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Am J Physiol Cell Physiol 290: C472-C483, 2006. First published August 17, 2005; doi:10.1152/ajpcell.00508.2004
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Cytosolic COOH terminus of the peptide transporter PEPT2 is involved in apical membrane localization of the protein

Maja Klapper,1 Hannelore Daniel,2 and Frank Döring1

1Research Group Molecular Nutrition, University of Kiel, Kiel; and 2Physiology of Nutrition, Technical University of Munich, Freising-Weihenstephan, Germany

Submitted 19 October 2004 ; accepted in final form 15 August 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The peptide transporter PEPT2 is a polytopic transmembrane protein that mediates the cellular uptake of di- and tripeptides and a variety of peptidomimetics. It is widely expressed in mammalian tissues, including kidney, lung, mammary gland, choroid plexus, and glia cells. In renal tubular cells, PEPT2 is exclusively found at the apical membrane. The molecular mechanisms underlying this polarized expression and targeting to the brush-border membrane are not known. We have explored the role of the 36 COOH-terminal amino acid residues in PEPT2 trafficking and apical expression. EGFP-tagged PEPT2 wild-type transporter and various truncated and mutant proteins were expressed in the polarized proximal tubule cell lines SKPT and OK, and the cellular distribution of the fusion proteins was assessed using confocal microscopy. Whereas deletion of the last seven amino acids (delC7) did not alter PEPT2 surface expression, deletion of the next residue (delC8) or up to 30 terminal amino acids resulted in impaired apical expression and distinct accumulation of mutant proteins in endosomal and lysosomal vesicles. Truncation of more amino acids (delC36) containing tyrosine-based motifs led to a rather diffuse intracellular distribution pattern. Mutations introduced at isoleucine-720 (I720A) and leucine-722 (I722A) also caused an impaired surface appearance. Internalization assays revealed a higher endocytotic rate of the PEPT2 mutants I720A, L722A, and delC36. Our data suggest that a three-amino acid stretch (INL) and tyrosine-based motifs within the COOH tail of PEPT2 are involved in PEPT2's apical membrane localization and membrane steady-state level.

di- and tripeptide transport; polarized epithelial cells; lysosomes

TWO PROTON-COUPLED PEPTIDE transporters (PEPTs) are expressed in mammals. Both transporters are able to transport a huge number of di- and tripeptide structurally related peptidomimetics. The high-capacity, low-affinity type PEPT1 is found mainly in the intestine, with lower expression levels observed in the kidney (4, 18, 47), whereas the high-affinity isoform PEPT2 (3, 45) is found in tissues such as kidney (3, 35), lung (22), mammary gland (1, 13, 21), glia cells (12, 15, 20), and epithelia of choroid plexus (2, 11, 51, 56). Renal tubular expression is highest in S3 segments (49, 52). By coupling of substrate transport to proton movement down an electrochemical proton gradient, the localization of PEPT2 in apical membranes of epithelial cells allows efficient uphill transport of peptide-bound amino acids and xenobiotics. The molecular determinants that control the cell surface expression, subcellular distribution, and dynamics of PEPT2 remain undefined.

The asymmetric distribution of apical transmembrane proteins is not completely understood. Direct or indirect sorting of newly synthesized proteins occurs in the trans-Golgi network (TGN), where proteins are assembled in specific vesicles. Information for basolateral targeting appears to be encoded in short signals in intracellular tails, such as tyrosine-based YXXØ or NPXY (with X representing any amino acid and Ø representing hydrophobic) and dileucine-based motifs (24), whereas apical targeting seems to be mediated by a wider range of signals. Protein-based signals are present in the transmembrane (16) or cytoplasmic domains. COOH-terminal postsynaptic density-95/Drosophila discs large/zonula occludens-1 (PDZ) motifs (TXL/F) mediating interaction with PDZ domain-containing proteins are suggested to play a role in apical targeting or anchoring of integral proteins in the apical membrane (25, 39, 50). More recently, new apical sorting motifs comprising a {beta}-turn structure were identified in Na+-dependent transporter proteins (53, 54). Other mechanisms of apical targeting require either clustering of proteins into glycolipid- and cholesterol-enriched rafts, O-glycosylation (8, 27), or N-glycosylation (26) of extracellular domains.

