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Am J Physiol Gastrointest Liver Physiol 293: G1155-G1165, 2007. First published October 11, 2007; doi:10.1152/ajpgi.00334.2007
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MUCOSAL BIOLOGY

Association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells

Hang Thi Thu Nguyen,1 Laetitia Charrier-Hisamuddin,1 Guillaume Dalmasso,1 Abel Hiol,2 Shanthi Sitaraman,1 and Didier Merlin1

1Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, Georgia; and 2Université Paul Cezanne, Equipe Biochimie Alimentaire, Faculte des Sciences et Techniques, Marseille, France

Submitted 23 July 2007 ; accepted in final form 8 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The transporter PepT1, apically expressed in intestinal epithelial cells, is responsible for the uptake of di/tripeptides. PepT1 is also expressed in nonpolarized immune cells. Here we investigated the localization of PepT1 in lipid rafts in small intestinal brush border membranes (BBMs) and polarized and nonpolarized cells, as well as functional consequences of the association of PepT1 with lipid rafts. Immunoblot analysis showed the presence of PepT1 in low-density fractions isolated from mouse intestinal BBMs, polarized intestinal Caco2-BBE cells, and nonpolarized Jurkat cells by solubilization in ice-cold 0.5% Triton X-100 and sucrose gradient fractionation. PepT1 colocalized with lipid raft markers GM1 and N-aminopeptidase in intestinal BBMs and Caco2-BBE cell membranes. Disruption of lipid rafts with methyl-β-cyclodextrin (MβCD) shifted PepT1 from low- to high-density fractions. Remarkably, we found that MβCD treatment increased PepT1 transport activity in polarized intestinal epithelia but decreased that in intestinal BBM vesicles and nonpolarized immune cells. Mutational analysis showed that phenylalanine 293, phenylalanine 297, and threonine 281 in transmembrane segment 7 of the human di/tripeptide transporter, hPepT1, are important for the targeting to lipid rafts and transport activity of hPepT1. In conclusion, the association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells.

brush border; Caco2-BBE; mice

AN IMPORTANT FUNCTION OF intestinal epithelial cells (IECs) is the absorption of small peptides from the diet by the apical membrane oligopeptide transporter PepT1 (1, 2, 26, 40). Human PepT1 (hPepT1), which mediates this peptide transport activity (6, 8, 9), cotransports peptides with H+ (12, 17, 21, 24) and has a broad specificity including many di/tripeptides and peptide-derived drugs (42, 43). Functional expression of hPepT1 in Xenopus laevis oocytes induces H+-dependent peptide-transport activity, therefore recapitulating the transport activity observed in natural gut epithelia (30, 40). The hPepT1 protein expressed in the acidic environment of polarized IECs exhibits optimal transport activity under low extracellular pH conditions. hPepT1 is appropriately expressed in the apical membrane of the human intestinal epithelial cell line Caco2 (5, 6, 25, 26), which can be used as an in vitro model for biological analyses of this transporter. The cDNA encoding hPepT1 is 2,263 base-pair (bp) long, with an open reading frame of 2,127 bp, and encodes a 708-amino acid protein with a predicted molecular mass of 78 kDa. The hydropathy plot of hPepT1 indicates that there are 12 putative membrane-spanning domains (21, 40).

PepT1 is mainly expressed in brush border membranes (BBMs) of enterocytes in small intestine, in proximal tubular cells of the S1 segment of kidney, and in bile duct epithelial cells (1, 9, 17, 34, 35, 38). Expression of PepT1 mRNA or protein is low (46) or not detected (25, 34) in the colon but is transiently expressed in normal rat colon cells during the first few days after birth (35). Interestingly, we have recently demonstrated that hPepT1 is also expressed in immune cells (6). Furthermore, we have shown that the characteristics of hPepT1 transport activities differ depending on whether hPepT1 is expressed in polarized cells such as enterocytes or in nonpolarized cells such as immune cells (6).

Lipid rafts, defined as cholesterol/sphingolipids enriched membrane microdomains that can be isolated by their resistance to nonionic detergent solubilization at cold, may provide specialized lipid environments that are understood to regulate the organization and function of many plasma membrane proteins (4, 37). In polarized cells, lipid rafts are predominantly localized in the apical domains. The BBM of small intestinal enterocytes is a rich source of lipid rafts, and there is an increasing body of evidence showing that some digestive enzymes, trafficking molecules, and signaling proteins are present in lipid rafts. Indeed, the major brush border digestive enzymes, including sucrase and isomaltase, and several other types of membrane proteins have been found in microvillar lipid rafts. These include peripheral membrane proteins such as annexin A2 (15), annexins IV and XIIIb, glutamate receptor, guanine nucleotide binding protein G{alpha}q and G{alpha}11, G protein-coupled receptor 7 (29), as well as galectin-4 (11), the epithelial sodium channel (16), melanotransferrin-a glycosylphosphatidyl inositol-linked iron receptor (10), prominin (32), and stomatin (39). A component of the ileal villus cell BBM, the Na+/H+ exchanger NHE3, was also found to be partially associated with lipid rafts (20) and its activity and trafficking is lipid raft dependent (28). A functional coupling between hPepT1 and NHE3 has previously been shown (44). It is therefore reasonable to hypothesize that hPepT1 could also be present in lipid rafts. Lipid rafts are also found in nonpolarized cells that express hPepT1. In contrast to polarized cells, under resting conditions, lipid rafts are not segregated into a particular membrane domain in nonpolarized cells (23, 37). Membrane transporters associated with lipid rafts in polarized and nonpolarized cells might therefore have different functional characteristics. In the present study, we investigated the presence of PepT1 in the lipid rafts in small intestinal BBMs and polarized and nonpolarized cells, as well as functional consequences of the association of PepT1 with lipid rafts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All experiments were carried out using male C57BL6 mice (6 wk, 12–14 g) obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were group housed in standard cages under a controlled temperature (25°C) and photoperiod (12:12-h light-dark cycle) and were given standard chow and tap water ad libitum. Mice were allowed to acclimate to these conditions for at least 7 days before inclusion in experiments. All animal procedures were approved by the Animal Care Committee of Emory University and were conducted in accordance to the Guide for the Care of Use of Laboratory Animals from the US Public Health Service.

