Interactions of the AP-1 Golgi Adaptor with the Polymeric Immunoglobulin Receptor and Their Possible Role in Mediating Brefeldin A-sensitive Basolateral Targeting from the trans-Golgi Network
1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês
10.1074/jbc.274.4.2201
ISSN1083-351X
AutoresEna Orzech, Karni Schlessinger, Aryeh Weiss, Curtis T. Okamoto, Benjamin Aroeti,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoWe provide morphological, biochemical, and functional evidence suggesting that the AP-1 clathrin adaptor complex of the trans-Golgi network interacts with the polymeric immunoglobulin receptor in transfected Madin-Darby canine kidney cells. Our results indicate that immunofluorescently labeled γ-adaptin subunit of the adaptor complex and the polymeric immunoglobulin receptor partially co-localize in polarized and semi-polarized cells. γ-Adaptin is co-immunoisolated with membranes expressing the wild-type receptor. The entire AP-1 adaptor complex could be chemically cross-linked to the receptor in filter-grown cells. γ-Adaptin could be co-immunoprecipitated with the wild-type receptor, with reduced efficiency with receptor mutant whose basolateral sorting motif has been deleted, and not with receptor lacking its cytoplasmic tail. Co-immunoprecipitation of γ-adaptin was inhibited by brefeldin A. Mutation of cytoplasmic serine 726 inhibited receptor interactions with AP-1 but did not abrogate the fidelity of its basolateral targeting from the trans-Golgi network. However, the kinetics of receptor delivery to the basolateral cell surface were slowed by the mutation. Although surface delivery of the wild-type receptor was inhibited by brefeldin A, the delivery of the mutant receptor was insensitive to the drug. Our results are consistent with a working model in which phosphorylated cytoplasmic serine modulates the recruitment of the polymeric immunoglobulin receptor into AP-1/clathrin-coated areas in the trans-Golgi network. This process may regulate the efficiency of receptor targeting from thetrans-Golgi network. We provide morphological, biochemical, and functional evidence suggesting that the AP-1 clathrin adaptor complex of the trans-Golgi network interacts with the polymeric immunoglobulin receptor in transfected Madin-Darby canine kidney cells. Our results indicate that immunofluorescently labeled γ-adaptin subunit of the adaptor complex and the polymeric immunoglobulin receptor partially co-localize in polarized and semi-polarized cells. γ-Adaptin is co-immunoisolated with membranes expressing the wild-type receptor. The entire AP-1 adaptor complex could be chemically cross-linked to the receptor in filter-grown cells. γ-Adaptin could be co-immunoprecipitated with the wild-type receptor, with reduced efficiency with receptor mutant whose basolateral sorting motif has been deleted, and not with receptor lacking its cytoplasmic tail. Co-immunoprecipitation of γ-adaptin was inhibited by brefeldin A. Mutation of cytoplasmic serine 726 inhibited receptor interactions with AP-1 but did not abrogate the fidelity of its basolateral targeting from the trans-Golgi network. However, the kinetics of receptor delivery to the basolateral cell surface were slowed by the mutation. Although surface delivery of the wild-type receptor was inhibited by brefeldin A, the delivery of the mutant receptor was insensitive to the drug. Our results are consistent with a working model in which phosphorylated cytoplasmic serine modulates the recruitment of the polymeric immunoglobulin receptor into AP-1/clathrin-coated areas in the trans-Golgi network. This process may regulate the efficiency of receptor targeting from thetrans-Golgi network. polymeric immunoglobulin receptor brefeldin A trans-Golgi network Madin-Darby