Correspondence to: Kai Simons, European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Programme, Postfach 102209, Meyerhofstrasse 1, D-69012 Heidelberg, Germany., Simons{at}EMBL-Heidelberg.de (E-mail), 49-6221-387-334 (phone), 49-6221-387-512 (fax)
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Abstract |
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Glycosyl-phosphatidylinositol (GPI)- anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells. It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts. We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells. The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein. However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretory proteins. In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly. Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery.
Key Words: lipid rafts, N-glycans, GPI-anchored proteins, Madin-Darby canine kidney cells, sorting
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Introduction |
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Avery heterogeneous subset of cellular surface proteins including several receptors, enzymes, and adhesion molecules is tethered to the outer leaflet of cellular membranes through a glycosyl-phosphatidylinositol (GPI)1 anchor. It has been found that most endogenous and exogenous GPI-anchored proteins and GPI-anchored fusion proteins are delivered predominantly to the apical surface of polarized epithelial cells (
However, the function of the GPI anchor as an apical targeting determinant has been questioned recently and it is possible that apical sorting information in the protein moiety accounts for the apical delivery of GPI-anchored proteins (
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To analyze the sorting information contained in a GPI anchor and thereby also the consequence of raft association for polarized sorting we expressed the nonglycosylated rat growth hormone (rGH0) linked to the GPI anchor signal of DAF (rGH0-DAF) in MDCK cells. rGH0 is secreted 40% apically and 60% basolaterally from MDCK cells (
We show that the GPI-anchored protein rGH0-DAF is delivered in an unpolarized fashion to the cell surface and that upon N-glycosylation rGH12-DAF is transported apically in MDCK cells. The nonraft-associated protein rGH0-LDL-R is transported inefficiently to the cell surface and accumulates intracellularly. The intracellular accumulation of rGH0-LDL-R can be prevented by the addition of N-glycans to the protein that act as apical sorting signals on both GPI-anchored and transmembrane proteins.
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Material and Methods |
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Cell Lines and Cell Culture
MDCK cells strain II were grown in MEM (GIBCO BRL) containing 10% FCS, supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM glutamine (GIBCO BRL). A MDCK cell line stably expressing human PLAP was obtained from D. Brown (State University of New York at Stony Brook, Stony Brook, NY) (
Antibodies
A rabbit polyclonal anti-PLAP antibody was from Dako, a rabbit polyclonal antibody against rGH was purchased from Biogenesis, and a rabbit anticaveolin-1 antibody was obtained from Santa Cruz Biotechnology. The rabbit anti-gp80 was described previously (
Preabsorbed secondary rhodamine-conjugated antirabbit and antimouse antibodies were from Dianova.
Recombinant Adenoviruses and Expression Constructs
The DNA construct pRc-CMV/rGH0-DAF (
Transfection and Viral Infections of MDCK Cells
MDCK II cells were transfected with the expression constructs pcDNA-3/rGH0-LDL-R and rGH12-LDL-R by electroporation. Stably transfected cells were selected by treatment with 0.5 mg/ml G-418 (GIBCO BRL) for 2 wk and expressing clones were identified by immunofluorescence microscopy.
Before viral infection, MDCK cells grown for 3 d on Transwell polycarbonate filters were washed once from the apical side with infection medium (MEM with 0.2% BSA, 10 mM Hepes, pH 7.3). Infection with recombinant adenoviruses was done from the apical side in a total volume of 125 µl of infection medium for 90 min. The cells were then washed once with medium, cultured for 1820 h and subsequently used either for surface transport assays or immunofluorescence microscopy.
