Abteilung Epithelphysiologie, Max-Planck-Institut für molekulare Physiologie, 44227 Dortmund, Germany
Submitted 28 January 2003 ; accepted in final form 27 May 2003
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ABSTRACT |
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endosome; microtubules; enterocyte; D-glucose transport; antibodies
The autosomal recessive disorder glucose galactose malabsorption syndrome is caused by missense mutations in the SGLT1 gene. Heterologous expression of these mutant SGLT1 genes in Xenopus laevis oocytes leads to complete loss of sodium-dependent D-glucose uptake into oocytes compared with oocytes expressing wild-type SGLT1, although in both cases the overall protein levels were comparable. Transport deficiency of the mutants was due to a trafficking defect of the SGLT1 protein, which did not reach the plasma membrane but was accumulated in intracellular compartments (30).
Trafficking of SGLT1 is not only an important issue for the explanation of SGLT1 malfunctions in disease states, but intracellular trafficking also seems to play a critical role in the regulation of sodium-D-glucose cotransport mediated by wild-type SGLT1. Sodium-dependent D-glucose uptake into Xenopus laevis oocytes expressing wild-type SGLT1 was increased by activation of protein kinase A and decreased by the activation of protein kinase C. These reversible changes in the maximum transport rate occurred within minutes and were accompanied by proportional changes in the number of SGLT1 transporters in the plasma membrane (18). These data suggest a mechanism by which sodium-dependent D-glucose transport can be rapidly upregulated by the recruitment of SGLT1 to the plasma membrane and can be downregulated by endocytosis of SGLT1. Such regulatory mechanisms would necessarily require intracellular pools, stores of SGLT1.
There are conflicting data in the literature with regard to the cellular localization of SGLT1 in epithelial cells. An exclusively apical localization is reported in SGLT1-transfected Madin-Darby canine kidney (MDCK) cells (40, 41) and in thin sections of rat small intestine (48). In LLC-PK1 cells, a cell line from the pig kidney proximal tubule, endogenous SGLT1 was not detected in the brush border membrane but "near" the apical plasma membrane (20). In excised loops from rabbit jejunum, SGLT1 was located in the brush border membrane and was also proposed in intracellular sites (9). Where exactly does SGLT1 reside in absorptive epithelial cells? Contradicting data in the literature may simply reflect a different SGLT1 distribution in different cells/tissues. However, a more likely explanation may be provided by the properties of the antibodies used to detect SGLT1 in different studies. In earlier work, antibodies raised against relatively small peptides from the COOH-terminal amino acid sequence of SGLT1 were used. Therefore, it is likely that the epitopes for these antibodies may be blocked in certain cellular locations due to the interaction of SGLT1 with the cytoskeleton or other proteins, for example, with the chaperone heat shock protein 70, which was recently identified to form a complex with SGLT1 (20).
To circumvent these obstacles, we raised antibodies against relatively large parts of three extramembranous loops of the SGLT1 protein for the present study. We investigated the distribution of endogenous SGLT1 in Caco-2 cells, a model for human enterocytes (7), using immunological methods after cell fractionation by free-flow electrophoresis (FFE) and by immunofluorescence microscopy.
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EXPERIMENTAL PROCEDURES |
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All chemicals were of the highest purity available and were purchased from
Sigma (Deisenhofen, Germany). Other reagents and antibodies were also from
Sigma unless other sources are indicated. Proteins were separated on 10% gels
by SDS-PAGE according to Laemmli
(25). For Western blotting,
polypeptides were electrotransferred
(42) on polyvinylidene
difluoride membrane (Schleicher & Schuell). Primary antibodies were
detected by incubation with horseradish peroxidase (HRP)-conjugated secondary
antibodies followed by detection with an enhanced chemiluminescence system
from NEN Life Science Products. Sodium-dependent uptake of
-[U-14C]methyl-D-glucose (300 mCi/mmol; NEN) into
Caco-2 cells was carried out as described earlier
(5,
31).
Generation of Anti-SGLT1 Antibodies QIS30, LYT30, and RNS71
Glutathione S-transferase (GST) fusion proteins containing fragments of rabbit SGLT1 (QIS30: amino acids 243-272, starting with Q, I, S; LYT30: amino acids 338-367, starting with L, Y, T; and RNS71: amino acids 564-634, starting with R, N, S) were used as antigens to raise antibodies. The corresponding coding DNA fragments were amplified from full-length cDNA (32) by polymerase chain reaction (PCR) using the following oligonucleotides: QIS30, 5'-AAT GGA TCC TGC AGA TCT CCT ACG GA-3' (containing a BamHI site) and 5'-ATT GTC GAC TAC CCA GTG ATG GCA TC-3' (containing a SalI site); LYT30, 5'-AAT GGA TCC TGC TGT ACA CAG ACA AA-3' (containing a BamHI site) and 5'-ATT GTC GAC TAT GGG AAG GCA ATG TT-3' (containing a SalI site); and RNS71, 5'-ATT GGA TCC TGC GTA ATA GCA AAG AG-3' (containing a BamHI site) and 5'-ATT GTC GAC TAG GAG GTG TCT GTG AG-3' (containing a SalI site). The PCR products were digested with BamHI and SalI and ligated to the BamHI/SalI sites of pGEX-5X-3 vector (Amersham Pharmacia Biotechnology). In-frame cloning was confirmed by DNA sequencing. Expression of the GST fusion proteins in Escherichia coli BL21 cells and purification using glutathione-Sepharose beads were performed according to protocols provided by Amersham Pharmacia Biotechnology. A commercial service was employed to raise polyclonal antibodies in rabbits (Biotrend, Köln, Germany) with the use of a standard immunization and bleeding schedule protocol. Antibodies directed against GST were removed from the antisera by a passage through a Sepharose-GST column. Peptide-specific antibodies were then purified using Sepharose-fusion protein columns.