Cell culture. The human enterocyte-like Caco2-BBE cell line and Chinese hamster ovary (CHO) cells [from the American Type Culture Collection (ATCC)] were grown in high-glucose Dulbecco's Vogt-modified Eagle's medium (DMEM, Invitrogen) supplemented with 14 mmol/l NaHCO3, 10% (vol/vol) heat-inactivated fetal bovine serum (FBS, GIBCO-BRL), and 1.5 µg/ml plasmocin (Invitrogen). Cells were kept at 37°C in 5% CO2 and 90% humidity. Caco2-BBE cells were grown on filters (area 1 cm2; pore size 0.4 µm; Transwell-Clear polyester membranes, Costar). CHO cells were grown on plastic flasks (Costar). The human T-lymphoblastoid Jurkat E6-1 cell line was obtained from the ATCC. The cells were propagated in RPMI-1640 (GIBCO-BRL) supplemented with 10% FBS and 1.5 µg/ml plasmocin.

Isolation of BBMs and lipid rafts from mouse small intestine. BBMs and lipid rafts were isolated as previously described (29). Briefly, small intestine from 6-wk C57BL6 male mice was rinsed and mucosa was scraped in ice-cold homogenization buffer (HB) containing 20 mM Tris·HCl pH 7.4, 150 mM NaCl, 5% sucrose and protease inhibitor mixture (Roche Diagnostics, Mannheim, Germany). Unless otherwise stated, all experiments were performed at 4°C. Mucosa (4 g) was homogenized in 8 vol (wt/vol) of HB using a Potter-Elvehjem homogenizer. The homogenate was filtered and centrifuged at 1,000 rpm for 5 min. The resulting supernatant was centrifuged at 10,000 rpm for 10 min, and the recovered supernatant containing 2 mg of protein was incubated with 10 mM CaCl2 or MgCl2 for 10 min and then adjusted to 40% sucrose in buffer containing 20 mM Tris·HCl pH 7.4, 1 mM MgCl2 in a centrifuge tube. The tube was overlaid with 0.5 vol of 5% sucrose in the same buffer. After centrifugation at 40,000 rpm for 1 h in a Sorval SW 41 Ti rotor (Beckman Coulter), the floating membrane at the interface, defined as BBMs, was collected.

To isolate lipid rafts, the purified BBMs were incubated with 0.5% Triton X-100 (TX-100) for 30 min on ice. The extract containing 2 mg of protein was adjusted to 40% sucrose in 3 ml of buffer containing 20 mM Tris·HCl pH 7.4, 1 mM MgCl2 in a 12-ml centrifuge tube. Discontinuous gradients were prepared by overlaying the tube with 2 vol of 30% sucrose followed by 1 vol of 5% sucrose in the same buffer. After centrifugation at 40,000 rpm for 18 h at 4°C in the Sorval SW 41 Ti rotor, 12 fractions (1 ml each) were harvested gently from the top of the gradients. Protein concentration was measured by using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Cholesterol content of the gradient fractions was enzymatically determined by the colorimetric method from Boehringer-Mannheim following manufacturer's instructions.

Isolation of lipid rafts from Caco2-BBE cells. Confluent Caco2-BBE cells were scraped from filters into ice-cold phosphate-buffered saline (PBS; Invitrogen) and centrifuged at 1,500 rpm for 5 min at 4°C. The cell pellet was resuspended and lysed for 20 min with ice-cold lysis buffer [1% TX-100 in Tris-NaCl-EDTA (TNE) buffer (25 mM Tris·HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, protease inhibitors)]. Cells were then disrupted by passing through a 25-gauge needle 30 times. The resulting cell extract containing 2 mg of protein was adjusted to 40% sucrose in TNE buffer, placed in the 12-ml centrifuge tube, overlaid with 2 vol of 30% sucrose followed by 1 vol of 5% sucrose both in TNE buffer. After centrifugation at 40,000 rpm for 18 h at 4°C, gradient fractions were collected as described above.

Miscellaneous procedures. For cholesterol depletion, the mucosa scraped from mouse intestine was treated with 10 mM methyl-β-cyclodextrin (MβCD) in HB for 30 min at 37°C as previously described (29). Caco2-BBE monolayers were apically incubated with 10 mM MβCD in FBS-free DMEM for 30 or 60 min at 37°C. For cholesterol replenishment, MβCD-treated mucosa or MβCD-treated Caco2-BBE monolayers were incubated with 2 mM water-soluble cholesterol in HB or DMEM for 30 min at 37°C, respectively.

Western blot analysis. Equal amounts (15 µg) of total protein from the gradient fractions were analyzed by SDS-PAGE on 10% polyacrylamide gels (Bio-Rad) and then transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBST) and then incubated for 1 h at room temperature with rabbit polyclonal antibody to N-aminopeptidase (NAP; Santa Cruz Biotechnology, CA), or mouse polyclonal antibody raised against mouse or human PepT1 (6, 25). Control experiments were performed using goat polyclonal antibody to villin (Santa Cruz Biotechnology) or rabbit polyclonal antibody to human monocarboxylate transporter 1 (MCT-1) (Alpha Diagnostic, San Antonio, TX). After washes with PBST, membranes were incubated for 1 h at room temperature with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody: anti-rabbit IgG (Amersham, Piscataway, NJ) or anti-goat IgG (Santa Cruz Biotechnology). Membranes were then washed, and immunoreactive proteins were detected by use of an enhanced chemiluminescence (ECL) detection kit according to the manufacturer's instructions (Amersham, Piscataway, NJ). The band intensities of the Western blots were quantified by using a gel documentation system (Alpha Innotech, San Leandro, CA).

To detect ganglioside GM1 in the gradient fractions, 2 µg of protein from each fraction was spotted on nitrocellulose membranes. Membranes were blocked, washed as described above for Western blot analysis, and then incubated with HRP-conjugated cholera toxin B (CTB; Sigma, St. Louis, MI). Blots were developed using ECL detection kit.

Plasmid construction and transfections. The hPepT1/pEGFP-C3 plasmid (Clontech, Palo Alto, CA) constructed as previously described (25) was used as the template to generate hPepT1/pEGFP-C3 construct variants.

The F293A-hPepT1/pEGFP-C3 mutant was generated by changing the codon at 934–936 (TTC->GCC) position from phenylalanine (F) to alanine (A) using specific primers (forward, 5'-TT CCA CTC CCC ATG GCC TGG GCC TTG-3'; reverse, 5'-CA AGG CCC AGG CCA TGG GGA GTG GAA-3'). (Codon positions are based on NCBI AF 043233 numbering; 1st met start of translation = 57.) The F297A-hPepT1/pEGFP-C3 mutant was generated by changing the codon at 945–947 (TTT->GCT) position from phenylalanine to alanine using specific primers (forward, 5'-G TTC TGG GCC CTG GCT GAC CAG CAG-3'; reverse, 5'-C TGC TGG TCA GCC AGG GCC CAG AAC-3'). The T281A-hPepT1/pEGFP-C3 mutant was generated by changing the codon at 897–899 (ACG->GCC) position from threonine (T) to alanine using specific primers (forward, 5'-T AAG ATG GTT GCC CGG GTG ATG TT-3'; reverse, 5'-A ACA TCA CCC GGG CAA CCA TCT TA-3'). The L296A-hPepT1/pEGFP-C3 mutant was generated by changing the codon at 942–944 (TTG->GCG) position from leucine (L) to alanine using specific primers (forward, 5'-TG TTC TGG GCT GCG TTT GAC CAG-3'; reverse, 5'-CT GGT CAA ACG CAG CCC AGA ACA-3') by site-directed mutagenesis using PCR-mediated overlap extension to facilitate fusion of the DNA sequence by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The constructed plasmids were verified by sequencing.