canine kidney endoplasmic reticulum secretory component cation-dependent mannose 6-phosphate receptor ADP-ribosylation factor polyacrylamide gel electrophoresis fluorescein isothiocyanate tetramethylrhodamine isothiocyanate post-nuclear supernatant phosphate-buffered saline minimal Eagle's medium hemagglutinin bovine serum albumin dimeric IgA protein kinase A 3,3′-dithiobis[sulfosuccinimidyl propionate. Polarized epithelial cells possess two surface domains as follows: the apical plasma membrane that faces the external environment and the basolateral plasma membrane that is in contact with internal milieu. The apical and basolateral plasma membranes have very different protein and lipid compositions. It has been proposed that sorting and targeting events of membrane proteins and lipids largely contribute to the polarized phenotype of the cell (1Rodriguez-Boulan E. Powell S.K. Annu. Rev. Cell Biol. 1992; 8: 327-395Crossref Scopus (354) Google Scholar, 2Nelson W.J. Science. 1992; 258: 948-955Crossref PubMed Scopus (174) Google Scholar, 3Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8092) Google Scholar). Although in recent years we have learned a great deal about relatively small stretches of amino acids encoding sorting signals that mediate polarized trafficking of membrane proteins (4Aroeti B. Okhrimenko H. Reich V. Orzech E. Biochim. Biophys. Acta. 1998; 1376: 57-90Crossref PubMed Scopus (51) Google Scholar, 5Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Crossref PubMed Scopus (391) Google Scholar, 6Mostov K. Apodaca G. Aroeti B. Okamoto C. J. Cell Biol. 1992; 116: 577-583Crossref PubMed Scopus (191) Google Scholar, 7Mostov K.E. Cardone M.H. BioEssays. 1995; 17: 129-138Crossref PubMed Scopus (118) Google Scholar), almost nothing is known about the sorting machinery that decodes these signals and confers polarized trafficking to a specific surface domain (1Rodriguez-Boulan E. Powell S.K. Annu. Rev. Cell Biol. 1992; 8: 327-395Crossref Scopus (354) Google Scholar, 2Nelson W.J. Science. 1992; 258: 948-955Crossref PubMed Scopus (174) Google Scholar, 6Mostov K. Apodaca G. Aroeti B. Okamoto C. J. Cell Biol. 1992; 116: 577-583Crossref PubMed Scopus (191) Google Scholar, 7Mostov K.E. Cardone M.H. BioEssays. 1995; 17: 129-138Crossref PubMed Scopus (118) Google Scholar, 8Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1336) Google Scholar). Polarized trafficking steps are modulated by signal transduction processes (4Aroeti B. Okhrimenko H. Reich V. Orzech E. Biochim. Biophys. Acta. 1998; 1376: 57-90Crossref PubMed Scopus (51) Google Scholar, 7Mostov K.E. Cardone M.H. BioEssays. 1995; 17: 129-138Crossref PubMed Scopus (118) Google Scholar, 9Weimbs T. Low S.-H. Chapin S.J. Mostov K.E. Trends Cell Biol. 1997; 7: 393-399Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 10Luton F. Cardone M.H. Zhang M. Mostov K.E. Mol. Biol. Cell. 1998; 9: 1787-1802Crossref PubMed Scopus (47) Google Scholar), whose activity is thought to be superimposed on the activity of the sorting machinery, but the mechanisms through which these processes facilitate protein incorporation into a particular pathway are largely unknown. Presently, we know four distinct features shown to determine sorting of apical proteins to the apical domain as follows: first is the glycosylphosphatidylinositol anchor of membrane proteins (11Brown D.A. Crise B. Rose J.K. Science. 1989; 245: 1499-1501Crossref PubMed Scopus (304) Google Scholar, 12Lisanti M.P. Caras I.W. Davitz M.A. Rodriguez-Boulan E. J. Cell Biol. 1989; 109: 2145-2156Crossref PubMed Scopus (375) Google Scholar); the second is the mannose-rich core of N-glycans present in the luminal portion of proteins (13Scheiffele P. Peranen J. Simons K. Nature. 