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• W2019908099 abstract "The invariant chain (Ii) targets major histocompatibility complex class II molecules to an endocytic processing compartment where they encounter antigenic peptides. Analysis of Ii-transferrin receptor chimeras expressed in polarized Madin-Darby canine kidney (MDCK) cells shows that the Ii cytoplasmic tail contains a dihydrophobic basolateral sorting signal, Met16-Leu17, which is recognized in both the biosynthetic and endocytic pathways. Pro15-Met16-Leu17 has previously been identified as one of two dihydrophobic Ii internalization signals active in non-polarized cells. Pro15 is also required for endocytosis in MDCK cells but not for basolateral sorting, indicating that the internalization signal recognized at the plasma membrane is distinct from the sorting signal recognized by basolateral sorting machinery. Another dihydrophobic sequence, Leu7-Ile8, is required for rapid internalization of the chimeric receptors in MDCK cells but not for basolateral sorting, providing further evidence that the structural requirements for basolateral sorting and internalization differ. Deletion analysis suggests that basolateral sorting of newly synthesized Ii-TR chimeras is also mediated by the membrane-proximal region of the Ii cytoplasmic tail. However, this region does not promote polarized basolateral recycling, indicating that the structural requirements for polarized sorting in the biosynthetic and endocytic pathways are not identical. The invariant chain (Ii) targets major histocompatibility complex class II molecules to an endocytic processing compartment where they encounter antigenic peptides. Analysis of Ii-transferrin receptor chimeras expressed in polarized Madin-Darby canine kidney (MDCK) cells shows that the Ii cytoplasmic tail contains a dihydrophobic basolateral sorting signal, Met16-Leu17, which is recognized in both the biosynthetic and endocytic pathways. Pro15-Met16-Leu17 has previously been identified as one of two dihydrophobic Ii internalization signals active in non-polarized cells. Pro15 is also required for endocytosis in MDCK cells but not for basolateral sorting, indicating that the internalization signal recognized at the plasma membrane is distinct from the sorting signal recognized by basolateral sorting machinery. Another dihydrophobic sequence, Leu7-Ile8, is required for rapid internalization of the chimeric receptors in MDCK cells but not for basolateral sorting, providing further evidence that the structural requirements for basolateral sorting and internalization differ. Deletion analysis suggests that basolateral sorting of newly synthesized Ii-TR chimeras is also mediated by the membrane-proximal region of the Ii cytoplasmic tail. However, this region does not promote polarized basolateral recycling, indicating that the structural requirements for polarized sorting in the biosynthetic and endocytic pathways are not identical. Polarized epithelial cells have structurally and functionally distinct plasma membrane domains with distinct protein and lipid compositions (reviewed in Refs. 1Mostov K. Apodaca G. Aroeti B. Okamoto C. J. Cell Biol. 1992; 116: 577-583Google Scholar and 2Rodriguez-Boulan E. Powell S.K. Annu. Rev. Cell Biol. 1992; 8: 395-427Google Scholar). In simple epithelia, the apical membrane faces the external environment and is specialized for secretion and nutrient absorption, while the basolateral membrane is in contact with the underlying tissue and performs fundamental cellular functions, such as intercellular adhesion and nutrient uptake from the blood supply. To generate and maintain polarity, membrane proteins which function at either the apical or basolateral border must be delivered to the appropriate cell-surface domain. In Madin-Darby canine kidney (MDCK) 1The abbreviations used are: MDCK, Madin-Darby canine kidney; Ii, human invariant chain; TR, human transferrin receptor; FcR, Fc receptor II B2 isoform; CEF, chicken embryo fibroblasts; RSV(A), Rous sarcoma virus subtype A; mAb, monoclonal antibody; Tf, human transferrin; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IiCT, Ii cytoplasmic tail; DMEM, Dulbecco's modified Eagle's