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Deletion of the cytoplasmic domain of the polymeric immunoglobulin receptor prevents basolateral localization and endocytosis
Cell, Vol. 47, 359-364,
November 7, 1986, Copyright
0 1986 by Cell Press
eletion of the Cytoplasmic Domain of the lmmunoglobulin Receptor Prevents asolateral Localization and Endocytosis ith E. ostov, Anne de Bruyn Kops, and David L. Deitcher itehead Institute e Cambridge Center mbridge, Massachusetts 02142
Summary We deleted the cytoplasmic domain of the polymeric immunoglobulin receptor. When expressed in fibroblasts, the truncated receptor, like the wild-type, reaches the ceil surface, can bind ligand, and is cleaved to secretory component. Unlike the wild-type, it is not endocytosed. When expressed in polarized MadinDarby canine kidney cells, the mutant receptor is transported from the Golgi apparatus directly to the apical surface and cleaved to secretory component. In contrast, the wild-type receptor travels from the Golgi to the basolateral surface and is then endocytosed and sent to the apical surface. These results suggest toplasmic domain of the receptor is necesth basolateral localization and endocytosis.
Many types of epithelial cells produce a receptor that transports polymeric IgA and IgM across the cell and into external secretions (Mostov and Simister, 1985). After synthesis, this receptor is sent to the basolateral surface where it binds polymeric immunoglobulins (poly-lg). The receptor-ligand complex is then endocytosed, transported across the cells in vesicles, and exocytosed at the apical surface (Sztul et al., 1983, 1985; Solari and Kraehenbuhl, 1984; Hoppe et al., 1985; Limet et al., 1985). During this process of transcytosis, or after exocytosis at the apical surface, the ligand-binding domain of the receptor is proteolytically cleaved and subsequently discharged into the external secretion in association with the poly-lg (Mostov et al., 1980; Mostov and Blobel, 1982). Thus, the receptor is used for only one round of transport. The ligand-binding domain that is released into the secretion is known as secretory component (SC). We have been investigating the pathway of this receptor as a model system for studying the protein sorting that occurs in endocytosis and the generation and maintenance of cell polarity (Matlin and Simons, 1984; Misek et al., 1984; Pfeiffer et al., 1985; Simons and Fuller, 1985). Little is known about the signals on membrane proteins that direct them to their correct location in the cell (Blobel, 1980). It has long been suspected that the cytoplasmic tails of transmembrane proteins may contain such signals, since these tails could easily interact with other proteins in the cytoplasm. One recent approach to identifying sorting sig-
* Present address: Committee on Cell and Developmental Biology, Division of Medical Sciences, Harvard University, 25 Shattuck St., Boston, Massachusetts 02115.
nals has been to analyze altered proteins, which are the result of either natural mutations or site-directed mutagenesis of cloned genes (for review, see Garoff, 1985). Perhaps the most informative work has been on the low density lipoprotein receptor (Goldstein et al., 1985). Alterations of the 50 amino acid cytoplasmic tail of this receptor (either certain single amino acid substitutions or large deletions) result in receptors that reach the cell surface normally but are not endocytosed (Lehrman et al., 1985; Davis et al., 1986). The cytoplasmic domains of other plasma membrane proteins have been altered, and the results have been inconsistent, ranging from no effect on transport to proteins that remain stuck in the rough endoplasmic reticulum or the Golgi (Doyle et al., 1985; Zuniga et al., 1983). We have recently expressed the cloned cDNA for the poly-lg receptor in both nonpolarized fibroblasts (Deitcher et al., 1986) and polarized Madin-Darby canine kidney (MDCK) epithelial cell line (Mostov and Deitcher, 1986). In fibroblasts the receptor reaches the cell surface where it can bind poly-lg. At least 30% of the bound poly-lg is rapidly endocytosed and then re-exocytosed. The receptor is cleaved to SC, which is released into the medium. The exact site of cleavage in the cell is unknown. In the MDCK cells the receptor travels first to the basoiateral surface where it can bind poly-lg. The receptor (with or without iigand) is then endocytosed, transcytosed, and exocyiosed at the apical surface. The receptor is cleaved to SC, so SC and poly-lg are released into the apical medium. ‘Thus, as far as can be determined, the pathway of the exogenous receptor expressed in MDCK cells resembles its pathway in vivo. We can quantitatively assay the various steps of receptor transport in these cell culture systems. This provides an excellent opportunity to identify possible sorting sequences in the receptor by in vitro mutagenesis. We have now deleted virtually the entire cytoplasmic tail of the poly-lg receptor by oligonucleotide-directed mutagenesis and have examined the effects on receptor transport. In fibroblasts the receptor reaches the cell surface, ican bind poly-lg, and is cleaved to SC. However, there is no detectable endocytosis of the receptor. In MDCK cells ahe receptor never reaches the basoiateral surface. Instead, it goes directly to the apical surface, and SC is released into the apical medium. Results After removal of the signal peptide, the intact rabbit poly-lg receptor consists of 755 amino acids (Mostov et al., 19S4). Starting at the N-terminus, residues l-629 are extracellular, residues 630-652 span the membrane, and residues 653-755 are cytoplasmic. Using a mutagenic oligonucleotide and the standard single-stranded nique (Zoller and Smith, 1983) we introduced a stop codon in place of residue 655. This deleted 101 residues from the C-terminus and left a cytoplasmic domain of only 2 residues, Arg-Ala. We designated this the “minus-tail receptor?
Cell 360
Control
Figure1. Pulse-ChaseAnalysisof Processing of Wild-Typeand Mutant Receptor in Fibro-
Minus Tail
blasts Cells expressing the wild-type or mutant recep tor were pulse-labeled for 15 min with [35S]cysteine, then chased for the indicated period. Cells and media were then immunoprecipitated and analyzed by SDS-PAGE and fluorography.