1995; 378: 96-98Crossref PubMed Scopus (417) Google Scholar); the third isO-glycosylated stalk domain of transmembrane protein (14Yeaman C. Le Gall A.H. Baldwin A.N. Monlauzeur L. Le Bivic A. Rodriguez-Boulan E. J. Cell Biol. 1997; 139: 929-940Crossref PubMed Scopus (246) Google Scholar); and the fourth is a proteinaceous signal encoded by either the transmembrane domain or the ectodomain (15Alonso M.A. Fan L. Alarcon B. J. Biol. Chem. 1997; 272: 30748-30752Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Many studies agree that basolateral sorting of plasma membrane proteins is mediated by the presence of relatively short but specific cytoplasmic sorting motifs (reviewed in Refs. 4Aroeti B. Okhrimenko H. Reich V. Orzech E. Biochim. Biophys. Acta. 1998; 1376: 57-90Crossref PubMed Scopus (51) Google Scholar and 5Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Crossref PubMed Scopus (391) Google Scholar). Extensive mutagenesis studies have uncovered two general types of basolateral sorting motifs. First there are basolateral sorting signals for localization to clathrin-coated membranes that rely either on a critical tyrosine residue, such as those found in the low density lipoprotein receptor (low density lipoprotein receptor proximal determinant, (16Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (304) Google Scholar)), the vesicular stomatitis virus G protein (17Thomas D.C. Brewer C.B. Roth M.G. J. Biol. Chem. 1993; 268: 3313-3320Abstract Full Text PDF PubMed Google Scholar), or on a di-leucine motif (18Hunziker W. Fumey C. EMBO J. 1994; 13: 2963-2967Crossref PubMed Scopus (220) Google Scholar). The second are basolateral sorting signals unrelated to localization to clathrin-coated membranes. These signals can rely either on a Tyr motif, e.g. the distal determinant in the low density lipoprotein receptor, or rely on non-aromatic residues such as found for the polymeric immunoglobulin receptor (pIgR1 (4Aroeti B. Okhrimenko H. Reich V. Orzech E. Biochim. Biophys. Acta. 1998; 1376: 57-90Crossref PubMed Scopus (51) Google Scholar, 19Aroeti B. Kosen P.A. Kuntz I.D. Cohen F.E. Mostov K.E. J. Cell Biol. 1993; 123: 1149-1160Crossref PubMed Scopus (118) Google Scholar)). Interestingly, either the same or a closely related basolateral sorting signal can mediate basolateral sorting from the TGN and recycling from endosomes, after endocytosis from the basolateral plasma membrane (20Aroeti B. Mostov K.E. EMBO J. 1994; 13: 2297-2304Crossref PubMed Scopus (89) Google Scholar,21Matter K. Whitney J.A. Yamamoto E.M. Mellman I. Cell. 1993; 74: 1053-1064Abstract Full Text PDF PubMed Scopus (133) Google Scholar). We use the pIgR expressed in Madin-Darby canine kidney (MDCK) cells as a model system for studying the mechanisms that regulate polarized trafficking of membrane proteins in epithelial cells. Previous studies have intensively analyzed the intracellular trafficking of the pIgR in MDCK cells. According to the simplest model, the pIgR is synthesized in the endoplasmic reticulum (ER) as a single spanning type I membrane protein. It is then targeted from the ER to the Golgi apparatus and from the trans-Golgi network (TGN) pIgR molecules are directed to the basolateral surface. However, the exact pathway that newly synthesized membrane proteins take to the basolateral cell surface is currently not understood. Although in the simplest model, basolateral proteins are selectively sorted (packed) into basolateral transport vesicles that are subsequently directly delivered to the basolateral surface, recent studies have provided evidence that targeting to the cell surface may be indirect, i.e.involving passage through an endosomal compartment prior to cell-surface delivery (22Futter C.E. Connolly C.N. Cutler D.F. Hopkins C.R. J. Biol. Chem. 1995; 270: 