medium; MHC, major histocompatibility complex. cells, most newly synthesized apical and basolateral membrane proteins comigrate through the Golgi complex and are selectively delivered to either the apical or basolateral surface (3Rindler M.J. Ivanov I.E. Plesken H. Rodriguez-Boulan E. Sabatini D.D. J. Cell Biol. 1984; 98: 1304-1319Google Scholar, 4Fuller S.D. Bravo R. Simons K. EMBO J. 1985; 4: 297-307Google Scholar). Studies during the past few years have established that polarized biosynthetic delivery to the basolateral surface requires distinct sorting signals located within the cytoplasmic domains of trafficking membrane proteins (5Mostov K.E. de Bruyn Kops A. Deitcher D.L. Cell. 1986; 47: 359-364Google Scholar, 6Brewer C.B. Roth M.G. J. Cell Biol. 1991; 114: 413-421Google Scholar, 7Casanova J.E. Apodaca G. Mostov K. Cell. 1991; 66: 65-77Google Scholar, 8Hunziker W. Harter C. Matter W. Mellman I. Cell. 1991; 66: 907-920Google Scholar). Basolateral sorting signals which have been identified within the cytoplasmic domains of trafficking membrane proteins can be distinguished based upon whether or not they are colinear with internalization signals (reviewed in Ref. 9Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Google Scholar). Most basolateral sorting signals which overlap with internalization signals are dependent for activity upon the same tyrosine residue found to be important for endocytosis. In some cases, however, the structural requirements for basolateral sorting and internalization differ (10Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Google Scholar, 11Prill V. Lehmann L. von Figura K. Peters C. EMBO J. 1993; 12: 2181-2193Google Scholar), demonstrating that the signal recognized by the basolateral sorting machinery is not identical to the signal recognized within plasma membrane clathrin-coated pits. Members of the other class of basolateral sorting signal, which are spatially separate from endocytic motifs, include the membrane-distal basolateral sorting signal of the low density lipoprotein receptor (10Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Google Scholar) and the basolateral sorting signal of vesicular stomatitis virus glycoprotein G (12Thomas D.C. Brewer C.B. Roth M.G. J. Biol. Chem. 1993; 268: 3313-3320Google Scholar), both of which are tyrosinedependent, as well as the basolateral sorting signal of the polymeric immunoglobulin receptor, which does not depend upon tyrosine for activity (13Aroeti B. Kosen P.A. Kuntz I.D. Cohen F.E. Mostov K.E. J. Cell. Biol. 1993; 123Google Scholar). Two basolateral sorting signals have been identified which are similar to dihydrophobic sorting signals that are known to mediate delivery to the endocytic pathway directly from the biosynthetic pathway or from the plasma membrane via endocytosis (reviewed in Ref. 14Sandoval I.V. Bakke O. Trends Cell Biol. 1994; 4: 292-297Google Scholar). A dileucine sequence in the cytoplasmic tail of the Fc receptor II B2 isoform (FcR) is required for both basolateral sorting and endocytosis (15Hunziker W. Fumey C. EMBO J. 1994; 13: 2963-2969Google Scholar, 16Matter K. Yamamoto E.M. Mellman I. J. Cell Biol. 1994; 126: 991-1004Google Scholar), demonstrating that this signal belongs to the class of basolateral sorting signals which are colinear with internalization signals. In contrast, a Leu-Val sequence targets newly synthesized CD44 to the basolateral surface but does not promote internalization (17Sheikh H. Isacke C.M. J. Biol. Chem. 1996; 271: 12185-12190Google Scholar). However, endocytosis of CD44 may be inhibited by direct submembranous interactions between its cytoplasmic domain and the cytoskeleton, preventing clustering in plasma membrane clathrin-coated pits (18Lokeshar V.B. Fregien N. Bourguignon L.Y.W. J. Cell Biol. 1994; 126: 1099-1109Google Scholar). The trans-Golgi network is generally considered to be the primary site for polarized basolateral sorting in MDCK cells (2Rodriguez-Boulan E. Powell S.K. Annu. Rev. Cell Biol. 1992; 8: 395-427Google Scholar, 19Wandinger-Ness A. Bennett M.K. Antony C. Simons K. J. Cell Biol. 1990; 111: 987-1000Google Scholar), although direct evidence for this is lacking. However, to maintain their