Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Abstract Full Text (PDF, 4256K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Liu, S.-H. Articles by Brodsky, F. M. Search for Related Content PubMed PubMed Citation Articles by Liu, S.-H. Articles by Brodsky, F. M. Social Bookmarking                            What's this?

© The Rockefeller University Press, 0021-9525/1998//1023 $5.00
The Journal of Cell Biology, Volume 140, Number 5, , 1998 1023-1037

A Dominant-negative Clathrin Mutant Differentially Affects Trafficking of Molecules with Distinct Sorting Motifs in the Class II Major Histocompatibility Complex (MHC) Pathway

Shu-Hui Liu^*, Michael S. Marks, and Frances M. Brodsky^*

^* The G.W. Hooper Foundation, Department of Microbiology and Immunology, and Department of Biopharmaceutical Sciences, and Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0552; and Department of Pathology and Laboratory Medicine, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104-6082

The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA–DM and the invariant chain (I chain) complexed to HLA–DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR–I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR–I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR–I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR–I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR–I chain until their intended rendezvous in late endocytic compartments.

Abbreviations used in this paper: AP, adaptor protein; CCV, clathrin-coated vesicle; CI-M6PR, cation-independent mannose-6-phosphate receptor; CMV, cytomegalovirus; COP, coat protein; FITC-Tfn, fluorescein-conjugated transferrin; I chain, invariant chain; IL, interleukin; LC, light chain; LRSC, lissamine rhodamine; PM, plasma membrane; TfnR, transferrin; TfnR, transferrin receptor.

CLATHRIN-COATED vesicles (CCVs)¹ can mediate selective budding of receptors and ligands from one cellular membrane for transport to another (Brodsky, 1988; Pley and Parham, 1993; Schmid, 1997). This is achieved by self-assembly of clathrin into a polyhedral membrane coat that incorporates the adaptor protein (AP)1 or AP2 molecules that bind receptors at the TGN and plasma membrane (PM), respectively. There is strong evidence that CCV formation is responsible for receptor-mediated endocytosis at the PM and for the sorting of lysosomal hydrolases in the TGN. However, there is an increasing need for the precise definition of the role of CCVs in other intracellular pathways given the proliferation of specialized sorting pathways, novel receptor sorting motifs, and the recent discovery of a novel adaptor molecule, AP3, that recognizes conventional CCV sorting motifs but is apparently not a component of CCVs (Dell'Angelica et al., 1997a,b; Simpson et al., 1997). Furthermore, there are cellular sites to which clathrin has been localized, such as maturing secretory granules (Tooze and Tooze, 1986; Dittié et al., 1996) and endosomes (Stoorvogel et al., 1996), where the function of clathrin is not clearly understood. Here we establish a system to probe clathrin's intracellular function in mammalian cells by transfecting cells with a dominant-negative mutant of clathrin. We then apply this approach to defining the role of CCVs in the specialized sorting pathways that control antigen presentation by class II molecules of the major histocompatibility complex (MHC).

Class II MHC molecules are targeted to the endocytic pathway where they bind antigenic peptides that are then displayed on the cell surface. This so-called "antigen presentation" by class II MHC molecules activates helper T cells that stimulate B cells to produce antibody. Two of the accessory molecules required for functional antigen presentation by class II MHC molecules bear sorting motifs that suggest a role for CCVs in their intracellular targeting, but sorting steps at which CCVs are involved in this process have yet to be completely defined. Endocytic localization of the invariant chain (I chain), which is responsible for transport of the class II molecule itself, depends on a dileucine-containing sequence (Bakke and Dobberstein, 1990; Lotteau et al., 1990; Pieters et al., 1993; Odorizzi et al., 1994), whereas localization of the HLA–DM molecule, which is a catalyst for peptide loading, depends on a tyrosine-containing sequence (Lindstedt et al., 1995; Marks et al., 1995; Ohno et al., 1995; Copier et al., 1996). These sequences conform to two of the general consensus motifs associated with binding to adaptor components of CCVs (Johnson and Kornfeld, 1992; Letourneur and Klausner, 1992; Marks et al., 1997). In vitro studies show that sequences with the tyrosine-containing motif interact with the µ1 or µ2 subunits of the AP1 or AP2 adaptors (Ohno et al., 1995, 1996; Rapoport et al., 1997), and there is evidence that the dileucine motif can mediate binding of intact AP1 and AP2 molecules to receptor tails (Heilker et al., 1996; Salamero et al., 1996; Dietrich et al., 1997). However, the intracellular locations where CCVs actually sequester receptors with the different motifs and perform their sorting function are more specific than these binding studies indicate. Here we establish where CCVs play a role in targeting the I chain with associated class II molecules and HLA–DM to the endocytic pathway, defining the actual intracellular steps where these motifs contribute to clathrin-mediated sorting and where CCVs play a role in antigen presentation.

There is considerable successful precedent for using mutants to analyze clathrin function. Clathrin-deficient mutants of Saccharomyces cerevisiae (Lemmon et al., 1991; Seeger and Payne, 1992), Dictyostelium discoideum (Niswonger and O'Halloran, 1997), and Drosophila melanogaster (Bazinet et al., 1993) have phenotypes that confirm roles for CCVs in endocytosis, retention of TGN proteins, and sorting of lysosomal hydrolases and reveal roles in spermatogenesis and cytokinesis. Mutation of the GTPase dynamin, which plays a specific role in the scission of CCVs during receptor-mediated endocytosis, has been effectively applied to dissecting clathrin's role at the PM in Drosophila and mammalian cells (Herskovits et al., 1993; van der Bliek et al., 1993; Damke et al., 1995; Wang et al., 1997). To design a dominant-negative mutant of clathrin that would enable analysis of clathrin-mediated transport throughout the cell, we took advantage of our understanding of the domain structure of clathrin. Clathrin is a triskelion-shaped molecule composed of three heavy chains (192-kD) and three light chains (25–29 kD) of which there are two types, LCa and LCb, in mammalian cells (for review see Brodsky, 1988). Using a bacterial expression system, we have previously demonstrated that the carboxy-terminal third of the clathrin heavy chain trimerizes and folds to reproduce the central portion of the triskelion, forming the hub fragment (Liu et al., 1995). The hub molecules bind clathrin light chains and can self-assemble with the same kinetics as intact clathrin but they polymerize into an open-ended lattice instead of a closed polyhedron. The potential ability of hubs to interact with endogenous clathrin heavy and light chains and the fact that they assemble into nonfunctional structures, made the hub fragment a good candidate for a dominant-negative mutant that could disrupt clathrin-dependent functions. Here we demonstrate that expression of clathrin hub fragments by transfection of mammalian cells perturbs receptor-mediated endocytosis, lysosomal targeting, and clathrin-mediated intracellular sorting in the TGN, without pleiotropic effects on other intracellular transport pathways. Expression of hub fragments was found to inhibit the intracellular targeting of HLA–DM molecules to the endocytic pathway. In contrast, the direct targeting of I chain to the endocytic pathway was unaffected in the presence of hubs. The surface levels of both molecules, however, were elevated when clathrin-mediated endocytosis was blocked in hub-transfected cells. Thus, CCVs participate in the differential sorting of the class II molecule/I chain complex and HLA–DM. Because the sorting of these complexes is known to be controlled by either dileucine- or tyrosine-based motifs, these findings also suggest that whereas CCVs sort both motifs at the PM, the dileucine-based motif is sorted independently of CCVs in the TGN.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Antibodies
Antibodies obtained from commercial sources were anti-T7 epitope tag mAb and its biotinylated version (subtype IgG_2b; Novagen, Inc., Madison, WI), anti–β-coat protein (COP) mAb M3A5 (Sigma Chemical Co., St. Louis, MO), anti-CD63 mAb (Immunotech Inc., Westbrook, ME). FITC- and rhodamine-conjugated goat anti–rabbit and FITC-conjugated goat anti–mouse Igs were from Organon Teknika Corp. (West Chester, PA). Lissamine rhodamine (LRSC)-conjugated goat anti–mouse Ig and TRITC-conjugated streptavidin were purchased from Jackson ImmunoResearch Laboratories, Inc., (West Grove, PA). FITC-conjugated goat anti–mouse IgG₁ and IgG_2a–specific secondary antibodies were obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Antibodies produced in the laboratory, using standard methods were anti-LC (anticlathrin light chain polyclonal rabbit antiserum recognizing the consensus sequence in all LCs, residues 23–40; Acton and Brodsky, 1990), X32 mAb (anticlathrin heavy chain, which does not react with the clathrin heavy chain hub region; Blank and Brodsky, 1986), AP.6 mAb (anti– chain of the AP2 adaptor; Chin et al., 1989), KESL (polyclonal rabbit antiserum, recognizing the carboxy-terminal residues in the lumenal domain of human I chain), and W6/32 mAb (anti-MHC class I [HLA-A, -B, and -C]; Barnstable et al., 1978). Antibodies obtained as gifts from colleagues were 100/3 mAb (anti– chain of the AP1 adaptor, gift from E. Ungewickell, Washington University, St. Louis, MO; Ahle et al., 1988), anti-ER (rabbit polyclonal antiserum, gift from D. Louvard, Institut Curie, Paris, France; Louvard et al., 1982), anti-Tac (rabbit polyclonal antiserum, gift from W. Greene, University of California, San Francisco, CA), anti-DM (rabbit polyclonal antiserum, gift from D. Zaller, Merck Research Laboratories, Rahway, NJ; Sloan et al., 1995), PIN.1.1 mAb (anti-I chain cytoplasmic domain, gift from P. Cresswell, Yale University, New Haven, CT; Roche et al., 1991) and anti–cation-independent mannose-6-phosphate receptor (CI-M6PR, polyclonal rabbit antiserum; gift from L. Traub, Washington University).

