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Am J Physiol Cell Physiol 294: C233-C240, 2008. First published November 14, 2007; doi:10.1152/ajpcell.00468.2007
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia

Veedamali S. Subramanian,1,2,4 Jonathan S. Marchant,3 and Hamid M. Said1,2,4

Departments of 1Medicine and 2Physiology and Biophysics, University of California, Irvine, California; 3Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota; and 4Department of Veterans Affairs Medical Center, Long Beach, California

Submitted 5 October 2007 ; accepted in final form 7 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The human proton-coupled folate transporter (hPCFT) is a recently discovered intestinal transporter involved in folate uptake in epithelia (and possibly other cells). Little is currently known about the structure-function relationship of the different domains of this transporter, particularly which regions are important for substrate transport as well as targeting of the transporter to the apical cell surface of polarized cells. Here we have investigated the role of the COOH-terminal domain and a well-conserved sequence separating transmembrane (TM) domains TM2 and TM3 (DXXGRR; amino acids 109–114) speculated by others to be important for transport function. Using live cell imaging approaches, we show that 1) an hPCFT-yellow fluorescent protein construct is functionally expressed at the apical membrane domain and is localized differentially to the human reduced folate carrier; 2) the predicted cytoplasmic COOH-terminal region of hPCFT is not essential for apical targeting or transporter functionality; 3) mutations that ablate a consensus β-turn sequence separating predicted TM2 and TM3 abolished apical [3H]folic acid uptake as a consequence of endoplasmic reticulum retention of mutant, likely misfolded, transporters; and 4) cell surface delivery of hPCFT is disrupted by microtubule depolymerization or by overexpression of the dynactin complex dynamitin (p50). For the first time, our data present information regarding structure-function and membrane targeting of the hPCFT polypeptide, as well as the mechanisms that control its steady-state expression in polarized cells.

folate

FOLATE IS AN ESSENTIAL MICRONUTRIENT for many cellular functions and especially for growth and development. Deficiency in folate is a significant nutritional problem that can occur via impairments in intestinal folate uptake [including the condition of hereditary folate malabsorption syndrome (11, 13, 14, 26, 34, 37)], drug interactions, and chronic alcohol consumption (10, 12, 23, 38). The optimization of folate homeostasis decreases the incidence of neural tube defects and omphalocele (4, 5, 9) and may also reduce the incidence of cardiovascular disease (2), decrease the risk of Alzheimer disease (29), and protect against certain types of cancer (11, 19). Humans and other mammals have lost their ability to synthesize folate de novo and must therefore obtain the vitamin from exogenous sources via intestinal absorption. Elimination of folate from the body is prevented by reabsorption of the filtered folate in the kidneys. Both the intestinal folate absorption and the renal reabsorption processes occur via specific uptake mechanisms (26, 28).

Three predominant systems for folate transport have been reported in mammals, namely, the reduced folate carrier (RFC), the folate receptors (FRs), and the recently identified proton-coupled folate transporter PCFT ("h" is prefixed to these abbreviations to specify the human gene product) (1, 24, 26, 28). The level of expression of each of these folate transporters is tissue-specific. For example, all three systems are expressed in the kidney, but only the RFC and the PCFT are expressed in the normal small intestine. The transporters hRFC and hPCFT share only ~14% identity at the amino acid level, although both belong to the major facilitator superfamily of transport proteins that have a predicted topology of 12 transmembrane (TM) domains with intracellular cytoplasmic NH2 and COOH termini (22, 30, 37). The FRs are a family of glycosylphosphatidylinositol membrane-anchored proteins, intensively investigated as a route for tumor-specific delivery of chemotherapeutic agents. Significant advances have been made in understanding the cell biology and physiology of hFRs and hRFC (6, 22, 24, 28, 30); however, much less is currently known about the mechanism(s) that control the functionality and targeting of hPCFT to the epithelial cell surface.