Polymerase Chain Reaction
mRNA was isolated from Caco-2 cells by using the RNeasy kit from Qiagen (Hilden, Germany). cDNA was reverse transcribed from mRNA with oligo(dT)15 primer (Promega) and Superscript II reverse transcriptase (Invitrogen). Reversed transcribed cDNA was used as template to identify message for SGLT1 and the two SGLT2 sequences (see Table 1) in Caco-2 cells by PCR. The following sequence-specific primers were used: SGLT1 (sense), 5'-GCC CTG GTT TTG GTG GTT G-3'; SGLT1 (antisense), 5'-CGA GAT CTT GGT GAA AAT GTA GAG C-3'; SGLT2a (sense), 5'-CAC CAT AGC TGA GAC CCC AGA G-3'; SGLT2a (antisense), 5'-TAT AGT ACC TCG GTT GGT CTT CAG C-3'; SGLT2b (sense), GAG CAC ACA GAG GCA GGC TC-3'; and SGLT2b (antisense), GCC TCT GTT GGT TCT GCA CAT-3'. Genomic DNA was employed as template for the positive control, which was prepared from Caco-2 cells with the QIAamp DNA kit from Qiagen.
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Cell Culture
Caco-2 cells were acquired from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (Braunschweig, Germany). Cells were grown in 75-cm2 tubes (Falcon, Heidelberg, Germany) at 37°C and 5% CO2 in minimal essential medium (Life Technologies) that was supplemented with 10% fetal calf serum, 1% nonessential amino acids, and 1% glutamine. Every 2 days, the culture medium was renewed. For the preparation of the cellular organelle fraction (COF) and immunoprecipitation, Caco-2 cells (passages 15-25) were seeded (2 x 105 cells/dish) on 5-cm petriPerm hydrophilic dishes (Vivascience, Hannover, Germany) and grown until a confluent, polarized monolayer was formed (>14 days). Alternatively, Caco-2 cells were grown (1 x 106 cells/filter) on 0.9-cm2 polyethylene terephthalate (PET) filter inserts (0.4-µm average pore size; Becton Dickinson, Heidelberg, Germany).
Endosome Labeling and Preparation of Cellular Organelle Fraction
Endosomes of Caco-2 cells were labeled according to a pulse-chase protocol, which was used in previous studies to label early and late endosomes of HeLa cells (3). Late endosomes were labeled by incubation of Caco-2 cells with medium containing 20 mg/ml FITC-dextran (70 kDa, dialyzed against PBS) for 3 min at 37°C, followed by a chase of 12 min in marker-free medium. HRP was then added to a final concentration of 10 mg/ml, and cells were incubated for 3 min at 37°C to label early endosomes. Typically, three petriPerm dishes with Caco-2 cells pretreated with endosome markers were used for the preparation of a COF. Cells were washed four times with ice-cold FFE buffer (10 mM triethanolamine, 10 mM acetic acid, 1 mM EDTA, and 0.25 M sucrose, pH 7.4 adjusted with NaOH), removed with a rubber policeman, and suspended in 12 ml of FFE buffer. The suspension was equilibrated in a high-pressure chamber (Parr Instrument model 4639) with 900 lb./in.2 nitrogen at 4°C for 15 min. The cell suspension was rapidly expanded against atmospheric pressure and centrifuged at 850 g for 10 min to remove cell debris and nuclei. The supernatant was layered on top of a 4-ml sucrose cushion (40% sucrose in FFE buffer) and centrifuged for 45 min at 100,000 g in a swinging bucket rotor. The interphase between the 40% sucrose and the 0.25 M sucrose layers was collected (COF). Protein in an aliquot of the COF was precipitated with trichloroacetic acid, and the protein concentration was measured according to the method of Lowry et al. (27), using bovine serum albumin as standard. For further analysis by FFE, the COF was adjusted to a protein concentration of 1 mg/ml by the addition of FFE buffer.
FFE and Fraction Analysis
The COF (1 mg/ml protein) was subjected to gentle trypsin treatment by
addition of 0.5% TPCK trypsin/mg protein and incubation for 5 min at 37°C.
The reaction was stopped by addition of a 10-fold excess of soybean trypsin
inhibitor and rapid cooling to 4°C. Trypsin-treated COF were injected (1
ml/h) into an Octopus FFE apparatus (chamber height 0.5 mm; Dr. Weber, Munich,
Germany), which was maintained at 4°C at 145 mA constant and 1,200 V
with a flow rate of the chamber buffer (FFE buffer) of 300 ml/h. A total of 96
fractions was collected (
2 ml/fraction). Aliquots of the fractions (100
µl) were transferred to 96-well plates and analyzed for alkaline
phosphatase (44), Na/K-ATPase
(16), HRP (assay kit from
Sigma), FITC-dextran (fluorescence plate reader, Fluoroskan Ascent FL;
Labsystems, Frankfurt, Germany), and SGLT1 (ELISA assay).