Plasmid Construction
The cDNA encoding bovine clathrin heavy chain residues 1073–1675 (Liu et al., 1995) was first cloned into the BamHI and HindIII sites of the vector pET23d (Novagen, Inc.) after the T7 gene 10-leader peptide sequence. The T7Hub cDNA fragment was then cut out and inserted between the HindIII and XhoI sites of the vector pCDM8 (Invitrogen, Carlsbad, CA) under the cytomegalovirus (CMV) promoter to generate pCDM8T7Hub for transfection. HLA–DM and β subunit cDNAs were obtained from the XhoI fragments of pCDM8DM and pCDM8DMβ (Kelly et al., 1991; Marks et al., 1995), respectively, and then subcloned into the vector pMT302 containing the modified human metallothionein IIA promoter (gift from S. Haskill, University of North Carolina, Chapel Hill, NC; Makarov et al., 1994) to produce pMT302DM and pMT302DMβ. The DMβHSS construct, lacking the carboxy-terminal 16 residues of the 23-residue cytoplasmic domain that eliminates the DMβ internalization motif, was in pCDM8.1 and its expression was regulatable by cadmium when coexpressed with pMT302DM. I chain p33 (Ip33) cDNA was derived from pCDM8Ip33 (gift from P. Roche, National Institutes of Health, Bethesda, MD; Roche et al., 1992) and inserted into pMT302 to generate plasmid pMT302Ip33. Detailed cloning procedures will be provided upon request. Tac (interleukin [IL]-2 receptor subunit) chimeric constructs TTM.GSTY (mouse H2M cytoplasmic tail fused to the lumenal and transmembrane domain of Tac; Marks et al., 1995) and Tac-DKQTLL (the T cell receptor CD3 dileucine sequence DKQTLL attached to the carboxyl terminus of Tac, also called TTt3-t2; Letourneur and Klausner, 1992), both in the vector pCDM8.1 (Invitrogen), were used as described previously (Marks et al., 1996, 1997). Plasmids containing MHC class II DR and β chain cDNA, pCDM8–DRA, and pCDM8–DRB (pCDM8– DRB1*0101) were obtained from E. Long, National Institutes of Health; Long et al., 1994.

Cell Culture and Transfection
HeLa 229 cells (American Type Culture Collection, Rockville, MD) were maintained in DME supplemented with 10% fetal bovine serum. Cells were plated the day before transfection and then transfected by the standard calcium phosphate method at 30–50% confluency. Typically, 7.2 µg per 60-mm plate or 0.7 µg per coverslip of T7Hub plasmid was used for transfection. 12 h later the transfection mixture was removed and then replaced with fresh medium. Cells were generally assayed at 40–48 h after transfection. To induce expression of transfected genes under the metallothionein promoter, 10 µM CdCl₂ was added to the culture 20–24 h before analysis.

Endocytosis Assay
For transferrin (Tfn) uptake, pCDM8T7Hub–transfected HeLa cells were changed to transferrin uptake buffer PBS, with 10 mM Tris-HCl, 10 mM Pipes, 5 mM glucose, 1 mg/ml BSA, 0.1 g/L MgCl₂· 6 H₂O, 0.1 g/L CaCl₂· 2 H₂O, pH 7.4) for 3 h at 37°C. Fluorescein-conjugated human Tfn (FITC-Tfn; Molecular Probes, Eugene, OR) at 50 µg/ml in the uptake buffer was then added to the cells for 10 min at 37°C, after which the medium was removed and then the cells were washed three times with ice-cold PBS and processed for indirect immunofluorescent microscopy. To measure bulk fluid-phase endocytosis, 1 mg/ml lysine-fixable FITC-dextran (70K; Molecular Probes) was added to the cells for 3 h at 37°C in PBS buffer (with Ca²⁺ and Mg²⁺).

Immunoprecipitation and Immunoblotting
Transfected cells on 60-mm plates were harvested and then lysed in cell lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1 mM EDTA, pH 7.4) plus protease inhibitors. For immunoprecipitation of T7Hub from transfected cells, 5 µl of 1 mg/ml purified anti-T7 mAb was added to the cell lysate for at least 1 h at 4°C, followed by 50 µl of protein G–Sepharose slurry for another hour (Pharmacia Diagnostics AB, Uppsala, Sweden). The unbound fractions were removed and then the precipitates were washed three times with lysis buffer at 4°C. Both supernatants and immunoprecipitates were then resolved by SDS-PAGE (Laemmli, 1970), transferred to nitrocellulose, and then analyzed by immunoblotting (Towbin et al., 1979). Antibody binding was detected with secondary antibodies conjugated to horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA) using the enhanced chemiluminescence substrate (Amersham Corp., Arlington Heights, IL).

Indirect Immunofluorescent Microscopy
HeLa cells plated on 12-mm coverslips were transfected as described above. 40–48 h after transfection, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, and then permeabilized with 0.04% saponin (Sigma Chemical Co.) for another 10 min. Cells were then incubated with blocking buffer (PBS, 0.02% SDS, 0.1% NP-40, 0.02% NaN₃ with 5% goat serum or with 0.66% gelatin and 1% BSA when labeling with anti–CI-M6PR) for 30 min, followed by incubation with various primary and fluorochrome-conjugated secondary antibodies diluted in appropriate blocking buffer, for 1 h each. After the final wash, the samples were mounted in 0.1% p-phenylene diamine (Sigma Chemical Co.) in Fluoromount G (Fisher Scientific Co., Pittsburgh, PA) and then viewed with a Zeiss Axiophot fluorescence microscope (Thornwood, NY). For cytosol depletion, transfected cells were treated with 0.004% digitonin (Calbiochem-Novabiochem Corp., La Jolla, CA) in permeabilization buffer (12.5 mM Hepes–KOH, 50 mM Pipes, 1 mM MgSO₄, 4 mM EGTA, pH 7.0) for 5 min on ice before fixation. To detect the surface level of certain antigens, the transfected cells were incubated with primary antibodies in PBS (with Ca²⁺ and Mg²⁺) on ice for 30 min, the antibody solution was removed, and then the cells were washed three times with ice-cold PBS before fixation.

Surface Biotinylation
The surface biotinylation procedure was modified from an earlier method (Bonnerot et al., 1995). Transfected HeLa cells were washed three times with ice-cold PBS and then incubated with freshly prepared 0.5 mg/ml sulfo-NHS-LC-Biotin (Pierce Chemical Co., Rockford, IL) in PBS for 15 min on ice. The reaction was quenched with 50 mM NH₄Cl/PBS for another 15 min. Cells were lysed and then the biotinylated proteins were isolated with avidin–agarose (Pierce Chemical Co.) in the cell lysis buffer.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Expression of Hubs Affects the Intracellular Distribution of Both Clathrin Heavy and Light Chains but Not Adaptors
The hub fragment of clathrin, comprising the carboxy-terminal third of the clathrin heavy chain, can bind clathrin LCs in vitro (Liu et al., 1995) and can interact with intact clathrin (data not shown). Therefore, we predicted that expression of the hub in mammalian cells would affect CCV formation in vivo. To test whether hub would bind endogenous clathrin LCs during intracellular expression, HeLa cells were transfected with a mammalian expression vector encoding the hub fragment of bovine clathrin modified with a T7 epitope-tag at the amino terminus (T7Hub). The T7Hub protein was immunoisolated from the cell lysate, using anti-T7 epitope antibody, 48 h after transfection. Endogenous clathrin light chains, LCa and LCb, were both observed associated with the T7Hub proteins (Fig. 1 A, lane 4). Hubs presumably compete with endogenous clathrin heavy chains for newly synthesized LCs, since LCs do not readily dissociate from preassembled complexes with clathrin heavy chains (Acton and Brodsky, 1990).

