Fig. 1B) were fixed, permeabilized, stained with primary Tac antibody and secondary Cy3, and visualized by confocal fluorescence microscopy. Images in A–C represent XY scans at the apical membrane level, and images in DF represent scans at the basolateral membrane level. Images in G–I represent a confocal cross section (XZ scatter) through the focal plane.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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The sat-1 is located at the BLM of the proximal tubule in the kidney, where it is proposed to function as a sulfate/oxalate exchanger (20). However, no studies to date have looked at the sorting pathway of sat-1 to the BLM. The aim of the present study was to characterize the trafficking of the rsat-1 protein in renal epithelial cell lines and to identify potential sorting motifs responsible for its BLM expression.

LLC-PK1 and MDCK cells were able to correctly traffic rsat-1 to BLM, as in vivo, suggesting that these cells, derived from renal proximal tubular and distal tubular origins, respectively, contain the correct machinery for rsat-1 plasma membrane targeting. The addition of BFA, a reagent that perturbs vesicular transport, to rsat-1 WT-expressing MDCK cells showed a disruption of the polarized expression of rsat-1. This finding suggests that rsat-1 is sorted to the BLM via a BFA-sensitive basolateral pathway, similar to the BLM sorting of the human liver bile acid transporter NTCP (45). To identify the rsat-1 BLM sorting determinant, we targeted its cytoplasmic COOH terminus, from which we generated a series of truncations and looked at their expression in LLC-PK1 and MDCK cells. Sequence analysis of rsat-1 COOH terminus revealed the presence of a few potential sorting motifs, including a PDZ-binding domain, a tyrosine-based sorting motif, and two dileucine motifs.

The last three amino acids (SAL) of rsat-1 strongly resemble the PDZ-interacting/binding domain S/T-X-L/V/M/F (8, 44). PDZ domain proteins are known to be involved in the targeting of proteins, as well as interacting with the actin cytoskeleton, which may play a role in stabilizing proteins at the plasma membrane level (8, 9). The PDZ-interacting domain of CFTR is required for its trafficking to the apical membrane in MDCK cells, and the PDZ protein EBP50 (NHERF) has been shown to interact with CFTR and is expressed at the apical membrane (33, 34). The three PDZ proteins lin2, lin7, and lin10 are required for basolateral localization of the receptor tyrosine kinase let23 in vulval precursor cells of the Caenorhabditis elegans (17). In the present study, we demonstrated that removal of the putative PDZ domain of rsat-1 had no effect on its basolateral expression in MDCK cells or on its plasma membrane function in Xenopus oocytes, suggesting that this motif is not required for rsat-1 plasma membrane sorting. However, this does not rule out the possibility that an interaction between some PDZ protein(s) and the last three amino acids (SAL) of rsat-1 or other parts of the rsat-1 protein does not occur (6, 46); further work is required for this to be determined.

The COOH terminus of rsat-1 contains a putative motif at position 641 (YRAL) that conforms with the consensus tyrosine-based targeting signal YXXØ, which has been shown to direct the LDL receptor to the BLM in MDCK cells (29). However, this motif does not seem to be involved in basolateral sorting of rsat-1, because the {Delta}30 truncation that included the putative tyrosine-based motif was targeted incorrectly (intracellular localization). In agreement with this, two cytoplasmic tyrosine-based motifs were found to play no role in the targeting of E-cadherin to the BLM of LLC-PK1 and MDCK cells (4). Roush et al. (41) showed inverted membrane polarity in LLC-PK1 cells, resulting in incorrect membrane sorting of the H+-K+-ATPase {beta}-subunit to the apical membrane. LLC-PK1 cells lack the µ1B-subunit of the AP-1 adapter complex, which directs proteins to the BLM (36). The absence of the µ1B-subunit results in the inverted expression of proteins relying on tyrosine-based signals, but expression of recombinant µ1B protein in LLC-PK1 cells was sufficient to restore correct plasma membrane sorting (10). Although rsat-1 contains a putative tyrosine-based sorting motif, the rsat-1 protein was targeted to the correct plasma membrane in LLC-PK1 cells, suggesting that the tyrosine-based motif is not required for rsat-1 sorting to the BLM.