PEPT2 is an integral membrane protein with 12 predicted transmembrane domains (TMDs) and cytoplasmic NH2 and COOH termini (3, 35). Because the cytosolic COOH-terminal regions of transmembrane proteins are more frequently involved in polarized expression, we have analyzed the COOH terminus of PEPT2 with respect to protein domains that are important to its apical expression. Cellular distribution of enhanced green fluorescent protein (EGFP)-tagged wild-type PEPT2 (PEPT2-WT) and a series of proteins with COOH-terminal truncations and amino acid substitutions were analyzed using confocal microscopy after expression in the polarized proximal tubule cell lines SKPT and OK. In the present study, we have demonstrated that the amino acids isoleucine-720 (I720) and leucine-722 (L722) are important for the apical localization of PEPT2. Deletion or exchange of these amino acids by alanine resulted in impaired apical cell surface expression and accumulation of the transporter mutants mainly in lysosomal structures. Tyrosine-based motifs near the last TMD seem also to be involved in the sorting of PEPT2. A mutant lacking the whole COOH terminus displayed a more diffuse intracellular accumulation pattern in contrast to a mutant that still contained these motifs. Internalization studies revealed that PEPT2 mutants were internalized at a higher rate than wild-type transporters.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
PEPT2-GFP fusion plasmids. After deletion of the stop codon and generation of a COOH-terminal AgeI restriction enzyme digestion site using the QuickChange in vitro mutagenesis kit (Stratagene), the cDNA encoding the rabbit PEPT2-WT was subcloned into the SalI/AgeI sites of the EGFP-N1 vector (Clontech), resulting in PEPT2-EGFP. The following primer and the complementary oligonucleotide containing the mutation in the middle were used (5' -> 3'): CCAAGAAGACAAAGCTCTCACCGGTCCCAGGACTCT.

To introduce an AgeI site at the desired last COOH-terminal codon, PEPT2 truncation mutants were generated by performing PCR amplification using the forward primer (5' -> 3') TCGTCGTATCTCCAAGTGTGG and the following specific reverse primers (5' -> 3'): delC7, CCACCGGTAAGTTGATCATGTTCCC; delC8, CCACCGGTCCGTTGATCATGTTCCC; delC30, CCACCGGTATGGGAATATAGTAGTAGC; and delC36, CAACCGGTCCCATGATGGAGAAGATC. After EcoRV/AgeI restriction enzyme digestion of PCR products and PEPT2-EGFP, the PEPT2-WT COOH terminus was replaced by truncated COOH termini by subcloning of the PCR products into the EcoRV/AgeI sites of PEPT2-EGFP.

Construction of single-amino acid substitution mutants was performed using the QuickChange in vitro mutagenesis kit. The following primers and the complementary oligonucleotides containing the mutation in the middle were used (5' -> 3'): L722A, CCATATGCAAGGGAACATGATCAACGCAGAGACCAAG; I720A, CCATATGCAAGGGAACATGGCCAACTTAGAGACCAAG; L722I, CCATATGCAAGGGAACATGATCAACATAGAGACCAAG; I720L, CCATATGCAAGGGAACATGCTCAACTTAGAGACCAAG; and ILA, CCATATGCAAGGGAACATGATCCTCGCAGAGACCAAG. All constructs were verified by performing sequencing.

Cell culture. The renal proximal tubule cell lines from rat (SKPT-0193 Cl.2) and opossum (OK) were kindly provided by M. Brandsch (Martin-Luther-University, Halle, Germany) and H. Murer (University of Zurich, Zurich, Switzerland), respectively. Culture media, antibiotics, FBS, and trypsin solution were purchased from Life Technologies; apo-transferrin and dexamethasone were obtained from Sigma; and EGF was purchased from Promega.

OK cells were cultured in 1:1 DMEM-Ham's F-12 medium supplemented with 10% FBS and penicillin-streptomycin. SKPT cells were cultured in 1:1 DMEM-Ham's F-12 medium supplemented with 10% FBS, insulin, EGF, apo-transferrin, dexamethasone, and gentamicin. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. SKPT and OK cells were transiently transfected with a FuGENE 6 transfection reagent (Roche) mixture at a ratio of 2 µg of DNA to 3 µl of FuGENE 6 in a final 100-µl volume of serum-free medium per well according to the manufacturer's instructions. SKPT cells were seeded into six-well plates with coverslips and transfected 24 h after seeding at 70% confluence. OK cells were transiently transfected 24–48 h after seeding under the same conditions. Stable transfections of OK cells were performed in six-well tissue culture plates. At ~70% confluence, cells were transfected by adding the DNA-FuGENE 6 reagent mixture at a ratio of 2 µg of linearized DNA to 6 µl of FuGENE 6 in a final 100-µl volume of serum-free medium per well. After transfection (24 h), cells were split 1:9 into six-well plates and selected in OK medium containing 0.4 mg/ml G418 (Life Technologies) for 3 wk. Single colonies were picked.

Confocal microscopic immunofluorescence analysis. Cells were washed with PBS, fixed with 4% paraformaldehyde-PBS for 15 min, and permeabilized with 0.2% Triton X-100-PBS. Paraformaldehyde was quenched with 20 mM glycine-PBS and washed three times for 5 min with PBS before being blocked with 3% serum-0.1% Triton X-100-PBS for 30 min. Incubation with mouse MAb (BD Transduction Laboratories) against early endosomal antigen 1 (EEA1; 1:100 dilution) or Rab11 (1:50 dilution) and cathepsin D (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 3% serum-0.1% Triton X-100-PBS was performed at 4°C overnight. After three 10-min washes with 3% serum-0.1% Triton X-100-PBS, cells were incubated with Cy3-conjugated secondary goat anti-mouse antibodies (1:600 dilution; Dianova) and donkey anti-rabbit antibodies (1:500 dilution; Dianova), respectively, for 1 h; washed three times for 10 min with PBS; and embedded in glycerol medium. Confocal images were obtained using a Leica TCS SP2 confocal laser-scanning microscope equipped with a x63 magnification oil-immersion lens objective. EGFP was excited with a 488-nm laser line and imaged at 500–530 nm. Cy3 was excited at 543 nm and imaged at 560–600 nm. EGFP and Cy3 were measured sequentially. LysoTracker Red DND-99 (Molecular Probes) staining was performed according to the manufacturer's instructions. Cells were grown on coverslips and incubated for 30–60 min with 50 nM LysoTracker Red DND-99. Excitation was induced at 488/543 nm, and imaging was performed between 620 and 700 nm. Actin was stained with Alexa Fluor 633 phalloidin (Molecular Probes) after fixation and permeabilization of cells according to the manufacturer's instructions. Phalloidin was excited at 633 nm and imaged at 640–700 nm. Images were analyzed using Leica confocal software version 2.5 and produced using Adobe Photoshop version 7.0 software.