View larger version (76K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Transfected hubs alter the intracellular distribution of both clathrin heavy chains and LCs. (A) HeLa cells were transfected with control vector (pCDM8 without the T7Hub insert, lanes 1 and 2) or with T7Hub (pCDM8T7Hub vector, lanes 3 and 4). 48 h after transfection, cells were lysed and then the T7Hub protein was immunoprecipitated with anti-T7 mAb bound to protein G–Sepharose. The unbound (UB) and bound (B) fractions were then analyzed by SDS-PAGE and immunoblotting. The T7Hub protein and clathrin LCs (LCa and LCb) were detected with anti-T7 mAb and anti-LC antiserum, respectively. IgH and IgL are the subunits of the mAb used for immunoprecipitation, detected by the secondary anti-immunoglobulin antibody. (B) HeLa cells were transfected with T7Hub. 48 h after transfection, cells were processed for immunofluorescent microscopy and stained with anti-T7 mAb and anti-LC antiserum (anti-LC) followed by LRSC-conjugated goat anti–mouse IgG and FITC-conjugated goat anti–rabbit IgG. (C) T7Hub-transfected HeLa cells were permeabilized with 0.004% digitonin to remove cytosol before fixation and thereby visualize membrane-associated clathrin. The cells were stained with anti-LC antiserum (anti-LC) and antiheavy chain mAb X32 (anti-HC) followed by rhodamine- conjugated goat anti–rabbit IgG and FITC-conjugated goat anti–mouse IgG. The T7Hub-transfected cells were identified by the loss of punctate clathrin LC staining (arrowheads) and are the ones with increased staining of clathrin heavy chain at the PM.

Binding of clathrin to cellular membranes is mediated through AP2 and AP1 adaptors (Keen, 1990; Pearse and Robinson, 1990). To establish whether the increase of clathrin at membranes in the presence of hubs caused recruitment of additional adaptors, the distribution of adaptors was assessed in hub-transfected cells. Labeling with mAbs specific for AP1 or AP2 revealed that the general distribution of adaptors was not affected by hub expression. In the T7 Hub-transfected cells, AP2 remained in peripheral punctate structures and AP1 staining was still mostly perinuclear (Fig. 2). The lack of effect on adaptor distribution suggests that the excess clathrin associated with membranes in hub-transfected cells represents hyperassembly due to altered clathrin–clathrin interactions that are adaptor independent.

View larger version (80K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Expression of T7Hub in transfected cells does not alter the general distribution of adaptor molecules. HeLa cells were transfected with T7Hub and after 48 h were then analyzed by immunofluorescence for the distribution of the AP1 and AP2 adaptors. (A) Cells stained with anti-AP2 mAb AP.6 (AP2) and biotinylated anti-T7 mAb followed by FITC-conjugated anti–mouse IgG1–specific secondary antibody (for AP.6) and TRITC-conjugated streptavidin. (B) Cells stained with anti-AP1 mAb 100/3 (AP1) and anti-LC (anti-LC) antiserum followed by LRSC-conjugated goat anti–mouse IgG and FITC-conjugated goat anti– rabbit IgG. The T7Hub– transfected cells in B were identified by the loss of punctate clathrin LC staining (arrowheads).

View larger version (96K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Expression of T7Hub in transfected cells inhibits receptor-mediated endocytosis but not bulk fluid-phase uptake and alters the intracellular distribution of the CI-M6PR. (A) HeLa cells were transfected with T7Hub. 48 h after transfection, cells were allowed to internalize FITC-Tfn for 10 min at 37°C, and then were processed for immunofluorescent microscopy. Hub-transfected cells were visualized by anti-T7 mAb and LRSC-conjugated goat anti–mouse IgG. (B) HeLa cells were transfected with T7Hub and, after 48 h, were allowed to internalize FITC-dextran at 37°C for 3 h before being processed for anti-T7 mAb staining to visualize cells with hubs. (C) HeLa cells were transfected with T7Hub and after 48 h were processed for immunofluorescent microscopy to visualize the CI-M6PR, with specific rabbit antiserum and FITC-conjugated goat anti–rabbit IgG, and hub-transfected cells, with anti-T7 mAb and LRSC-conjugated goat anti–mouse IgG.

To ascertain that hub expression does not cause a general pleiotropic effect on organelle integrity, the ER and Golgi structures of cells transfected with T7Hub cDNA were examined by immunofluorescent staining with anti-ER antibody and a mAb against the Golgi-associated β-COP (Allan and Kreis, 1986). In cells expressing T7Hub, no change of the ER staining was observed, and β-COP remained concentrated in the apparently intact Golgi apparatus (Fig. 4, A and B). The cellular distribution of class I MHC molecules, which are efficiently transported through the secretory pathway to their site of accumulation on the PM, with no known requirement for sorting, was also not altered in cells expressing hubs (Fig. 4 C).

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Hub expression does not have pleiotropic effects on organelle integrity or the constitutive secretory pathway. HeLa cells were transfected with T7Hub and, after 48 h, were stained with (A) anti-ER antiserum and FITC-conjugated goat anti– rabbit IgG, (B) anti–β-COP mAb M3A5 and FITC-conjugated goat anti–mouse IgG1–specific secondary antibody, or (C) anti-MHC class I mAb W6/32 and FITC-conjugated anti–mouse IgG2a–specific secondary antibody. The T7Hub-transfected cells in each field were visualized either by (A) anti-T7 mAb followed by LRSC-conjugated goat anti–mouse IgG or (B and C) biotinylated anti-T7 mAb followed by TRITC-conjugated streptavidin.

Chimeric molecules were transfected into HeLa cells with or without cotransfection of T7Hub and their steady-state levels were measured by immunoblotting with Tac antibody (Fig. 5). Surface biotinylation was also performed on the transfected cells to assess the surface distribution of the chimeras by isolating surface molecules with avidin and then immunoblotting with Tac. When either of the Tac chimeras were expressed alone in transfected cells, only low levels were detectable in total cell lysate and virtually none were detectable in the cell surface-biotinylated pool (Fig. 5), consistent with lysosomal targeting. Cotransfection with T7Hub cDNA caused dramatic increases in the cellular level of either chimera. In addition, a detectable proportion (20%) appeared in the cell surface-biotinylated pool (Fig. 5), a ratio that is probably an underestimate due to the inefficiency of biotinylation. Intracellular distribution of the chimeric proteins was further examined by immunofluorescent microscopy. In accordance with the biochemical analysis, the fluorescent signals from staining the Tac constructs were greatly enhanced upon cotransfection with T7Hub, compared to the generally weak staining obtained in the control cells (Fig. 6). The bulk of the chimeric proteins in hub-transfected cells also seemed to be predominantly localized on the PM (Fig. 6), whereas those in cells without hubs had a punctate intracellular distribution. These results indicate that blocking CCV function results in the mistargeting of proteins normally destined for lysosomes and because of a block in receptor-mediated endocytosis, they were accumulating on the cell surface. It is not possible to determine whether these proteins were expressed on the cell surface as part of their normal transport pathway to lysosomes or whether they were diverted to the cell surface because of missorting in the TGN. However, these results clearly show that chimeric molecules with either the tyrosine or dileucine motif rely on CCVs for their endocytosis. They also confirm that hub transfection blocks CCV function at the cell surface.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Expression of T7Hub increases steady-state levels of cotransfected Tac chimeras bearing tyrosine- and dileucine-based sorting motifs and causes their cell surface accumulation. HeLa cells were transfected with control vector without the hub insert (V) or T7Hub (H). Each population of cells was cotransfected with vector encoding the Tac chimera with the tyrosine-based sorting signal of H2M (Y) or vector encoding the Tac chimera with the dileucine-based sorting motif of CD3 (LL). 48 h after transfection, cells were surface biotinylated and lysates were prepared. Surface molecules were removed from the lysate with avidin–agarose and then internal molecules were left in the unbound fraction. The presence of Tac chimeras in each fraction of the lysate was established by immunoblotting with anti-Tac antiserum after SDS-PAGE. For each sample, 100% of the surface fraction and 25% of the internal fraction were analyzed.