Subconfluent CHO cells were plated 24 h prior to transfection by using Lipofectin (Invitrogen, Carlsbad, CA) in Opti-MEM I serum-reduced medium (Invitrogen) for 20 h. Serum was added for the subsequent 48 h, and transfectants were selected in culture medium supplemented with 1.2 mg/ml Geneticin (Sigma). Clones with the highest levels of wild-type (WT) hPepT1-GFP, mutated hPepT1-GFP and GFP expression as shown by Western blot analysis were selected for this study.

Pictures of stable transfected CHO cells were taken on live cells via inverted fluorescent microscope (Nikon Eclipse TS100).

Uptake experiments. Caco2-BBE cells were grown on filters and used 14 days postconfluency. Caco2-BBE monolayers were washed twice with Hanks' balanced salt solution (HBSS; Sigma) supplemented with 10 mM HEPES (pH 7.2) in the basolateral compartment, and with HBSS-10 mM 2-[N-Morpholine] ethanesulfonic acid (MES) (pH 5.2 or pH 6.2) or HBSS-10 mM HEPES (pH 7.2) in the apical compartment, and stabilized in the same buffers for 15 min at 37°C. Cells were then incubated with HBSS-10 mM (MES) (pH 5.2 or pH 6.2) or HBSS-10 mM HEPES (pH 7.2) containing 20 µM [14C]Gly-Sar (specific activity of 50 mCi/mM, American Radiolabeled Chemicals, St. Louis, MO) ± 20 mM Gly-Leu in the apical compartment, and with HBSS-10 mM HEPES (pH 7.2) in the basolateral compartment for 15 min at room temperature. Filters were washed twice with ice-cold PBS and cut. Radioactivity was determined by using a β-counter (1219 Rackbeta, Wallac, Gaithersburg, MD).

The MCT-1-mediated butyrate uptake in Caco2-BBE monolayers was performed as described above except that cells were incubated with HBSS-10 mM MES (pH 6.2) containing 20 µM [14C]butyrate (specific activity of 16 mCi/mmol, Sigma) ± 1 mM {alpha}-cyano-4-hydroxycinnamate (CHC) in the apical compartment, and with HBSS-10 mM HEPES (pH 7.2) in the basolateral compartment for 1 h at room temperature.

Uptakes of [14C]Gly-Sar in CHO cells stably transfected with WT hPepT1-GFP and its construct variants were performed using confluent cells grown on 12-well plastic plates (Costa). Cells were washed, stabilized, and incubated with HBSS-10 mM MES (pH 6.2) containing 20 µM [14C]Gly-Sar ± 20 mM Gly-Leu for 15 min at room temperature. Total radioactivity was measured as described above.

For Jurkat cells, 5 x 106 cells were used per assay. Cells were pretreated with or without 10 mM MβCD in FBS-free RPMI for 30 min at 37°C. Cells were washed twice with PBS, stabilized in HBSS-10 mM HEPES (pH 7.2) for 15 min at 37°C, and then incubated in the same buffer containing 20 µM [14C]Gly-Sar ± 20 mM Gly-Leu for 1 h at room temperature. Cells were then washed twice and resuspended in ice-cold PBS, and total radioactivity was measured as described above.

Specific uptake of [14C]Gly-Sar mediated by PepT1 was calculated as follows: (uptake of [14C]Gly-Sar) – (uptake of [14C]Gly-Sar + Gly-Leu). Specific uptake of [14C]butyrate mediated by MCT-1 was calculated as follows: (uptake of [14C]butyrate) – (uptake of [14C]butyrate + CHC).

Preparation of BBMVs and in vivo uptake experiments. BBM vesicles (BBMVs) were prepared from intact mucosa, MβCD-treated mucosa, or mucosa pretreated with MβCD and then replenished with cholesterol by a magnesium-precipitation technique as previously described (8) with some modifications. All experiments were performed at 4°C. Briefly, mucosa was homogenized in a buffer containing 60 mM mannitol, 12 mM Tris·HCl pH 7.4, 10 mM EGTA, and protease inhibitor mixture. The homogenate was centrifuged at 3,000 g for 15 min (step 1). The resulting supernatant was incubated with 10 mM MgCl2 for 15 min and centrifuged at 27,000 g for 30 min (step 2). The pellet was resuspended in 35 ml of the homogenization buffer. Steps 1 and 2 were repeated and the resulting pellet was homogenized in 10 ml of preloading buffer (100 mM KCl, 100 mM mannitol, 20 mM HEPES-Tris pH 7.4, protease inhibitor mixture). The final suspension was centrifuged at 27,000 g for 30 min. The purified BBMVs were resuspended in the preloading buffer at a protein concentration of 10 mg/ml. The BBMVs were stored in liquid nitrogen until further use.

In vivo uptake experiments were performed by a rapid filtration technique using Millipore filters (HAWP type, 0.45-µm pore size) as described previously (27). Uptake of [14C]Gly-Sar was monitored in 200 µl of transport buffer (HBSS-10 mM MES pH 6.2, 100 mM mannitol) containing 300 µg of protein from BBMVs and 20 µM [14C]Gly-Sar ± 20 mM Gly-Leu. The mixture was incubated at room temperature for 10 s and terminated by an addition of 5 ml ice-cold stop solution (2 mM HEPES-Tris pH 7.4, 210 mM KCl) followed by filtration. Filters were washed twice with 5 ml of stop solution and the radioactivity was determined by use of a β-counter. The difference in [14C]Gly-Sar uptake values, in picomoles of [14C]Gly-Sar per milligram protein per second, measured in the presence and absence of 20 mM Gly-Leu, was defined as the [14C]Gly-Sar-specific uptake mediated by hPepT1.

Measurement of surface hPepT1. Total amount of hPepT1 on the apical surface of Caco2-BBE cells was determined by cell-surface biotinylation (25) and modified ELISA (28). Caco2-BBE cells were grown on filters and used 14 days postconfluency. Cells were treated with or without 10 mM MβCD in the apical compartment for 30 min at 37°C.