Immunofluorescence Microscopy
MDCK cells, either filter-grown or grown on coverslips, were washed once in PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2 (PBS+) and fixed for 30 min in 4% paraformaldehyde, washed with PBS+, and quenched for 15 min with 10 mM NH4Cl in PBS containing 0.1% TX-100 to permeabilize cells. Subsequently, the cells were washed twice in PBS+ with 0.2% BSA and incubated for 1 h at room temperature. Next, the cells were incubated for 45 min at 37°C with the anti-rGH antibody diluted 1:100 in PBS/0.2% BSA. Excess antibody was removed by four washes with PBS/0.2% BSA. Primary antibodies were detected with TRITC-conjugated secondary antibodies diluted 1:200 in PBS/0.2% BSA for 45 min at 37°C. Finally, the cells were washed five times for 5 min with PBS under vigorous shaking and mounted in 90% glycerol in PBS containing 4% pyrogallol as an antifading reagent. Confocal microscopy was done on a LSM 510 Zeiss confocal microscope.
Floatation of DIGs
Cells grown on a 3-cm dish or on a 12-mm Transwell filter were scraped thoroughly in PBS and pelleted. Detergent extractions were done on ice with prechilled solutions. Cells were resuspended in 100 µl 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA (TNE) with CLAP (chymostatin, leupeptin, antipain, and pepstatin A, 25 µg/ml each final), and then 1 vol of 2% TX-100 in the same buffer was added. After 30 min of incubation the lysate was adjusted to 40% Optiprep (Nycomed Pharma As), overlaid with 30% and 5% Optiprep, and spun for 4 h in a SW-60 rotor at 28,000 rpm at 4°C. The fractions were collected from the top, precipitated in 10% TCA, separated by SDS-PAGE, and the distribution of individual proteins in the gradient was detected by Western blotting.
Selective Biotinylation of Apical and Basolateral Cell Surface Proteins
Filter-grown MDCK cells, either stable cell lines or virus infected, were washed three times for 10 min with PBS+ at 4°C. Cells were then biotinylated with 1 mg/ml sulfo-NHS-LC-biotin (Pierce) in PBS+ from the apical or basolateral side for 30 min at 4°C with PBS+ containing 1% BSA present on the other side of the filter. After three washes with PBS+ and quenching with 10 mM glycine in PBS+ the filters were cut out and cells were lysed in TNE containing CLAP, 1% TX-100, and 0.2% SDS and were sonicated in a waterbath sonicator for 10 min at room temperature.
Cell Surface Transport Assay
1820 h after viral infection filter-grown MDCK II cells were labeled with [35S]methionine (2.5 mCi/ml) in methionine-free medium for 15 min and chased in the presence of cycloheximide (10 µg/ml) and excess methionine for 040 min. Subsequently, the cells were cooled to 4°C and washed three times for 10 min with ice-cold PBS+. Surface biotinylation was performed as described above. rGH-GPI was immunoprecipitated from the lysate with 2 µl of the anti-rGH antibody and PLAP with 3 µl of the anti-PLAP antibody and with 25 µl of protein ASepharose CL-4B (Pharmacia) overnight. Beads were washed twice in buffer A (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% TX-100), three times in buffer A with 500 mM NaCl, and once in 10 mM Tris-HCl, pH 7.4. Proteins were eluted from the beads by boiling the sample twice for 5 min in 150 µl 0.6% SDS in TNE and the supernatant (300 µl) was mixed with 600 µl TNE containing 1.5% TX-100. Biotinylated proteins were precipitated with 10 µl streptavidin-agarose for 4 h at 4°C. Finally, the beads were washed twice in buffer A containing 500 mM NaCl, once in 10 mM Tris-HCl, pH 7.4, and bound proteins were analyzed by SDS-PAGE and autoradiography. Gp80 was immunoprecipitated from the apical and basolateral medium with 1 µl of the anti-gp80 antibody.