10999-11003Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 23Leitinger B. Hille-Rehfeld A. Spiess M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10109-10113Crossref PubMed Scopus (90) Google Scholar). Whether a similar indirect route exists in MDCK cells is currently unknown nor is the mechanism of sorting to these endosomes known. Upon arrival at the basolateral cell surface, polymeric Igs (e.g. dimeric IgA; dIgA) specifically bind to the pIgR. The pIgR·dIgA complexes are endocytosed via clathrin-coated pits, delivered to basolateral endosomes, and then delivered to the opposite pole of the cell via vesicular intermediates in a process termed transcytosis. Transcytotic pIgR·dIgA complexes are not directly routed from basolateral endosomes to the apical surface. Rather, pIgR·Ig complexes are first targeted to, and subsequently accumulate at, a pericentriolar subapical endosomal compartment that is active in recycling of apical and perhaps basolateral membrane-bound ligands (24Apodaca G. Katz L.A. Mostov K.E. J. Cell Biol. 1994; 125: 67-86Crossref PubMed Scopus (345) Google Scholar,25Barroso M. Sztul E. J. Cell Biol. 1994; 124: 83-100Crossref PubMed Scopus (181) Google Scholar). This compartment has been termed the "apical recycling endosome." The final step of transcytosis involves pIgR targeting from the apical recycling endosome to the apical surface. There, the pIgR is cleaved by an endogenous protease, and the extracellular ligand-binding domain (i.e. the secretory component, SC) is released together with dIgA to external (mucosal) secretions, such as milk, saliva, bile, tears, and intestinal fluids, where they form the first immunological response against infections. The pIgR is also transcytosed constitutively (i.e. in the absence of the ligand), and this process is regulated by phosphorylation of cytoplasmic Ser-664 (26Casanova J.E. Breitfeld P.P. Ross S.A. Mostov K.E. Science. 1990; 248: 742-745Crossref PubMed Scopus (174) Google Scholar). Mutagenesis studies revealed that the cytoplasmic domain of the pIgR contains several discrete sorting signals that mediate as follows: its targeting from the TGN to the basolateral surface; endocytosis; avoidance of lysosomes; and transcytosis (4Aroeti B. Okhrimenko H. Reich V. Orzech E. Biochim. Biophys. Acta. 1998; 1376: 57-90Crossref PubMed Scopus (51) Google Scholar, 6Mostov K. Apodaca G. Aroeti B. Okamoto C. J. Cell Biol. 1992; 116: 577-583Crossref PubMed Scopus (191) Google Scholar). Basolateral sorting from the TGN is mediated by an autonomous and dominant 17-residue membrane-proximal basolateral sorting signal (27Casanova J.E. Apodaca G. Mostov K.E. Cell. 1991; 66: 65-75Abstract Full Text PDF PubMed Scopus (226) Google Scholar), whose activity depends mainly on three amino acids (His-656, Arg-657, and Val-660) contained within the 17-residue signal (19Aroeti B. Kosen P.A. Kuntz I.D. Cohen F.E. Mostov K.E. J. Cell Biol. 1993; 123: 1149-1160Crossref PubMed Scopus (118) Google Scholar). Structural studies of oligopeptides corresponding to the 17-residue basolateral sorting signal provided evidence for a β-turn secondary structure, which might constitute a general feature of endocytotic and other sorting signals (7Mostov K.E. Cardone M.H. BioEssays. 