For biotinylation, cells were washed twice with PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 pH 7.4 (PBS-Ca/Mg). Apical or basolateral filter compartments were incubated with 150 µl or 500 µl, respectively, of 1 mg/ml sulfo-NHS-biotin (Pierce, Rockford, IL) in PBS-Ca/Mg for 30 min on ice. The reaction was quenched with ice-cold 50 mM NH4Cl for 5 min and cells were lysed with lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, 5 mM EDTA, 1% TX-100, 0.2% BSA, and protease inhibitor mixture). After 30-min incubation on ice, the protein extract was collected by centrifugation at 13,000 rpm for 30 min at 4°C. The protein solution was then incubated with Neutravidin-agarose beads (Pierce) overnight at 4°C to bind biotinylated proteins. Beads were recovered by centrifugation at 12,000 g for 20 s. The resulting supernatant was retained as the intracellular fraction. Neutravidin-agarose beads were washed twice with PBS, once with washing buffer (500 mM NaCl, 20 mM Tris pH 8.0, 0.5% TX-100, 0.2% BSA), and then once more with PBS. Finally, biotinylated proteins were recovered from beads in Laemmli buffer by boiling at 100°C for 5 min. Intracellular and surface fractions were separated by 10% SDS-PAGE and analyzed by Western blot.

For ELISA, cells were incubated with anti-hPepT1 antibody or isotype IgG1 for 1.5 h at 4°C, washed six times with cold growth medium-PBS (1:9, vol/vol), and fixed with PBS containing 3% paraformaldehyde (Electron Microscopy Sciences, Washington, PA) for 10 min at room temperature. Cells were then washed three times with PBS, incubated with PBS containing 100 mM glycine for 15 min at room temperature, and blocked with PBS containing 5% FBS and 1% BSA for 30 min at room temperature. Cells were then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and washed six times with growth medium-PBS. For detection of peroxidase activity, cells were incubated with 0.5 ml of 0.4 mg/ml o-phenylenediamine dihydrochloride in 0.05 M phosphate-citrate buffer (25.7 ml of 0.2 M Na2HPO4, 24.3 ml of 0.1 M citric acid, pH 5.0) containing 0.03% H2O2 for 15 min at room temperature in the dark. The reaction was stopped by addition of 0.75 M HCl. Supernatants were collected and absorbance at 492 nm was measured.

Immunofluorescence staining. Caco2-BBE cells were grown on filters and used postconfluency. Caco2-BBE monolayers were washed twice with PBS-Ca/Mg and stained with 5 µg/ml Alexa Fluor 594-conjugated CTB (Invitrogen) for 2 h at 4°C. After GM1 labeling, cells were fixed with 4% paraformaldehyde in PBS-Ca/Mg for 15 min at room temperature and washed three times with PBS-Ca/Mg. Cells were then blocked with PBS-Ca/Mg containing 0.1% TX-100 and 3% BSA for 30 min at room temperature and incubated with anti-PepT1 antibody for 1 h at room temperature. After washes with PBS-Ca/Mg, cells were incubated with an Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) for 1 h at room temperature. After washes with PBS-Ca/Mg, filters were mounted by use of Slowfade kit (Molecular Probes). Negative controls were performed in the same condition except for the omission of the primary antibody. Microscopy was performed using a Zeiss epifluorescence microscope equipped with a Bio-Rad MRC600 confocal unit, computer, and laser scanning microscope image analysis software (Carl Zeiss, Jena, Germany).

Statistical analysis. Values are expressed as means ± SE. Statistical analysis was performed using unpaired Student's t-test. A P value <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PepT1 is present in the lipid raft fractions isolated from mouse intestinal BBMs. Lipid rafts were isolated from purified mouse intestinal BBMs as previously described (29). After 0.5% TX-100 solubilization at cold and sucrose-gradient fractionation, homogenous floating membranes (lipid raft-like membrane) were recovered at fractions 4–5. Cholesterol content of the gradient fractions was determined (Fig. 1A). As expected, low-density fractions (LDFs, fractions 4–5) were significantly enriched in cholesterol (4). As shown by immunoblot and dot-blot analyses, high amounts of PepT1 and known intestinal lipid raft markers, NAP and GM1, were found in LDFs (Fig. 1B). Densitometric analysis of PepT1 immunoblots showed that ~48% of membrane PepT1 was present in LDFs (data not shown). In contrast, villin, previously reported to be tightly associated with BBMs (14), was not found in LDFs and was only found in HDFs (Fig. 1B). Therefore villin was defined here as the non-lipid raft marker of intestinal BBMs.


Figure 1
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  		Fig. 1. PepT1 is present in lipid raft fractions isolated from mouse intestinal brush border membranes (BBMs). Mouse intestinal mucosa was pretreated with or without 10 mM methyl-β-cyclodextrin (MβCD) for 30 min at 37°C. BBMs prepared from native or MβCD-treated mucosa were used for lipid raft isolation by solubilization in ice-cold 0.5% Triton X-100 (TX-100) and sucrose-gradient fractionation. A: MβCD treatment reduced cholesterol content of gradient fractions (1-ml each) by ~70%. Values represent means ± SE of 4 independent experiments. *P < 0.05 vs. control; **P < 0.005 vs. control. B: equal amounts of total protein (15 µg) from the gradient fractions were separated by 10% SDS-PAGE and analyzed by Western blot using antibodies against N-aminopeptidase (NAP), PepT1, and villin. The distribution of GM1 in the gradient fractions was determined using 2 µg of protein of each fraction and horseradish peroxidase (HRP)-conjugated cholera toxin B (CTB). LDF, low-density fraction; HDF, high-density fraction.

 
We then examined the effect of cholesterol depletion on the distribution of lipid raft-resident proteins in the gradient fractions. Pretreating mouse mucosa with 10 mM MβCD for 30 min at 37°C decreased cholesterol content of LDFs by 70% (Fig. 1A). As shown by immunoblot analysis, cholesterol depletion caused a complete shift of PepT1 from LDFs 4–5 to HDFs 9–12. NAP and GM1 were also partially or completely shifted from LDFs (Fig. 1B). Together, these results strongly suggest that PepT1 localizes in lipid raft-like membranes of mouse intestinal brush border.

hPepT1 is present in the lipid raft fractions isolated from polarized Caco2-BBE monolayers. Caco2-BBE cells, a commonly used polarized epithelial cell line expressing a well-developed brush border, were used for the isolation of lipid rafts. After sucrose-density gradient fractionation, low-density floating fraction 4 was considered as the lipid raft fraction because of the colocalization of NAP and GM1 (Fig. 2). Immunoblot analysis showed that ~40% of membrane hPepT1 was present in LDFs. In contrast, the non-lipid raft marker villin was not found in LDFs and was only found in HDFs (Fig. 2). MβCD treatment resulted in a complete shift of hPepT1 from lipid raft fractions 4–5 to HDFs 8 and 12. NAP and GM1 were also partially or completely removed from LDFs 4–5 upon MβCD treatment (Fig. 2). Together, these results suggest that hPepT1 is present in lipid rafts in Caco2-BBE monolayers.