Our aims in the present investigation were, therefore, to examine the role of the hPCFT COOH-terminal domain (amino acids 450–459) and a well-conserved β-turn sequence separating TM domains TM2 and TM3 (DXXGRR; amino acids 109–114) recently speculated by others to be important for transport function (27, 36). The rationale for focusing on these regions was as follows. First, studies with many other membrane transporters have shown that the COOH-terminal motifs/signals play an important role in transporter function and/or targeting of the polypeptide to the plasma membrane (7, 15, 31, 33, 35). Second, five nucleotide substitutions that result in amino acid point mutations in hPCFT have recently been characterized in patients exhibiting hereditary folate malabsorption (37). All these mutations are localized within sequence, or closely proximal to sequence, predicted to form TM-spanning domains. Most notably, a loss-of-function R113S mutation is localized to the cytoplasmic interface at the start of TM3, within a broader motif DXXGR113R that is conserved in many cell surface transporters (27, 36). In metal-tetracycline transporters, mutation of the glycine residue has been shown to abolish transport function (36).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. DsRed-ER, DsRed-monomer-N1, and YFP-N1 were from BD Biosciences (Palo Alto, CA). Cell lines were obtained from American Type Culture Collection (Manassas, VA). PCB6-HA-p50 was a generous gift from Prof. T. A. Schroer (The Johns Hopkins University, Baltimore, MD). DNA oligonucleotide primers (Table 1) were from Sigma Genosys (Woodlands, TX). [3H]folic acid (specific activity of 20 Ci/mmol) was obtained from ARC (St. Louis, MO).


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  		Table 1. Primer sequence and combinations used to prepare human proton-coupled folate transporter constructs by PCR

 
Generation of hPCFT-YFP-N1 and truncated/mutant constructs. The open reading frame of hPCFT (1377 bp) was amplified by RT-PCR from human small intestinal total RNA by using gene-specific primers (Table 1). The full-length hPCFT-YFP and truncated constructs were generated by PCR by using the primer combinations shown in Table 1 and conditions described previously (22, 31). The PCR products and the YFP-N1 or GFP-C3 or DsRed-monomer-N1 vectors were subsequently digested with the restriction enzymes HindIII and SacII, and the products were gel separated and then ligated together to generate in-frame fusion proteins with the yellow (YFP) or red fluorescent (DsRed) protein fused to the COOH terminus or green fluorescent protein (GFP) fused to the NH2 terminus of each construct. The Quik Change site-directed mutagenesis kit (Stratagene; La Jolla, CA) was used to introduce or delete nucleotides into the open reading frame of hPCFT. Sense and antisense oligonucleotides primers encompassing the specified mutation sites (Table 1), together with hPCFT-YFP as a template, were used for PCR mutagenesis. The resulting nucleotide sequence of each construct was verified by sequencing (Laragen).

Cell culture and transient and stable transfections. Madin-Darby canine kidney (MDCK) cells and human duodenal cells (HuTu-80) were maintained in minimal essential medium (MEM), and human colon adenocarcinoma cells (Caco-2) were maintained in Dulbecco's modified Eagle's medium (DMEM). The media were supplemented with 10% fetal bovine serum, glutamine (0.29 g/l), sodium bicarbonate (2.2 g/l for MEM and 3.7 g/l for DMEM), penicillin (100,000 U/l), and streptomycin (10 mg/l). For transient transfection, cells were grown on sterile glass-bottomed petri dishes (MatTek) or 12-well culture plates (Corning ) and were transfected at 90% confluency with 2 µg plasmid DNA by using Lipofectamine 2000 (Invitrogen). After 24–48 h, cells were analyzed by confocal microscopy or an uptake assay was performed. For the generation of stable cell lines, transiently transfected MDCK cells were selected by using G418 (0.8 mg/ml) for 6–8 wk.

RT-PCR. Total RNA (5 µg) was isolated from mock or stable hPCFT-YFP-transfected MDCK cells and was primed with oligo-dT primers to synthesize first-strand cDNA (First-Strand Synthesis RT-PCR kit; Invitrogen). Primers specific for hPCFT (Table 1) and β-actin (forward 5'-TTGTAACCAACTGGGACGATATGG-3'; reverse 5'-GATCTTGATCTTCATGGTGCTAGG-3') were used to identify hPCFT and β-actin mRNA by using established PCR conditions (22, 31).

