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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
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
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-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 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 
-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.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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3), which conform to a PDZ consensus binding motif (SAL), the last 30 residues (30), and the last 65 residues (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 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 -chain (hIL2R, 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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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.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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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-13 protein in Xenopus oocytes and tested them for radiotracer sulfate uptake: rsat-13 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-130) or the last 65 (EGFP/rsat-165) 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 65 and 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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(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 (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 (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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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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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 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 -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 -chain of the hIL2R (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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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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 J. Zheng, G.-G. Du, K. Matsuda, A. Orem, S. Aguinaga, L. Deak, E. Navarrete, L. D. Madison, and P. Dallos The C-terminus of prestin influences nonlinear capacitance and plasma membrane targeting J. Cell Sci., July 1, 2005; 118(13): 2987 - 2996. [Abstract] [Full Text] [PDF]
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