68-
As previously described, we used the retroviral vector DO-L and the w packaging system to express this truncated receptor in murine fibroblasts (Deitcher et al., 1986). Cells expressing either the wild-type receptor (control) or the minus-tail mutant were pulse-labeled with [ssS]cysteine and chased for various periods. Cells and media were then immunoprecipitated with antiserum to rabbit SC, then analyzed by SDS-PAGE and fluorography. As seen in Figure 1 (and as previously reported), in the control cells a single polypeptide of m90 kd is present after the pulse and is converted to a poorly resolved group of species of ml00 kd due to modification of the carbohydrates to the complex type. These are cleaved to a group of products of ~70 kd, which appear in the medium and comigrate with authentic rabbit milk SC. As previously described, the heterogeneity of SC is due, at least in part, to variability in the exact site of cleavage of SC from the receptor (Eiffert et al., 1984; Deitcher et al., 1986). With the minus-tail mutant, a protein of ~75 kd is initially present. This does not shift up appreciably with time, but does acquire complex carbohydrates, as judged by endoglycosidase H resistance (data not shown). The mutant receptor is also cleaved to a group of species of m70 kd that appear in the medium and comigrate with authentic SC. The kinetics of release of SC appear roughly similar to the control. These results do not directly show that the receptor is reaching the cell surface, but do indicate that it is cleaved to SC, which is released into the medium. We studied the binding of a ligand, 1251-labeled human dimeric IgA (dIgA), to the cells at 4%. The results in the cells expressing the minus-tail mutant were in good agreement with our previously reported data in control cells: approximately 9,000-10,000 receptors per cell with an apparen? Ko of 8 x 1O-g M (data not shown). This strongly suggests that the mutant receptor is present on the cell surface and is capable of binding ligand. We previously found that at least 30% of dlgA bound at 4% could be rapidly internalized, as assayed by acquisition of protease resistance, when cells were warmed to 37%. The internalized dlgA returned to the cell surface within lo-15 min. All of the bound dIgA, whether or not it had been internalized, was released into the medium within 20 min. When we carried out this experiment with
.o--
,;
5
IO
D
15
20
Time (Minutes) Figure 2. Internalization of [‘asl]dlgA by Fibroblasts WildType or Mutant Receptor
Expressing
the
[1z51]dlgA (0.4 pglmi, 4 x 10s cpm/ug) was bound at 4% for 2 hr to cells expressing either the wild-type (A) or mutant (B) receptor. Unbound ligand was washed away at 4°C and the cells were warmed to 37°C for the indicated time. The amount of ligand released into the warmup medium is indicated by the triangles. Cells were then cooled to 4%, digested with pronase for 2 hr, and sedimented. Radioactivity released into the digestion buffer is defined as cell surface ligand and is indicated by the closed circles. Ligand remaining associated with the cell pellet is defined as internalized ligand and is indicated by the open circles. Values are expressed as fraction of the total dlgA initially bound, which average 1,240 pg per 35 mm dish for the wild type and 1,397 pg per 35 mm dish for the mutant. Points are mean of triplicates, and standard errors of the means were less than ?O%.
Cytoplasmic 381
Domain of poly-lg Receptor
Control
Minus Tail
Figure 3. Pulse-Chase Analysis of Processing of Wild-Type and Mutant Receptor in MDCK
Cells MDCK cells expressing the wild-type or mutant receptor and grown on Miilipore filters were pulse-labeled for 15 min with [%]cysteine, then chased for the indicated periods. Cells and apical (A) or basal (8) media were then immunoprecipitated and analyzed by SDS-PAGE and fluorography. When electrophoresed in adjacent lanes, the material released into apical media from both types of cells comigrates. This is not apparent in this figure due to curvature
of the gel.
the minus-tail mutant, we did not detect any internalized dlgA (Figure 2, compare open circles for control [A] and minus-tail [B]). instead, all of the bound dlgA was rapidly released into the medium (triangles). This result suggests that the minus-tail poly-lg receptor cannot be endocytosed. We also expressed the minus-tail receptor in MDCK cells and compared it with the control in a pulse-chase experiment (Figure 3). The wild-type receptor is cleaved to SC, which is released almost entirely into the apical medium. Processing of the mutant receptor in MDCK cells was similar to that observed in fibroblasts except that at the earliest time point (0 hr chase) two distinct species are seen. Treatment of this sample with endoglycosidase H converts both species to a single species of faster mobility, suggesting that in MDCK cells two differently glycosyfated forms of the same polypeptide are produced (data not shown). During the chase period, these two species do not undergo a clear-cut change in mobility. Nevertheless, they do become endoglycosidase H-resistant, suggesting that modification of the carbohydrate to the complex form does occur. The receptor is cleaved to SC, which is released into the medium. As in the wild-type, SC is released almost entirely into the apical medium, although release is slightly faster than in the wild-type. We previously obtained direct evidence that the wildtype receptor reaches the basolateral surface before being released as SC in the apical medium (Mostov and Deitcher, 1988). Cells were pulse-labeled, and during the 4 hr chase, goat antiserum against rabbit SC was included in the basal medium. These antibodies bound the receptor exposed at the basolateral surface and remained attached to the receptor as it was transcytosed across the ceil. Hence the cleaved SC released into the apical medium already had antibodies bound to it and was immunoprecipitated by simply adding protein A-Sepharose to the medium. Not all of the SC released into the apical medium had bound anti-SC antibodies at the basolateral surface, either because the receptor was never exposed at the basolateral surface or because of other reasons. This “residual” SC could be recovered by adding anti-SC antibodies to the apical medium (after first absorbing it inrith protein A-Sepharose) and isolating the immune complexes with additional protein A-Sepharose. We found the ceils expressing
that in the wild-type cells, roughly 90% of the SC released into the apical medium had bound antibodies added ?othe basolateral medium. When we performed the same experiments on the minus-tail mutant, we observed that almost none of the SC released into the apical medium was bound to antibodies added basally (Figure 4, compare antibody-bound SC in the apical medium of the cells expressing the minus-tail receptor [lane 51 with that in the wild type [lane I]). Virtually all of the SC was recovered as residual SC (Figure 4, compare residual SC in the mutant [lane l] and in the wildtype [lane 31).These data are quantitated in Table 1,where the total SC recovered from the apical medium is taken as ‘100%. Performing the chase for 8 hr instead of 4 hr did not substantially affect the results. This experiment suggests that the minus-tail receptor is sent directly to the apical surface, without an intermediate stop at the basolateral surface. It is possible that the receptor is transiently exposed at the basolateral surface, but fails to bind the antibodies either because it is too rapidly internalized or because it is in a conformation not recognized by the antiserum. The former seems unlikely because of the defect in endocytosis described above, while the latter is also improbable because the SC released into the medium is recognized by the antiserum. Several indirect lines of evidence aiso support the hypothesis that the minus-tail receptor is sent directly to the apical surface. If the receptor were to reach the basoiateral surface it might accumulate there, since the receptor cannot be endocytosed. (We have only directly shown that ligand bound to the receptor is not endocylosed in fibroblasts, but it seems likely that the receptor itself is not endocytosed and that this would also be true in MDCK cells. We could not assay internalization at the basolateral surface of MDCK cells by protease protection, due to interference from protein bound to the filter.) In the pulse-chase experiments (Figure 3) the receptor does not accumulate at the basolateral surface, but rather is almost entirely chased to SC in the apical medium. Another possibility is that the receptor is slowly cleaved to SC at the oasolateral surface. However, only traces of SC are released into the basal medium. We previously observed that MDCK cells expressing the wild-type receptor could transcytose [‘25l]dlgA (Mostov