ELISA Assay
A 40-µl aliquot of each fraction obtained from FFE was coated to individual wells of a 96-well plate by mixing with 40-µl coating buffer (NaHCO3/Na2CO3 buffer, pH 10.3) and incubation overnight at 4°C. After blocking with 2% bovine serum albumin in TBS (170 mM NaCl, 0.05% Tween 20, 20 mM Tris, pH 7.4 adjusted with HCl) for 60 min at room temperature, the wells were washed four times with TBS and then incubated with anti-QIS30 antibody (1:2,000) in 0.2% bovine serum albumin in TBS for 90 min at room temperature. The wells were washed four times with TBS and were then incubated with goat anti-rabbit IgG linked to biotin (1:4,000) in 0.2% bovine serum albumin in TBS for 60 min at room temperature. The wells were washed four times with TBS and were then incubated with avidin linked to alkaline phosphatase (1:10,000) for 30 min at room temperature. The wells were washed four times with TBS, and 200 µl of alkaline phosphatase assay mixture (100 mM glycine, 1 mM MgCl2, 0.1 mM ZnSO4, 5 mM p-nitrophenolphosphate, pH 10.5 adjusted with NaOH) were added per well. The plate was incubated for 1 h at 37°C, and the absorption at 405 nm was read.
Metabolic Labeling and Immunoprecipitation
Caco-2 cells grown on 5-cm petriPerm dishes were washed twice with PBS and metabolically labeled with 0.1 mCi/ml Tran35S-Label (1175 Ci/mmol; ICN Biomedicals) in methionine- and cysteine-free minimal essential medium (Life Technologies) supplemented with 10% dialyzed fetal calf serum for 1 h at 37°C. The cells were washed twice with PBS and maintained at 37°C with regular growth medium for the chase times indicated. Immunoprecipitation of SGLT1, using 10 µg of anti-QIS30 antibody, was carried out as described elsewhere (22).
Conventional and Confocal Microscopy
Caco-2 cells grown to confluence on poly-L-lysine-coated
coverslips were fixed with 3% paraformaldehyde in PBS (15 min), permeabilized
with 0.1% Triton X-100 in PBS (3 min), and blocked for 60 min with PBG (0.2%
gelatin, 0.5% BSA in PBS, pH 7.4). Cells were then incubated for 60 min at
room temperature with primary antibodies directed against SGLT1 (QIS30, rabbit
polyclonal, 1:400) and -tubulin (mouse monoclonal, 1:800) in PBG. The
cells were washed with PBS and then incubated for 60 min at room temperature
with a cocktail of Cy2-conjugated goat anti-mouse IgG (1:120 in PBG; Jackson
ImmunoResearch) and Cy3-conjugated goat anti-rabbit IgG (1:480 in PBG, Jackson
ImmunoResearch). After being washed with PBS, DNA was stained by incubation
with 4',6-diamidino-2-phenylindole (DAPI; 25 ng/ml in PBS) for 10 min.
Labeling of the plasma membrane was achieved by incubation of the cells with 2
mg/ml Sulfo-NHSBiotin (Pierce) in PBS, pH 8.0, for 10 min at 4°C. After
being washed with the same PBS, the cells were incubated with Cy2-labeled
Streptavidin (1:500; Amersham Life Science) for 60 min to detect the biotin
bound to integral membrane proteins. The specimens were mounted with Mowiol
(CalBiochem) and analyzed using conventional and confocal microscopy. For
conventional fluorescence microscopy, cells were viewed under a Zeiss Axiophot
microscope equipped with a Zeiss x40/0.9 objective lens, a Zeiss
x63/1.25 oil-immersion objective lens, and a Zeiss AxioCam
charge-coupled device camera. Images from triple or double staining were
obtained at the same focal planes. Image acquisition and analysis were
performed using Axiovision 2.05 and Image Pro Plus 4.5 software, respectively.
For confocal microscopy, a Noran OZ laser scanning confocal imaging system
connected to a Nikon Eclipse TE200 inverted microscope was used. Magnification
was set to x400 under a Nikon x40/1.3 oil-immersion objective
lens. The microscope was connected to an argon laser (Omnichrome series 43;
457/488/514 nm), a diode pumped solid state Nd:YAG laser (Coherent DPSS 532;
532 nm), and a Coherent Enterprise UV laser (352/361 nm). The system was
controlled by a Silicon Graphics workstation. Images of Cy2, Cy3, and DAPI
were captured from the same optical section using the Intervision acquisition
software. The captured images were then pseudocolored: green for Cy2, red for
Cy3, and blue for DAPI. Regions of colocalization of Cy2 and Cy3 appear in
yellow.
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RESULTS |
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Three peptides (QIS30, LYT30, and RNS71) representing extramembranous loops (34) of the rabbit SGLT1 (32) amino acid sequence were expressed as GST fusion proteins in E. coli and subsequently used to immunize rabbits for the production of antisera. Peptide-specific antibodies were purified from the respective antisera by affinity chromatography. Because of the high homology between rabbit and human SGLT1, the peptide-specific antibodies QIS30, LYT30, and RNS71 also recognized the human isoform of SGLT1 present in Caco-2 cells. In Western blots, all three peptide-specific antibodies reacted with a single antigen present in Caco-2 cell homogenates at 73 kDa, the expected size of mature SGLT1. IgG from the respective preimmune sera did not recognize antigens in Caco-2 cell homogenates at all (Fig. 1). Use of all anti-SGLT1 antibodies (QIS30, LYT30, RNS71) led to essentially the same results in the experiments presented. However, most of the experiments were carried out using QIS30 peptide-specific antibody, simply because affinity chromatography of QIS30 antiserum gave the highest yield in peptide-specific antibodies and anti-QIS30 antibody was, therefore, available in the largest amount.