polarized cell-surface distribution, basolateral membrane proteins which are rapidly internalized via clathrin-coated pits must be efficiently recycled back to this surface. Recent studies have shown that mutations which impair the polarized basolateral delivery of newly synthesized low density lipoprotein receptor and polymeric immunoglobulin receptor also lead to an increase in basolateral-to-apical transcytosis of these proteins, suggesting that in each protein, the same basolateral sorting signal is recognized in both the biosynthetic and endocytic pathways (20Matter K.J. Whitney A.J. Yamamoto E.M. Mellman I. Cell. 1993; 74: 1053-1064Google Scholar, 21Aroeti B. Mostov K.E. EMBO J. 1994; 13: 2297-2304Google Scholar). The MHC class II-associated invariant chain (Ii) assembles with newly synthesized class II αβ dimers in the endoplasmic reticulum and targets them to an endocytic processing compartment in which antigenic peptides are encountered (22Wolf P.R. Ploegh H.L. Annu. Rev. Cell Dev. Biol. 1995; 11: 267-306Google Scholar). Ii contains within its cytoplasmic domain two independent sorting signals, Leu7-Ile8 and Pro15-Met16-Leu17, related to dihydrophobic signals. Each sorting signal in Ii promotes rapid endocytosis (23Pieters J. Bakke O. Dobberstein B. J. Cell Sci. 1993; 106: 831-846Google Scholar, 24Bremnes B. Madsen T. Gedde-Dahl M. Bakke O. J. Cell Sci. 1994; 107: 2021-2032Google Scholar, 25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar), and the same or closely related signals are required for intracellular sorting and direct delivery of Ii to the endocytic pathway, which is the predominant route along which class II αβ-Ii complexes are transported (25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar, 26Benaroch P. Yilla M. Raposo G. Ito K. Miwa K. Geuze H.J. Ploegh H.L. EMBO J. 1995; 14: 37-49Google Scholar). Ii and MHC class II molecules are normally restricted to professional antigen-presenting cells, such as B lymphocytes, macrophages, and dendritic cells. However, expression of both Ii and class II molecules in non-immune epithelial cells can be induced by interferon-γ (27Baudeau C. Delarue F. He C.J. Nguyen G. Adida C. Peraldi M.N. Sraer J.D. Rondeau E. Exp. Nephrol. 1994; 2: 306-312Google Scholar,28Colgan S.P. Parkos C.A. Matthews J.B. D'Andrea L. Awtrey C.S. Lichtman A.H. Delp-Archer C. Madara J.L. Am. J. Physiol. 1994; 267: C402-410Google Scholar). We have investigated the sorting of Ii in epithelial cells by analyzing the trafficking of Ii-transferrin receptor (TR) chimeras expressed in MDCK cells. We find that the cytoplasmic domain of Ii targets newly synthesized chimeric receptors to the basolateral surface, where they are rapidly internalized. Endocytosed chimeras which recycle to the surface are selectively delivered to the basolateral border, indicating that the cytoplasmic tail of Ii is also recognized by the basolateral sorting machinery in the endocytic pathway. By analysis of mutant Ii-TR chimeras, we demonstrate that a dihydrophobic sorting signal consisting of Met16-Leu17 functions as a basolateral sorting signal which is required for polarized sorting in the endocytic pathway and is involved in basolateral sorting in the biosynthetic pathway. Deletion analysis suggests additional basolateral sorting information resides in the membrane-proximal region of the Ii cytoplasmic tail that is recognized within the biosynthetic pathway but not the endocytic pathway. Both dihydrophobic sequences Leu7-Ile8 and Pro15-Met16-Leu17 that independently mediate efficient endocytosis in nonpolarized cells (23Pieters J. Bakke O. Dobberstein B. J. Cell Sci. 1993; 106: 831-846Google Scholar, 24Bremnes B. Madsen T. Gedde-Dahl M. Bakke O. J. Cell Sci. 1994; 107: 2021-2032Google Scholar, 25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar) are required for internalization activity in MDCK cells. However, neither Leu7-Ile8 nor Pro15 are required for basolateral sorting of Ii-TR chimeras in MDCK cells providing clear evidence that there are different structural requirements for basolateral sorting and internalization in these cells. Thus, dihydrophobic sorting signals within the cytoplasmic tail of Ii are differentially recognized by the basolateral sorting machinery located within the biosynthetic and endocytic pathways and by plasma membrane clathrin-coated pits. Construction of human Ii-TR chimeras and expression in chick embryo fibroblasts (CEF) using the retroviral vector RCAS-BP(A) derived from Rous sarcoma virus subtype A (RSV(A)) (29Hughes S. Petropoulos C. Federspiel M. Sutrave P. Forry-Schaudies S. Bradac J. J. Reprod. Fert. Suppl. 