Ceil 362
12345678
I i
ABAB
R
Figure 4. Effect of Including Medium during the Chase
ABAB
A nt iserum
R
against
SC in the Basal
MDCK cells expressing either the minus-tail or the wild-type receptor were grown on Millipore filters and pulse-labeled for 1TT5 min. During the 4 hr chase, 3 ~1 of goat antiserum against rabbit SC was included in the basal medium. However, the apical (A) and basal (B) media were adsorbed with protein A-Sepharose without adding additional antiserum. These immunoprecipitates were then analyzed by SDS-PAGE and fluorography (lanes 5 and 6 for minus-tail, lanes 1 and 2 for wild type). Residual SC (designated R) was recovered from the supernatants of these immunoprecipitations by adding 3 nl of additional antiserum, incubating at 22% for2 hr, and adsorbing the immune complexes with additional protein A-Sepharose (lanes 7 and 8 for minus-tail, lanes 3 and 4 for wild type).
and Deitcher, 1986). We have now compared dlgA transcytosis by the cells expressing the wild-type receptor (Figure 5A) and the minus-tail receptor (Figure 58). Transcytosis from the basal medium to the apical medium was roughly 10 times greater in cells expressing the wild-type receptor than in cells expressing the minus-tail receptor (Figures 5A and 5B, compare open circles). In cells expressing the wild-type receptor, this transport could be largely competed away by an excess of unlabeled dIgA, whereas transport was not competed away in the cells expressing the minus-tail receptor (Figures 5A and 58, com-
Table 1. Quantitation
of lmmunoprecipitation
pare squares). In both cell types there was little transport from the apical medium to the basal medium (Figure 5, closed circles), and this was not competed away by unlabeled dlgA (triangles). This low level of transport was similar to that observed in either direction in the parent cell line that does not express the receptor (data not shown). This suggests that the small amount of transport seen in the cells expressing the minus-tail receptor was due to leakage and/or fluid-phase transcytosis (von Bonsdorff et al., 1985) and that the mutant receptor does not function in transcytosis. Since the minus-tail receptor can bind ligand, this result further supports the hypothesis that the receptor does not travel to the basolateral surface before reaching the apical surface. We also attempted to localize the receptor by fixing the cells on filters, applying antiserum to SC to either the apical or the basolateral surface, and then visualizing with fluorescent second antibody. We observed weak fluorescence at the apical surface, similar to that seen with the wild-type receptor. No specific fluorescence was seen at the basolateral surface. However, there was a moderately high background, due to nonspecific sticking of the antibodies to the nitrocellulose filter. Hence a weak basolateral signal may have escaped detection (data not shown). This problem has also hampered the development of a sufficiently sensitive radioimmunoassay for detecting basolateral receptor. Discussion We have deleted virtually the entire cytoplasmic domain of the poly-lg receptor and expressed this mutant protein in both nonpolarized fibroblasts and polarized MDCK cells. In fibroblasts the mutant receptor, like the wild-type, is transported to the cell surface, can bind ligand, and is cleaved to SC and released. However, unlike the wild-type, the minus-tail receptor cannot internalize ligand. Naturally occurring mutations in the cytoplasmic domain of the low density lipoprotein receptor have shown that its cytoplasmic tail is important for clustering into coated pits and endocytosis (Lehrman et al., 1985; Davis et at., 1986). Our data extend this to a second receptor system, suggesting that the cytoplasmic tail may be generally involved in endocytosis. In polarized MDCK cells the minus-tail receptor reaches the apical surface and is cleaved to SC, which is released almost exclusively into the apical medium. However, unlike the wild-type, the minus-tail receptor apparently never makes an intermediate stop at the basolateral surface, but rather goes directly to the apical surface. There are S@V-
of Apical Medium % of SC in Apical Medium ( f SEM)
Cells
Sample
4 hr Chase
8 hr Chase
Wild-type
First immtmoprecipitation Residual immunoprecipitation
92 (17)
89 (12)
8 (5)
11 (6)
First immunoprecipitation Residual immunoprecipitation
6 (3) 94 (21)
3 (2) 97 (15)
Mutant
;?3oplasmic
Domain of poly-ig Receptor
20
5
Time (Hours) Figure 5. Transcytosis of dlgA by MDCK Cells Expressing Type or Mutant Receptor
the Wild-
MDCK cells expressing the wild-type (A) or minus-tail (B) receptor were grown on Millipore filters. [1251]dlgA (0.2 bglml) with or without unlabeled dlgA (50 pglml) was added to the basolateral or apical medium. After the indicated time, the medium on the side opposite to which the labeled dlgA was added was collected, TCA-precipitated, and counted in a gamma counter. Open circles, [‘251]dlgA alone added to basal medium; squares, [lnsl]dlgA and unlabeled dlgA added to the basal medium; triangles, [1*51]dlgA added to the apical medium; closed circles, [1251]dlgAand unlabeled dlgA added to the apical medium. Standard errors of the mean of triplicate points were less than 20%.
era1 possible interpretations of this result. Most likely, the cytoplasmic domain contains a positive signal directing the molecule to the basolateral surface. The information for reaching the apical surface may reside in a signal in the noncytoplasmic (either extracellular or transmembrane) portion of the molecule. Alternatively, in the absence of a signal to go to the basolateral surface, the receptor goes to the apical surface by default, without needing any specific signal. When exogenous secretory proteins (e.g., chicken lysozyme) are expressed in MDCK cells, they are released roughly equally at both surfaces, suggesting that the default pathway for secretory proteins is to both surfaces at random (Kondor-Koch et al., 1985; Gottlieb et al., 1986). Whether there is a default pathway for membrane proteins leading exclusively to one surface or the other is not known. One could also imagine that the mutant protein has an altered conformation that somehow prevents it from going to the basolateral surface, but causes it to go to the apical surface. (However, the conformation is not so altered as to prevent ligand binding or cleavage.) This change could, for instance, be from an oligomer to a monomer or vice versa, although there is no evidence that the receptor is ever oligomeric. Larkin, et al. (1986) have recently shown that the cytoplasmic tail of the poly-lg receptor becomes phosphorylated on serine. It is tempting to speculate that this phosphorylation plays a role in targeting the protein to the correct location.