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Expression of T7Hub results in cellular redistribution of cotransfected Tac chimeras bearing tyrosine- and dileucine-based sorting motifs. HeLa cells were transfected with (A and D) control vector (–hub) or (B, C, E, and F) T7Hub (+hub) together with (A–C) vector encoding the Tac chimera with a tyrosine-based sorting signal (Y motif) or (D–F) vector encoding the Tac chimera with a dileucine-based sorting signal (LL motif). 48 h after transfection, cells were processed for immunofluorescence and stained with (A, B, D, and E) anti-Tac antiserum followed by FITC-conjugated goat anti–rabbit IgG and (C and F) anti-T7 mAb followed by LRSC-conjugated goat anti–mouse IgG.

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Hub expression results in surface accumulation of coexpressed HLA–DM molecules. HeLa cells were transfected with either (A) control vector (no hub) or (B and C) T7Hub and (A–C) cotransfected with vectors encoding the HLA–DM /β dimer (pCDM8DM and pCDM8DMβ). (A and B) Surface expression of DM was analyzed by incubating nonpermeabilized, live cells with anti-DM antiserum for 30 min on ice. Cells were then fixed and processed for immunofluorescent microscopy using FITC-conjugated goat anti–rabbit IgG to detect surface DM. (C) Cells in B were labeled with anti-T7 mAb followed by LRSC-conjugated goat anti–mouse IgG to identify cells with hubs.

View larger version (80K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Direct delivery of HLA–DM molecules from the TGN to lysosome-like compartments is inhibited by hubs. HeLa cells were transfected with (A–C) control vector or (D–F) T7Hub. (A, B, D, and E) were cotransfected with vectors encoding the HLA–DM /β dimer driven by the inducible human metallothionein promoter (pMT302DM and pMT302DMβ). C and F were cotransfected with a vector encoding the DMβ chain without the YTPL internalization motif (pCDM8.1.DMβ-HSS) and the inducible DM chain (pMT302DM). 24 h after transfection, expression of DM molecules was induced by exposure to 10 µM CdCl2 for 20 h. Cells were fixed, permeabilized, and then processed for intracellular immunofluorescence. Colocalization of DM relative to the lysosomal marker CD63 was analyzed by double-staining cells with (A and D) anti-DM antiserum followed by FITC-conjugated goat anti–rabbit IgG and (B and E) anti-CD63 mAb followed by LRSC-conjugated goat anti–mouse IgG. Note that in A and D, DM is detected only in transfected cells that are cotransfected with control vector or hub, whereas CD63 (B and E) is detected in all cells. The surface staining of DM in comparable samples was barely detectable for both control and hub-transfected cells (data not shown). In C and F the surface expression of the DMβ chain without the YTPL internalization motif occured when expression of cotransfected DM was induced and was detected in control or hub-transfected cells by labeling with anti-DM antiserum followed by FITC-conjugated goat anti– rabbit IgG.

CCVs Mediate Endocytosis but Not the Intracellular Targeting of MHC Class II DR–I Chain Complex
The dileucine signal of the cytoplasmic tail of the I chain is responsible for the targeting of the I chain complexed with class II MHC molecules to the endocytic pathway (Pieters et al., 1993; Odorizzi et al., 1994). To determine whether CCVs play a role in this targeting and to establish what steps depend on their participation, I chain–class II MHC complexes were coexpressed with hubs. For this study, a plasmid encoding the Ip33 form of the I chain, which is the most abundant form responsible for class II molecule targeting, was chosen for transfection along with plasmids encoding the and β chain of HLA–DR, a human class II MHC molecule. In initial experiments all these cDNAs were under transcriptional control of the constitutive CMV promoter, and then cotransfected into HeLa cells, with or without cotransfection of T7Hub. First, the surface expression of these molecules was examined by immunofluorescent microscopy. I chain, coexpressed with HLA– DR, was not detectable on the surface of control cells, (Fig. 9 A) but was significantly increased on the surface of cells cotransfected with T7Hub (Fig. 9, B and C). This observation demonstrates that surface I chain–DR is normally endocytosed through CCVs and is consistent with the conclusions of a recent study of the effects of a dynamin mutant on I chain–DR distribution (Wang et al., 1997), a finding that we have confirmed independently by transfection with mutant dynamin (Marks, M., unpublished results). Hub-transfected cells were also analyzed by intracellular immunofluorescent microscopy for the distribution of I chain–DR complexes (data not shown), revealing a normal distribution in the endocytic pathway. This normal localization could be a function of targeting during hub accumulation or could be due to the fact that I chain transport to the endocytic pathway may not depend on CCVs.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 9 Hub expression results in the surface accumulation of coexpressed I chain. HeLa cells were transfected with (A) control vector (no hub) or (B and C) T7Hub and (A, B, and C) cotransfected with vectors encoding I chain and HLA–DR and β chains (pCDM8Ip33, pCDM8DR, pCDM8DRβ). (A and B) Surface expression of I chain was analyzed by incubating nonpermeabilized, live cells with anti-I chain antiserum KESL for 30 min on ice. Cells were then fixed and processed for immunofluorescent microscopy using FITC-conjugated goat anti–rabbit IgG to detect surface I chain. (C) Cells in B were labeled with anti-T7 mAb followed by LRSC-conjugated goat anti–mouse IgG to identify cells with hubs.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 10 Direct delivery of I chain (coexpressed with DR) from the TGN to the endocytic pathway is not affected by expression of hubs. HeLa cells were transfected with (A) control vector (no hub) or (B and C) T7Hub and (A–C) cotransfected with vectors encoding HLA-DR and β chains (pCDM8DR and pCDM8DRβ) and the I chain driven by human metallothionein promoter (pMT302Ip33). 24 h after transfection, I chain expression was induced by exposure to 10 µM CdCl2 for 20 h. Cells were fixed, permeabilized, and then processed for immunofluorescence. The cells were stained with (A and B) anti-I chain mAb PIN.1.1, followed by FITC-conjugated goat anti–mouse IgG1– specific secondary antibody and then (C) biotinylated anti-T7 mAb followed by TRITC-conjugated streptavidin.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Previous studies from the laboratory established that the hub fragment, formed by the carboxy-terminal third of the clathrin heavy chain, trimerizes and folds to mimic the central portion of the clathrin triskelion. Here, we show that the hub fragment expressed in mammalian cells binds endogenous clathrin LCs. Accompanying this behavior is a hyperassembly of LC-free endogenous triskelions onto cellular membranes. This can be explained by the fact that LCs prevent clathrin assembly at physiological pH in the absence of adaptors (Liu et al., 1995), so triskelions without LCs could be adding onto existing clathrin lattices, without the need for adaptors. Indeed, although we observe increased membrane recruitment of endogenous clathrin heavy chain in hub-transfected cells, we see no change in the distribution of either AP1 or AP2 adaptors on cellular membranes. Therefore, we hypothesize that hubs inhibit clathrin function primarily by LC depletion and thus induce hyperassembly at the edge of existing clathrin-coated pits. Hubs themselves may also be incorporated into existing clathrin lattices, as we can detect some membrane-associated hub molecules. The outcome of this inhibition mechanism is that clathrin-coated pits are frozen at the PM and TGN, trapping associated adaptors and receptors. This results in a phenotype in which CCV-mediated import or export of receptors, which are not overexpressed, is blocked, rather than the missorting of the phenotype observed in clathrin-deficient yeast mutants (Seeger and Payne, 1992). Characteristic of this phenotype is the patchy accumulation of molecules on the cell surface when endocytosis is blocked as seen in Figs. 7 and 9, and the apparent backup of molecules in the secretory pathway when TGN sorting is blocked by hubs, as seen in Fig. 3 C for the CI-M6PR and in Fig. 8 D for HLA–DM.

It has not yet been possible to establish permanent transfectants with the hub construct under an inducible promoter, likely due to a long-term toxic effect of even low levels of hub expression from leaky promoters. Analysis of the distribution of existing pools of molecules after hub transfection showed varying degrees of effects on steady-state distribution. For example, when the distribution of TfnR was analyzed in hub-expressing cells, we observed a variable, but notable, reduction of TfnR on the cell surface (data not shown). This correlates with previous observations that the bulk of cellular transferrin receptor is endosomal (Futter et al., 1995). It also suggests that hubs are probably inhibiting clathrin-dependent recycling (Stoorvogel et al., 1996), as well as clathrin-dependent endocytosis. The distribution of endogenous CI-M6PR was also affected to varying degrees in different cells, after 24 h of hub expression.