Cell surface biotinylation and internalization assay. OK cells were grown on 9.2-cm2 petri dishes or 24-well plates for 8 days. Postconfluent (5 days) monolayers were preincubated with the lysosomal inhibitor leupeptin (100 µg/ml; Sigma) for internalization assays and washed three times with ice-cold PBS-CM (1.25 mM MgCl2 and 0.5 mM CaCl2, pH 7.5), and cell surface proteins were biotinylated by gentle shaking with 0.75 mg/ml EZ-Link sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin) for internalization assays or EZ-Link sulfo-NHS-long chain (LC)-biotin (Pierce) in PBS-CM (pH 7.5) for 30 min on ice. Cells were washed three times with quenching buffer (100 mM glycine in PBS-CM) to remove nonreacted biotin. Subsequently, cells were washed twice with PBS-CM. For internalization assays, cells were incubated with medium at 37°C for the indicated time intervals. Remaining cell surface biotin was cleaved with 50 mM glutathione buffer (in mM: 90 NaCl, 1.25 MgCl2, and 1.25 CaCl2, pH 8.6). Unreacted glutathione was quenched by three washes with iodoacetamide (5 mg/ml PBS-CM; pH 7.4). Cells were scraped and solubilized using RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris·HCl, pH 8.0, and 0.1 mg/ml PMSF) supplemented with Complete Mini protease inhibitors (Roche), sonicated for 60 s, agitated on a shaker at 4°C for 30 min, and centrifuged at 14,000 rpm for 10 min to remove insoluble cellular debris. Cell lysate (100 µg) was incubated with Streptavidin Sepharose High Performance (Amersham Biosciences) in 300 µl of RIPA buffer for 3 h at 4°C. After brief centrifugation, supernatants were assumed to represent the intracellular basolateral pool, precipitated with acetone, and boiled in Laemmli buffer. Streptavidin-bound proteins were washed three times with RIPA buffer. Biotinylated proteins were eluted by boiling in Laemmli buffer and analyzed using SDS-PAGE and Western blot analysis.

SDS-PAGE, Western blot analysis, and ECL detection. Proteins were separated on 8% SDS polyacrylamide gels and transferred to PVDF membranes. Membrane blocking and incubation with antibodies were performed with 3% nonfat dry milk-TBST (20 mM Tris·HCl, pH 7.4, 137 mM NaCl, and 0.05% Tween 20). PEPT2-EGFP was detected with PAb against EGFP (1:250–1:500 dilution, Living Colors A.V. peptide antibody; Clontech) and horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000 dilution; Santa Cruz Biotechnology) and developed using ECL (Amersham Biosciences). Western blots were analyzed densitometrically using ImageJ public domain software (available at: http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, MD).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Localization of EGFP-tagged PEPT2 wild-type and COOH-terminal deletion mutants of PEPT2. Topological models of PEPT2 predict 12 TMDs with intracellular COOH- and NH2-terminal tails (3). The COOH terminus consists of 36 amino acids (3) and contains different motifs that could be involved in specific membrane targeting. Recently, the COOH-terminal amino acid sequence TAL was shown to operate as a PDZ motif (TXL/F, where X is any amino acid) that interacts with the PDZ domain-containing protein PDZK1 (29). Interactions with PDZ proteins have been described as playing a role in the polarized distribution of apical transmembrane proteins (25, 39, 50). Moreover, next to the last TMDs, two overlapping potential tyrosine-based signals YXXØ can be identified. These motifs appear to be involved in multiple sorting steps, including targeting of proteins from TGN to the basolateral plasma membrane as well as internalization and lysosomal sorting (6). Table 1 shows the various motifs found in the sequence of PEPT2 in four different mammalian species.