Confocal imaging of hPCFT constructs. MDCK or Caco-2 cell monolayer grown on glass-bottomed petri dishes was imaged for construct expression by using a Nikon C-1 confocal scanner head attached to Nikon inverted phase contrast microscope. Fluorophores were excited by using the 488-nm line from an argon ion-laser, and emitted fluorescence was monitored with a 530 ± 20-nm band-pass (YFP or GFP) or a 620-nm long-pass (DsRed) filter.

Folic acid uptake analysis. For [3H]folic acid uptake measurements on filters, stable hPCFT-YFP-expressing MDCK cells were seeded onto collagen-coated filters (12-well filter insert) and were grown to ~5-day postconfluency as described previously (3, 31). The cell monolayer was incubated for 3 min at 37°C in Krebs-Ringer buffer (pH 5.0) in the presence of [3H]folic acid (9 nM) added to either the apical or basolateral surface of the monolayer. For uptake experiments on solid support (12-well plates), MDCK cells that were stably or transiently expressing constructs were incubated for 3 min at 37°C in Krebs-Ringer buffer (pH 5.0) in the presence of [3H]folic acid (9 nM). Uptake assays were terminated by the addition of 5 ml ice-cold Krebs-Ringer buffer, and accumulated radioactivity was determined by using scintillation counting.

Depolymerization of microtubules. MDCK cells were treated with the microtubule depolymerizing drug nocodazole by using minor modifications of the method described by Kreitzer et al. (18). Briefly, cells were treated in serum-free medium on ice for 30 min to depolymerize the cold-labile, nocodazole-resistant microtubules. MDCK cells were then incubated with 20 µM nocodazole (30 min, on ice) and thereafter were transfected at room temperature with hPCFT-YFP cDNA. The monolayer was then maintained in 10 µM nocodazole (37°C, overnight) until confocal imaging (18–24 h later).

Flow cytometric analysis. Flow cytometry was performed by using a FACSCalibur benchtop cytometer (BD Biosciences). hPCFT full-length, truncated/mutant stably or transiently expressing MDCK cells were grown within T25 tissue culture flasks. Monolayers were trypsinized, and cells were pelleted and resuspended in 1-ml aliquots of Ca2+-free media at a density of 1 x 106 cells/ml as described previously (22, 31). In all flow cytometry experiments, the samples of mock and YFP alone-transfected MDCK cells were run in parallel with hPCFT, truncated/mutant-expressing samples, to calibrate optical parameters for identifying the intact, transfected cell population.

Statistical analysis. Results are expressed as means ± SE. All experiments were repeated a minimum of 3–5 times to ensure reproducibility, and representative experiments are presented.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Figure 1A shows a schematic diagram of the full-length hPCFT-YFP fusion construct to illustrate the structural domain organization of the protein, including the location of amino acid substitutions (unfilled circles) between TM2 and TM3. The coding sequence encompasses an NH2-terminal domain (amino acid residues 1–24), followed by a predicted TM domain region (amino acid residues 25–449) and a short cytoplasmic COOH-terminal tail (amino acid residues 450–459) to which YFP was fused. Currently, two alternative predictions for the TM topology of hPCFT have been proposed, suggesting either 9 (27) or 12 TM helices (37) and differing on the localization of NH2 terminus (i.e., in both cases, the COOH-terminal region is proposed to be cytosolic). Here, we have used the Zhao et al. (37) prediction of 12 TM helices on the basis of homology with other transporters of the major facilitator superfamily.


Figure 1
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  		Fig. 1. Apical targeting of human proton-coupled folate transporter (hPCFT)-yellow fluorescent protein (YFP) in renal and intestinal epithelial cells. A: schematic representation of the full-length hPCFT protein with YFP fused to the COOH terminus (hPCFT-YFP). B and C: targeting of hPCFT-YFP (left) and YFP (right) in single MDCK (top) and Caco-2 (bottom) cells in lateral (xy) and axial (xz) sections. D: confocal images of a stable MDCK cell line expressing hPCFT-YFP. E: [3H]folic acid uptake assays in the hPCFT-YFP stable MDCK cell line grown on permeable filter supports after addition of [3H]folic acid to either the apical (solid bars) or basal (open bars) side of the chamber. Inset: RT-PCR detection of hPCFT mRNA expression from mock-transfected (control) and the hPCFT-YFP stably expressing MDCK cell line.