Figure 2
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  		Fig. 2. Human PepT1 (hPepT1) is present in lipid raft fractions isolated from polarized Caco2-BBE monolayers. Caco2-BBE cells were grown on filters and used 14 days postconfluency. Cells pretreated with (+) or without (–) 10 mM MβCD for 30 min at 37°C were lysed with ice-cold lysis buffer containing 1% TX-100 and subjected to sucrose-gradient fractionation. Equal amounts of total protein (15 µg) from the gradient fractions were separated by 10% SDS-PAGE and analyzed by Western blot using antibodies against hPepT1, NAP, and villin. The distribution of GM1 in the gradient fractions was determined using 2 µg of protein of each fraction and HRP-conjugated CTB as described in MATERIALS AND METHODS.

 
PepT1 colocalizes with GM1 in polarized Caco2-BBE cell membranes and intestinal BBMs. To evaluate the distribution of PepT1 and its possible colocalization with GM1, we performed immunofluorescence staining, followed by scanning confocal microscopy analysis. Caco2-BBE monolayers were stained with CTB, which binds to GM1, and then with hPepT1 antibody. Analyses of GM1 expression and distribution in Caco2-BBE monolayers (Fig. 3A) revealed an uneven staining of GM1 over the cell surface, similar to the pattern of hPepT1 fluorescence. A merged image of two distribution patterns clearly revealed orange areas, resulting from the overlap of green and red fluorescences representing hPepT1 and GM1, respectively, which correspond to colocalization areas. Furthermore, we examined the immunoprecipitation of hPepT1 with GM1 in Caco2-BBE cells by exposure of hPepT1 and IgG1 immunoprecipitates to HRP-conjugated CTB. We found that hPepT1 immunoprecipitates but not IgG1 immunoprecipitates exhibited immunoreactivity, accounting for the presence of GM1 (Fig. 3B). Lipid raft fraction 4, used as a positive control, also displayed immunoreactivity (Fig. 3B). In addition, we showed the specificity of hPepT1 immunoprecipitates by immunoblot analysis (Fig. 3C). Similar results were obtained for PepT1 immunoprecipitates from mouse intestinal BBMs (Fig. 3B). Together, these results indicate that PepT1 and GM1 colocalize at the plasma membrane of polarized Caco2-BBE cells and intestinal BBMs, strongly supporting the finding that hPepT1 is associated with GM1-enriched lipid raft microdomains.


Figure 3
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  		Fig. 3. PepT1 colocalizes with GM1 in Caco2-BBE cell membranes and mouse small intestinal brush border membranes (SI BBMs). A: Caco2-BBE monolayers were stained with Alexa Fluor 594-conjugated CTB for 2 h at 4°C. After GM1 labeling, cells were fixed, blocked, and incubated with anti-hPepT1 antibody and then with Alexa Fluor 488-conjugated secondary antibody both for 1 h at room temperature. Cells were imaged by confocal laser-scanning microscopy. Horizontal sections of Caco2-BBE monolayers were taken. Confocal images of separated channels show the localization of hPepT1 (green channel) and GM1 (red channel) in Caco2-BBE cell membranes. Merged images indicate the colocalization (orange areas) of hPepT1 and GM1 in the apical membrane of Caco2-BBE cells. B: Caco2-BBE cell lysate and SI BBMs were used for immunoprecipitation (IP) using anti-PepT1 antibody or isotype IgG1. Detection of GM1 in IP eluates and the lipid raft fraction was performed using HRP-conjugated CTB. C: equal amounts of total protein (15 µg) from Caco2-BBE cell lysate, hPepT1, or IgG1 immunoprecipitates were separated by 10% SDS-PAGE and analyzed by Western blot using anti-hPepT1 antibody.

 
MβCD does not affect the surface amount of hPepT1 and the barrier function in Caco2-BBE cells. To examine whether MβCD treatment affects the amount of hPepT1 in the apical membrane of Caco2-BBE cells, we determined the surface amount of hPepT1 in cells apically treated with or without 10 mM MβCD for 30 min at 37°C, using protein biotinylation and modified ELISA. ELISA results show that MβCD treatment did not affect the surface amount of hPepT1 (107.68% ± 1.54% of control cells, nonsignificant, Fig. 4A). As a negative control, the surface expression of isotype IgG1 was not significantly detected (Fig. 4A). Representative immunoblots of surface and intracellular hPepT1 are shown in Fig. 4B, and densitometric analysis of hPepT1 immunoblots is shown in Fig. 4C. Similar 100-kDa immunoreactive bands corresponding to hPepT1 were detected in the surface fractions from untreated and MβCD-treated Caco2-BBE monolayers. A low amount of hPepT1 was also found in the cytoplasm of untreated and MβCD-treated Caco2-BBE cells at similar levels (Fig. 4, B and C).


Figure 4
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  		Fig. 4. Treatment of Caco2-BBE monolayers in the apical compartment with 10 mM MβCD for 30 min at 37°C does not affect the surface amount of hPepT1. A: surface amount of hPepT1 in untreated or MβCD-treated Caco2-BBE monolayers quantified by ELISA. Cells were incubated with anti-hPepT1 antibody or isotype IgG1 for 1.5 h at 4°C. Cells were washed, fixed, blocked, and then incubated with HRP-conjugated secondary antibody. Peroxidase activity bound to surface hPepT1 was quantified as described in MATERIALS AND METHODS. Data are means ± SE of 4 independent experiments with the control set at 100%. B: representative immunoblots of surface or intracellular hPepT1. The biotinylated surface and cytoplasm fractions containing equal amounts of total protein (10 µg) from untreated and MβCD-treated cells were analyzed by Western blot using anti-hPepT1 antibody. C: densitometric analysis of surface and intracellular hPepT1 immunoblots. Values represent the means ± SE of 3 analyses.