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  		Table 1. Conserved putative trafficking motifs in cytosolic COOH terminus of PEPT2

 
To examine the importance of these motifs within the COOH terminus of PEPT2 for its delivery to the apical membrane, sequential deletions were performed (Table 2). PEPT2-WT and mutants were fused to EGFP, and the cellular localization of the fusion proteins was studied after expression in the rat proximal tubule cell line SKPT. As shown using confocal laser scanning microscopy (Fig. 1), in postconfluent monolayers, tagged PEPT2-WT was almost exclusively found in the apical membrane. The deletion of the last seven COOH-terminal amino acids to E723 (delC7) and including the putative PDZ motif did not impair the membrane localization and yielded essentially a wild-type form of apical targeting (Fig. 2, A, E, and I). This suggests that the PDZ motif may not be essential for apical surface expression of PEPT2. However, the additional removal of L722 resulted in impaired apical membrane localization and an accumulation in intracellular compartments of the deletion mutant (delC8) as shown in Fig. 2, B, F, and J. Polarized membrane distribution of delC8 was maintained; however, vesicles were detected in close proximity to the basolateral membrane. The deletion of further amino acids up to K700 (delC30) did not substantially change the distribution compared with delC8 (Fig. 2, C, G, and K). Subsequently, the entire COOH terminus (delC36) was removed, including the two putative tyrosine-based signals YXXØ. In contrast to delC8 and delC30, which still contained the tyrosine-based motifs, delC36 displayed a more diffuse intracellular distribution that was not restricted to vesicular structures. The overall expression was reduced, in contrast to overall expression observed in all other mutant proteins (Fig. 2, D, H, and L).


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  		Table 2. Amino acid sequence of COOH-terminal truncation mutants of PEPT2

 


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  		Fig. 1. Cellular distribution of a mutant encoding an enhanced green fluorescent protein (EGFP) fusion protein of peptide transporter 2 (PEPT2-EGFP) in polarized SKPT cells. SKPT cells were transiently transfected with a vector encoding PEPT2-EGFP, and 72 h after transfection, postconfluent cells were processed for confocal microscopy. Focal planes of the apical (top) and basolateral sides (middle) and an x-z cross section (bottom) are shown. Bars, 10 µm.

 


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  		Fig. 2. Cellular distribution of PEPT2-EGFP deletion mutants in SKPT cells. Cells were transfected with a PEPT2 mutant protein in which the last seven amino acids were deleted (delC7; shown in A, E, and I), a PEPT2 mutant protein in which the last eight amino acids were deleted (delC8; shown in B, F, and J), a PEPT2 mutant protein in which the last 30 amino acids were deleted (delC30; shown in C, G, and K), and a PEPT2 mutant protein in which the last 36 amino acids were deleted (delC36; shown in D, H, and L). After transfection (72 h), postconfluent cells were processed for confocal microscopy. Focal planes (A–D) and x-z cross sections (E–L) are shown. EGFP staining is shown in green, and actin staining is shown in red. Merged fluorescence signals are shown in A–D and I–L. Bars, 10 µm.

 
Branched chain amino acid residues in the 720 and 722 positions are essential for apical targeting. To assess the role of amino acid residue L722 and the region upstream of L722 for membrane localization, we generated a series of PEPT2 mutants with single-amino acid substitutions. The amino acids N718 to L722 were examined by individually exchanging them with alanine (Table 3). Substitutions I720A and L722A led to essentially the same distribution observed for delC8, i.e., decreased cell surface expression relative to the accumulation in intracellular, and often enlarged, vesicular structures (Fig. 3). Modification of amino acid residues N718, M719, and N721 did not induce similar distribution patterns (Fig. 3). To investigate the role of amino acid residues 720 and 722 in the sequence I720N721L722 in more detail, we generated mutants by replacing the parent residues with other branched-chain amino acids leading to I720L and L722I (Fig. 3). The localization of both mutants was not distinguishable from that of the wild-type protein, thus remaining completely in the correct apical localization. Furthermore, a mutant (ILA) with displacement of leucine from position 722 to position 721, leading to the sequence I720L721A722, also showed wild-type expression (Fig. 3). On the basis of these data, we conclude that a crucial signal for proper expression is encoded by the branched-chain amino acid residues I720 and L722, whereas the distance between these residues seems to be of less importance.


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  		Table 3. Amino acid sequence of COOH-terminal mutants of PEPT2 with single-amino acid substitutions

 


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  		Fig. 3. Cellular distribution of PEPT2-EGFP single-amino acid substitution mutants in SKPT cells. Cells were transfected with PEPT2 N718A (A, I, and Q), M719A (B, J, and R), I720A (C, K, and S), N721A (D, L, and T), L722A (E, M, and U), I720L (F, N, and V), L720I (G, O, and W), and ILA (H, P, and X). After transfection (72 h), postconfluent cells were processed for confocal microscopy. Focal planes are shown in A–H, and x-z cross sections are shown in I–T and M–X. EGFP staining is shown in green, actin staining is shown in red, and merged images are shown in A–H and Q–X. Bars, 10 µm.