The mutant receptor apparently goes directly from the Golgi to the apical surface and probably follows the same pathway as other proteins that are found exclusively on the apical surface, such as the ~emagglut~nin of influenza virus. The wild-type receptor travels to the apical surface anly after endocytosis at the basoiateral surface. If positive signals to direct the proteins to the apical surface are involved in both pathways, we can speculate that the same recognition machinery is used in both paths. Moreover, regardless of whether or not positive signals are involved, it is possible that apically directed proteins coming from either the Golgi or the endosomes pass through a common compartment. This data is, to the best of our knowledge, the clearest indication that the cytoplasmic domain of a plasma membrane protein is involved in directing the protein to one surface or the other of a polarized cell. The pathway of the poly-lg receptor is unusually complex, gaing first to one surface, then to the other. It is possible that this role of the cytoplasmic tail is unique to the poly-lg receptor and that other basolateral membrane proteins are targeted to that surface by signals located elsewhere.
Experimental
Procedures
of Mutant poly-lg Receptor Ciones Construction All recombinant DNA steps used standard procedures (Maniatis et al., 1982). The previously described fragment (nucleotides 96-2,451) that contains the entire coding region of the poly-lg receptor and to which Bglll linkers had been added (Deitcher et al., 1986) was filled in with the Klenowfragment of DNApoymerase I. Replicativeform of M13mp8 phage was cut with Sall, filled in with Klenow, and treated with calf intestinal phosphatase. The poly-lg receptor coding fragment was bluntend-ligated into this mp8 vector. This ligation regenerates the Bglll sites at the ends of the coding fragment, allowing its easy recovery ‘ram the replicative form of the phage. A 17-mer oligonucleotide, TCCTGTGCTAGGCTCTG, was synthesized on an Applied Biosystems synthesizer, purified on a 20% acrylamide, 7 M ureagel, eluted by diffusion, and ethanol-precipitated. This oiigonucleotide is complementary to nucieotides 2282-2298 of the cDNA, except for two changes that convert the Arg residue 655 to 2 stop codon. The standard oligonucleotide mutagenesis procedure was performed (Zoller and Smith, 1983), and mutant clones were identified by hybridization with the mutagenic oligonucleotide using washes with 3 M tetramethylammonium chloride at 50°C (Wood et al., 1985). The mutation was verified by dideoxy sequencing. To ensure that no additional mutations were introduced, we resequenced the entire cDNA insert, using a series of sequencing primers spaced approximately every 300 nucleotides. Replicative form DNA from phage containing the mutant was prepared, then digested with 3glll. The cDNA insert was ligated into the BamHI site of the DOL vector. Retroviral vector DNA was transfected into vAM cells, and the transiently produced virus was used to infect ~2 cells (a derivative of NIH 3T3 fibroblasts) and MDCK strain II cells. Six G418-resistant clones of each were picked, expanded, and screened for mutant poly-lg receptor production by pulse labeling with [%]cysteine, immunoprecipitation, SDS-PAGE, and fluorography. All clones produced receptor. One ~2 clone and one MDCK clone were selected for further study. The MDCK clone was checked for polarized growth on Millipore filters by polarity of [%]methionine uptake and by immunofluorescence with an endogencus 60 kd membrane protein (Herzlinger and Ojakian, 1984) and was determined to be 2s polar as the parent MDCK strain. Other Procedures All other procedures used in analysis of the poly-lg receptor transport have been previously described (Deitcher et al., 1986; Mostov and Deitcher, 1986). These include growth on filters, pulse-chase, immuno-
Cell 364
precipitation, ligand binding, internalization, transcytosis, of anti-SC antibodies to the basolateral surface.
and binding
Acknowledgments We thank Drs. l-l. Lodish, K. Matlin, and E. Rodriguez-Boulan for useful discussions, J.-P Kraehenbuhl and G. Ojakian for antibodies, and Ms. Devon Young for manuscript preparation. This work was supported by an NIH New Investigator Award to K. M. A. de B. K. was supported by an NSF predoctoral fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received June 10, 1986; revised August 12, 1986 References Blobel, G. (1980). Intracellular Sci. USA 77, 1496-1500.