 
hPCFT mediates [3H]folic acid accumulation at the apical cell surface in renal and intestinal cells. To visualize the targeting of hPCFT in both renal and intestinal cell lines, the cDNA of the full-length hPCFT-YFP construct was transiently transfected into polarized MDCK and Caco-2 cells, and the resulting fluorescence distribution was imaged (~48 h later) by using confocal microscopy. In transfected monolayers of both cell lines, grown either on filters or coverglass, axial sections revealed that hPCFT-YFP was localized at the apical cell surface (Fig. 1, B and C). YFP-transfected cells showed fluorescence distribution throughout the entire cytoplasmic volume (Fig. 1, B and C). Similar apical targeting phenotypes were observed with NH2-terminal fluorescent protein fusions (e.g., GFP-hPCFT) in MDCK and Caco-2 cells (data not shown). In a nonpolarized human duodenally derived cell line (HuTu-80), which is well suited for imaging intracellular trafficking vesicles (22, 31, 32), real-time movies of hPCFT-YFP revealed a dynamic, heterogeneous population of intracellular vesicular structures containing hPCFT-YFP (see supplemental movie 1 in the online version of this article).

To assess transporter functionality, a stable hPCFT-YFP-expressing MDCK cell line (MDCK cells express endogenous PCFT; data not shown) was generated via antibiotic selection. After selection, confocal imaging revealed strong expression of hPCFT-YFP at the apical membrane domain (Fig. 1D). RT-PCR experiments demonstrated strong hPCFT mRNA expression (Fig. 1E, inset). Functional studies of [3H]folic acid accumulation showed that hPCFT-YFP overexpression resulted in an approximately fivefold increase (P < 0.01) in the rate of [3H]folic acid accumulation after the addition of substrate to the apical compartment, compared with an insignificant increase over endogenous levels when substrate was added basally (Fig. 1E).

Role of the cytoplasmic COOH-terminal tail of hPCFT. To delimit region(s) within the hPCFT polypeptide important for targeting and function, we first deleted the COOH terminus of the hPCFT protein. Complete truncation of the COOH-terminal tail of hPCFT (hPCFT [1-449]-YFP) did not disrupt apical cell surface targeting (Fig. 2A) or functional uptake of [3H]folic acid (Fig. 2B) or affect the mean fluorescence intensity of the transfected cell population when assessed by flow cytometry (Fig. 2C). These results suggest that the short COOH-terminal domain is not essential for transporter targeting to the apical cell surface or functionality.


Figure 2
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  		Fig. 2. The COOH-terminal region of hPCFT is not essential for apical membrane targeting or transporter functionality. A: apical targeting of a COOH-truncated construct (hPCFT[1-449]-YFP) in transiently transfected MDCK cells (top) and Caco-2 cells (bottom). B: [3H]folic acid uptake assay in transiently expressing hPCFT-YFP and hPCFT[1-449]-YFP MDCK cells. C: flow cytometry analysis of mean population fluorescence intensities of MDCK cells positively transfected with either full-length or the hPCFT[1-449]-YFP construct.

 
Apical expression of hPCFT is prevented by mutations between TM2 and TM3. Two recent publications have underscored the potential importance of residues between TM2 and TM3 [a cytoplasmic loop or an extracellular loop in the predictions of Zhao et al. (37) and Shayeghi et al. (27), respectively]. Shayeghi et al. (27) suggest that the DXXGR113R motif is potentially important for substrate transport. Zhao et al. (37) demonstrate the clinical relevance of this region by resolving a point mutation (R113S) that is associated with hereditary folate malabsorption. The application of independent topology prediction algorithms for this loop region (8, 16) suggests the formation of a β-turn around the tetrapeptide DSVG (Fig. 3). Furthermore, comparison of the DSVG sequence with known β-turn motifs collated in searchable databases shows significant similarity of this sequence to verified β-turns [e.g., DSGG, DSFG, DAVG, DSDG (8)]. This is not entirely unexpected given the propensity of TM-connecting peptides to form loops that stabilize helices for membrane insertion (21).