 
Mutant proteins I720A and L722A accumulate predominantly in lysosomes. Next, we sought to identify the intracellular compartments at which the mistargeted mutants were targeted. Therefore, we examined the cellular localization of the mutant proteins I720A and L722A compared with WT using the specific subcellular markers binding protein (BiP)/glucose-regulated protein of 78 kDa (GRP78), a Golgi matrix protein of 130 kDa (GM130), EEA1, Rab11, and cathepsin D in SKPT cells. BiP/GPR78 is a chaperone of the endoplasmic reticulum (ER) lumen (23). GM130 functions as a structural element of the Golgi apparatus (40). Rab11 is localized in the TGN, secretory vesicles, and pericentriolar recycling endosomes and is involved in regulating traffic at the Golgi complex (57, 58). EEA1 localizes to early endosomes and is required for vesicular transport of proteins through early endosomes (37). Cathepsin D is a lysosomal aspartyl protease (17). Neither PEPT2-WT nor the mutant proteins colocalized with BiP/GRP78 and GM130 (data not shown), suggesting that the mutants were not retained in the ER and the cis-Golgi, which are involved in quality control of newly synthesized proteins. Although PEPT2-WT was found mainly in the apical membranes, a small proportion of the wild-type protein was detected in Rab11 (Fig. 4, a–c) - and EEA1 (Fig. 5, a–c) -positive vesicles belonging to TGN and/or pericentriolar recycling endosomes and early endosomes, respectively. The large vesicles found in the case of mutant proteins did not colocalize with Rab11-positive structures (Fig. 4, d–i). Because we also did not observe colocalization of the mutants with GM130 as a marker for the cis-Golgi, we conclude that the proteins are transported correctly through the cis-Golgi and the trans-Golgi. Furthermore, the detection of PEPT2 mutants at the plasma membrane and most of the intracellular accumulated proteins in lysosomes or multivesicular bodies, where proteins can be delivered either from the plasma membrane or directly from the TGN to lysosomes (38), argues for a proper exit of mutants from the TGN.



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  		Fig. 4. Immunostaining of SKPT cells expressing PEPT2-EGFP wild-type (WT) and mutant proteins I720A and L722A with Rab11. Cells were transiently transfected with PEPT2-EGFP-WT (a–c), I720A (d–f), and L722A (g–i). After transfection (72 h), postconfluent monolayers were stained with an antibody against Rab11 [trans-Golgi network (TGN) and recycling endosomes] and analyzed using confocal microscopy. EGFP staining (green) is shown in a, d, and g; Rab11 staining (red) is shown in b, d, and h; and merged images are shown in c, f, and i. Overlapping staining is shown in yellow. Overlapping structures are marked with arrows. Bars, 10 µm.

 


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  		Fig. 5. Immunostaining with early endosomal antigen 1 (EEA1) of SKPT cells expressing PEPT2-EGFP-WT and mutant proteins I720A and L722A. Cells were transiently transfected with PEPT2-EGFP WT (a–c), I720A (d–f), and L722A (g–i). After transfection (72 h), postconfluent monolayers were stained with an antibody against EEA1 and analyzed using confocal microscopy. EGFP staining (green) is shown in a, d, and g; EEA1 staining (red) is shown in b, d, and h; and merged images are shown in c, f, and i. Overlapping staining is shown in yellow, and overlapping structures are marked with arrows. Bars, 10 µm.

 
As was true of PEPT2-WT, a proportion of mutant proteins was detected in stained structures of the endocytic pathway in early endosomes (Fig. 5, d–i). As shown in Fig. 6, the mutants I720A and L722A, but neither the wild-type protein nor the mutant ILA, colocalized with lysosomes stained with cathepsin D. In enlarged cathepsin D-positive vesicles, a membrane-like distribution was detected.



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  		Fig. 6. Immunostaining with cathepsin D of SKPT cells expressing PEPT2-EGFP-WT and mutant proteins I720A, L722A, and ILA. Cells were transiently transfected with PEPT2-EGFP-WT (a–c), I720A (d–f), L722A (g–i) and ILA (j–l). After transfection (72 h), postconfluent monolayers were stained with an antibody against cathepsin D (late endosomes and lysosomes) and analyzed using confocal microscopy. EGFP staining (green) is shown in a, d, g, and j; cathepsin D staining (red) is shown in b, e, h, and k; and merged images are shown in c, f, i, and l. Overlapping staining is shown in yellow, and overlapping structures are marked with arrows. Bars, 10 µm.

 
Cellular distribution of the transporter variants in stably transfected OK cells. To verify the results in SKPT cells and to perform further analysis of mutants, we established OK cell lines stably expressing PEPT2-WT and mutant PEPT2-EGFP fusion proteins. The OK cell lines were also used because we were unable to establish stable SKPT cell lines that expressed the proteins. Microvilli can be detected as distinct patches in polarized OK cells (42), and the PEPT2-WT protein was predominantly found in these membrane structures, with only a minor fraction detected in basolateral membranes (Fig. 7A). As was true in SKPT cells, the ILA mutant in OK cells also localized to the apical membrane (Fig. 7B). For the mutant proteins I720A and L722A (Fig. 7B), the expression pattern was also similar to that observed in SKPT cells with an accumulation in acidic compartments, i.e., late endosomes and lysosomes, as revealed upon searching for colocalization using LysoTracker Red (Fig. 7C). The only difference from SKPT cells was that in OK cells, the mutant delC36 (Fig. 7B) displayed a polarized apical membrane distribution, which could not be clearly shown in SKPT cells. However, in this truncated transporter form, an enrichment in lysosomal structures also was detectable (Fig. 7C). Altogether, in both cell lines, the mutant proteins I720A and L722A as well as delC36 accumulated in late endosomal and/or lysosomal compartments. However, they also appeared in the apical membrane.