For a more definitive application of hub expression to studying the role of clathrin in the sorting of particular molecules, we took the approach of cotransfecting cells with novel molecules to be analyzed, so that these molecules were expressed either during hub accumulation or, when the test molecule was under the control of an inducible promoter, it was expressed after hub accumulation. This approach made it possible to analyze clearly the role of clathrin in the intracellular targeting of test molecules. Cotransfection of hubs with chimeric molecules bearing cytoplasmic domains with predicted CCV recognition signals attached to the Tac extracellular domain of the IL-2 receptor -chain, revealed that hub expression blocked the internalization of the chimeric molecules with either the tyrosine-containing signal of the H2M β chain or the dileucine signal of the CD3 chain. This is, in fact, the first functional demonstration of a role for clathrin in internalizing molecules with this particular YXXØ-tyrosine signal. It is also the first confirmation of the role of CCVs in uptake of molecules with the dileucine signal, which has been implicated in endocytosis. In sum, these experiments characterizing the clathrin hub expression system revealed its utility in studying the role of clathrin-dependent processes in mammalian cells.

The Role of CCVs in the Class II MHC–Molecule Antigen Presentation Pathway
Class II MHC molecules acquire peptides for presentation to helper T cells when they intersect the endocytic pathway after biosynthesis. Targeting of class II molecules to the endocytic pathway and their peptide loading depends on two important accessory molecules that have predicted CCV association signals in their cytoplasmic domains, the I chain, and HLA–DM. By analyzing the effects of hub expression on the endocytic delivery of these molecules, we can establish where CCVs are involved in sorting molecules with dileucine- or tyrosine-based CCV association signals, and where such sorting is critical for antigen presentation.

A dileucine signal in the I chain is responsible for targeting associated class II molecules to the endocytic pathway, where the I chain is degraded and its residual CLIP peptide, which occupies the class II molecule's peptide binding site, is replaced by an antigenic peptide. How the I chain–class II complex is targeted to the endocytic pathway and whether it is first transported to the PM and then internalized has not yet been established. It is clear that a substantial proportion of newly synthesized class II–I chain complexes appears on the cell surface before their processing in the endocytic pathway (Roche et al., 1993; Odorizzi et al., 1994; Wang et al., 1997) and morphological evidence indicates intact I chain can be detected in early, as well as late, endocytic compartments (Guagliardi et al., 1990; Castellino and Germain, 1995). Clathrin does not colocalize with I chain–class II complexes in the TGN (Glickman et al., 1996) but overexpression of I chain–class II complexes results in an increase in AP1 binding sites on internal cellular membranes (Salamero et al., 1996). Taken together, these results suggest that clathrin may be involved in uptake and targeting of the I chain–class II complex and could also play a role at some stage in its intracellular sorting.

A tyrosine-based signal in the β chains of HLA–DM or the mouse equivalent H2Mb (Lindstedt et al., 1995; Marks et al., 1995; Ohno et al., 1995; Copier et al., 1996) is required for targeting these molecules to lysosome-like endocytic compartments where they interact with class II molecules (Amigorena et al., 1994; Tulp et al., 1994; West et al., 1994). DM serves as a catalyst to antigen presentation by stabilizing the empty form of class II molecules (Denzin et al., 1996; Kropshofer et al., 1997), thereby promoting the exchange of the CLIP fragment of I chain for peptides derived from exogenous internalized proteins (Denzin and Cresswell, 1995; Sherman et al., 1995; Sloan et al., 1995; Weber et al., 1996). Yeast two-hybrid interaction studies suggest a possible role for CCVs in sorting DM to the endocytic pathway at both the PM and the TGN. However, under steady-state conditions, DM cannot be detected on the surface of cells in which it is expressed (Sanderson et al., 1994).

The distribution of both I chain–class II and DM complexes is altered by hub expression, making it possible to define the steps where CCVs play a role in targeting these molecules to the endocytic pathway and the routes by which they are transported (Fig. 11). Expression of I chain–class II complexes or HLA–DM heterodimers in the presence of clathrin hubs resulted in accumulation of both complexes on the cell surface. In the absence of hubs, both complexes were undetectable on the cell surface, confirming that they are preferentially targeted to the endocytic pathway, and that their targeting mechanism was not saturated at these levels of expression. These results indicate that at some point in their transport, both I chain–class II complexes and HLA–DM access the cell surface and are internalized in CCVs. When HLA–DM or I chain–class II complexes were expressed from an inducible promoter in cells that had already accumulated hubs, HLA–DM molecules were trapped in the secretory pathway and never reached endocytic compartments, whereas endocytic targeting of the I chain–class II complexes was unaffected by hubs. Thus, CCVs are required for DM sorting in the TGN but sorting of I chain–class II complexes is clathrin independent. In addition, when HLA–DM was expressed after hub accumulation, none appeared on the cell surface and when I chain–class II complexes were expressed under the same conditions, only low levels were detectable on the cell surface. This implies both complexes are primarily targeted to the endocytic pathway before surface expression, otherwise they would have accumulated on the cell surface, since hubs block their endocytosis.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 11 The role of clathrin and routes of transport in targeting I chain–class II MHC complexes and HLA–DM to the endocytic pathway. Analysis of DM and I chain–class II targeting, in the presence of hub molecules that inhibit CCV–mediated transport, suggests that both DM and I chain–DR are directly transported to the endocytic pathway from the TGN, without initial routing via the PM. In the TGN, DM is sorted in CCV and I chain–DR by a clathrin-independent mechanism. Our results also indicate that both complexes can subsequently appear on the cell surface and are then internalized by CCV, hence we propose they can follow routes 2 and 3, as shown. Solid arrows indicate pathways determined by the data reported, with clathrin-dependence shown in black and clathrin-independent pathways depicted in gray. Dashed arrows indicate speculated pathways where the role of clathrin is unknown. The detailed rationale behind this transport scheme is presented in the Discussion.

	Acknowledgments

 
We thank members of the Brodsky lab for helpful comments and our colleagues, mentioned in the Materials and Methods, for antibodies and DNA constructs.

This work was supported by National Institutes of Health grants (GM38093, AI39152, and GM57657) to F.M. Brodsky, and American Cancer Society grants (ACS RPG-97-003-01-BE and IRG-135) to M.S. Marks.

Submitted: 11 November 1997

Revised: 6 January 1998

Address all correspondence to Frances M. Brodsky, The G.W. Hooper Foundation, Department of Microbiology and Immunology and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0552. Tel.: (415) 476-6406. Fax: (415) 476-6185. E-mail: fmarbro{at}itsa.ucsf.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Acton S & Brodsky FM. Predominance of clathrin light-chain LC_bcorrelates with the presence of a regulated secretory pathway, J Cell Biol, 1990, 111, 1419–1426.[Abstract/Free Full Text]

Ahle S, Mann A, Eichelsbacher U & Ungewickell E. Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane, EMBO (Eur Mol Biol Organ) J, 1988, 7, 919–929.[Medline]

Allan VJ & Kreis TE. A microtubule-binding protein associated with membranes of the Golgi apparatus, J Cell Biol, 1986, 103, 2229–2239.[Abstract/Free Full Text]

Amigorena S, Drake JR, Webster P & Mellman I. Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B lymphocytes, Nature, 1994, 369, 113–120.[Medline]

Bakke O & Dobberstein B. MHC class II-associated invariant chain contains a sorting signal for endosomal compartments, Cell, 1990, 63, 707–716.[Medline]

Barnstable CJ, Bodmer WF, Brown G, Galfré G, Milstein C, Williams AF & Ziegler A. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens—new tools for genetic analysis, Cell, 1978, 14, 9–20.[Medline]

Bazinet C, Katzen AL, Morgan M, Mahowald AP & Lemmon SK. The Drosophilaclathrin heavy chain gene: clathrin function is essential in a multicellular organism, Genetics, 1993, 134, 1119–1134.[Abstract]

Blank GS & Brodsky FM. Site-specific disruption of clathrin assembly produces novel structures, EMBO (Eur Mol Biol Organ) J, 1986, 5, 2087–2095.[Medline]

Bonnerot C, Lankar D, Hanau D, Spehner D, Davoust J, Salamero J & Fridman WH. Role of B cell receptor Ig and Igβ subunits in MHC class II-restricted antigen presentation, Immunity, 1995, 3, 335–347.[Medline]

Brodsky FM. Living with clathrin: its role in intracellular membrane traffic, Science, 1988, 242, 1396–1402.[Abstract/Free Full Text]