1995; 17: 129-138Crossref PubMed Scopus (118) Google Scholar). Residues involved in basolateral sorting of the pIgR from the TGN also control polarized sorting in endosomes (20Aroeti B. Mostov K.E. EMBO J. 1994; 13: 2297-2304Crossref PubMed Scopus (89) Google Scholar), suggesting that common mechanisms regulate polarized trafficking in the TGN and in endosomes. The pIgR thus appears to contain multiple cytoplasmic signals that mediate distinct intracellular transport events; these signals are probably decoded by a cytoplasmic sorting machinery located at specific compartments through which the pIgR passes en route to a target organelle. Coat proteins such as clathrin and clathrin adaptor proteins (AP), AP-1 and AP-2, are thought to be involved in membrane protein sorting into coated membrane domains and promote the budding of transport vesicles from the trans-Golgi network (TGN) and from the plasma membrane, respectively (for recent reviews see Refs. 28Lewin D.A. Mellman I. Biochim. Biophys. Acta. 1998; 1401: 129-145Crossref PubMed Scopus (50) Google Scholar and 29Robinson M.S. Trends Cell Biol. 1997; 7: 99-102Abstract Full Text PDF PubMed Scopus (123) Google Scholar). The AP-1 and AP-2 adaptor complexes are composed of two large subunits (γ and β1 for AP-1 or α and β for AP-2), a medium-sized subunit (μ1 or μ2), and a small subunit (ς1 and ς2). Although in a few cases the interactions between AP-binding and membrane proteins have been resolved (for a recent review see Ref. 30Marks M.S. Ohno H. Kirchhausen T. Bonifacino J.S. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar), the mechanistic relations between coat binding and membrane protein targeting has not been fully elucidated. One common feature is that many of the coat proteins involved in protein sorting are sensitive to the action of the fungal metabolite brefeldin A (BFA). This drug is thought to act, in part, by blocking the binding to Golgi membranes of at least four coat proteins as follows: coatomer (e.g. COP-I) involved in ER-Golgi transport and in endocytosis (8Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1336) Google Scholar, 29Robinson M.S. Trends Cell Biol. 1997; 7: 99-102Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 31Aridor M. Balch W.E. Trends Cell Biol. 1996; 6: 315-320Abstract Full Text PDF PubMed Scopus (80) Google Scholar); the TGN clathrin-coated vesicle-associated protein AP-1 adaptor complex (32Wong D.H. Brodsky F.M. J. Cell Biol. 1992; 117: 1171-1179Crossref PubMed Scopus (84) Google Scholar) involved in sorting of the mannose 6-phosphate receptor (MPR) to endosomes; p200, a protein of 200,000 daltons that is now known to be type II myosin (33Narula N. McMorrow I. Plopper G. Doherty J. Matlin K.S. Burke B. Stow J.L. J. Cell Biol. 1992; 117: 27-38Crossref PubMed Scopus (88) Google Scholar, 34Musch A. Cohen D. Rodriguez-Boulan E. J. Cell Biol. 1997; 138: 291-306Crossref PubMed Scopus (158) Google Scholar); and the recently discovered AP-3 adaptor protein (35Dell' Angelica E.C. Ohno H. Ooi C.E. Rabinovich E. Roche K.W. Bonifacino J.S. EMBO J. 1997; 16: 917-928Crossref PubMed Scopus (330) Google Scholar, 36Simpson F. Peden A.A. Christopoulou L. Robinson M.S. J. Cell Biol. 1997; 137: 835-845Crossref PubMed Scopus (306) Google Scholar) whose association with clathrin coats in the Golgi is controversial (36Simpson F. Peden A.A. Christopoulou L. Robinson M.S. J. Cell Biol. 1997; 137: 835-845Crossref PubMed Scopus (306) Google Scholar, 37Dell'Angelica E.C. Kluperman J. Stoorvogel W. Bonifacino J.S. Science. 1998; 280: 431-434Crossref PubMed Scopus (308) Google Scholar). Both, AP-1 and COP-I bind to target membranes in association with a small GTPase ARF1 (for ADP-ribosylation factor 1) (38Donaldson J.G. Klausner R.D. Curr. Opin. Cell Biol. 1994; 6: 527-532Crossref PubMed Scopus (232) Google Scholar). GDP-GTP exchange on ARF occurs concomitant with binding; inhibition of exchange by BFA blocks the assembly of these coat proteins. Do coat proteins participate in polarized membrane trafficking? Recent in vitro binding studies revealed that AP-1 adaptors interact with tyrosine-based basolateral sorting motif and with a di-leucine signal artificially introduced into the influenza hemagglutinin's (HA) C-terminal domain (39Heliker R. Manning-Krieg U. Zuber J.