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Results |
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N-Glycans as Apical Sorting Signals on GPI-anchored rGH
The expression of GPI-anchored forms of wild-type and doubly glycosylated rGH (rGH0 and rGH12) allowed us to analyze the targeting information contained in the GPI anchor in the presence or absence of additional sorting information in the protein. We used recombinant adenoviruses to express rGH0-DAF and rGH12-DAF (Figure 1) in MDCK cells. The steady-state distribution of the fusion proteins in filter-grown MDCK cells was analyzed by confocal immunofluorescence microscopy. As can be seen in Figure 2, rGH0-DAF was detectable at both the apical (Figure 2 A) and the basolateral (Figure 2 B) surface. The N-glycosylated rGH12-DAF showed a predominant apical distribution (Figure 2 C) and was hardly detectable on the basolateral side (Figure 2 D). Both GPI-anchored proteins were almost exclusively detected at the cell surface, and the presence of a significant intracellular pool was not observed. We further analyzed the steady-state distribution of the proteins in filter-grown MDCK cells by selective biotinylation of the apical or basolateral cell surface 18 h after adenoviral infection. As can be seen in Figure 3 A, rGH0-DAF can be detected as expected on a Western blot as a single band of 29 kD (
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We next analyzed the detergent insolubility of the apical and basolateral pools of the rGH0-DAF separately. Surface proteins of filter-grown cells were biotinylated from the apical or basolateral side and the cells were extracted with TX-100 on ice. The detergent-resistant fraction was floated in an Optiprep gradient centrifugation and analyzed for the presence of biotinylated rGH0-DAF (Figure 3 B). Two fractions collected from the gradient are shown: the 530% Optiprep interface containing the DIGs (I), and the 40% Optiprep bottom fraction containing the solubilized material (S). We found that 90% of both the apical and the basolateral pool of the protein were floating to the 530% Optiprep interface, indicating that the large basolateral pool of rGH0-DAF was raft-associated also. The small basolateral pool of PLAP in MDCK cells has also reported to be resistant to TX-100 extraction (
Next we analyzed the biosynthetic surface delivery of the proteins in pulsechase experiments. Based on autoradiography, the nonglycosylated GPI-anchored rGH0-DAF was found to be delivered predominantly to the basolateral side of MDCK cells (Figure 3 C). Quantification showed that after 40 min of chase only 40 ± 5% (n = 12) of rGH0-DAF was delivered to the apical surface and 60 ± 5% of the protein was delivered directly to the basolateral side (Figure 4). In contrast, the mono- and doubly glycosylated forms of rGH12-DAF were both delivered 63 ± 5% (n = 12) to the apical surface (Figure 3 C). Similar results were obtained in time course experiments at 20, 30, and 60 min of chase (data not shown). These results show that the sorting of GPI-anchored rGH0 is similar to that of secretory rGH0 in MDCK cells. The addition of N-glycans to GPI-anchored rGH-DAF clearly leads to increased apical delivery as it has been previously shown for the secretory form.
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As a control we analyzed in parallel the apical delivery of PLAP in a stable MDCK cell line (
Our data demonstrate that the attachment of a GPI anchor to a protein is sufficient for raft association but not sufficient for predominant apical delivery. Furthermore, the experiments provide evidence that N-glycans can act as apical targeting signals on GPI-anchored proteins.
Reduced Cell Surface Transport of a NonRaft-associated, Nonglycosylated Membrane Protein
To address the question of how a nonraft-associated, nonglycosylated protein is transported in MDCK cells, we constructed chimeric transmembrane proteins consisting of rGH0 or rGH12 as the ectodomain, and the TMD and the CT12 truncation of the cytoplasmic tail of the human LDL-R (Figure 1). The CT12 mutation of the human LDL-R (
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The distribution of the fusion proteins was analyzed by immunofluorescence microscopy in unpolarized MDCK cells. As can be seen in Figure 6 A, rGH0-LDL-R was detected at steady state in the perinuclear region, resembling the Golgi complex, and to a lower extent at the plasma membrane. In contrast, the glycosylated rGH12-LDL-R shows a clear cell surface staining and only a minor fraction is visible in internal structures (Figure 6 B).