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of epithelial cell polarity: intracellular sorting and vectoral exocytosis of an apical plasma membrane glycoprotein. Cell 39, 537-546. Mostov, K. E., and Simister, N. E. (1985). Transcytosis. Cell 43,389-390. Mostov, K. E., and Blobel, G. (1982). A transmembrane precursor of secretory component. J. Biol. Chem. 257; 11816-11821. Mostov, K. E., and Deitcher, D. L. (1986). Polymeric immunoglobulin receptor expressed in MDCK cells transcytoses IgA. Cell 46, 613-621. Mostov, K. E., Kraehenbuhl, J.-P., and Blobel, G. (1980). Receptormediated transcellular transport of immunoglobulins: synthesis of secretory component as multiple and larger transmembrane forms. Proc. Natl. Acad. Sci. USA 77, 7257-7261. Mostov, K. E., Friedlander, M., and Blobel, G. (1984). The receptor for transepithelial transport of IgA and IgM contains multiple immunoglobulin-like domains. Nature 308, 37-43. Pfeiffer, S., Fuller, S. D., and Simons, K. (1985). lntracellularsortingand basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells. J. Cell Biol. 107, 470-476. Simons, K., and Fuiler, S. D. (1985). Cell surface polarity in epithelia. Ann. Rev. Cell Biol. 7, 243-288. Solari, R., and Kraehenbuhl, J.-P (1984). Biosynthesis of the IgA antibody receptor: a model for the transepithelial sorting of a membrane giycoprotein. Cell 36, 61-71. Sztul, E. S., Howell, K. E., and Palade, G. E. (1983). Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes. J. Cell Biol. 97; 1582-1591. Sztul, E. S., Howell, K. E., and Palade, G. E. (1985). Biogenesis of the polymeric IgA receptor in rat hepatocytes. II. Localization of its intracellular forms by cell fractionation studies. J. Cell Biol. 700, 1255-1261. von Bonsdorff, C.-H., Fuller, S. D., and Simons, K. (1985). Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters. EMBO J. 4, 2781-2792. Wood, W., Gitschier, J., Lasky, L., and Lawn, R. (1985). Base composition independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc. Natl. Acad. Sci. USA 82, 1585-1588. Zoller, M., and Smith, M. (1983). Oligonucleotide-directed mutagenesis of DNA fragments cloned into Ml3 vectors. Meth. Enzymol. 700, 468-500. Zuniga, M. C., Malissen, B., McMillan, M., Brayton, P. R., Clark, S. S., Forman, J., and Hood, L. (1983). Expression and function of transplantation antigens with altered or deleted cytoplasmic domains. Cell 34, 535-544.
November 7, 1986, Copyright
0 1986 by Cell Press
eletion of the Cytoplasmic Domain of the lmmunoglobulin Receptor Prevents asolateral Localization and Endocytosis ith E. ostov, Anne de Bruyn Kops, and David L. Deitcher itehead Institute e Cambridge Center mbridge, Massachusetts 02142
Summary We deleted the cytoplasmic domain of the polymeric immunoglobulin receptor. When expressed in fibroblasts, the truncated receptor, like the wild-type, reaches the ceil surface, can bind ligand, and is cleaved to secretory component. Unlike the wild-type, it is not endocytosed. When expressed in polarized MadinDarby canine kidney cells, the mutant receptor is transported from the Golgi apparatus directly to the apical surface and cleaved to secretory component. In contrast, the wild-type receptor travels from the Golgi to the basolateral surface and is then endocytosed and sent to the apical surface. These results suggest toplasmic domain of the receptor is necesth basolateral localization and endocytosis.
Many types of epithelial cells produce a receptor that transports polymeric IgA and IgM across the cell and into external secretions (Mostov and Simister, 1985). After synthesis, this receptor is sent to the basolateral surface where it binds polymeric immunoglobulins (poly-lg). The receptor-ligand complex is then endocytosed, transported across the cells in vesicles, and exocytosed at the apical surface (Sztul et al., 1983, 1985; Solari and Kraehenbuhl, 1984; Hoppe et al., 1985; Limet et al., 1985). During this process of transcytosis, or after exocytosis at the apical surface, the ligand-binding domain of the receptor is proteolytically cleaved and subsequently discharged into the external secretion in association with the poly-lg (Mostov et al., 1980; Mostov and Blobel, 1982). Thus, the receptor is used for only one round of transport. The ligand-binding domain that is released into the secretion is known as secretory component (SC). We have been investigating the pathway of this receptor as a model system for studying the protein sorting that occurs in endocytosis and the generation and maintenance of cell polarity (Matlin and Simons, 1984; Misek et al., 1984; Pfeiffer et al., 1985; Simons and Fuller, 1985). Little is known about the signals on membrane proteins that direct them to their correct location in the cell (Blobel, 1980). It has long been suspected that the cytoplasmic tails of transmembrane proteins may contain such signals, since these tails could easily interact with other proteins in the cytoplasm. One recent approach to identifying sorting sig-
* Present address: Committee on Cell and Developmental Biology, Division of Medical Sciences, Harvard University, 25 Shattuck St., Boston, Massachusetts 02115.
nals has been to analyze altered proteins, which are the result of either natural mutations or site-directed mutagenesis of cloned genes (for review, see Garoff, 1985). Perhaps the most informative work has been on the low density lipoprotein receptor (Goldstein et al., 1985). Alterations of the 50 amino acid cytoplasmic tail of this receptor (either certain single amino acid substitutions or large deletions) result in receptors that reach the cell surface normally but are not endocytosed (Lehrman et al., 1985; Davis et al., 1986). The cytoplasmic domains of other plasma membrane proteins have been altered, and the results have been inconsistent, ranging from no effect on transport to proteins that remain stuck in the rough endoplasmic reticulum or the Golgi (Doyle et al., 1985; Zuniga et al., 1983). We have recently expressed the cloned cDNA for the poly-lg receptor in both nonpolarized fibroblasts (Deitcher et al., 1986) and polarized Madin-Darby canine kidney (MDCK) epithelial cell line (Mostov and Deitcher, 1986). In fibroblasts the receptor reaches the cell surface where it can bind poly-lg. At least 30% of the bound poly-lg is rapidly endocytosed and then re-exocytosed. The receptor is cleaved to SC, which is released into the medium. The exact site of cleavage in the cell is unknown. In the MDCK cells the receptor travels first to the basoiateral surface where it can bind poly-lg. The receptor (with or without iigand) is then endocytosed, transcytosed, and exocyiosed at the apical surface. The receptor is cleaved to SC, so SC and poly-lg are released into the apical medium. ‘Thus, as far as can be determined, the pathway of the exogenous receptor expressed in MDCK cells resembles its pathway in vivo. We can quantitatively assay the various steps of receptor transport in these cell culture systems. This provides an excellent opportunity to identify possible sorting sequences in the receptor by in vitro mutagenesis. We have now deleted virtually the entire cytoplasmic tail of the poly-lg receptor by oligonucleotide-directed mutagenesis and have examined the effects on receptor transport. In fibroblasts the receptor reaches the cell surface, ican bind poly-lg, and is cleaved to SC. However, there is no detectable endocytosis of the receptor. In MDCK cells ahe receptor never reaches the basoiateral surface. Instead, it goes directly to the apical surface, and SC is released into the apical medium. Results After removal of the signal peptide, the intact rabbit poly-lg receptor consists of 755 amino acids (Mostov et al., 19S4). Starting at the N-terminus, residues l-629 are extracellular, residues 630-652 span the membrane, and residues 653-755 are cytoplasmic. Using a mutagenic oligonucleotide and the standard single-stranded nique (Zoller and Smith, 1983) we introduced a stop codon in place of residue 655. This deleted 101 residues from the C-terminus and left a cytoplasmic domain of only 2 residues, Arg-Ala. We designated this the “minus-tail receptor?