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The specificity of the anti-peptide antibodies was further analyzed by a BLAST search for human amino acid sequences similar to the QIS30, LYT30 and RNS71 peptides in the National Center for Biotechnology Information (NCBI) protein database. Six proteins, in addition to SGLT1, were identified that may be recognized by the antibodies (Table 1). Candidate proteins 4-7 share only 5-10 consecutive matching amino acids with one of the SGLT1 peptides, which is probably a minimum requirement for an appropriate antibody epitope. Furthermore, the sequence of each of the candidate proteins 4-7 matched with only one of the three antibody peptides QIS30, LYT30, and RNS71, respectively. Because all three anti-peptide antibodies recognized only a single antigen at 73 kDa in Western blots, it is unlikely that one of the candidate proteins 4-7 is recognized by one of the antibodies. Likely proteins to be recognized by the antibodies are the two low-affinity sodium-D-glucose cotransporters (SGLT2) in the database (proteins 2 and 3); therefore, we tested for the expression of SGLT2 in Caco-2 cells by RT-PCR. As shown in Fig. 2, mRNA for neither of the two SGLT2 transporters was detected in Caco-2 cells; thus in Caco-2 cells our antibodies are specific for SGLT1.
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SGLT1 Distribution in Caco-2 Cells
Free-flow electrophoresis. SGLT1 distribution was analyzed by separation of a COF prepared from Caco-2 cells. Cells were disrupted by nitrogen decompression, by equilibration with nitrogen at 950 lb./in.2 and rapid expansion against atmospheric pressure (39). The cellular organelles and plasma membranes were then collected by centrifugation on a sucrose cushion. The Caco-2 cell preparation obtained by this procedure was depleted of nuclei, mitochondria, and cytosol and was termed the COF. The COF was resolved in an electric field by FFE. This method utilizes differences in the surface charges of organelles and plasma membrane vesicles for the separation of subcellular material and was successfully applied to the separation of apical and basolateral plasma membranes from proximal tubule cells of rat kidney (16) and to the preparation of lysosomes (15) and functional endosomes from cultured cells (29, 36) and animal tissue (13).
After separation of the COF by FFE, the obtained fractions were analyzed
for the endogenous marker enzymes activities of alkaline phosphatase, which is
only located at the apical plasma membrane of Caco-2 cells
(17), and Na/K-ATPase, which
is only located at the basolateral plasma membrane of Caco-2 cells
(43). To identify endosomal
compartments, the exogenous markers HRP (early endosomes) and FITC-dextran
(late endosomes) were determined, which had been applied to the cells
according to a pulse-chase protocol (see EXPERIMENTAL PROCEDURES)
before subcellular fractionation. It was observed in earlier studies that
separation of endosomal compartments from the bulk of plasma membranes
required gentle trypsin digestion of the cell preparation subjected to FFE,
probably to remove cytoskeletal interconnections between cellular organelles
(29,
37). Therefore, we performed
FFE of the COF with and without prior trypsin treatment; typical results are
summarized in Fig. 3. Apical
and basolateral plasma membranes were readily separated by FFE without trypsin
treatment of the sample, as indicated by the distribution of alkaline
phosphatase and Na/K-ATPase (Fig.
3A), whereas basolateral membranes (Na/K-ATPase), early
endosomes (HRP), and late endosomes (FITC-dextran) were not resolved under
these conditions (Fig. 3, A and
B). Separation of the endosomal compartments and the
basolateral membrane was achieved by gentle trypsin treatment (0.5% trypsin/mg
protein for 5 min) of the COF before FFE. Trypsin-digested samples were
resolved into clearly distinguishable peaks of alkaline phosphatase activity
(apical membrane), Na/K-ATPase activity (basolateral membrane), HRP activity
(early endosomes), and FITC-dextran fluorescence (late endosomes)
(Fig. 3, D and
E). With the distribution of marker enzymes among the
fractions separated by FFE having been established, SGLT1 was detected by an
ELISA assay using the peptide-specific antibody QIS30. Only a minor fraction
of the total amount of SGLT1 present in Caco-2 cells was located in the apical
membrane; most of the SGLT1 resided in intracellular compartments, which
copurified in FFE with early endosomes
(Fig. 3, C and
F). The distribution ratio between intracellular
compartments and apical plasma membrane was estimated by comparison of the
areas below the respective peaks and was 2:1. Two peaks for
SGLT1-distribution were observed in trypsin-treated and nontreated samples,
indicating that the second peak is not an artifact caused by trypsin
treatment.
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Epifluorescence and confocal microscopy. The subcellular fractionation studies using FFE indicated that a substantial amount of SGLT1 was located in intracellular compartments. We next attempted to visualize and further characterize this intracellular population in whole Caco-2 cells by immunofluorescence microscopy using epitope-specific antibodies against SGLT1. In nonpermeabilized Caco-2 cells, SGLT1 was detected as sparse punctuated structures, which represent transporters (or clusters of transporters) at the apical cell surface (Fig. 4A). By contrast, in permeabilized cells, in which the antibody also accessed intracellular compartments containing SGLT1, significantly more immunostaining was visible (Fig. 4B). These results are in good accordance with the results from the biochemical cell fractionation studies, which suggested the same SGLT1 distribution: a small amount in the apical plasma membrane and a larger pool of transporters inside the cell.