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The strong internal staining of rGH0-LDL-R (Figure 6 A) prompted us to compare the amount of the protein present on the cell surface with the amount of protein that accumulated within the cells at steady state. Polarized filter-grown cells were surface-biotinylated simultaneously from the apical and basolateral sides. The biotinylated surface proteins were precipitated from the cell lysate with streptavidin-agarose. The unbound nonbiotinylated proteins in the depleted supernatant were precipitated with TCA. The presence of the fusion proteins was analyzed in both fractions on Western blots and quantified using NIH Image software. Only 30 ± 6% (n = 4) of the total rGH0-LDL-R were precipitated by streptavidin-agarose (Figure 7 A, lane 1), whereas 70 ± 6% of the molecules were left in the supernatant (Figure 7 A, lane 2) and therefore are considered as being accumulated within the cells. Thus, the nonglycosylated, non-raft membrane protein rGH0-LDL-R accumulated intracellularly and was transported inefficiently to the cell surface. In contrast, the majority of rGH12-LDL-R (82 ± 4%, n = 3) was biotinylated and precipitated by streptavidin-agarose. We did not find a significant difference between the efficiency of surface transport of mono- and doubly glycosylated rGH-LDL-R.
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Finally, we analyzed the surface distribution of rGH12-LDL-R by confocal immunofluorescence microscopy in polarized MDCK cells and found that the glycosylated fusion protein was almost exclusively localized at the apical surface (Figure 6 C), and only little basolateral staining was detectable (Figure 6 D). The steady-state distribution of both fusion proteins was further analyzed by surface biotinylation of filter-grown cells. We found that the majority of the cell surface fraction of rGH0-LDL-R was at the basolateral cell surface (Figure 7 B, lane 2), whereas the glycosylated rGH12-LDL-R was predominantly detected at the apical side (Figure 7 B, lane 3). This shows that N-glycans can act as apical sorting signals on non-raft proteins.
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Discussion |
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In this paper, we have analyzed whether association of proteins to lipid rafts by a GPI anchor leads to predominant apical delivery from the TGN in MDCK cells. Here, we show that when rGH0 is GPI-anchored the basolateral and the apical surface delivery is 60 and 40%, respectively. Previous studies in our lab showed that the secretory form of rGH0 is secreted 60% basolaterally and 40% apically in MDCK cells (
Nevertheless, mechanisms different from glycan-mediated sorting may also lead to preferential apical delivery of secretory and GPI-anchored proteins. Lisanti and co-workers found that GPI-anchored nonglycosylated human growth hormone was apically localized in MDCK cells at steady state (
Apical Delivery of Glycosylated Proteins
One interesting observation presented in this paper is the intracellular accumulation of the nonraft-associated rGH0-LDL-R which can be overcome by the addition of N-glycans. The same phenomenon was seen previously by
Alternative models for the role of glycans in apical delivery have also been forwarded based on glycans affecting the folding of proteins and stabilizing a transport-permissive conformation (
VIP36 was a candidate for a lectin involved in apical sorting (
How Are Lipid Rafts Transported Basolaterally?
We found that 60% of rGH0-DAF is delivered to the basolateral surface of MDCK cells. This shows that rafts are not restricted to the apical pathway. Clearly, the basolateral plasma membrane contains raft lipids, but in lower concentrations than in the apical membrane (
One important conclusion is that raft-based sorting is not an all or none phenomenon, demonstrated in this paper by the fact that raft association via a GPI anchor is not sufficient for predominant apical delivery. Several layers of interactions with raft platforms can be envisaged that lead to efficient surface-specific delivery of rafts and their associated proteins.
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Acknowledgements |
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We thank P. Keller for advice during the generation of recombinant adenoviruses, K. Ekroos for cell culture, and T. Kurzchalia and C. Koch-Brandt for the generous gift of cDNAs and antibodies. We thank Suzanne Eaton and Derek Toomre for critical reading of the manuscript. Many thanks to Iris Ansorge and Konrad for valuable discussions and support.
J. Benting and A. Rietveld were supported by an EMBO long-term fellowship. This work was supported by the Commission of the European Community and grant SFB 352.
Submitted: May 11, 1999; Revised: June 8, 1999; Accepted: June 11, 1999.
1.used in this paper: DAF, decay acceleration factor; DIG, detergent-insoluble glycosphingolipid complex; GPI, glycosyl-phosphatidylinositol; LDL-R, human low density lipoprotein receptor; PLAP, placental alkaline phosphatase; rGH, rat growth hormone; TMD, transmembrane domain; TX-100, Triton X-100
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