Cell 360
Control
Figure1. Pulse-ChaseAnalysisof Processing of Wild-Typeand Mutant Receptor in Fibro-
Minus Tail
blasts Cells expressing the wild-type or mutant recep tor were pulse-labeled for 15 min with [35S]cysteine, then chased for the indicated period. Cells and media were then immunoprecipitated and analyzed by SDS-PAGE and fluorography.
68-
As previously described, we used the retroviral vector DO-L and the w packaging system to express this truncated receptor in murine fibroblasts (Deitcher et al., 1986). Cells expressing either the wild-type receptor (control) or the minus-tail mutant were pulse-labeled with [ssS]cysteine and chased for various periods. Cells and media were then immunoprecipitated with antiserum to rabbit SC, then analyzed by SDS-PAGE and fluorography. As seen in Figure 1 (and as previously reported), in the control cells a single polypeptide of m90 kd is present after the pulse and is converted to a poorly resolved group of species of ml00 kd due to modification of the carbohydrates to the complex type. These are cleaved to a group of products of ~70 kd, which appear in the medium and comigrate with authentic rabbit milk SC. As previously described, the heterogeneity of SC is due, at least in part, to variability in the exact site of cleavage of SC from the receptor (Eiffert et al., 1984; Deitcher et al., 1986). With the minus-tail mutant, a protein of ~75 kd is initially present. This does not shift up appreciably with time, but does acquire complex carbohydrates, as judged by endoglycosidase H resistance (data not shown). The mutant receptor is also cleaved to a group of species of m70 kd that appear in the medium and comigrate with authentic SC. The kinetics of release of SC appear roughly similar to the control. These results do not directly show that the receptor is reaching the cell surface, but do indicate that it is cleaved to SC, which is released into the medium. We studied the binding of a ligand, 1251-labeled human dimeric IgA (dIgA), to the cells at 4%. The results in the cells expressing the minus-tail mutant were in good agreement with our previously reported data in control cells: approximately 9,000-10,000 receptors per cell with an apparen? Ko of 8 x 1O-g M (data not shown). This strongly suggests that the mutant receptor is present on the cell surface and is capable of binding ligand. We previously found that at least 30% of dlgA bound at 4% could be rapidly internalized, as assayed by acquisition of protease resistance, when cells were warmed to 37%. The internalized dlgA returned to the cell surface within lo-15 min. All of the bound dIgA, whether or not it had been internalized, was released into the medium within 20 min. When we carried out this experiment with
.o--
,;
5
IO
D
15
20
Time (Minutes) Figure 2. Internalization of [‘asl]dlgA by Fibroblasts WildType or Mutant Receptor
Expressing
the
[1z51]dlgA (0.4 pglmi, 4 x 10s cpm/ug) was bound at 4% for 2 hr to cells expressing either the wild-type (A) or mutant (B) receptor. Unbound ligand was washed away at 4°C and the cells were warmed to 37°C for the indicated time. The amount of ligand released into the warmup medium is indicated by the triangles. Cells were then cooled to 4%, digested with pronase for 2 hr, and sedimented. Radioactivity released into the digestion buffer is defined as cell surface ligand and is indicated by the closed circles. Ligand remaining associated with the cell pellet is defined as internalized ligand and is indicated by the open circles. Values are expressed as fraction of the total dlgA initially bound, which average 1,240 pg per 35 mm dish for the wild type and 1,397 pg per 35 mm dish for the mutant. Points are mean of triplicates, and standard errors of the means were less than ?O%.
Cytoplasmic 381
Domain of poly-lg Receptor
Control
Minus Tail
Figure 3. Pulse-Chase Analysis of Processing of Wild-Type and Mutant Receptor in MDCK
Cells MDCK cells expressing the wild-type or mutant receptor and grown on Miilipore filters were pulse-labeled for 15 min with [%]cysteine, then chased for the indicated periods. Cells and apical (A) or basal (8) media were then immunoprecipitated and analyzed by SDS-PAGE and fluorography. When electrophoresed in adjacent lanes, the material released into apical media from both types of cells comigrates. This is not apparent in this figure due to curvature
of the gel.