Castellino F & Germain RN. Extensive trafficking of MHC class II-invariant chain complexes in the endocytic pathway and appearance of peptide-loaded class II in multiple compartments, Immunity, 1995, 2, 73–88.[Medline]

Chin DJ, Straubinger RM, Acton S, Näthke I & Brodsky FM. 100-kDa polypeptides in peripheral clathrin-coated vesicles are required for receptor-mediated endocytosis, Proc Natl Acad Sci USA, 1989, 86, 9289–9293.[Abstract/Free Full Text]

Copier J, Kleijmeer MJ, Ponnambalam S, Oorschot V, Potter P, Trowsdale J & Kelly A. Targeting signal and subcellular compartments involved in the intracellular trafficking of HLA–DMB, J Immunol, 1996, 157, 1017–1027.[Abstract]

Cowles CR, Odorizzi G, Payne GS & Emr SD. The AP-3 adaptor complex is essential for cargo-selective transport to the yeast vacuole, Cell, 1997, 91, 109–118.[Medline]

Damke H, Baba T, van der Bliek AM & Schmid SL. Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin, J Cell Biol, 1995, 131, 69–80.[Abstract/Free Full Text]

Dell'Angelica EC, Ohno H, Ooi CE, Rabinovich E, Roche KW & Bonifacino JS. AP-3: an adaptor-like protein complex with ubiquitous expression, EMBO (Eur Mol Biol Organ) J, 1997a, 16, 917–928.[Medline]

Dell'Angelica EC, Ooi CE & Bonifacino JS. β3A-adaptin: a subunit of the adaptor-like complex AP-3, J Biol Chem, 1997b, 272, 15078–15084.[Abstract/Free Full Text]

Denzin LK & Cresswell P. HLA–DM induces CLIP dissociation from MHC class II β dimers and facilitates peptide loading, Cell, 1995, 82, 155–165.[Medline]

Denzin LK, Hammond C & Cresswell P. HLA–DM interactions with intermediates in HLA-DR maturation and a role for HLA–DM in stabilizing empty HLA-DR molecules, J Exp Med, 1996, 184, 2153–2165.[Abstract/Free Full Text]

Dietrich J, Kastrup J, Nielsen BL, Ødum N & Geisler C. Regulation and function of the CD3 DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro, J Cell Biol, 1997, 138, 271–281.[Abstract/Free Full Text]

Dittié AS, Hajibagheri N & Tooze SA. The AP-1 adaptor complex binds to immature secretory granules from PC12 cells, and is regulated by ADP-ribosylation factor, J Cell Biol, 1996, 132, 523–536.[Abstract/Free Full Text]

Doxsey SJ, Brodsky FM, Blank GS & Helenius A. Inhibition of endocytosis by anti-clathrin antibodies, Cell, 1987, 50, 453–463.[Medline]

Futter CE, Connolly CN, Cutler DF & Hopkins CR. Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface, J Biol Chem, 1995, 270, 10999–11003.[Abstract/Free Full Text]

Glickman JN, Morton PA, Slot JW, Kornfeld S & Geuze HJ. The biogenesis of the MHC class II compartment in human I–cell disease B lymphoblasts, J Cell Biol, 1996, 132, 769–785.[Abstract/Free Full Text]

Guagliardi LE, Koppelman B, Blum JS, Marks MS, Cresswell P & Brodsky FM. Co-localization of molecules involved in antigen processing in an early endocytic compartment, Nature, 1990, 343, 133–139.[Medline]

Heilker R, Manning-Krieg U, Zuber J-F & Spiess M. In vitro binding of clathrin adaptors to sorting signals correlates with endocytosis and basolateral sorting, EMBO (Eur Mol Biol Organ) J, 1996, 15, 2893–2899.[Medline]

Herskovits JS, Burgess CC, Obar RA & Vallee RB. Effects of mutant rat dynamin on endocytosis, J Cell Biol, 1993, 122, 565–578.[Abstract/Free Full Text]

Johnson KF & Kornfeld S. The cytoplasmic tail of the mannose 6–phosphate/insulin-like growth factor II receptor has two signals for lysosomal enzyme sorting in the Golgi, J Cell Biol, 1992, 119, 249–257.[Abstract/Free Full Text]

Karlsson L, Péléraux A, Lindstedt R, Liljedahl M & Peterson PA. Reconstitution of an operational MHC class II compartment in nonantigen-presenting cells, Science, 1994, 266, 1569–1573.[Abstract/Free Full Text]

Keen JH. Clathrin and associated assembly and disassembly proteins, Annu Rev Biochem, 1990, 59, 415–438.[Medline]

Kelly AP, Monaco JJ, Cho S & Trowsdale J. A new human HLA class II-related locus, DM. , Nature, 1991, 353, 571–573.[Medline]

Kornfeld S. Structure and function of the mannose-6-phosphate/insulin-like growth factor II receptors, Annu Rev Biochem, 1992, 61, 307–330.[Medline]

Kropshofer H, Arndt SO, Moldenhauer G, Hammerling GJ & Vogt AB. HLA–DM acts as a molecular chaperone and rescues empty HLA-DR molecules at lysosomal pH, Immunity, 1997, 6, 293–302.[Medline]

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, 227, 680–685.[Medline]

Lemmon SK, Pellicena-Palle A, Conley K & Freund CL. Sequence of the clathrin heavy chain from Saccharomyces cerevisiaeand requirement of the COOH terminus for clathrin function, J Cell Biol, 1991, 112, 65–80.[Abstract/Free Full Text]

Letourneur F & Klausner RD. A novel di-leucine motif and a tyrosine based motif independently mediate lysosomal targeting and endocytosis of CD3 chains, Cell, 1992, 69, 1143–1157.[Medline]

Lindstedt R, Liljedahl M, Péléraux A, Peterson PA & Karlsson L. The MHC class II molecule H2-M is targeted to an endosomal compartment by a tyrosine-based targeting motif, Immunity, 1995, 3, 561–572.[Medline]

Liu S-H, Wong ML, Craik CS & Brodsky FM. Regulation of clathrin assembly and trimerization defined using recombinant triskelion hubs, Cell, 1995, 83, 257–267.[Medline]

Long E, LaVaute T, Pinet V & Jaraquemada D. Invariant chain prevents the HLA-DR restricted presentation of a cytosolic peptide, J Immunol, 1994, 153, 1487–1494.[Abstract]

Lotteau V, Teyton L, Peleraux A, Nilsson T, Karlsson L, Schmid SL, Quaranta V & Peterson PA. Intracellular transport of class II MHC molecules directed by invariant chain, Nature, 1990, 348, 600–605.[Medline]

Louvard D, Reggio H & Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum, J Cell Biol, 1982, 92, 92–107.[Abstract/Free Full Text]

Makarov SS, Jonat C & Haskill S. Hyperinducible human metallothionein promoter with a low level basal activity, Nucleic Acids Res, 1994, 22, 1504–1505.[Free Full Text]

Marks MS, Roche PA, van Donselaar E, Woodruff L, Peters PJ & Bonifacino JS. A lysosomal targeting signal in the cytoplasmic tail of the β chain directs HLA–DM to MHC class II compartments, J Cell Biol, 1995, 131, 351–369.[Abstract/Free Full Text]

Marks MS, Woodruff L, Ohno H & Bonifacino JS. Protein targeting by tyrosine and dileucine-based signals: evidence for distinct saturable components, J Cell Biol, 1996, 135, 341–354.[Abstract/Free Full Text]

Marks MS, Ohno H, Kirchhausen T & Bonifacino JS. Protein sorting by tyrosine based signals: adapting to the the Ys and wherefores, Trends Cell Biol, 1997, 7, 124–128.[Medline]

Niswonger ML & O'Halloran TJ. A novel role of clathrin in cytokinesis, Proc Natl Acad Sci USA, 1997, 94, 8575–8578.[Abstract/Free Full Text]

Odorizzi CG, Trowbridge IS, Xue L, Hopkins CR, Davis CD & Collawn JF. Sorting signals in the MHC class II invariant chain cytoplasmic tail and transmembrane region determine trafficking to an endocytic processing compartment, J Cell Biol, 1994, 126, 317–330.[Abstract/Free Full Text]

Ohno H, Stewart J, Fournier M-C, Bosshart H, Rhee I, Miyatake S, Saito T, Galluser A, Kirchhausen T & Bonifacino JS. Interaction of tyrosine-based sorting signals with clathrin-associated proteins, Science, 1995, 269, 1872–1875.[Abstract/Free Full Text]

Ohno H, Fournier M-C, Poy G & Bonifacino JS. Structural determinants of interaction of tyrosine-based sorting signals with the adaptor medium chains, J Biol Chem, 1996, 271, 29009–29015.[Abstract/Free Full Text]