1990; 41: 39-49Google Scholar) has been previously described (25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar). Since both Ii and TR are type II membrane proteins, the polarity of the polypeptide sequence with respect to the membrane is maintained. Recombinant virus produced by transfected CEF was concentrated by centrifugation of 10 ml of tissue culture supernatant at 23,000 rpm for 2.5 h at 4 °C in a Beckman SW40 Ti rotor. The pelleted virus was resuspended in 1 ml of DMEM, then passed through a 0.45-μm filter. The receptor for RSV(A) (30Bates P. Young J.A.T. Varmus H.E. Cell. 1993; 74: 1043-1051Google Scholar) has been stably expressed in MDCK II cells, rendering them susceptible to infection by RSV(A) (31Odorizzi G. Pearse A. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1996; 135: 139-152Google Scholar). MDCK cells derived from a clone expressing the RSV(A) receptor were plated at 105 cells/well of a 24-well tissue culture dish (Costar Corp., Cambridge, MA) and 12 h later were incubated with concentrated recombinant RCAS-BP(A) virus for 12 h at 37 °C. Afterward, 1 ml of growth medium was added, and the cells were grown to confluency. Expression of mutant human Ii-TR chimeras was analyzed by immunofluorescence using B3/25, a monoclonal antibody (mAb) against the extracellular domain of the receptor, and a goat anti-mouse secondary antibody conjugated to fluorescein isothiocyanate (Cooper Biomedical, Malvern, PA). Individual clones of MDCK cells expressing human Ii-TR chimeras were isolated by limited dilution. MDCK cells expressing human Ii-TR chimeras were plated at high density (1.5 × 106) onto 24-mm diameter Costar Transwell polycarbonate filters (0.4 μm pore size) (Costar Corp., Cambridge, MA) and cultured for 3 days, with the media changed on the second day. 125I-Labeled diferric human transferrin (Tf) (ICN Biomedicals, Irvine, CA) was prepared by incubating 500 μg of Tf with 40 μg of chloramine T (Sigma) and 0.5–1.0 mCi of Na125I (Amersham) in a total volume of 150 μl of phosphate-buffered saline (PBS). The reaction was stopped by adding 80 μg of sodium metabisulfite (Fisher Scientific, Fair Lawn, NJ) in 10 μl of PBS. 125I-Labeled Tf was separated from free125I on a Sephadex G-25 column equilibrated in PBS. To determine the steady-state cell-surface distribution of human Ii-TR chimeras, filter-grown cells were incubated in DMEM for 1 h at 37 °C, then shifted to 4 °C and washed with PBS+ (PBS with 1 mm CaCl2 and 1 mmMgCl2) containing 0.5% bovine serum albumin (BSA) (BSA-PBS+). Cells were then incubated for 1 h at 4 °C with 4 μg/ml 125I-labeled Tf in BSA-PBS+ added to either side of the monolayer (150 μl basolaterally or 350 μl apically). Under these conditions, less than 0.1% of the 125I-labeled Tf crossed the monolayers. Cells were then washed 3 times at 4 °C with BSA-PBS+ (2 ml/monolayer surface), and the amount of radioactivity specifically bound to the cells was determined by excising the filters and counting them in a γ-counter. MDCK cells expressing human Ii-TR chimeras were plated at a density of 7.5 × 104 cells/cm2 in a 24-well Costar tissue culture plate and cultured at 37 °C for 1 day. The cells were then incubated for 1 h at 37 °C in 1 ml of serum-free DMEM, then incubated with 150 μl of 4 μg/ml 125I-labeled Tf in DMEM containing 0.5% BSA (BSA-DMEM) for 1 h at 37 °C. The media was removed, and the cells were washed three times at 4 °C with 1 ml of BSA-PBS+, then incubated at 4 °C twice for 3 min with 0.5 ml of 0.2 m acetic acid, 0.5 mNaCl (pH 2.4) to remove surface-bound 125I-labeled Tf (32Hopkins C.R. Trowbridge I.S. J. Cell Biol. 1983; 97: 508-521Google Scholar). Cells were removed from the wells with 2 washes in 0.5 ml of 1m NaOH, and radioactivity in the acid wash and the cell lysate was determined. Prolonged