the minus-tail mutant, we did not detect any internalized dlgA (Figure 2, compare open circles for control [A] and minus-tail [B]). instead, all of the bound dlgA was rapidly released into the medium (triangles). This result suggests that the minus-tail poly-lg receptor cannot be endocytosed. We also expressed the minus-tail receptor in MDCK cells and compared it with the control in a pulse-chase experiment (Figure 3). The wild-type receptor is cleaved to SC, which is released almost entirely into the apical medium. Processing of the mutant receptor in MDCK cells was similar to that observed in fibroblasts except that at the earliest time point (0 hr chase) two distinct species are seen. Treatment of this sample with endoglycosidase H converts both species to a single species of faster mobility, suggesting that in MDCK cells two differently glycosyfated forms of the same polypeptide are produced (data not shown). During the chase period, these two species do not undergo a clear-cut change in mobility. Nevertheless, they do become endoglycosidase H-resistant, suggesting that modification of the carbohydrate to the complex form does occur. The receptor is cleaved to SC, which is released into the medium. As in the wild-type, SC is released almost entirely into the apical medium, although release is slightly faster than in the wild-type. We previously obtained direct evidence that the wildtype receptor reaches the basolateral surface before being released as SC in the apical medium (Mostov and Deitcher, 1988). Cells were pulse-labeled, and during the 4 hr chase, goat antiserum against rabbit SC was included in the basal medium. These antibodies bound the receptor exposed at the basolateral surface and remained attached to the receptor as it was transcytosed across the ceil. Hence the cleaved SC released into the apical medium already had antibodies bound to it and was immunoprecipitated by simply adding protein A-Sepharose to the medium. Not all of the SC released into the apical medium had bound anti-SC antibodies at the basolateral surface, either because the receptor was never exposed at the basolateral surface or because of other reasons. This “residual” SC could be recovered by adding anti-SC antibodies to the apical medium (after first absorbing it inrith protein A-Sepharose) and isolating the immune complexes with additional protein A-Sepharose. We found the ceils expressing
that in the wild-type cells, roughly 90% of the SC released into the apical medium had bound antibodies added ?othe basolateral medium. When we performed the same experiments on the minus-tail mutant, we observed that almost none of the SC released into the apical medium was bound to antibodies added basally (Figure 4, compare antibody-bound SC in the apical medium of the cells expressing the minus-tail receptor [lane 51 with that in the wild type [lane I]). Virtually all of the SC was recovered as residual SC (Figure 4, compare residual SC in the mutant [lane l] and in the wildtype [lane 31).These data are quantitated in Table 1,where the total SC recovered from the apical medium is taken as ‘100%. Performing the chase for 8 hr instead of 4 hr did not substantially affect the results. This experiment suggests that the minus-tail receptor is sent directly to the apical surface, without an intermediate stop at the basolateral surface. It is possible that the receptor is transiently exposed at the basolateral surface, but fails to bind the antibodies either because it is too rapidly internalized or because it is in a conformation not recognized by the antiserum. The former seems unlikely because of the defect in endocytosis described above, while the latter is also improbable because the SC released into the medium is recognized by the antiserum. Several indirect lines of evidence aiso support the hypothesis that the minus-tail receptor is sent directly to the apical surface. If the receptor were to reach the basoiateral surface it might accumulate there, since the receptor cannot be endocytosed. (We have only directly shown that ligand bound to the receptor is not endocylosed in fibroblasts, but it seems likely that the receptor itself is not endocytosed and that this would also be true in MDCK cells. We could not assay internalization at the basolateral surface of MDCK cells by protease protection, due to interference from protein bound to the filter.) In the pulse-chase experiments (Figure 3) the receptor does not accumulate at the basolateral surface, but rather is almost entirely chased to SC in the apical medium. Another possibility is that the receptor is slowly cleaved to SC at the oasolateral surface. However, only traces of SC are released into the basal medium. We previously observed that MDCK cells expressing the wild-type receptor could transcytose [‘25l]dlgA (Mostov
Ceil 362
12345678
I i
ABAB
R
Figure 4. Effect of Including Medium during the Chase
ABAB
A nt iserum
R
against
SC in the Basal
MDCK cells expressing either the minus-tail or the wild-type receptor were grown on Millipore filters and pulse-labeled for 1TT5 min. During the 4 hr chase, 3 ~1 of goat antiserum against rabbit SC was included in the basal medium. However, the apical (A) and basal (B) media were adsorbed with protein A-Sepharose without adding additional antiserum. These immunoprecipitates were then analyzed by SDS-PAGE and fluorography (lanes 5 and 6 for minus-tail, lanes 1 and 2 for wild type). Residual SC (designated R) was recovered from the supernatants of these immunoprecipitations by adding 3 nl of additional antiserum, incubating at 22% for2 hr, and adsorbing the immune complexes with additional protein A-Sepharose (lanes 7 and 8 for minus-tail, lanes 3 and 4 for wild type).
and Deitcher, 1986). We have now compared dlgA transcytosis by the cells expressing the wild-type receptor (Figure 5A) and the minus-tail receptor (Figure 58). Transcytosis from the basal medium to the apical medium was roughly 10 times greater in cells expressing the wild-type receptor than in cells expressing the minus-tail receptor (Figures 5A and 5B, compare open circles). In cells expressing the wild-type receptor, this transport could be largely competed away by an excess of unlabeled dIgA, whereas transport was not competed away in the cells expressing the minus-tail receptor (Figures 5A and 58, com-
Table 1. Quantitation
of lmmunoprecipitation
pare squares). In both cell types there was little transport from the apical medium to the basal medium (Figure 5, closed circles), and this was not competed away by unlabeled dlgA (triangles). This low level of transport was similar to that observed in either direction in the parent cell line that does not express the receptor (data not shown). This suggests that the small amount of transport seen in the cells expressing the minus-tail receptor was due to leakage and/or fluid-phase transcytosis (von Bonsdorff et al., 1985) and that the mutant receptor does not function in transcytosis. Since the minus-tail receptor can bind ligand, this result further supports the hypothesis that the receptor does not travel to the basolateral surface before reaching the apical surface. We also attempted to localize the receptor by fixing the cells on filters, applying antiserum to SC to either the apical or the basolateral surface, and then visualizing with fluorescent second antibody. We observed weak fluorescence at the apical surface, similar to that seen with the wild-type receptor. No specific fluorescence was seen at the basolateral surface. However, there was a moderately high background, due to nonspecific sticking of the antibodies to the nitrocellulose filter. Hence a weak basolateral signal may have escaped detection (data not shown). This problem has also hampered the development of a sufficiently sensitive radioimmunoassay for detecting basolateral receptor. Discussion We have deleted virtually the entire cytoplasmic domain of the poly-lg receptor and expressed this mutant protein in both nonpolarized fibroblasts and polarized MDCK cells. In fibroblasts the mutant receptor, like the wild-type, is transported to the cell surface, can bind ligand, and is cleaved to SC and released. However, unlike the wild-type, the minus-tail receptor cannot internalize ligand. Naturally occurring mutations in the cytoplasmic domain of the low density lipoprotein receptor have shown that its cytoplasmic tail is important for clustering into coated pits and endocytosis (Lehrman et al., 1985; Davis et at., 1986). Our data extend this to a second receptor system, suggesting that the cytoplasmic tail may be generally involved in endocytosis. In polarized MDCK cells the minus-tail receptor reaches the apical surface and is cleaved to SC, which is released almost exclusively into the apical medium. However, unlike the wild-type, the minus-tail receptor apparently never makes an intermediate stop at the basolateral surface, but rather goes directly to the apical surface. There are S@V-
of Apical Medium % of SC in Apical Medium ( f SEM)
Cells
Sample
4 hr Chase
8 hr Chase
Wild-type
First immtmoprecipitation Residual immunoprecipitation
92 (17)
89 (12)
8 (5)
11 (6)
First immunoprecipitation Residual immunoprecipitation
6 (3) 94 (21)
3 (2) 97 (15)
Mutant
;?3oplasmic
Domain of poly-ig Receptor
20
5
Time (Hours) Figure 5. Transcytosis of dlgA by MDCK Cells Expressing Type or Mutant Receptor
the Wild-
MDCK cells expressing the wild-type (A) or minus-tail (B) receptor were grown on Millipore filters. [1251]dlgA (0.2 bglml) with or without unlabeled dlgA (50 pglml) was added to the basolateral or apical medium. After the indicated time, the medium on the side opposite to which the labeled dlgA was added was collected, TCA-precipitated, and counted in a gamma counter. Open circles, [‘251]dlgA alone added to basal medium; squares, [lnsl]dlgA and unlabeled dlgA added to the basal medium; triangles, [1*51]dlgA added to the apical medium; closed circles, [1251]dlgAand unlabeled dlgA added to the apical medium. Standard errors of the mean of triplicate points were less than 20%.