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  		Fig. 7. Cellular distribution of PEPT2-EGFP-WT and mutant proteins in stably transfected opossum kidney (OK) cells. A: EGFP fluorescence of postconfluent OK cells stably expressing PEPT2-EGFP-WT is shown in the focal planes of apical and basolateral domains and an x-z cross section. B: EGFP staining of postconfluent OK cells stably expressing PEPT2-EGFP-WT, I720A, L722A, ILA, and delC36 proteins is shown in x-z cross sections. C: postconfluent OK cells expressing the mutant proteins PEPT2-EGFP-WT (Ca–Cc), I720A (Cd–Cf), L722A (Cg–Ci), ILA (Cj–Cl), and delC36 (Cm–Co) were stained with LysoTracker Red (late endosomes and lysosomes). Focal planes are presented. EGFP staining (green) is shown in Ca, Cd, Cg, Cj, and Cm; LysoTracker Red (red) staining is shown in Cb, Ce, Ch, Ck, and Cn; and merged images are shown in Cc, Cf, Ci, Cl, and Co. Overlapping staining is shown in yellow. Bars, 10 µm.

 
Quantification of protein levels in apical membrane. We quantified the relative amount of PEPT2-WT and mutants I720A, L722A, and delC36 by performing apical cell surface biotinylation experiments with OK cells stably expressing the PEPT2-EGFP fusion constructs. Initial experiments with untransfected cells using peptide N-glycosidase F treatment of whole cell lysates and deglycosylation of PEPT2-EGFP using an antibody against EGFP revealed a specific band in Western blots for PEPT2-EGFP protein in its glycosylated form at a mass of 150–250 kDa (data not shown). Western blot analysis was performed using biotinylated precipitated proteins and supernatants incubated with an antibody against actin to exclude the biotinylation of intracellular proteins. Signals were observed only in lanes that had been loaded with supernatants (data not shown). In Fig. 8, representative blots for PEPT2-WT and mutants I720A, L722A, ILA, and delC36 are shown. The majority of PEPT2-WT (mean ± SE, 88.4 ± 3.5%; n = 3) was present at the apical membrane, whereas only 45.3 ± 18.3% (mean ± SE; n = 3) and 31.3 ± 4.0% (mean ± SE; n = 4) of mutant transporters I720A and L722A, respectively, were localized apically. The mutant ILA was also localized predominantly in the apical membrane, like the wild type. In the case of the truncation mutant delC36, 46.0 ± 5.4% of proteins (mean ± SE; n = 4) were found in the apical membrane. These results therefore confirm at the protein level the protein distribution patterns observed using confocal microscopy.



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  		Fig. 8. Steady-state levels of PEPT2-EGFP-WT and mutant proteins I720A, L722A, ILA, and delC36 in apical membranes were determined using biotinylation. Apical cell surface proteins of postconfluent OK cells that stably expressed PEPT2-EGFP WT, L722A, I720A, ILA, and delC36 proteins were biotinylated from the apical side. Biotinylated cell surface proteins were precipitated from cell lysates with streptavidin-conjugated Sepharose beads. Precipitates and supernatants were analyzed using SDS-PAGE and immunoblotting with an antibody against EGFP. Representative Western blots are shown. Supernatants (lane 1) represent intracellular and basolateral PEPT2 levels, and biotinylated proteins (lane 2) represent the apical PEPT2 levels.

 
Constitutive internalization rate of mutant proteins is increased. To assess whether the amino acid residues I720 and L722 play a role in protein retention in the apical membrane, the apparent endocytosis rate of the mutants was determined. Apical membranes of polarized OK cells were biotinylated with sulfo-NHS-SS-biotin, and protein density was estimated using Western blot analysis of proteins isolated at various intervals from cells incubated at 37°C. The data obtained in this manner are shown in Fig. 9. Analysis revealed that in the case of PEPT2-WT, only 8.3 ± 4.1% (n = 4, means ± SE) of the protein was endocytosed within 2 h, whereas in the case of I720A and L722A, 28.7 ± 5.3% (n = 3) and 25.4 ± 3.1% (n = 4) of the proteins, respectively, were detected in intracellular compartments after 2 h. However, the initial endocytosis rate of the L722A was higher than that of L720A. The mutant ILA was internalized at a low rate that was comparable to that of the wild-type protein (8.1 ± 3.2%; n = 4). In case of the truncated protein delC36, the internalization rate was highest at 57.1 ± 7.6% (n = 5) within 2 h.



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  		Fig. 9. Constitutive internalization of PEPT2-WT and mutant proteins I720A, L722A, ILA, and delC36. Cell surface proteins of OK cells 5 days postconfluency that stably expressed PEPT2-EGFP WT, L722A, I720A, ILA, and delC36 proteins were incubated with the lysosomal inhibitor leupeptin (10 µg/ml), which was biotinylated from the apical side using sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin) and incubated for different time intervals at 37°C. Thereafter cells were stripped with glutathione (GSH), and biotinylated cell surface proteins were precipitated from cell lysates with streptavidin-conjugated Sepharose beads. Precipitates were analyzed using SDS-PAGE and immunoblotting with antibody against EGFP. n = 3–5 independent experiments. A: representative Western blots showing total apical PEPT2 labeled at time 0, apical proteins of GSH-treated cells at time 0, and internalized protein at the time points shown. B: quantification of the internalized proteins using densitometric analysis of the protein bands of the Western blots. Results are expressed as %protein detected in intracellular basolateral compartments at a given time point compared with time 0. Data are means ± SE of 3–5 independent experiments.