incubation with the acid wash did not affect the radioactivity released (33Jing S. Spencer T. Miller K. Hopkins C. Trowbridge I.S. J. Cell Biol. 1990; 110: 283-294Google Scholar). The internalization efficiencies of mutant human Ii-TR chimeras relative to the wild-type human TR were determined from the steady-state intracellular distribution of 125I-labeled Tf and calculated as described previously (25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar, 33Jing S. Spencer T. Miller K. Hopkins C. Trowbridge I.S. J. Cell Biol. 1990; 110: 283-294Google Scholar). Filter-grown MDCK cells expressing human Ii-TR chimeras were incubated for 30 min at 37 °C in methionine- and cysteine-free DMEM (2 ml/monolayer surface) containing 0.5 mCi/ml35S-labeled methionine-cysteine (ICN Biomedicals, Irvine, CA) and 1% dialyzed fetal bovine calf serum. Monolayers were then washed 2 times with DMEM and chased for 20 or 40 min at 37 °C in DMEM containing a 10-fold excess of unlabeled methionine and cysteine. The cells were then chilled with 2 washes in ice-cold DMEM at 4 °C and incubated for 30 min in DMEM containing 100 μg/ml trypsin added to either the apical or the basolateral surface (Worthington Biochemical Corp., Freehold, NJ). DMEM containing 100 μg/ml trypsin inhibitor (Sigma) was added to the opposite surface. Tryptic cleavage of surface TR generates a soluble ∼70-kDa extracellular domain fragment which was immunoprecipitated from the apical and basolateral media using B3/25 mAb and analyzed on 10% SDS-polyacrylamide gels (34Omary M.B. Trowbridge I.S. J. Biol. Chem. 1981; 256: 12888-12892Google Scholar). Dried gels were exposed to flashed XAR film (Eastman Kodak, Rochester, NY), and quantitation of radioactivity was performed on a model 425 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). No TR tryptic fragments were detected in immunoprecipitates of media containing trypsin inhibitor. Filter-grown MDCK cells expressing human Ii-TR chimeras were incubated for 1 h at 37 °C in DMEM, then for 1 h at 37 °C with 4 μg/ml 125I-labeled Tf in BSA-DMEM (150 μl basolaterally and 350 μl apically). Cells were then washed 3 times at 4 °C with BSA-DMEM (2 ml/monolayer surface), and surface-bound Tf was removed with >95% efficiency using deferoxamine mesylate as described previously (31Odorizzi G. Pearse A. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1996; 135: 139-152Google Scholar, 33Jing S. Spencer T. Miller K. Hopkins C. Trowbridge I.S. J. Cell Biol. 1990; 110: 283-294Google Scholar). Cells were then washed 3 times with BSA-DMEM at 4 °C and incubated at 37 °C for 90 min in BSA-DMEM containing 100 μg/ml unlabeled Tf (1 ml/monolayer surface). Afterward, the radioactivity released into the apical and basolateral media, as well as the cell associated radioactivity, was determined. To investigate the trafficking of Ii in polarized epithelial cells, we expressed in MDCK cells wild-type and mutant Ii-TR chimeras (Fig. 1) that had previously been expressed in CEF (25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar) using a retroviral vector derived from RSV(A) (29Hughes S. Petropoulos C. Federspiel M. Sutrave P. Forry-Schaudies S. Bradac J. J. Reprod. Fert. Suppl. 1990; 41: 39-49Google Scholar, 35Odorizzi G. Trowbridge I.S. Methods Cell Biol. 1994; 43: 79-98Google Scholar). Recombinant retrovirus produced by CEF was used to infect MDCK cells in which the 157-amino acid isoform of the receptor for RSV(A) (30Bates P. Young J.A.T. Varmus H.E. Cell. 1993; 74: 1043-1051Google Scholar) had been expressed (31Odorizzi G. Pearse A. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1996; 135: 139-152Google Scholar). The steady-state cell-surface distributions of Ii-TR chimeras in filter-grown MDCK monolayers were subsequently determined by measuring the binding of 125I-labeled human Tf at the apical and basolateral surfaces at 4 °C. As shown in Fig. 2, a chimera (IiCT) consisting of the 30-residue amino-terminal cytoplasmic tail of Ii (p33 isoform) and the transmembrane and extracellular domains of TR was localized predominantly at the basolateral surface, similar to the polarized cell-surface distribution of the wild-type TR (31Odorizzi G. Pearse A. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1996; 135: 139-152Google Scholar), demonstrating that the cytoplasmic tail of Ii contains basolateral sorting information. Deletion of residues 2–11 of the Ii cytoplasmic tail resulted in a modest decrease in the polarized steady-state basolateral cell-surface expression of chimeric receptors (Fig. 2). However, deletion of residues 2–17 led to almost a complete loss of basolateral polarity (Fig. 2), although a small but significantly greater fraction of this mutant chimera was found at the basolateral border when compared with the distribution of the tailless TR (Δ3–59) in MDCK cells (31Odorizzi G. Pearse A. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1996; 135: 139-152Google Scholar).Figure 2Sorting information in the Ii cytoplasmic tail targets Ii-TR chimeras to the basolateral surface of MDCK cells. Filter-grown MDCK cells expressing IiCTchimeras were incubated either apically or basolaterally for 1 h at 4 °C with 125I-labeled Tf (4 μg/ml). After washing away unbound Tf, the amount of 125I-labeled Tf specifically bound on each surface was determined (mean ± S.E. of three independent experiments). Values for the distributions of wild-type and tailless (Δ3–59) TR, shown for comparison, are from Odorizziet al. (31Odorizzi G. Pearse A. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1996; 135: 139-152Google Scholar).View Large Image Figure ViewerDownload (PPT) Located within residues 2–17 of the Ii cytoplasmic tail are two dihydrophobic sorting signals (Leu7-Ile8 and Pro15-Met16-Leu17) which independently promote rapid internalization in non-polarized cells (23Pieters J. Bakke O. Dobberstein B. J. Cell Sci. 1993; 106: 831-846Google Scholar, 24Bremnes B. Madsen T. Gedde-Dahl M. Bakke O. J. Cell Sci. 1994; 107: 2021-2032Google Scholar, 25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar). However, Leu7-Ile8 are not required for basolateral localization, as alteration of both residues to alanine did not affect the polarized steady-state expression of IiCT chimeras at the basolateral surface (Fig. 2). We next investigated whether Pro15-Met16-Leu17 is involved in basolateral targeting by examining the cell-surface distributions of previously constructed IiCT chimeras in which each of these residues had been individually altered to alanine in the context of an Ii cytoplasmic tail in which Leu7-Ile8 had also been changed to alanine (25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar). Fig. 2 shows that alteration of Pro15 to alanine resulted in only a small decrease in the selective steady-state basolateral distribution of IiCTchimeras, suggesting that this residue has, at most, a minor role in basolateral sorting. However, a random cell-surface distribution resulted from the substitution of alanine for Met16. A similar result was obtained if Met16 had been altered to alanine in the context of the wild-type Ii cytoplasmic tail in which Leu7-Ile8 were intact (Fig. 2), confirming that Met16, but not Leu7-Ile8, is required for selective basolateral expression of IiCTchimeras. Fig. 2 shows that Leu17 is required for basolateral polarity as well, although alteration of this residue more significantly impaired basolateral localization. The steady-state level of the L17A mutant chimera on the basolateral surface is lower than predicted from the independent measurements of the biosynthetic delivery and recycling of the mutant (see Figs. 4 and5) and, therefore, may reflect differences in either the rates of internalization or degradation of the mutant chimera from the basolateral and apical surfaces. Previous studies of Ii-TR chimeras in non-polarized cells indicated that Gly18 has no role in high efficiency endocytosis (25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar). Similarly, substitution of alanine for Gly18 had no effect on the basolateral expression of chimeric receptors in MDCK cells (Fig. 2). These results, therefore, indicate that Met16 and Leu17 are important for the polarized basolateral localization of IiCT chimeras in MDCK cells, whereas Pro15 is not required.Figure 5Met16-Leu17, but not the membrane-proximal region, are required for basolateral sorting of Ii-TR chimeras in the