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In fixed and permeabilized Caco-2 cells, SGLT1 was detected throughout the cell with prominent perinuclear staining (Fig. 5A). However, SGLT1 seemed to be not randomly distributed, which was particularly obvious in regions where Caco-2 cells were torn apart due to the preparation process (Fig. 5, B and C). In these regions, SGLT1-containing intracellular compartments had an appearance of small vesicles, which were assembled like "pearls on an invisible string." This observation led to the hypothesis that SGLT1-containing vesicles were associated with cytoskeletal elements, probably microtubules.
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To test this hypothesis, we double-stained Caco-2 cells for SGLT1 (red) and
-tubulin (microtubules, green). SGLT1 was located in structures that
were lined up along microtubules (Fig. 6,
A-C). Again, association of SGLT1-containing compartments
was best investigated in cells that were torn apart during the preparation
process and is most impressively demonstrated in
Fig. 6B.
Interestingly, on microtubules that were mechanically stretched
(Fig. 6C, arrow),
associated SGLT1-containing compartments were also stretched simultaneously
and became tubular-shaped themselves, indicating a very tight association of
SGLT1 compartments with microtubules. Colocalization of SGLT1 compartments and
microtubules was further confirmed by analysis of the same specimens with a
confocal laser scanning microscope (Fig. 6,
D-F). When images of labeled
-tubulin (green) and
SGLT1 (red) were merged, the vesicles containing SGLT1 appeared yellow,
reflecting the additive effect of superimposing red and green pixels
(Fig. 6F).
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Polarity Status of Caco-2 Cell Cultures
Intracellular compartments containing membrane transporters may be due to
epithelial cells not yet being polarized. Thus, membrane proteins associated
with the apical or basolateral plasma membrane of polarized MDCK cells were
found in intracellular compartments in nonpolarized MDCK cells
(26). Therefore, we
investigated the development of polarity of Caco-2 cells under our particular
growth conditions (seeding cell density, growth substratum). Polarity status
of Caco-2 cells was estimated by measuring the sodium-dependent
-methyl-D-glucose uptake into Caco-2 cells, because
sodium-dependent D-glucose uptake is a property typical of only
polarized Caco-2 cells (5).
Caco-2 cell cultures grown on petriPerm support reach polarity 8-11 days after
seeding (Fig. 7). For FFE
studies, Caco-2 cells that were grown for >14 days were used, indicating
that these studies were all performed on polarized Caco-2 cells. The fact that
these cells reach polarity earlier than those grown on filters is probably due
to the 200-fold higher amount of cells used for seeding.
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Caco-2 cells grown on PET filter inserts reach polarity 11-12 days after seeding (Fig. 7). The distribution of SGLT1 in polarized Caco-2 cells, which were grown for >14 days on PET filter inserts, was analyzed by confocal laser scanning microscopy. Integral proteins of the apical membrane were labeled with Sulfo-NHSBiotin and Streptavidin-Cy2 (green). A series of images from confocal planes (0.1-µm thickness) perpendicular to the z-axis starting from the apical membrane (0 µm) to below the nucleus (6.5 µm) are shown in Fig. 8. SGLT1 (red) can be detected not only in the apical membrane (green) but also in images from intracellular confocal planes (4.0, 5.0, and 6.5 µm), where the staining of the cell surface is no longer visible. These data indicate that an intracellular pool of SGLT1 is a property of the polarized Caco-2 cell.
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Distribution of SGLT1 in Caco-2 Cells With Switched Off Protein Synthesis
Our biochemical experiments and histological observations identified a large intracellular population of SGLT1 in Caco-2 cells that is dispersed in microtubule-associated vesicles. What is a large population of a plasma membrane transporter, typically fulfilling its physiological task in the apical plasma membrane, doing in the cell interior? Two scenarios are to be considered in this regard: 1) intracellular SGLT1-containing vesicles may be en route from biosynthesis to their cellular destination or 2) may represent an intracellular reserve pool, from which transporters can be recruited or stored to regulate apical abundance of SGLT1 by an endo/exocytosis mechanism. To discriminate between these two possibilities, we studied intracellular distribution and function of SGLT1 in Caco-2 cells in which protein biosynthesis was inhibited by cycloheximide.
De novo protein synthesis was efficiently blocked by incubation of Caco-2 cells with cycloheximide (10 µg/ml) for 60 min. Treatment with cycloheximide completely abolished metabolic labeling with [35S]methionine/cysteine of proteins in Caco-2 cell homogenates. Homogenates from 35S-labeled control cells and from cells incubated with cycloheximide showed similar protein patterns (Fig. 9A). However, proteins from the homogenate of control cells were intensively labeled with 35S, whereas no radioactivity was detected in proteins from the homogenate of cycloheximide-treated cells (Fig. 9B). These data indicate that the conditions for cycloheximide treatment of Caco-2 cells were sufficient to completely block de novo protein synthesis.
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Specificity of anti-QIS30 antibodies for the immunoprecipitation of SGLT1 was established by comparison of immunoprecipitations with anti-QIS30 antibodies and the respective preimmune IgG from [35S]methionine/cysteine-labeled Caco-2 cells. As shown in Fig. 9C, 35S-labeled SGLT1 was readily precipitated with anti-QIS30 antibodies, whereas the preimmune serum failed to precipitate SGLT1. Evidence that biosynthesis of SGLT1 in particular was also inhibited by cycloheximide was established by immunoprecipitation. No radioactivity was associated with SGLT1 in immunoprecipitates after cycloheximide treatment, whereas significant amounts of 35S-labeled SGLT1 were detected in metabolically labeled control cells (Fig. 9D).