 
Furthermore, we found that transepithelial resistance (TER), an indicator of the barrier function, of Caco2-BBE monolayers apically treated with 10 mM MβCD for 30 and 60 min (351.44 ± 16.75 and 327.11 ± 18.02 {Omega}·cm2, respectively) were not significantly decreased compared with TER of untreated Caco2-BBE monolayers (350.78 ± 36.37 {Omega}·cm2). These data suggest that, under our experimental conditions, MβCD treatment does not affect the asymmetric distribution of plasma membrane components that characterizes distinct apical and basolateral membranes and maintains the intestinal epithelial barrier function.

Association of hPepT1 with lipid rafts decreases its transport activity in polarized Caco2-BBE cells. To examine whether the association of hPepT1 with lipid rafts affects its transport activity, we measured the rate of [14C]Gly-Sar specific uptake in polarized Caco2-BBE cells treated with or without MβCD. We found that treatment of Caco2-BBE monolayers with 10 mM MβCD for 30 min resulted in a complete removal of hPepT1 from lipid raft fractions 4–5 (Fig. 5A). As shown in Fig. 5B, hPepT1 transport activity at physiological pH 6.2 was significantly increased by ~156 and ~190% when Caco2-BBE monolayers were treated with MβCD for 30 and 60 min, respectively. Cholesterol replenishment of 30-min and 60-min MβCD-treated cells reduced hPepT1 activity in these cells by ~34 and ~29%, respectively (Fig. 5B). The decrease of hPepT1 transport activity upon cholesterol replenishment is due to the shift of hPepT1 from HDFs 9–12 back to lipid raft fraction 4 (Fig. 5A). In contrast, MβCD treatment did not affect the distribution (Fig. 5C) and transport activity (Fig. 5D) of the membrane transporter MCT-1 that was not found in lipid raft fractions from Caco2-BBE monolayers. Together, these results indicate that the delocalization of hPepT1 from lipid rafts increases its transport activity in polarized intestinal epithelia.


Figure 5
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  		Fig. 5. Methyl-β-cyclodextrin (MβCD) treatment increases hPepT1 transport activity in polarized Caco2-BBE cells. Cells were grown on filters and used 14 days postconfluency. Cells were apically treated with or without 10 mM MβCD for 30 or 60 min at 37°C. For cholesterol replenishment, MβCD-treated cells were incubated with 2 mM cholesterol in FBS-free DMEM for 30 min at 37°C. A: immunoblot analysis showed a shift of hPepT1 from LDFs to HDFs upon MβCD treatment and its return to LDFs upon cholesterol replenishment. C: immunoblot analysis showed the absence of MCT-1 in LDFs. MβCD treatment did not affect MCT-1 distribution in the gradient fractions. B and D: uptake of 20 µM [14C]Gly-Sar ± 20 mM Gly-Leu (B) and uptake of 20 µM [14C]butyrate ± 1 mM {alpha}-cyano-4-hydroxycinnamate (D) in Caco2-BBE monolayers at apical pH 6.2 and basolateral pH 7.2 for 15 min at room temperature. E: uptake of 20 µM [14C]Gly-Sar ± 20 mM Gly-Leu at different apical pH (5.2, 6.2, or 7.2) and basolateral pH 7.2 in Caco2-BBE monolayers for 15 min at room temperature. Values represent means ± SE of 3 determinations. *P < 0.05 vs. control; **P < 0.005 vs. control.

 
pH dependency of hPepT1 activity is lipid raft independent. It is known that the activity of hPepT1 expressed in the apical plasma membrane of intestinal epithelial cells is optimal at approximately pH 6.2 (6). Here, we demonstrated that hPepT1 transport activity in Caco2-BBE monolayers at pH 5.2, 6.2, or 7.2 was significantly increased upon 10 mM MβCD treatment for 30 min (Fig. 5E). Furthermore, hPepT1 transport activity in MβCD-treated Caco2-BBE monolayers was maximal at pH 6.2 (Fig. 5E). Together, these results indicate that the dissociation of hPepT1 from lipid rafts upon MβCD treatment increases its transport activity but does not change its optimal working pH in polarized Caco2-BBE cells.

Association of PepT1 with lipid rafts increases its transport activity in mouse BBMVs. We next examined the effect of PepT1 association with lipid rafts on PepT1 transport function in mouse intestinal BBMs. BBMVs were prepared from intact mouse intestinal mucosa, MβCD-treated mucosa, or mucosa pretreated with MβCD and then replenished with cholesterol, and PepT1 transport activity in the BBMVs was determined. We found that treatment of mucosa with 10 mM MβCD for 30 min at 37°C induced a complete shift of PepT1 from LDFs 4–5 to HDFs 9–12 (Fig. 6A), and reduced PepT1 transport activity in BBMVs by ~62% (Fig. 6B). The reduction of PepT1 transport activity was partially reversed (~28% of control) by cholesterol replenishment (Fig. 6B), which induced a shift of PepT1 from HDFs back to the LDF 4 (Fig. 6A). Together, these results indicate that the dissociation of PepT1 from lipid rafts decreased its transport activity in BBMVs, which stands in contrast with what we found in polarized intestinal epithelia.


Figure 6
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  		Fig. 6. MβCD treatment decreases PepT1 transport activity in mouse BBM vesicles (BBMVs). Mouse intestinal mucosa was pretreated with or without 10 mM MβCD for 30 min at 37°C and subsequently incubated with 2 mM cholesterol for 30 min at 37°C. A: isolation of BBMs and lipid rafts from mucosa was performed as described in MATERIALS AND METHODS. Equal amounts of total protein (15 µg) from the gradient fractions were analyzed by Western blot using anti-PepT1 antibody. B: BBMVs were prepared from native mucosa, MβCD-treated mucosa, or mucosa treated with MβCD and then replenished with cholesterol. In vivo uptake of [14C]Gly-Sar in BBMVs was performed by using 300 µg of protein from BBMVs and 20 µM [14C]Gly-Sar ± 20 mM Gly-Leu in 200 µl of transport buffer (HBSS-10 mM MES pH 6.2, 100 mM mannitol) for 10 s at 25°C. Values represent means ± SE of 3 determinations. *P < 0.05 vs. control; **P < 0.005 vs. control.

 
Association of hPepT1 with lipid rafts in nonpolarized immune cells increases its transport activity. Recently, we showed that hPepT1 is expressed in the plasma membrane of nonpolarized cells such as immune cells (6). Using Jurkat cells as a model of nonpolarized immune cells, we demonstrated that the LDFs 4–5 extracted from Jurkat cells, defined as the lipid rafts, were enriched in GM1 and hPepT1 (~34% of membrane hPepT1) (Fig. 7A). Treatment of Jurkat cells with MβCD shifted hPepT1 from LDFs to HDFs and the removal of hPepT1 from lipid raft fractions was recovered by cholesterol replenishment (Fig. 7A). Furthermore, we showed that hPepT1 coimmunoprecipitates with GM1 in Jurkat cells (Fig. 7B). These results strongly suggest that hPepT1 localizes in the lipid rafts of nonpolarized immune cells.