-F. Spiess M. EMBO J. 1996; 15: 2893-2899Crossref PubMed Scopus (156) Google Scholar), but the function of these interactions in basolateral trafficking of the HA mutant is unknown. Basolateral targeting of the pIgR from the TGN is significantly inhibited by BFA (40Hunziker W. Whitney J.A. Mellman I. Cell. 1991; 57: 1-20Google Scholar, 41Apodaca G. Aroeti B. Tang K. Mostov K.E. J. Biol. Chem. 1993; 268: 20380-20385Abstract Full Text PDF PubMed Google Scholar, 42Reich V. Mostov K. Aroeti B. J. Cell Sci. 1996; 109: 2133-2139Crossref PubMed Google Scholar), suggesting that BFA-sensitive coat proteins regulate pIgR exocytosis from the TGN to the basolateral surface. The cytoplasmic tail of pIgR contains a phosphorylated Ser, Ser-726, that functions in rapid internalization of the pIgR in the basolateral plasma membrane via clathrin-coated pits (43Okamoto C.T. Song W. Bomsel M. Mostov K.E. J. Biol. Chem. 1994; 269: 15676-15682Abstract Full Text PDF PubMed Google Scholar). This Ser-based motif resides in a putative CKII/PKA phosphorylation site upstream to a di-leucine motif with yet undefined function (see Fig.1 A and Ref. 43Okamoto C.T. Song W. Bomsel M. Mostov K.E. J. Biol. Chem. 1994; 269: 15676-15682Abstract Full Text PDF PubMed Google Scholar). In this respect this motif is interesting as it resembles to the putative CKII/PKA motif present in the cytoplasmic tail of cation-dependent mannose 6-phosphate receptor (CD-MPR), shown to serve as an AP-1-binding site (44Johnson K.F. Kornfeld S. J. Biol. Chem. 1992; 267: 17110-17115Abstract Full Text PDF PubMed Google Scholar, 45Mauxion F. Le Borgne R. Munier-Lehmann H. Hoflack B. J. Biol. Chem. 1996; 271: 2171-2178Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). These observations led us to propose that AP-1 adaptors bind to the Ser-726 motif of pIgR and that these interactions may facilitate certain steps in basolateral exocytosis of the receptor. The goal of the work reported herein was to identify these interactions and to examine their role in pIgR trafficking from the TGN to the basolateral cell surface in polarized MDCK cells. We find that the AP-1 adaptor complex associates with the wild-type pIgR cytoplasmic tail in vivo and that the interactions are diminished when Ser-726 is mutated to Ala. The interactions with AP-1 seem to commence in the TGN, persist, and/or are even enhanced in post-TGN compartments. Interestingly, the TGN to basolateral delivery of pIgR-Ser-726 to Ala mutant appears to be significantly slower than that of the wild-type receptor. In addition, unlike the wild-type receptor, the basolateral pathway of this pIgR mutant is completely insensitive to the action of BFA. Our results indicate that AP-1 interactions play a regulatory role in prompting efficient basolateral transport of the pIgR from the TGN, and they also suggest the existence of multiple mechanisms that direct membrane proteins from the TGN to the basolateral surface in MDCK cells. MDCK cells expressing the wild-type or mutant receptors were maintained for up to 10 passages in minimal Eagle's medium (MEM, Biological Industries Co, Beit Haemek, Israel) supplemented with 5% (v/v) fetal bovine serum (Biological Industries Co, Beit Haemek, Israel), 100 units/ml penicillin, and 0.1 mg/ml streptomycin in 5% CO2, 95% air. In our experiments we used three MDCK cell lines that stably express the wild-type pIgR ("pIgR-WT"). One previously described cell line generated by cell transfection with the retroviral pWE vector has been used (26Casanova J.E. Breitfeld P.P. Ross S.A. Mostov K.E. Science. 