Figure 3
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  		Fig. 3. A predicted β-turn motif between transmembrane (TM) domains TM2 and TM3. Results of a β-turn predictive algorithm (8) applied to a 14-amino acid stretch spanning the region between TM2 and TM3 of wild-type hPCFT (shaded bars) or the alanine substitution mutants (DSVGRA, {square}; DSVAAA, {circ}; AAAAAA, {triangleup}). Positive values are indicative of an increased likelihood of forming a β-turn, and increasingly negative values are unsupportive of a β-turn conformation. Data suggest both the wild type and single alanine substitution for β-turns, because the key DSVG tetrapeptide is unaffected in these constructs.

 
Progressive alanine-substitution mutagenesis of amino acid residues within this region [hPCFT(DSVGR-A)-YFP, hPCFT(DSV-AAA)-YFP and hPCFT(AAAAAA)-YFP] was performed to assess the effects on hPCFT targeting and functionality. Results of the β-turn prediction algorithm for these constructs confirmed that neither the single alanine substitution nor the clinically identified mutant hPCFT[R113S] interfered with the predicted β-turn (DSVG), whereas the two multiple alanine substitutions (hPCFT[DSV-AAA]-YFP and hPCFT[AAAAAA]-YFP) were predicted to abolish the formation of the β-turn and not introduce any further β-turn topology (Fig. 3). In live cells, replacement of the terminal arginine (R114) with alanine [hPCFT(DSVGR-A)-YFP] did not affect apical membrane targeting in MDCK and Caco-2 cells as resolved by live cell confocal imaging (Fig. 4, A and B). Functionality was also preserved (apical [3H]folic acid uptake 0.597 ± 0.08 vs. 0.580 ± 0.07 pmol·mg protein–1·3 min–1, for hPCFT-YFP and hPCFT[DSVGR-A]-YFP, respectively; Fig. 4D). Finally, flow cytometry measurements revealed that the mean population fluorescence intensity of a stable hPCFT(DSVGR-A)-YFP-expressing MDCK cell line was similar to controls. In contrast, hPCFT(DSV-AAA)-YFP [further alanine substitutions of arginine (R113) and glycine (G112)] displayed a pleiotropic phenotype with a majority of cells showing intracellular localization, with only a few (<10%) cells displaying residual apical targeting in both MDCK and Caco-2 cells (Fig. 4, A and B). Enhanced [3H]folic acid uptake in MDCK cells overexpressing hPCFT(DSV-AAA)-YFP was abolished, with the rate of [3H]folic acid accumulation similar to untransfected or mock-transfected cells. Measurements of the mean population fluorescent intensity of a hPCFT(DSV-AAA)-YFP stable MDCK cell line revealed that this mutation significantly decreased the steady-state expression level of this construct (~90% of full-length; Fig. 4E). The hPCFT(AAAAAA)-YFP mutant was retained intracellularly within the endoplasmic reticulum (Fig. 4C) in both MDCK and Caco-2 cells. Functional uptake experiments were not performed with this construct owing to the observed intracellular location.


Figure 4
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  		Fig. 4. Effect of mutations between TM2 and TM3 of hPCFT on MDCK and Caco-2 cells. A and B: lateral (xy) and axial (xz) confocal sections of indicated hPCFT mutants in MDCK and Caco-2 cells, respectively. C: lateral (xy) confocal section of MDCK cells expressing hPCFT-YFP (top), hPCFT(DSVGRR109-114AAAAAA)-YFP (bottom), and DsRed-ER. YFP (left) and DsRed (middle) fluorescence channels were overlaid to generate a superimposed composite image (right). D: measurements of [3H]folic acid uptake by stable cell lines expressing the indicated constructs grown in filter supports. [3H]folic acid was introduced to either the apical or the basolateral side of the chamber. Results are expressed as the means ± SE of uptake values obtained from multiple inserts from at least three independent experiments. E: flow cytometry analysis of the mean fluorescence intensity of population of MDCK cells stably expressing indicated constructs.

 
Taken together, these results suggest a correlation between mutations that abrogate formation of the β-turn and efficient delivery of hPCFT to the cell surface. Disruption of the β-turn structure likely prevents proper protein folding and results in endoplasmic reticulum retention of the mutant protein.