Figure 7
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  		Fig. 7. MβCD treatment decreases PepT1 transport activity in nonpolarized immune cells. Jurkat cells (5 x 106 cells/ml) were treated with or without 10 mM MβCD for 30 min at 37°C, and subsequently incubated with 2 mM cholesterol for 30 min at 37°C. A: untreated or MβCD-treated cells were lysed and subjected to sucrose-gradient fractionation. Equal amounts of total protein (15 µg) from the gradient fractions were analyzed by 10% SDS-PAGE and Western blot using anti-hPepT1 antibody. B: Jurkat cell lysate was used for IP using anti-PepT1 antibody or isotype IgG1. Detection of GM1 in the gradient fractions (A) and IP eluates (B) was performed using HRP-conjugated CTB. C: uptake of [14C]Gly-Sar in Jurkat cells pretreated with or without MβCD. Cells were washed, stabilized with HBSS-10 mM HEPES (pH 7.2) for 15 min at 37°C, and incubated with the same buffer containing 20 µM [14C]Gly-Sar ± 20 mM Gly-Leu for 15 min at room temperature. Values represent means ± SE of 3 determinations. *P < 0.05 vs. control.

 
A pH value of 7.2 has been shown to be the optimal pH for hPepT1-mediated di/tripeptide uptake in nonpolarized immune cells (6). Here, we showed that hPepT1 transport activity in Jurkat cells at physiological pH 7.2 was significantly reduced by ~44% upon MβCD treatment (Fig. 7C). These results indicate that the association of hPepT1 with lipid rafts increases its transport activity in nonpolarized immune cells.

Transmembrane segment 7 of hPepT1 contains lipid raft sorting determinants. An important unanswered question in lipid raft research is how transmembrane proteins are targeted to lipid rafts. Recently, it has been demonstrated that the transmembrane helix 7 of hPepT1 is important for hPepT1 transport activity (19). Interestingly, it was found that mutation at F293, F297, and L296 in transmembrane segment 7 of hPepT1 dramatically decreased the transport activity without affecting the delivery to membrane of this transporter (19). In contrast, substitution of T281 did not decrease hPepT1 transport activity (19). It has also been hypothesized that some hydrophobic amino acid residues such as phenylalanine in transmembrane domains (TMDs) of transmembrane proteins may be important for their interaction with lipid rafts (41). To investigate the determinants for the association of hPepT1 with lipid raft, four alternative forms of the protein with site mutations in the TMD 7 were constructed: F293A-hPepT1-GFP, F297A-hPepT1-GFP, T281A-hPepT1-GFP, and L296A-hPepT1-GFP (Fig. 8A). The mutants were stably expressed in the CHO cell line. Fluorescent microscopy pictures taken on live cells showed that WT and mutated hPepT1-GFP mainly localize in the plasma membrane of stably transfected CHO cells, whereas GFP is evenly distributed throughout the cells (Fig. 8B and data not shown). The transfected CHO cells were used to isolate lipid rafts, and immunoblot analysis was performed using anti-hPepT1 antibody (Fig. 7C). Transport activity of WT or mutated hPepT1 was determined by measuring [14C]Gly-Sar-specific uptake in these CHO cells (Fig. 7D). We found that mutation at L296 did not affect the association of hPepT1 with lipid rafts (Fig. 8C, lanes WT and L296A) or its transport activity (Fig. 8D). However, mutation at F293 or F297 decreased the association of hPepT1 with lipid rafts (Fig. 8C, lanes F293A and F297A) as well as significantly reduced its transport activity by ~29% or ~40%, respectively (Fig. 8D). Remarkably, mutation at T281 resulted in a total shift of hPepT1 from LDFs to HDFs (Fig. 8C, lane T281A) and a significant reduction of hPepT1 transport activity by ~51% (Fig. 8D). Together, these results suggest that the hydrophobic amino acids F293 and F297 and the hydrophilic amino acid T281 in TMD 7 of hPepT1 are essential for its targeting to lipid rafts as well as its transport activity.


Figure 8
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  		Fig. 8. Transmembrane segment 7 of hPepT1 contains lipid raft sorting determinants. A: schematic diagram of hPepT1-GFP in the plasma membrane and mutated amino acids (shown in red) in transmembrane domain 7 (TMD7) of hPepT1. B: fluorescent microscopy pictures taken on Chinese hamster ovary (CHO) cells stably transfected with pEGFP-C3 vector, wild-type (WT) hPepT1-GFP, or mutated T281A-hPepT1-GFP. C: distribution of WT and mutated hPepT1-GFP in the gradient fractions extracted from stably transfected CHO cells was analyzed by 10% SDS-PAGE and Western blot using anti-hPepT1 antibody. GM1 distribution was analyzed using HRP-conjugated CTB. D: uptake of [14C]Gly-Sar in CHO cells stably transfected with WT hPepT1-GFP and its construct variants. Cells were grown on 12-well plastic plates and used postconfluency. Cells were washed, stabilized with HBSS-10 mM MES (pH 6.2) for 15 min at 37°C, and incubated with the same buffer containing 20 µM [14C]Gly-Sar ± 20 mM Gly-Leu for 15 min at room temperature. Cells were then washed and lysed, and radioactivity was determined by use of a β-counter. Values represent means ± SE of 3 determinations. **P < 0.005 vs. control.

 

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Many studies have shown the functional characteristics of the major brush border di/tripeptide transporter PepT1 in polarized epithelial cells. However, no previous study has directly investigated the functional targeting of PepT1 in lipid raft microdomains. Here, we show a specific colocalization of GM1 and PepT1 in lipid rafts in brush border intestinal epithelia. This observation suggests that PepT1 could functionally interact with GM1. Clearly, further work is needed to evaluate the functional role of the association of PepT1 with the cholera toxin receptor. Interestingly, the membrane transporter NHE3 that is functionally coupled to PepT1 (44) has also been found associated with lipid rafts in BBMs (20). The potential colocalization of the two membrane transporters PepT1 and NHE3 into specific membrane microdomains, such as lipid rafts, could provide the functional coupling between them in polarized intestinal epithelia.