Two dileucine motifs are found in the COOH terminus of the rsat-1 protein (positions 648/649 and 677/678). Dileucines are proposed to play an important role in endocytosis and BLM targeting (30). They have been suggested to be BLM sorting determinants for a variety of proteins, including E-cadherin (31), the Fc receptor (15), and the human norepinephrine transporter (hNET) (11). Conversely, a dileucine motif in the human equilibrative nucleoside transporter (hENT2) did not affect targeting to the BLM in MDCK cells but was implicated to play a role in its surface expression (24). On the other hand, a leucine residue in a dileucine motif in the COOH-terminal tail of the type IIb NaPi cotransporters was shown to be involved in the apical targeting in opossum kidney (OK) cells (19). The loss of the last 30 residues of the rsat-1 COOH terminus led to intracellular staining, suggesting that a possible rsat-1 BLM sorting motif lies between the last 30 and 3 residues. This region includes a dileucine motif at position 677/678 (26/27 residues from its COOH-terminal end) but excludes the more upstream dileucine motif, at position 648/649 (55–56 from the COOH terminus), as a possible sorting motif. Conversion of the dileucine motif at position 677/678 to alanines resulted in an intracellular expression, suggesting a potential role of these two leucine residues in targeting of rsat-1 to the BLM. Dileucine motifs with an acidic amino acid at position –4 (D/EXXXLL) were shown to function in the endocytic pathway (7, 37). rsat-1, however, has a nucleophilic threonine at position –4, suggesting that this dileucine motif is unlikely to act as a signal for endocytosis.

To verify the above-described findings, we fused the last 77 amino acids of the rsat-1 cytoplasmic COOH terminus with the {alpha}-chain of the hIL2R{alpha} (Tac) (23, 31, 38), a protein that sorts to the apical membrane in MDCK cells (38). The Tac/rsat-1 fusion protein was sorted to the BLM in MDCK cells; however, deletion of the last 30 amino acids and alanine substitution of the 677/678 dileucine motif led to a return of apical membrane expression, suggesting that the 677/678 dileucine motif is the basolateral sorting signal in the cytoplasmic tail of rsat-1.

The potential significance of our findings is that leucine motifs found within intracellular COOH termini of membrane proteins could be very important sorting determinants for trafficking to plasma membranes of epithelial cells. The list of proteins that require such leucine residues for proper membrane sorting include hNET, type IIb NaPi cotransporter, human aquaporin-2, the Fc receptor, and E-cadherin (11, 15, 19, 31). Naturally occurring mutations, if found in such leucine residues, may lead to inappropriate sorting, which not only will disrupt vectorial solute and fluid transport in epithelial cells but also could form the molecular basis of disease.

In conclusion, the present study shows that rsat-1 is expressed at the basolateral membranes of MDCK and LLC-PK1 cells. No requirement for the PDZ-binding domain was observed for basolateral expression and function. A potential basolateral sorting signal was proposed to be located within the last 3 and 30 residues of the rsat-1 intracellular COOH terminus, which was narrowed down to a dileucine motif at position 677/678. This information is important for understanding the mechanism(s) underlying the basolateral sorting of the sat-1 protein, which will provide greater knowledge of sat-1 expression in vivo in the renal proximal tubule and of its involvement in sulfate reabsorption and oxalate excretion.


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This work was supported in part by the Australian Research Council and National Health and Medical Research Council (to D. Markovich).


    ACKNOWLEDGMENTS
 
We thank Dr. J. L. Stow for providing the pCMVIL2R vector.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. Markovich, Dept. of Physiology and Pharmacology, School of Biomedical Sciences, Univ. of Queensland, St. Lucia, QLD 4072, Australia (E-mail: d.markovich{at}uq.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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