1990; 248: 742-745Crossref PubMed Scopus (174) Google Scholar). Two additional cell lines were generated by transfecting MDCK cells with the wild-type pIgR cDNA subcloned into the BglII sites of cytomegalovirus-based pCB6 or pCB7 vectors, as described previously (19Aroeti B. Kosen P.A. Kuntz I.D. Cohen F.E. Mostov K.E. J. Cell Biol. 1993; 123: 1149-1160Crossref PubMed Scopus (118) Google Scholar, 46Brewer C.B. Methods Cell Biol. 1994; 43: 233-245Crossref PubMed Scopus (84) Google Scholar). In some cases, MDCK cells expressing pIgR mutants at levels comparable to those of the wild-type receptor were generated by transfecting the cells with cDNA encoding mutant receptors subcloned into the pCB6 vector. Polarized MDCK cells expressing receptors were isolated and characterized as described (19Aroeti B. Kosen P.A. Kuntz I.D. Cohen F.E. Mostov K.E. J. Cell Biol. 1993; 123: 1149-1160Crossref PubMed Scopus (118) Google Scholar, 20Aroeti B. Mostov K.E. EMBO J. 1994; 13: 2297-2304Crossref PubMed Scopus (89) Google Scholar). Two clones expressing the "pIgR R654stop" mutant, where all but two residues of the cytoplasmic tail have been deleted (47Mostov K.E. de Bruyn Kops A. Deitcher D.L. Cell. 1986; 47: 359-364Abstract Full Text PDF PubMed Scopus (108) Google Scholar), have been isolated and their exocytic transport characteristics were analyzed and found to be identical to those described for the earlier characterized expressors (47Mostov K.E. de Bruyn Kops A. Deitcher D.L. Cell. 1986; 47: 359-364Abstract Full Text PDF PubMed Scopus (108) Google Scholar). The pIgR-Δ 655–668 mutant whose 15 of the 17 residues comprising the basolateral sorting signal have been deleted in-frame is delivered from the TGN directly to the apical surface (27Casanova J.E. Apodaca G. Mostov K.E. Cell. 1991; 66: 65-75Abstract Full Text PDF PubMed Scopus (226) Google Scholar). Newly isolated pIgR-Δ655–668 expressing MDCK clones revealed identical exocytic transport properties to the originally described clone (27Casanova J.E. Apodaca G. Mostov K.E. Cell. 1991; 66: 65-75Abstract Full Text PDF PubMed Scopus (226) Google Scholar). The previously described MDCK clones expressing pIgR S726A, whose Ser at position 726 in the cytoplasmic tail was mutated to Ala, have been used. This mutation does not abrogate pIgR sorting from the TGN to the basolateral surface, but it inhibits its endocytosis from that surface (43Okamoto C.T. Song W. Bomsel M. Mostov K.E. J. Biol. Chem. 1994; 269: 15676-15682Abstract Full Text PDF PubMed Google Scholar). Expression level was determined by SDS-PAGE analysis of equal protein amounts derived from SDS-cell lysates followed by immunoblotting and probing with polyclonal sheep anti-SC antibodies and appropriate horseradish peroxidase-labeled secondary antibodies. Protein bands were detected using the SuperSignal® chemiluminescent reagent (Pierce), according to the manufacturer's protocol. Autoradiograms were scanned at 300 dpi resolution using the HP ScanJet IIcx scanner, and band intensity was quantified by the NIH image 1.61 software. Mounting of figures was performed using the Adobe PhotoshopTM 3.05 (Adobe Systems, Inc., Mountain View, CA) and Aldus Freehand 5.02, Macromedia Inc. Autoradiograms showing the expression of selected pIgR mutants relative to the wild-type pIgR are depicted in Fig. 1 B. MDCK cells expressing the wild-type receptor were grown on Transwell filters (Costar, 0.4 μm) for 3–4 days. Cells on filters were fixed for 10 min in methanol at −20 °C, blocked, immunostained for pIgR and γ-adaptin, mounted, and stored as described previously (24Apodaca G. Katz L.A. Mostov K.E. J. Cell Biol. 1994; 125: 67-86Crossref PubMed Scopus (345) Google Scholar). The primary sheep anti-rabbit SC antibody was purified on protein-A Sepharose and used at 20 μg/ml concentration to label the pIgR. The 100/3 (Sigma Immunochemicals, Rehovot, Israel) antibody was used at 1:200 dilution to label the γ-adaptin. The R40.76 