Cycloheximide inhibits protein biosynthesis by inhibiting peptidyltransferase in ribosomes of eucaryotic cells; however, in some instances cycloheximide can also cause superinduction of certain proteins (10, 28). In homogenates from Caco-2 cells, no significant change in the total amount of proteins (Fig. 9A) and in the amount of SGLT1 detected by Western blot (Fig. 9E) was observed after treatment of the cells with cycloheximide, indicating that superinduction of proteins by cycloheximide is not a concern in our present study regarding SGLT1 distribution in Caco-2 cells.
Western blot analysis of homogenates from cycloheximide-treated cells and
control cells revealed no detectable change in the total amount of SGLT1
present in Caco-2 cells (Fig.
9E). Taking into account that our conditions for
cycloheximide treatment are sufficient to deplete proteins from the
biosynthetic pathway, these data indicated that newly synthesized SGLT1
contributed only a very small extent, which remained below detection limits,
to the total amount of SGLT1 present in Caco-2 cells. This scenario is in good
accordance with our further investigations. Depletion of proteins from the
biosynthetic pathway did not significantly alter SGLT1 distribution in Caco-2
cells as observed using immunofluoresence microscopy (data not shown),
indicating that the intracellular compartments containing SGLT1 are not
endoplasmic reticulum or Golgi. No significant differences between control
cells and cycloheximide-treated cells were observed when SGLT1 distribution
was examined by FFE (Fig.
10A). Furthermore, cycloheximide treatment for 2 h had no
significant effect on sodium-dependent -methyl-D-glucose
uptake into Caco-2 cells, a measure for transport active SGLT1 protein present
in the apical surface of the cell (Fig.
10B).
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Half-Life of SGLT1 in Caco-2 Cells
Our experiments regarding SGLT1 distribution in cycloheximide-treated Caco-2 cells suggest that newly synthesized SGLT1 did not significantly contribute to the total amount of SGLT1 present in Caco-2 cells. If this assumption is true, the overall half-life of SGLT1 in Caco-2 cells should be relatively "long" compared with the process of protein biosynthesis. Therefore, we measured the half-life of SGLT1 in Caco-2 cells. Caco-2 cells were pulse labeled with [35S]methionine/cysteine for 1 h and than chased for 1, 2, 3, 4, or 5 days, and the content of newly synthesized SGLT1 was determined by measuring the amount of radioactivity associated with immunoprecipitated SGLT1 (Fig. 11A). The half-life of SGLT1 was estimated from a semilogarithmic plot of 35S intensity vs. labeling time (Fig. 11B). Assuming first-order decay, SGLT1 had an apparent half-life of 2.5 days in Caco-2 cells.
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DISCUSSION |
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In Caco-2 cells, SGLT1 has an apparent half-life of 2.5 days as determined by metabolic labeling. The overall residence time of SGLT1 in Caco-2 cells is rather long compared with the time required for synthesis, processing, and membrane targeting of typical membrane proteins. The processing and passage of newly synthesized membrane proteins through endoplasmic reticulum and Golgi have been shown to be complete after 30-60 min (2, 22) in rat hepatocytes. Therefore, the relative long overall residence time of SGLT1 in Caco-2 cells suggests that most of the intracellular SGLT1 transporters are not en route from biosynthesis to their cellular destination. This assumption is supported by the observation that elimination of newly synthesized proteins from the biosynthetic path-way by cycloheximide did not significantly alter the size and appearance of the SGLT1 pool present in Caco-2 cells. Assuming a steady state at which protein synthesis and degradation proceed with the same rates, only a small SGLT1 amount is added to and retrieved from a large existing SGLT1 pool in a given time interval. The large existing pool of SGLT1 is distributed between a SGLT1 population, which is located in the apical plasma membrane, and a SGLT1 population, which is dispersed in intracellular, microtubule-associated vesicular structures. The quantification of immunodetectable SGLT1 in Caco-2 cells revealed a distribution ratio between apical plasma membrane and intracellular compartments of 1:2. Most of the SGLT1 actually resides inside the enterocyte!
What is SGLT1 doing inside the cell? Intracellular pools have been described for several membrane transporters: 1) the insulin-responsive glucose transporter 4 in rat adipocytes (35); 2) the aquaporin 2 water channel in LLC-PK1 cells (14); 3) the cystic fibrosis transmembrane conductance regulator (CFTR) in rat duodenal epithelium (1); 4) the proton/potassium ATPase in gastric parietal cells (47); and 5) canalicular ATP-binding cassette (ABC) transporters in rat hepatocytes (24). Common to all these examples is that these transporters are regulated in their plasma membrane abundance by the shifting of transporters between intracellular sites and the plasma membrane.
There are also indications for posttranslational regulation of SGLT1. SGLT1-mediated D-glucose uptake into cells was rapidly altered by prior treatment with forskolin (45), cAMP or the cAMP-raising agent cholera toxin (33), high D-glucose (38), and the peptide hormone glucagon-like peptide 2 (GLP-2) (8). In some of these examples, changes in D-glucose absorption have been shown to be accompanied by simultaneous changes in the plasma membrane abundance of SGLT1. Shifting of SGLT1 between cellular pools was recently directly demonstrated. Upon treatment of isolated blind loops of rabbit jejunum with epidermal growth factor, SGLT1 was increased in prepared brush border membrane vesicles and decreased in a prepared microsomal membrane fraction (9). We propose that the intracellular population of SGLT1 that we identified in Caco-2 cells is critically involved in the regulation of SGLT1 abundance at the cell surface. This is also supported by our finding that intracellular compartments containing SGLT1 are associated with microtubules. Because microtubules are intracellular "railroad tracks," this observation implies mobility of the intracellular SGLT1 pool.