 

   DISCUSSION
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 ABSTRACT
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PDZ motif seems not to be involved in apical localization of PEPT2. PDZ proteins that bind to the PEPT2 COOH terminus may function as scaffolding proteins, thus introducing PEPT2 into macromolecular complexes and an environment with other regulating proteins. Recently, the COOH-terminal motif TKL of PEPT2 was found to interact with the PDZ protein PDZK1 (29), which is expressed at the apical membranes of the kidney (31). In human embryonic kidney HEK-293 cells, PDZK1 increased the substrate uptake by PEPT2 (29). This finding might be explained by either functional regulation or regulation of trafficking. PDZK1 binds the PKA-binding protein dual specific A kinase-anchoring protein 2, which anchors PKA to PDZK1 and might bring it into close proximity to PEPT2 for possible regulation (19). Na+/H+ exchanger 3 interacts also with PDZK1 (19), which might be important for the transport activity of PEPT2 by increasing the proton gradient in close proximity to PEPT2 as a transport-activating force.

However, COOH-terminal tagging of PEPT2 with EGFP, and thus the elimination of the COOH-terminal group of the PDZ motif, which is important for interaction with PDZ domain-containing proteins (14), seems not to affect at least the apical expression of the protein. Moreover, deletion of the last seven COOH-terminal amino acids including the putative PDZ motif did not impair polarized distribution of PEPT2. However, we cannot exclude the possibility that binding of the PDZ motif to specific interaction partners such as PDZK1 might also be involved in the physiological or pathophysiological regulation mechanism of trafficking such as adaptation of the transport rate to the apical membrane (9), endocytosis (30), or recycling (34) and degradation (10).

Role of branched-chain amino acid residues in the COOH terminus. Whereas the truncation of seven amino acids did not change surface expression, the removal of one more residue (L722) to obtain delC8 led to a marked shift in this protein's steady-state distribution. Similarly, delC30 and delC36 proteins also showed less apical localization and an enrichment in intracellular compartments. Furthermore, we have shown that the exchange of single-amino acid residues of I720 or L722 to alanine altered the distribution of PEPT2 drastically. Similarly to deletion mutants, these single-point mutants accumulated intracellularly. Whereas ~90% of wild-type PEPT2 was found in the apical membrane of OK cells, less than one-half of mutant transporter proteins I720A and L722A were detected in this membrane domain. Surface biotinylation studies showed that the mutant proteins were internalized from the apical membrane at higher rates than WT or the ILA mutant. This might imply a function of I720 and L722 for membrane residence time and/or recycling of PEPT2. Because the mutant lacking the entire COOH terminus (delC36) showed a higher endocytosis rate than mutants I720A and I722A, other amino acid residues within COOH terminus also appear to be important to maintenance of membrane steady-state protein levels and/or recycling.

Generation of additional mutants I720L and L722I indicate that the amino acid positions 720 and 722 require branched-chain amino acids for correct localization. Moreover, the mutant ILA (720–722) and N721A displayed a WT-like distribution along with strong apical staining. On the basis of these studies, it may be concluded that the residue 721 within the sequence I720N721L722 is not essential, as well as that the distance between the two branched-chain amino acid residues I720 and L722 does not appear to be critical for proper apical localization. Nevertheless, the asparagine-flanking isoleucine and leucine residues seem to be crucial to these proteins' appearance and/or residence time in the apical membrane.

However, these experiments were performed with a PEPT2 that lacked a functional PDZ motif because of COOH-terminal tagging. We are aware that possible attachment of PEPT2 to the cytoskeleton and insertion of the transporter into a network of regulating proteins by binding to scaffolding PDZ proteins can influence the stability of PEPT2 in the apical membrane. We cannot rule out that in another protein environment, the branched-chain amino acids described herein could act in another way. The possible function of the branched-chain amino acids in retaining PEPT2 in the apical membrane could cross react with possible regulation by a PDZ protein, and these possibilities require further investigation.

Nevertheless, leucine residues of dileucine motifs have been described as playing a role in multiple cellular sorting events by interaction either with µ-chain subunits of adaptor proteins (APs) or with Golgi-localized, {gamma}-ear-containing, Arf (ADP ribosylation factor)-binding GGA proteins to allow binding of several proteins and to associate with clathrin complexes (5, 7). Furthermore, leucine residues seem to be crucial to the localization of apical transmembrane proteins. A dileucine motif was demonstrated to participate in the anchoring of insulin receptors to microvilli (48), and in the sodium phosphate transporter NaPi type IIb, a single leucine residue in the cytosolic COOH terminus was identified as essential for apical membrane expression in OK cells (28). I720 and L722 in PEPT2 do resemble the previously described motifs associated with adjacent amino acids 718–722/723. The sequence 718NMINL722 of PEPT2 mimics an N-not acidic-[IL]-X-Ø motif that was shown in plant cells to act as a vacuolar sorting signal in the NH2-terminal propeptide of prosporamin as well as at the COOH terminus of the mature protein (32, 36). Some similarity exists between PEPT2 amino acids 718NMINLE723 and a clathrin-binding motif called clathrin box pLØpØp (with P representing polar), whereas the leucine was observed to be nearly invariant (33). This observation demonstrates that signals similar to the PEPT2 COOH-terminal domain described herein are involved in several cellular sorting steps.