endocytic pathway of MDCK cells.Filter-grown MDCK cells expressing Ii-TR chimeras were incubated basolaterally at 37 °C for 1 h with 125I-labeled Tf. Monolayers were then washed at 4 °C, and surface-bound125I-labeled Tf was removed with deferoxamine mesylate. Cells were then incubated at 37 °C for 90 min, and the appearance of125I-labeled Tf in the apical and basolateral media was determined (mean ± S.E. of three independent experiments). More than 90% of the internalized 125I-labeled Tf recycled after 90 min at 37 °C.View Large Image Figure ViewerDownload (PPT) In non-polarized cells, both Leu7-Ile8 and Pro15-Met16-Leu17 independently mediate high efficiency endocytosis (23Pieters J. Bakke O. Dobberstein B. J. Cell Sci. 1993; 106: 831-846Google Scholar, 24Bremnes B. Madsen T. Gedde-Dahl M. Bakke O. J. Cell Sci. 1994; 107: 2021-2032Google Scholar, 25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar). Therefore, since only Met16-Leu17, but not Leu7-Ile8, were found to be important for basolateral localization, we next investigated whether rapid endocytosis in MDCK cells was mediated by the same internalization signals previously characterized in non-polarized cells. As in CEF (25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar), the IiCT chimera was rapidly internalized in MDCK cells with an efficiency similar to that of wild-type TR (Fig. 3). High efficiency endocytosis was completely abrogated by deletion of residues 2–17 in the Ii cytoplasmic tail. Interestingly, rapid internalization was also completely abolished in MDCK cells by independently altering either of the Leu7-Ile8 or Leu14-Pro15 dipeptides to two alanine residues (Fig. 3), mutations which in CEF impair internalization by only ∼50% (25Odorizzi C.G. Trowbridge I.S. Xue L. Hopkins C.R. Davis C.D. Collawn J.F. J. Cell Biol. 1994; 126: 317-330Google Scholar). Individual substitution of alanine for Met16 also completely abrogated rapid internalization in MDCK cells (Fig. 3), supporting the conclusion that the Leu7-Ile8and Pro15-Met16-Leu17 sequences identified as independent internalization signals in non-polarized cells can only act in concert to mediate rapid endocytosis in MDCK cells. The polarized basolateral steady-state cell-surface distribution of TR in MDCK cells has been demonstrated to result from the basolateral sorting of both newly synthesized and recycling receptors (31Odorizzi G. Pearse A. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1996; 135: 139-152Google Scholar). Therefore, we first investigated the biosynthetic cell-surface delivery of Ii-TR chimeras by pulse labeling newly synthesized chimeric receptors with35S-labeled methionine-cysteine and chasing them to the surface of filter-grown monolayers for 20 or 40 min at 37 °C. Subsequently, the apical or basolateral surface was incubated in trypsin at 4 °C, which cleaves the receptor near the transmembrane region, resulting in the release of an ∼70-kDa extracellular fragment which was immunoprecipitated using B3/25 mAb (34Omary M.B. Trowbridge I.S. J. Biol. Chem. 1981; 256: 12888-12892Google Scholar) and quantitated following SDS-polyacrylamide gel electrophoresis and PhosphorImager analysis. As shown in Fig. 4, newly synthesized IiCT chimeras were efficiently sorted to the basolateral surface, as were mutant chimeras in which residues 2–11 had been deleted from the cytoplasmic tail. As expected from their basolateral polarity seen at steady state (Fig. 2), mutant IiCT chimeras in which Leu7-Ile8 were altered to alanine were also sorted biosynthetically to the basolateral surface with high efficiency (Fig. 4). Interestingly, deletion of residues 2–17 resulted in only a modest decrease in the basolateral polarity of cell-surface delivery (∼75%), suggesting that" @default.
• W2019908099 created "2016-06-24" @default.
• W2019908099 creator A5009607649 @default.
• W2019908099 creator A5084440501 @default.
• W2019908099 date "1997-05-01" @default.
• W2019908099 modified "2023-09-28" @default.
• W2019908099 title "Structural Requirements for Major Histocompatibility Complex Class II Invariant Chain Trafficking in Polarized Madin-Darby Canine Kidney Cells" @default.
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