We have introduced a powerful method for the investigation of cellular distribution of membrane transporters in cultured epithelial cells. In contrast to classic biochemical subcellular fractionation techniques, preparation of a COF, followed by FFE, allowed the separation of a whole variety of membrane-enclosed cellular compartments in a single step, ready for analysis. Further analysis of the FFE fractions with immunochemical methods (ELISA) then revealed the distribution of SGLT1 (or any other protein of interest, against which a suitable antibody is available) over a large spectrum of cellular compartments. Because this method is also quantitative, it is suitable for detecting changes in the subcellular distribution of SGLT1 after exposure of cells to various stimuli before separation and analysis. In other words, this method has the potential to identify physiological signals affecting the intracellular distribution and trafficking of SGLT1 in epithelial cells, a project presently pursued in our laboratory.
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ACKNOWLEDGMENTS |
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REFERENCES |
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2. Bartles JR, Feracci HM, Stieger B, and Hubbard AL. Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation. J Cell Biol 105: 1241-1251, 1987.[Abstract]
3. Bayer N,
Schober D, Prchla E, Murphy RF, Blaas D, and Fuchs R. Effect of
bafilomycin A1 and nocodazole on endocytic transport in HeLa cells:
implications for viral uncoating and infection. J
Virol 72:
9645-9655, 1998.
4. Bissonnette P,
Gagne H, Blais A, and Berteloot A. 2-Deoxy-glucose transport and
metabolism in Caco-2 cells. Am J Physiol Gastrointest Liver
Physiol 270:
G153-G162, 1996.
5. Blais A, Bissonnette P, and Berteloot A. Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon. J Membr Biol 99: 113-125, 1987.[ISI][Medline]
6. Burckhardt G and Kinne RKH. Transport proteins. Cotransporters and countertransporters. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW and Giebisch G. New York: Raven, 1992, p. 64.
7. Chantret I, Barbat A, Dussaulx E, Brattain MG, and Zweibaum A. Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer Res 48: 1936-1942, 1988.[Abstract]
8. Cheeseman CI. Upregulation of SGLT-1 transport activity in
rat jejunum induced by GLP-2 infusion in vivo. Am J Physiol Regul
Integr Comp Physiol 273:
R1965-R1971, 1997.
9. Chung BM, Wallace LE, Hardin JA, and Gall DG. The effect of epidermal growth factor on the distribution of SGLT-1 in rabbit jejunum. Can J Physiol Pharmacol 80: 872-878, 2002.[ISI][Medline]
10. Cochran BH, Zullo J, Verma IM, and Stiles CD. Expression of the c-fos gene and of an fos-related gene is stimulated by platelet-derived growth factor. Science 226: 1080-1082, 1984.[ISI][Medline]
11. Dix CJ, Hassan IF, Obray HY, Shah R, and Wilson G. The transport of vitamin B12 through polarized monolayers of Caco-2 cells. Gastroenterology 98: 1272-1279, 1990.[ISI][Medline]
12. Faust RA and
Albers JJ. Regulated vectorial secretion of cholesteryl ester transfer
protein (LTP-I) by the CaCo-2 model of human enterocyte epithelium.
J Biol Chem 263:
8786-8789, 1988.
13. Fuchs R, Male
P, and Mellman I. Acidification and ion permeabilities of highly purified
rat liver endosomes. J Biol Chem
264: 2212-2220,
1989.
14. Fushimi K,
Sasaki S, and Marumo F. Phosphorylation of serine 256 is required for
cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel.
J Biol Chem 272:
14800-14804, 1997.
15. Harms E, Kern H, and Schneider JA. Human lysosomes can be purified from diploid skin fibroblasts by free-flow electrophoresis. Proc Natl Acad Sci USA 77: 6139-6143, 1980.[Abstract]
16. Heidrich HG,
Kinne R, Kinne-Saffran E, and Hannig K. The polarity of the proximal
tubule cell in rat kidney. Different surface charges for the brush-border
microvilli and plasma membranes from the basal infoldings. J Cell
Biol 54: 232-245,
1972.
17. Hidalgo IJ, Raub TJ, and Borchardt RT. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96: 736-749, 1989.[ISI][Medline]
18. Hirsch JR, Loo
DD, and Wright EM. Regulation of Na+/glucose cotransporter
expression by protein kinases in Xenopus laevis oocytes. J
Biol Chem 271:
14740-14746, 1996.
19. Hughes TE,
Sasak WV, Ordovas JM, Forte TM, Lamon-Fava S, and Schaefer EJ. A novel
cell line (Caco-2) for the study of intestinal lipoprotein synthesis.
J Biol Chem 262:
3762-3767, 1987.
20. Ikari A, Nakano M, Kawano K, and Suketa Y. Up-regulation of sodium-dependent glucose transporter by interaction with heat shock protein 70. J Biol Chem 2002.
21. Kinne-Saffran E and Kinne RK. Isolation of lumenal and contralumenal plasma membrane vesicles from kidney. Methods Enzymol 191: 450-469, 1990.[Medline]
22. Kipp H and
Arias IM. Newly synthesized canalicular ABC transporters are directly
targeted from the Golgi to the hepatocyte apical domain in rat liver.
J Biol Chem 275:
15917-15925, 2000.
23. Kipp H,
Kinne-Saffran E, Bevan C, and Kinne RK. Characteristics of renal
Na+-D-glucose cotransport in the skate (Raja
erinacea) and shark (Squalus acanthias). Am J Physiol
Regul Integr Comp Physiol 273:
R134-R142, 1997.