Microtubule disruption prevents steady-state apical expression of hPCFT. The role of the microtubule network in the apical membrane targeting of hPCFT in MDCK cells was examined. MDCK monolayers were incubated with the microtubule-depolymerizing drug nocodazole by using a protocol described previously (18, 32), after which hPCFT-YFP cDNA was transiently transfected in the continued presence of nocodazole (10 µM); the resulting construct distribution was subsequently imaged after 18–24 h (see MATERIALS AND METHODS). Nocodazole treatment disrupted the steady-state apical localization of hPCFT in the MDCK monolayer, producing a nonpolarized distribution of hPCFT-YFP across the entire cell surface (Fig. 5A).


Figure 5
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  		Fig. 5. Microtubule disruption inhibits steady-state expression of hPCFT in MDCK cells. A: microtubules were depolymerized by using nocodazole as described in MATERIALS AND METHODS, and hPCFT distribution was compared with parallel controls after 18–24 h. Representative images of control cells (left) and nocodazole-treated cells (right) are shown. B: lateral (xy; top) and axial (xz; bottom) confocal sections of MDCK cells transiently transfected with hPCFT-YFP alone and hPCFT-YFP and p50 together, imaged after 24 h.

 
Next, we investigated the role of the cytoplasmic dynein in regulating the polarized delivery of hPCFT to the apical membrane domain. Microtubules in well-differentiated epithelial cells are arranged longitudinally, with their minus ends oriented toward the apical plasma membrane. Cytoplasmic dynein, as a minus end-directed motor protein, binds to the microtubule and moves cargo toward the apical membrane. To impair dynein function, we expressed dynamitin (p50), which disrupts dynactin by binding to endogenous dynamitin subunits (25). MDCK monolayers were transiently transfected with either hPCFT-YFP alone or cotransfected with p50 and hPCFT-YFP, and they were imaged after 24 h. Overexpression of p50 disrupted the apical expression of hPCFT-YFP, which in cotransfected cells distributed throughout the apical, lateral, and basal membranes with more intracellular fluorescence compared with control cells (Fig. 5B). Both these results (nocodazole, p50 expression) demonstrate that the apical expression of hPCFT is critically dependent on an intact microtubule network in MDCK cells.

Differential cell surface targeting of hPCFT and hRFC in single polarized epithelial cells. Finally, in terms of relevance for vectorial transport of folate across polarized epithelial cells, we were interested in resolving the distribution of hPCFT and hRFC when coexpressed within single polarized cells. To do this, we fused hPCFT with a red fluorescent protein vector (DsRed-monomer-N1) and hRFC with GFP-N3 (30) and cotransfected both into MDCK cells. As shown in Fig. 6, when imaged after 24–48 h, hPCFT localized predominantly at the apical surface and hRFC localized basolaterally in the same cell. The differential targeting of both folate transporters to discrete membrane domains provides a potential route for transcellular folate transport across the renal epithelial monolayer.


Figure 6
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  		Fig. 6. Cellular localization of human reduced folate carrier (hRFC) and hPCFT in cotransfected MDCK cells. Lateral (xy; top) and axial (xz; bottom) confocal sections of MDCK cells transiently cotransfected with hRFC-GFP (left) and hPCFT-DsRed (middle) and a dual channel overlay (right) are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The protein product SLC46A1 (HUGO nomenclature) has recently been identified as a pH-independent intestinal heme transporter [heme carrier protein 1 (HCP-1) (27)] and independently as a PCFT (26) [there is a little structural similarity between the folate and the heme molecules, with the exception that both molecules possess two ionized (negatively charged) carboxyl groups]. Strong support for the proposal that folates comprise the major physiological substrates for this transporter is provided by studies of patients with the rare disorder of hereditary folate malabsorption that reveals several discrete mutations within the SLC46A1 gene that segregate with the disease phenotype in individual families (26, 37); it is also indicated by the significantly higher affinity to folate (apparent Km: 1.3 µM) compared with heme (apparent Km: 125 µM) (26, 27). Nonetheless, the reported differences in substrate specificity (indeed, the transporter may possess dual transport capabilities), regulation, and topology underscore the need for further studies of the cell biology of HCP-1/PCFT (referred to hereafter as PCFT) in epithelia, where it is endogenously expressed.