Pearse BMF & Robinson MS. Clathrin, adaptors and sorting, Annu Rev Cell Biol, 1990, 6, 151–171.[Medline]

Pieters J, Bakke O & Dobberstein B. The MHC class II-associated invariant chain contains two endosomal targeting signals within its cytoplasmic tail, J Cell Sci, 1993, 106, 831–846.[Abstract]

Pley U & Parham P. Clathrin: its role in receptor-mediated vesicular transport and specialized functions in neurons, Crit Rev Biochem Molec Biol, 1993, 28, 431–464.[Medline]

Rapoport I, Miyazaki M, Boll W, Duckworth B, Cantley LC, Shoelson S & Kirchhausen T. Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes, EMBO (Eur Mol Biol Organ) J, 1997, 16, 2240–2250.[Medline]

Roche PA, Marks MS & Cresswell P. Formation of a nine-subunit complex by HLA class II glycoproteins and the invariant chain, Nature, 1991, 354, 392–394.[Medline]

Roche PA, Teletski CL, Karp DR, Pinet V, Bakke O & Long EO. Stable surface expression of invariant chain prevents peptide presentation by HLA-DR, EMBO (Eur Mol Biol Organ) J, 1992, 11, 2841–2847.[Medline]

Roche PA, Teletski CL, Stang E, Bakke O & Long EO. Cell surface HLA-DR-invariant chain complexes are targeted to endosomes by rapid internalization, Proc Natl Acad Sci USA, 1993, 90, 8581–8585.[Abstract/Free Full Text]

Salamero J, Le Borgne R, Saudrais C, Goud B & Hoflack B. Expression of major histocompatibility complex class II molecules in HeLa cells promotes the recruitment of AP-1 Golgi-specific assembly proteins on Golgi membranes, J Biol Chem, 1996, 271, 30318–30321.[Abstract/Free Full Text]

Sanderson F, Kleijmeer MJ, Kelly A, Verwoerd D, Tulp A, Neefjes JJ, Geuze HJ & Trowsdale J. Accumulation of HLA–DM, a regulator of antigen presentation, in MHC class II compartments, Science, 1994, 266, 1566–1569.[Abstract/Free Full Text]

Schmid SL. Clathrin-coated vesicle formation and protein sorting: an integrated process, Annu Rev Biochem, 1997, 66, 511–548.[Medline]

Seeger M & Payne GS. Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae. , J Cell Biol, 1992, 118, 531–540.[Abstract/Free Full Text]

Sherman MA, Weber DA & Jensen PE. DM enhances peptide binding to class II MHC by release of invariant chain-derived peptide, Immunity, 1995, 3, 197–205.[Medline]

Silveira LA, Wong DH, Masiarz FR & Schekman R. Yeast clathrin has a distinctive light chain that is important for cell growth, J Cell Biol, 1990, 111, 1437–1449.[Abstract/Free Full Text]

Simpson F, Peden AA, Christopoulou L & Robinson MS. Characterization of the adaptor-related protein complex, AP3, J Cell Biol, 1997, 137, 835–845.[Abstract/Free Full Text]

Sloan VS, Cameron P, Porter G, Gammon M, Amaya M, Mellins E & Zaller DM. Mediation by HLA–DM of dissociation of peptides from HLA-DR, Nature, 1995, 375, 802–806.[Medline]

Stoorvogel W, Oorschot V & Geuze HJ. A novel class of clathrin-coated vesicles budding from endosomes, J Cell Biol, 1996, 132, 21–33.[Abstract/Free Full Text]

Tooze J & Tooze SA. Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells, J Cell Biol, 1986, 103, 839–850.[Abstract/Free Full Text]

Towbin H, Staehelin T & Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications, Proc Natl Acad Sci USA, 1979, 76, 4350–4354.[Abstract/Free Full Text]

Tulp A, Verwoerd D, Dobberstein B, Ploegh HL & Pieters J. Isolation and characterization of the intracellular MHC class II compartment, Nature, 1994, 369, 120–126.[Medline]

Ungewickell E & Ungewickell H. Bovine brain clathrin light chains impede heavy chain assembly in vitro, J Biol Chem, 1991, 266, 12710–12714.[Abstract/Free Full Text]

van der Bliek AM, Redelmeier TE, Damke H, Tisdale EJ, Meyerowitz EM & Schmid SL. Mutations in human dynamin block an intermediate stage in coated vesicle formation, J Cell Biol, 1993, 122, 553–563.[Abstract/Free Full Text]

Wang K, Peterson PA & Karlsson L. Decreased endosomal delivery of major histocompatibility complex class II-invariant chain complexes in dynamin-deficient cells, J Biol Chem, 1997, 272, 17055–17060.[Abstract/Free Full Text]

Weber DA, Evavold BD & Jensen PE. Enhanced dissociation of HLA-DR-bound peptides in the presence of HLA–DM, Science, 1996, 274, 618–620.[Abstract/Free Full Text]

West MA, Lucocq JM & Watts C. Antigen processing and class II MHC peptide-loading compartments in human B-lymphoblastoid cells, Nature, 1994, 369, 147–151.[Medline]

Ybe JA, Greene B, Liu S-H, Pley U, Parham P & Brodsky FM. Clathrin self-assembly is regulated by three light-chain residues controlling the formation of critical salt bridges, EMBO (Eur Mol Biol Organ) J, 1998, 17, 1297–1303.[Medline]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Facebook

Reddit

Technorati

Twitter

This article has been cited by other articles:

• Hood, F. E., Royle, S. J. (2009). Functional equivalence of the clathrin heavy chains CHC17 and CHC22 in endocytosis and mitosis. J. Cell Sci. 122: 2185-2190 [Abstract] [Full Text]  
• Kasprowicz, J., Kuenen, S., Miskiewicz, K., Habets, R. L.P., Smitz, L., Verstreken, P. (2008). Inactivation of clathrin heavy chain inhibits synaptic recycling but allows bulk membrane uptake. JCB 182: 1007-1016 [Abstract] [Full Text]  
• Tomlinson, C. C., Damania, B. (2008). Critical Role for Endocytosis in the Regulation of Signaling by the Kaposi's Sarcoma-Associated Herpesvirus K1 Protein. J. Virol. 82: 6514-6523 [Abstract] [Full Text]  
• Radulescu, A. E., Siddhanta, A., Shields, D. (2007). A Role for Clathrin in Reassembly of the Golgi Apparatus. Mol. Biol. Cell 18: 94-105 [Abstract] [Full Text]  
• Wang, Y., Li, D., Fan, H., Tian, L., Zhong, Y., Zhang, Y., Yuan, L., Jin, C., Yin, C., Ma, D. (2006). Cellular Uptake of Exogenous Human PDCD5 Protein. J. Biol. Chem. 281: 24803-24817 [Abstract] [Full Text]  
• Hyman, T., Shmuel, M., Altschuler, Y. (2006). Actin Is Required for Endocytosis at the Apical Surface of Madin-Darby Canine Kidney Cells where ARF6 and Clathrin Regulate the Actin Cytoskeleton. Mol. Biol. Cell 17: 427-437 [Abstract] [Full Text]  
• Cabezas, A., Bache, K. G., Brech, A., Stenmark, H. (2005). Alix regulates cortical actin and the spatial distribution of endosomes. J. Cell Sci. 118: 2625-2635 [Abstract] [Full Text]  
• McCormick, P. J., Martina, J. A., Bonifacino, J. S. (2005). Involvement of clathrin and AP-2 in the trafficking of MHC class II molecules to antigen-processing compartments. Proc. Natl. Acad. Sci. USA 102: 7910-7915 [Abstract] [Full Text]  
• Dugast, M., Toussaint, H., Dousset, C., Benaroch, P. (2005). AP2 Clathrin Adaptor Complex, but Not AP1, Controls the Access of the Major Histocompatibility Complex (MHC) Class II to Endosomes. J. Biol. Chem. 280: 19656-19664 [Abstract] [Full Text]  
• Karacsonyi, C., Knorr, R., Fulbier, A., Lindner, R. (2004). Association of Major Histocompatibility Complex II with Cholesterol- and Sphingolipid-rich Membranes Precedes Peptide Loading. J. Biol. Chem. 279: 34818-34826 [Abstract] [Full Text]  
• Moskowitz, H. S., Heuser, J., McGraw, T. E., Ryan, T. A. (2003). Targeted Chemical Disruption of Clathrin Function in Living Cells. Mol. Biol. Cell 14: 4437-4447 [Abstract] [Full Text]  
• Lu, J.-C., Scott, P., Strous, G. J., Schuler, L. A. (2002). Multiple Internalization Motifs Differentially Used by Prolactin Receptor Isoforms Mediate Similar Endocytic Pathways. Mol. Endocrinol. 16: 2515-2527 [Abstract] [Full Text]  
• Meier, O., Boucke, K., Hammer, S. V., Keller, S., Stidwill, R. P., Hemmi, S., Greber, U. F. (2002). Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake. JCB 158: 1119-1131 [Abstract] [Full Text]  
• Molinete, M., Dupuis, S., Brodsky, F. M., Halban, P. A. (2002). Role of clathrin in the regulated secretory pathway of pancreatic {beta}-cells. J. Cell Sci. 114: 3059-3066 [Abstract] [Full Text]  
• Hirst, J., Lindsay, M. R., Robinson, M. S. (2001). Golgi-localized, gamma-Ear-containing, ADP-Ribosylation Factor-binding Proteins: Roles of the Different Domains and Comparison with AP-1 and Clathrin. Mol. Biol. Cell 12: 3573-3588 [Abstract] [Full Text]  
• Stumptner-Cuvelette, P., Morchoisne, S., Dugast, M., Le Gall, S., Raposo, G., Schwartz, O., Benaroch, P. (2001). HIV-1 Nef impairs MHC class II antigen presentation and surface expression. Proc. Natl. Acad. Sci. USA 10.1073/pnas.221256498v1 [Abstract] [Full Text]  
• Bennett, E. M., Lin, S. X., Towler, M. C., Maxfield, F. R., Brodsky, F. M. (2001). Clathrin Hub Expression Affects Early Endosome Distribution with Minimal Impact on Receptor Sorting and Recycling. Mol. Biol. Cell 12: 2790-2799 [Abstract] [Full Text]  
• Pathak, S. S., Lich, J. D., Blum, J. S. (2001). Cutting Edge: Editing of Recycling Class II:Peptide Complexes by HLA-DM. J. Immunol. 167: 632-635 [Abstract] [Full Text]  
• Puertollano, R., Aguilar, R. C., Gorshkova, I., Crouch, R. J., Bonifacino, J. S. (2001). Sorting of Mannose 6-Phosphate Receptors Mediated by the GGAs. Science 292: 1712-1716 [Abstract] [Full Text]  
• Roseberry, A., Hosey, M. (2001). Internalization of the M2 muscarinic acetylcholine receptor proceeds through an atypical pathway in HEK293 cells that is independent of clathrin and caveolae. J. Cell Sci. 114: 739-746 [Abstract]  
• Zhao, X, Greener, T, Al-Hasani, H, Cushman, S., Eisenberg, E, Greene, L. (2001). Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin. J. Cell Sci. 114: 353-365 [Abstract]  
• Okamoto, C. T., Duman, J. G., Tyagarajan, K., McDonald, K. L., Jeng, Y. Y., McKinney, J., Forte, T. M., Forte, J. G. (2000). Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization. Am. J. Physiol. Cell Physiol. 279: C833-C851 [Abstract] [Full Text]  
• Denzer, K, Kleijmeer, M., Heijnen, H., Stoorvogel, W, Geuze, H. (2000). Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J. Cell Sci. 113: 3365-3374 [Abstract]  
• Ramm, G, Pond, L, Watts, C, Stoorvogel, W (2000). Clathrin-coated lattices and buds on MHC class II compartments do not selectively recruit mature MHC-II. J. Cell Sci. 113: 303-313 [Abstract]  
• Hofmann, M. W., Honing, S., Rodionov, D., Dobberstein, B., von Figura, K., Bakke, O. (1999). The Leucine-based Sorting Motifs in the Cytoplasmic Domain of the Invariant Chain Are Recognized by the Clathrin Adaptors AP1 and AP2 and their Medium Chains. J. Biol. Chem. 274: 36153-36158 [Abstract] [Full Text]  
• Altschuler, Y., Liu, S.-H., Katz, L., Tang, K., Hardy, S., Brodsky, F., Apodaca, G., Mostov, K. (1999). Adp-Ribosylation Factor 6 and Endocytosis at the Apical Surface of Madin-Darby Canine Kidney Cells. JCB 147: 7-12 [Abstract] [Full Text]  
• Davidson, H. W. (1999). Direct Transport of Newly Synthesized HLA-DR from the trans-Golgi Network to Major Histocompatibility Complex Class II Containing Compartments (MIICS) Demonstrated Using a Novel Tyrosine-sulfated Chimera. J. Biol. Chem. 274: 27315-27322 [Abstract] [Full Text]  
• Bresnahan, P. A., Yonemoto, W., Greene, W. C. (1999). Cutting Edge: SIV Nef Protein Utilizes Both Leucine- and Tyrosine-Based Protein Sorting Pathways for Down-Regulation of CD4. J. Immunol. 163: 2977-2981 [Abstract] [Full Text]  
• Brachet, V., Péhau-Arnaudet, G., Desaymard, C., Raposo, G., Amigorena, S. (1999). Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments. Mol. Biol. Cell 10: 2891-2904 [Abstract] [Full Text]  
• Vogler, O., Nolte, B., Voss, M., Schmidt, M., Jakobs, K. H., van Koppen, C. J. (1999). Regulation of Muscarinic Acetylcholine Receptor Sequestration and Function by beta -Arrestin. J. Biol. Chem. 274: 12333-12338 [Abstract] [Full Text]  
• Calvo, P. A., Frank, D. W., Bieler, B. M., Berson, J. F., Marks, M. S. (1999). A Cytoplasmic Sequence in Human Tyrosinase Defines a Second Class of Di-leucine-based Sorting Signals for Late Endosomal and Lysosomal Delivery. J. Biol. Chem. 274: 12780-12789 [Abstract] [Full Text]  
• Benmerah, A, Bayrou, M, Cerf-Bensussan, N, Dautry-Varsat, A (1999). Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. Cell Sci. 112: 1303-1311 [Abstract]  
• Samaan, A., Thibodeau, J., Mahana, W., Castellino, F., Cazenave, P. A., Kindt, T. J. (1999). Cellular distribution of a mixed MHC class II heterodimer between DR{alpha} and a chimeric DOß chain. Int Immunol 11: 99-111 [Abstract] [Full Text]  
• Altschuler, Y., Barbas, S. M., Terlecky, L. J., Tang, K., Hardy, S., Mostov, K. E., Schmid, S. L. (1998). Redundant and Distinct Functions for Dynamin-1 and Dynamin-2 Isoforms. JCB 143: 1871-1881 [Abstract] [Full Text]  
• Shi, G., Faundez, V., Roos, J., Dell'Angelica, E. C., Kelly, R. B. (1998). Neuroendocrine Synaptic Vesicles Are Formed In Vitro by Both Clathrin-dependent and Clathrin-independent Pathways. JCB 143: 947-955 [Abstract] [Full Text]  
• Trejo, J., Altschuler, Y., Fu, H.-W., Mostov, K. E., Coughlin, S. R. (2000). Protease-activated Receptor-1 Down-regulation. A MUTANT HeLa CELL LINE SUGGESTS NOVEL REQUIREMENTS FOR PAR1 PHOSPHORYLATION AND RECRUITMENT TO CLATHRIN-COATED PITS. J. Biol. Chem. 275: 31255-31265 [Abstract] [Full Text]  
• Carreno, S., Gouze, M.-E., Schaak, S., Emorine, L. J., Maridonneau-Parini, I. (2000). Lack of Palmitoylation Redirects p59Hck from the Plasma Membrane to p61Hck-positive Lysosomes. J. Biol. Chem. 275: 36223-36229 [Abstract] [Full Text]  
• Craven, S. E., Bredt, D. S. (2000). Synaptic Targeting of the Postsynaptic Density Protein PSD-95 Mediated by a Tyrosine-based Trafficking Signal. J. Biol. Chem. 275: 20045-20051 [Abstract] [Full Text]  
• Stumptner-Cuvelette, P., Morchoisne, S., Dugast, M., Le Gall, S., Raposo, G., Schwartz, O., Benaroch, P. (2001). HIV-1 Nef impairs MHC class II antigen presentation and surface expression. Proc. Natl. Acad. Sci. USA 98: 12144-12149 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF, 4256K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Liu, S.-H. Articles by Brodsky, F. M. Search for Related Content PubMed PubMed Citation Articles by Liu, S.-H. Articles by Brodsky, F. M. Social Bookmarking                            What's this?