Intracellular localization. Although PEPT2 clearly shows apical membrane distribution, a small proportion of the wild-type protein could be detected along the endocytotic recycling pathway by performing colocalization studies of EEA1- and Rab11-positive compartments. This finding and the low endocytotic rate of the wild-type protein suggest that PEPT2 could be recycled to the plasma membrane after endocytosis rather than being delivered via late endosomes to lysosomes for degradation. PEPT2 mutants I720A and L722A were found in early endosomes and predominantly in lysosome, and their internalization rate was found to be three to four times higher than that of the wild type. The increased internalization rate seems to account, at least in part, for the altered phenotypic distribution of both mutants, with lower membrane density, reduced membrane residence time, and increased accumulation in lysosomes. However, we cannot exclude a priori that the mutant proteins bypass apical membrane targeting by direct sorting from the TGN to lysosomal membranes.

The PEPT2 mutant proteins did show accumulation in membranes of enlarged vesicles that could represent multivesicular bodies (MVBs). The formation of MVBs occurs at the level of endosomes to invaginate membrane proteins into the endosomal lumen before fusion with lysosomes and subsequent degradation of the proteins (43). However, accumulation of PEPT2 mutants in the outer membranes of enlarged, acidic, cathepsin D-positive organelles suggests a nondegradative pathway to which these proteins are submitted. Although the residues I720 and L722 appear to determine membrane residence time and/or recycling of the transporter, other signals may exist that account for the enhanced lysosomal accumulation of the two mutant proteins I720A and L722A. In PEPT2, two overlapping YXXØ motifs could act as lysosomal targeting signals. Interestingly, this motif is also present in PEPT1, although the COOH termini of these two transporters share little sequence similarity. For PEPT1 and a PEPT1-like transporter, respectively a lysosomal localization has been shown in renal cells and a role of peptide transporters for export of short-chain peptides from intralysosomal protein degradation have been proposed (59). A lysosomal localization therefore could represent a normal state, depending on cell type and physiological conditions. However, in SKPT and OK cells and most likely in normal renal tubular cells as well, PEPT2 is found in the apical membrane. This steady-state localization of PEPT2 seems to be critically determined by I720 and L722, which appear to dominate the tyrosine-based motifs. Other transmembrane proteins show a distance of seven or eight amino acids between the last residue of the TMD and the COOH-terminal YXXØ motif that mediates lysosomal targeting (44, 46), whereas the putative YXXØ motifs of PEPT2 are located in +1 and +3 positions relative to the TMD.

The mutant delC36, which lacks these tyrosine-based motifs, did not show this pronounced accumulation in membranes of enlarged vesicles but instead displayed a more diffuse intracellular distribution pattern in SKPT cells. This mutant protein was also found to be expressed at lower levels compared with WT and all other mutants. Accelerated degradation and ineffective passing through sorting machinery at different cellular sorting levels may explain the odd distribution of mutants lacking the tyrosine-based motifs. Tyrosine-based motifs, i.e., NPXY-like motifs and a YXXØ motif known to mediate basolateral sorting, were shown to be involved in the apical localization of the megalin receptor (55). One of these tyrosine-based motifs in PEPT2 contains a conserved proline residue in the Y+2 position. Proline residues were found to be favored compared with other residues in this position of tyrosine-based motifs in the µ1-, µ2-, and µ3-subunits of APs that function at the level of the TGN and the plasma membrane as well as endosomes (41). Thus tyrosine-based motifs of PEPT2 may be involved in its sorting at different cellular levels of the complex sorting machinery.

In conclusion, this study is the first to be reported in which intrinsic protein signals were investigated for apical membrane localization of the peptide transporter PEPT2 in epithelial cells. Our findings demonstrate that the conserved regions within the cytosolic COOH-terminal tail of PEPT2 bear different signals, including two branched-chain amino acid residues and tyrosine-based motifs that are important for proper localization and determination of membrane residence time of the transporter in tubular cells. The specific roles of the different motifs in the complex sorting processes and the proteins interacting with these domains need to be identified to understand the apical targeting of PEPT2.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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 GRANTS
REFERENCES
 
This project was supported by Deutsche Forschungsgemeinschaft Grant DO 703/1.


   ACKNOWLEDGMENTS
 
We thank M. Brandsch and H. Murer for providing the SKPT and OK cell lines, respectively.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Döring, Research Group Molecular Nutrition, Univ. of Kiel, Hermann-Weigmann-Strasse 1, D-24103 Kiel, Germany (e-mail: doering{at}email.uni-kiel.de)

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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