Here we have used a live cell imaging approach to resolve the targeting and functionality of PCFT in intestinal (Caco-2) and renal (MDCK) epithelial cell lines that have been widely used as in vitro models for polarized transport (31, 32). Both epithelia express endogenous PCFT protein (20, 26, 27; unpublished observations), and, notably, the majority of constitutive folate uptake in Caco-2 cells can be suppressed by PCFT knockdown (26). Here, we provide independent confirmation that overexpression of hPCFT enhances [3H]folic acid uptake at low pH (Fig. 1E) and, for the first time (by using a GFP-tagging approach) in living polarized cells, that hPCFT targets to the apical membrane domain (Fig. 1). That the apical localization of hPCFT [and, indeed, the FR{alpha} (17)] contrasts with the basolateral bias of the hRFC when expressed in the same cells immediately suggests a route for vectorial transport of folate derivatives across the epithelial monolayer. Further studies of hPCFT and FR{alpha} transport kinetics, substrate specificity, and tissue distribution will be needed to interpret the extent of the redundancy between these two apical folate transport proteins, which share very little homology (8%). Delivery of hPCFT to the apical cell surface domain was observed to be dependent on an intact microtubule network and the motor protein dynein, consistent with previous observations made for other transporters when expressed in these cell lines (31, 32).

As a logical first step in exploring regions of the hPCFT polypeptide important for targeting, we focused on the extreme COOH terminus of the polypeptide [cytoplasmic in both topology predictions (27, 37)] and the region between TM2 and TM3 [an extracellular (27) or intracellular loop (37), according to the alternate topology predictions]. The TM2-TM3 loop, which secondary structure algorithms predict forming a β-turn (Fig. 3), has been speculated to be important in the transport functionality of HCP-1 (27) and contains a residue (R113) that is the site of a charge-nullifying point mutation (R113S) identified recently in a patient with hereditary folate malabsorption (37). First, disruption of the short COOH region had no effect on the apical targeting or functionality of hPCFT. Although COOH-terminal sequence motifs are often important for the subcellular targeting and functionality of proteins (7, 15, 33, 35), including members of the major facilitator superfamily of transporters (31), truncation of this sequence did not affect hPCFT behavior. Second, the region separating TM2 and TM3 is clearly important for transporter functionality. Five distinct point mutants (R113S, G147R, S318R, R376W, and P425R) have recently been identified in hPCFT, with R113S residing within this region between TM2 and TM3. Zhao et al. (37) have recently shown that all five point mutants impair hPCFT functionality; however, there is currently little insight into the cell biological basis of the hPCFT dysfunction associated with each clinical mutant. As the authors acknowledge, the polyclonal antibody used for the initial studies of these constructs in the HeLa cell line (cervical adenocarcinoma) had insufficient sensitivity to detect either endogenously expressed protein or even overexpressed mutants associated with residual transport activity (G147R, P425R) (37). Here, we show that mutations that abolish the β-turn between TM2 and TM3 abolish cell surface targeting of hPCFT. This β-turn sequence is likely important for stabilizing the formation of a helical hairpin that facilitates membrane insertion of the TM domains during protein synthesis. It is likely that mutations that disrupt this structure will impair overall protein folding, leading to endoplasmic reticulum retention of mutant protein. The alanine substitution at residue 113 (the site of the clinical R113S mutation) did not excessively disrupt apical cell surface targeting of the mutant construct [hPCFT(R113A); data not shown] nor the predicted occurrence of a β-turn; this is also consistent with the importance of the β-turn for hPCFT stability and implies that transport functionality is impaired in this clinical mutant owing to loss of function rather than impaired protein targeting.

In conclusion, our studies provide a first step in understanding the structure-function relationship of the newly discovered HCP-1/hPCFT protein and underscore its importance as a novel, apically targeted folate transporter.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Department of Veterans Affairs, National Institutes of Health [DK-56061 and DK-58057 (to H. M. Said), DK-63750 (to V. S. Subramanian), and NS-046783 (to J. S. Marchant)], and a National Science Foundation Career Fellowship [0237946 (to J. S. Marchant)].


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. M. Said, Dept. of Veterans Affairs Medical Center-151, Long Beach, CA 90822 (e-mail: hmsaid{at}uci.edu)

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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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