Polarized epithelial cells generally perform vectorial functions, which are reflected in the organization of their plasma membranes into apical and basolateral domains with unique lipid and protein compositions. The asymmetric distribution of plasma membrane components is maintained by tight junction proteins and is a fundamental characteristic of epithelial cells (31, 36). In polarized intestinal epithelia, PepT1 is specifically targeted to the apical plasma membrane (5, 6, 25, 26). Here we showed that hPepT1 mostly resides in lipid rafts in mouse intestinal BBMs and polarized Caco2-BBE cells (~48 and ~40% of membrane PepT1, respectively). It is important to discern whether the insolubility of PepT1 in cold TX-100 is due to its association with detergent-resistant lipid rafts and/or its anchoring to cytoskeletal elements. It should be noted that the results presented here are from detergent-resistant membranes (DRMs) isolated from highly purified intestinal BBMs. We previously felt to purify lipid rafts from total membrane fraction probably because of their lipid composition and cytoskeleton complexes (29). Proteomic analysis of DRMs isolated from total membrane by MALDI-TOF/MS showed the contamination mainly from mitochondrial and cytoskeleton proteins (29). In addition, Western blot analysis of DRMs extracted from total membrane exhibited two similar bands (data now shown). The first band is similar to what was found in DRMs from purified BBMs. The higher density band was enriched with actin cytoskeleton proteins and its extent can be decreased by sonication of the starting crude membrane.

As we have recently demonstrated, hPepT1 is also expressed in nonpolarized cells such as immune cells (6). Here we showed that hPepT1 is present in lipid rafts in nonpolarized Jurkat cell membranes (~34% of membrane hPepT1). The localization of PepT1 in lipid rafts in immune cells suggests that it may functionally aggregate with signaling molecules into lipid rafts at the immune response during cell signaling.

The transporter hPepT1 is a 12-transmembrane protein buried in the hydrophobic region of the plasma membrane. Since lipid rafts are enriched in cholesterol, it can be predicted that the transmembrane domains (TMDs) of transmembrane proteins that reside in lipid rafts may contain amino acid side chains particularly suited for their interacting with this sterol. Although the relative affinity of 20 amino acid side chains for cholesterol is not fully known yet, it can be anticipated from their chemical structure that phenylalanine and isoleucine residues would ideally fit with the aliphatic cycles and the isooctyl tail of cholesterol. In support of this hypothesis, we showed that amino acids F293 and F297 in TMD 7 of hPepT1 are important for the association of hPepT1 to lipid rafts but not for its overall membrane targeting. The assembly of cholesterol molecules around the TMD 7 enriched in phenylalanine residues may contribute to stabilize the interaction of hPepT1 with lipid rafts in the membrane liquid-ordered phase. These results are in agreement with previous studies showing the importance of TMDs for the association of transmembrane proteins with lipid rafts (7, 13, 18). Furthermore, we showed that F293 and F297 in TMD 7 are also important for hPepT1 transport activity in CHO cells. Together these observations suggest that these amino acids are important for the association of hPepT1 with lipid rafts and therefore its transport activity. In addition, we found that the association of hPepT1 with lipid rafts was T281-dependent. Threonine is a hydrophilic amino acid and is not expected to interact with cholesterol. However, we cannot rule out that mutation at T281 in TMD 7 of hPepT1 may cause a conformational change leading to the decrease of affinity of this transporter for lipid rafts. It should be noted that the TMD 7 might not be unique in determining the interaction of hPepT1 with lipid rafts. The importance of hydrophobic amino acids in 12 TMDs of hPepT1 for its association with lipid rafts needs to be further studied.

Changes in cholesterol content of the cell membrane might be expected to affect the transport activity of PepT1 in intestinal epithelia and in nonpolarized cells such as immune cells. In the present study we showed that cholesterol depletion of polarized Caco2-BBE monolayers with MβCD did not affect the surface amount of PepT1 as well as the intestinal barrier function but caused a significant increase in hPepT1-mediated transport events. These results are consistent with the relocation of a proportion of hPepT1 from the putative cholesterol-rich lipid raft microdomains to the surrounding cholesterol-poor liquid-disordered phase in the apical plasma membrane of intestinal epithelia. The mechanism of PepT1 activation by MβCD is presently unknown. It is likely that under resting state, PepT1 constitutively localizes in large macromolecular complexes that maintain its transport activity. Cholesterol removal may cause a breakup of these assemblies leading to a rearrangement of PepT1 in the membrane and therefore simultaneous removal of inhibitory interactions that hold PepT1 activity in the resting state. A similar mechanism has been recently proposed for adenylyl cyclase activation based on the observation that MβCD treatment augmented the enzyme activity (33). Another possibility is that the cholesterol depletion, which disrupts the interaction of hPepT1 with cholesterol molecules in lipid rafts, may change the overall conformational structure of this transporter in a manner that is favorable for its transport activity. Lipid rafts are also known to contain proteins that are involved in Ca2+ homeostasis (3, 22), and changes in [Ca2+]i due to lipid raft disruption could lead to the activation of PepT1. Indeed, alterations of [Ca2+]i levels, e.g., by Ca2+ channel blockers, affect pH regulatory systems, such as apical Na+ and H+ exchange, and thereby alter the H+ gradient that serves as the driving force for uptake of beta-lactams into polarized intestinal epithelial cells.

In contrast, MβCD treatment induced a significant decrease of PepT1 activity in BBMVs and nonpolarized immune cells. These results could be explained by the fact that BBMVs are uniquely composed of the apical plasma membrane of enterocytes and that immune cells are not differentiated into distinct apical and basolateral membrane domains. In other words, BBMVs and immune cells may not recapitulate the asymmetry distribution of lipids and proteins that exists in polarized intestinal epithelia. Similarly, it has been shown that cholesterol depletion of nonpolarized opossum kidney cell resulted in a decrease of NHE3 activity (28). This finding is consistent with our findings suggesting that the association of NHE3 and hPepT1 with lipid rafts in nonpolarized cells have similar effects on their activities. More data are required to understand the physiological mechanisms that regulate the transport activity of PepT1 by its association with lipid rafts.

In conclusion, we have recently demonstrated that 1) PepT1 is associated with lipid rafts in polarized epithelial cells and nonpolarized immune cells, 2) transmembrane segment 7 of hPepT1 contains determinants for its targeting to lipid rafts, and 3) the association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells.


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 ABSTRACT
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This work was supported by National Institutes of Health of Diabetes and Digestive and Kidney Diseases under a center grant (R24-DK-064399), RO1-DK061941 and RO1-KD071594 (to D. Merlin), and RO1-DK55850 (S. Sitaraman). L. Charrier-Hisamuddin is a recipient of a career development award from the Crohn's and Colitis Foundation of America.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Merlin, Emory Univ., Dept. of Medicine, Division of Digestive Diseases, 615 Michael St., Atlanta, GA 30322 (e-mail: dmerlin{at}emory.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.


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