24. Kipp H,
Pichetshote N, and Arias IM. Transporters on demand. Intrahepatic pools of
canalicular ATP binding cassette transporters in rat liver. J Biol
Chem 276:
7218-7224, 2001.
25. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970.[ISI][Medline]
26. Low SH, Miura
M, Roche PA, Valdez AC, Mostov KE, and Weimbs T. Intracellular redirection
of plasma membrane trafficking after loss of epithelial cell polarity.
Mol Biol Cell 11:
3045-3060, 2000.
27. Lowry OH,
Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin
phenol reagent. J Biol Chem
193: 265-275,
1951.
28. Ma Q, Renzelli
AJ, Baldwin KT, and Antonini JM. Super-induction of CYP1A1 gene
expression. Regulation of 2,3,7,
8-tetrachlorodibenzo-p-dioxin-induced degradation of Ah receptor by
cycloheximide. J Biol Chem 275:
12676-12683, 2000.
29. Marsh M, Schmid S, Kern H, Harms E, Male P, Mellman I, and Helenius A. Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis. J Cell Biol 104: 875-886, 1987.[Abstract]
30. Martin MG, Lostao MP, Turk E, Lam J, Kreman M, and Wright EM. Compound missense mutations in the sodium/D-glucose cotransporter result in trafficking defects. Gastroenterology 112: 1206-1212, 1997.[ISI][Medline]
31. Matosin-Matekalo M, Mesonero JE, Delezay O, Poiree JC, Ilundain AA, and Brot-Laroche E. Thyroid hormone regulation of the Na+/glucose cotransporter SGLT1 in Caco-2 cells. Biochem J 334: 633-640, 1998.[ISI][Medline]
32. Morrison AI, Panayotova-Heiermann M, Feigl G, Scholermann B, and Kinne RK. Sequence comparison of the sodium-D-glucose cotransport systems in rabbit renal and intestinal epithelia. Biochim Biophys Acta 1089: 121-123, 1991.[ISI][Medline]
33. Nath SK,
Rautureau M, Heyman M, Reggio H, L'helgoualc'h A, and Desjeux JF.
Emergence of Na+-glucose cotransport in an epithelial secretory
cell line sensitive to cholera toxin. Am J Physiol Gastrointest
Liver Physiol 256:
G335-G341, 1989.
34. Panayotova-Heiermann M, Eskandari S, Turk E, Zampighi GA, and Wright
EM. Five transmembrane helices form the sugar pathway through the
Na+/glucose cotransporter. J Biol Chem
272: 20324-20327,
1997.
35. Pessin JE,
Thurmond DC, Elmendorf JS, Coker KJ, and Okada S. Molecular basis of
insulin-stimulated GLUT4 vesicle trafficking. Location! Location! Location!
J Biol Chem 274:
2593-2596, 1999.
36. Schmid SL, Fuchs R, Male P, and Mellman I. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell 52: 73-83, 1988.[ISI][Medline]
37. Schmid SL and Mellman I. Isolation of functionally distinct endosome subpopulations by free-flow electrophoresis. Prog Clin Biol Res 270: 35-49, 1988.[Medline]
38. Sharp PA, Debnam ES, and Srai SK. Rapid enhancement of brush border glucose uptake after exposure of rat jejunal mucosa to glucose. Gut 39: 545-550, 1996.[Abstract]
39. Short CR, Maines MD, and Davis LE. Preparation of hepatic microsomal fraction for drug metabolism studies by rapid decompression homogenization. Proc Soc Exp Biol Med 140: 58-65, 1972.
40. Suzuki T, Fujikura K, Koyama H, Matsuzaki T, Takahashi Y, and Takata K. The apical localization of SGLT1 glucose transporter is determined by the short amino acid sequence in its N-terminal domain. Eur J Cell Biol 80: 765-774, 2001.[ISI][Medline]
41. Suzuki T, Fujikura K, and Takata K. Na+-dependent glucose transporter SGLT1 is localized in the apical plasma membrane upon completion of tight junction formation in MDCK cells. Histochem Cell Biol 106: 529-533, 1996.[ISI][Medline]
42. Towbin H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354, 1979.[Abstract]
43. Vieira-Coelho MA and Soares-da Silva P. Comparative study on sodium transport and Na+,K+-ATPase activity in Caco-2 and rat jejunal epithelial cells: effects of dopamine. Life Sci 69: 1969-1981, 2001.[ISI][Medline]
44. Walter K and Schütt C. Methods of Enzymatic Analysis. New York: Academic, 1974, vol. 2, p. 856-864.
45. Williams M and Sharp P. Regulation of jejunal glucose transporter expression by forskolin. Biochim Biophys Acta 1559: 179-185, 2002.[ISI][Medline]
46. Wright EM.
Renal Na+-glucose cotransporters. Am J Physiol Renal
Physiol 280:
F10-F18, 2001.
47. Yao X, Karam
SM, Ramilo M, Rong Q, Thibodeau A, and Forte JG. Stimulation of gastric
acid secretion by cAMP in a novel -toxin-permeabilized gland model.
Am J Physiol Cell Physiol 271:
C61-C73, 1996.
48. Yoshida A, Takata K, Kasahara T, Aoyagi T, Saito S, and Hirano H. Immunohistochemical localization of Na+-dependent glucose transporter in the rat digestive tract. Histochem J 27: 420-426, 1995.[ISI][Medline]