anti-ZO-1 rat monoclonal antibody (48Anderson J.M. Stevenson B.R. Jesatis L.A. Goodenough D.A. Mooseker M.A. J. Cell Biol. 1988; 106: 1141-1149Crossref PubMed Scopus (285) Google Scholar) was used at 1:250 dilution to label the tight junction-associated protein ZO-1. In some experiments, cells were treated with 10 μg/ml BFA for 15 min at 37 °C prior to fixation. Anti-sheep secondary antibodies conjugated to either fluorescein isothiocyanate (FITC), anti-rat-conjugated tetramethylrhodamine isothiocyanate (TRITC), or anti-mouse coupled to Texas Red were obtained from Jackson ImmunoResearch and used at recommended dilutions as described (24Apodaca G. Katz L.A. Mostov K.E. J. Cell Biol. 1994; 125: 67-86Crossref PubMed Scopus (345) Google Scholar). It should be noted that according to the manufacturer's comments, secondary antibodies were tested for minimal cross-reactivity for IgG and serum proteins of other species. In several experiments, the co-localization between γ-adaptin and internalized IgA was examined. In these experiments IgA was internalized into apical recycling endosome either from the basolateral or the apical plasma membrane as described previously (24Apodaca G. Katz L.A. Mostov K.E. J. Cell Biol. 1994; 125: 67-86Crossref PubMed Scopus (345) Google Scholar). Cells on filters were then fixed and stained with anti-human IgA antibodies (Sigma), and appropriate secondary antibodies were conjugated to FITC. γ-Adaptins were stained with 100/3 monoclonal antibodies and anti-mouse coupled to Texas Red. A Bio-Rad MRC-1024 confocal scanhead coupled to a Zeiss Axiovert 135M inverted microscope was used to acquire images of the stained cells, with a 63× oil objective (numerical aperture 1.4). Excitation light was provided by a 100-milliwatt air-cooled argon ion laser run in the multi-line mode. The excitation wavelength was 514 nm and was selected with a suitable interference filter. The relative excitation power level was set to 10% with a neutral density filter. The images presented in this paper were obtained by accumulating (summing) three scans. In order to detect three dyes (FITC, TRITC, and Texas Red) simultaneously, the three detection channels were configured as follows. The fluorescence emission was first split between two channels by a dichroic mirror (555DRLP, 50% point at 550 nm). The low wavelength side of the dichroic was followed by an HQ535/20 (535 nm ± 10 nm) band pass filter. The iris aperture was 2.5–3.0 mm. This channel detected FITC. However, when the TRITC emission was much stronger than the FITC emission, TRITC emission from the tight junctions was also detected in this channel. In post-processing, the output of the TRITC channel was subtracted from the FITC channel to eliminate the contribution of TRITC to this channel. This was particularly effective because the TRITC only stained tight junctions and was therefore well localized and distinct from the FITC emission. The long wavelength side of the first dichroic mirror was split by a second dichroic mirror (605DRHP, 50% point at 605 nm). The short wavelength side of this dichroic mirror was followed by an HQ570/30 (570 nm ± 15 nm) band pass filter. The iris aperture was 0.7–1.7 mm. This channel detected TRITC, as well as FITC. When necessary, the FITC signal as detected in the short wavelength channel described above was subtracted from the output of the TRITC channel. This subtraction was done using the mixer controls on the Bio-Rad MRC-1024. In some cases the TRITC emission was much stronger than the FITC emission, and this subtraction was not required. The long wavelength side of the second dichroic mirror was followed by an HQ655/90 (655 nm ± 45 nm) band pass filter. The iris aperture
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