era1 possible interpretations of this result. Most likely, the cytoplasmic domain contains a positive signal directing the molecule to the basolateral surface. The information for reaching the apical surface may reside in a signal in the noncytoplasmic (either extracellular or transmembrane) portion of the molecule. Alternatively, in the absence of a signal to go to the basolateral surface, the receptor goes to the apical surface by default, without needing any specific signal. When exogenous secretory proteins (e.g., chicken lysozyme) are expressed in MDCK cells, they are released roughly equally at both surfaces, suggesting that the default pathway for secretory proteins is to both surfaces at random (Kondor-Koch et al., 1985; Gottlieb et al., 1986). Whether there is a default pathway for membrane proteins leading exclusively to one surface or the other is not known. One could also imagine that the mutant protein has an altered conformation that somehow prevents it from going to the basolateral surface, but causes it to go to the apical surface. (However, the conformation is not so altered as to prevent ligand binding or cleavage.) This change could, for instance, be from an oligomer to a monomer or vice versa, although there is no evidence that the receptor is ever oligomeric. Larkin, et al. (1986) have recently shown that the cytoplasmic tail of the poly-lg receptor becomes phosphorylated on serine. It is tempting to speculate that this phosphorylation plays a role in targeting the protein to the correct location.
The mutant receptor apparently goes directly from the Golgi to the apical surface and probably follows the same pathway as other proteins that are found exclusively on the apical surface, such as the ~emagglut~nin of influenza virus. The wild-type receptor travels to the apical surface anly after endocytosis at the basoiateral surface. If positive signals to direct the proteins to the apical surface are involved in both pathways, we can speculate that the same recognition machinery is used in both paths. Moreover, regardless of whether or not positive signals are involved, it is possible that apically directed proteins coming from either the Golgi or the endosomes pass through a common compartment. This data is, to the best of our knowledge, the clearest indication that the cytoplasmic domain of a plasma membrane protein is involved in directing the protein to one surface or the other of a polarized cell. The pathway of the poly-lg receptor is unusually complex, gaing first to one surface, then to the other. It is possible that this role of the cytoplasmic tail is unique to the poly-lg receptor and that other basolateral membrane proteins are targeted to that surface by signals located elsewhere.
Experimental
Procedures
of Mutant poly-lg Receptor Ciones Construction All recombinant DNA steps used standard procedures (Maniatis et al., 1982). The previously described fragment (nucleotides 96-2,451) that contains the entire coding region of the poly-lg receptor and to which Bglll linkers had been added (Deitcher et al., 1986) was filled in with the Klenowfragment of DNApoymerase I. Replicativeform of M13mp8 phage was cut with Sall, filled in with Klenow, and treated with calf intestinal phosphatase. The poly-lg receptor coding fragment was bluntend-ligated into this mp8 vector. This ligation regenerates the Bglll sites at the ends of the coding fragment, allowing its easy recovery ‘ram the replicative form of the phage. A 17-mer oligonucleotide, TCCTGTGCTAGGCTCTG, was synthesized on an Applied Biosystems synthesizer, purified on a 20% acrylamide, 7 M ureagel, eluted by diffusion, and ethanol-precipitated. This oiigonucleotide is complementary to nucieotides 2282-2298 of the cDNA, except for two changes that convert the Arg residue 655 to 2 stop codon. The standard oligonucleotide mutagenesis procedure was performed (Zoller and Smith, 1983), and mutant clones were identified by hybridization with the mutagenic oligonucleotide using washes with 3 M tetramethylammonium chloride at 50°C (Wood et al., 1985). The mutation was verified by dideoxy sequencing. To ensure that no additional mutations were introduced, we resequenced the entire cDNA insert, using a series of sequencing primers spaced approximately every 300 nucleotides. Replicative form DNA from phage containing the mutant was prepared, then digested with 3glll. The cDNA insert was ligated into the BamHI site of the DOL vector. Retroviral vector DNA was transfected into vAM cells, and the transiently produced virus was used to infect ~2 cells (a derivative of NIH 3T3 fibroblasts) and MDCK strain II cells. Six G418-resistant clones of each were picked, expanded, and screened for mutant poly-lg receptor production by pulse labeling with [%]cysteine, immunoprecipitation, SDS-PAGE, and fluorography. All clones produced receptor. One ~2 clone and one MDCK clone were selected for further study. The MDCK clone was checked for polarized growth on Millipore filters by polarity of [%]methionine uptake and by immunofluorescence with an endogencus 60 kd membrane protein (Herzlinger and Ojakian, 1984) and was determined to be 2s polar as the parent MDCK strain. Other Procedures All other procedures used in analysis of the poly-lg receptor transport have been previously described (Deitcher et al., 1986; Mostov and Deitcher, 1986). These include growth on filters, pulse-chase, immuno-
Cell 364
precipitation, ligand binding, internalization, transcytosis, of anti-SC antibodies to the basolateral surface.
and binding
Acknowledgments We thank Drs. l-l. Lodish, K. Matlin, and E. Rodriguez-Boulan for useful discussions, J.-P Kraehenbuhl and G. Ojakian for antibodies, and Ms. Devon Young for manuscript preparation. This work was supported by an NIH New Investigator Award to K. M. A. de B. K. was supported by an NSF predoctoral fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received June 10, 1986; revised August 12, 1986 References Blobel, G. (1980). Intracellular Sci. USA 77, 1496-1500.
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