Trypanosoma brucei rhodesiense: membrane glycoproteins localized primarily in endosomes and lysosomes of bloodstream forms

Bloodstream forms of Trypanosoma brucei rhodesiense take up macromolecules in endocytic vesicles that form in a large coated pit called the flagellar pocket. Glycoproteins that bind to ricin are concentrated in the flagellar pocket and in intracellular vesicles. We purified Triton X-100-soluble ricin-binding glycoproteins by lectin affinity chromatography and immunized mice to generate hybridomas. Monoclonal antibody produced by the CB1 hybridoma recognized heterodisperse trypanosome components migrating with M(r) 84-140 kDa in immunoblots. CB1 binding was specifically inhibited by lactose. The CB1-reactive material was purified by sequential affinity chromatography on ricin- and CB1-Sepharose. N-Glycosidase F, but not endoglycosidase H, digestion destroyed CB1-reactivity of purified material. This suggests that N-linked oligosaccharides contribute to the CB1 epitope. Glycosidase digestion of biosynthetically radiomethionine-labeled, affinity purified, CB1-reactive material yielded two radiolabeled polypeptides, p57 and p42. Thirteen methionyl peptides were resolved in one-dimensional peptide maps of V8 protease digests of p57; p42 had 10 methionyl peptides with mobilities indistinguishable from those of peptides of p57. This suggests that p57 and p42 are closely related. In cryoimmunoelectron microscopy studies CB1 specifically labeled the interior surface of tubular and vesicular membranes located between the nucleus and the flagellar pocket. These membranes were morphologically identical to structures that have been previously identified as trans Golgi, lysosomal, and endosomal elements. In double-labeling studies endocytosed serum albumen-gold complexes were found in the lumen of vesicles that had CB1-reactive material in their membranes. This provides direct evidence that vesicles containing high levels of CB1-reactive material are part of the lysosome/endosomal system. Some CB1-reactive material was also detected in the flagellar pocket by cryoimmunoelectron microscopy. Corrolated flow cytofluorimetry and immunofluorescence analysis showed that 85-96% of the total CB1-reactive material was intracellular and inaccessible to antibody in living cells. The 4-15% of the total CB1-reactive material accessible to antibody in living cells was localized in the flagellar pocket. Bloodstream forms of Trypanosoma brucei brucei, Trypanosoma brucei gambiense, and T.b. rhodesiense all expressed the CB1 epitope. However, expression of this epitope is developmentally regulated during the parasite life cycle, for no CB1-reactive material was detected in procyclic forms. The trypanosome proteins detected by CB1 show some similarities to vertebrate lysosomal and endosomal membrane proteins.     

Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713-3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.     

Fluid-phase marker transport in rat liver: free-flow electrophoresis separates distinct endosome subpopulations

Free-flow electrophoresis (FFE) was used to investigate the intracellular compartments involved in fluid-phase marker, fluoresceine isothiocyanate (FITC)-dextran, transport in the isolated perfused rat liver. One to 2 min after uptake at 37 degrees C, FITC-dextran was found in endosomes with the same electrophoretic mobility as early sorting endosomes labeled either by the hepatocyte-specific marker asialoorosomucoid (ASOR) or by transferrin that enters all liver cells. Labeling at low temperature (16 degrees C) blocked transport of ASOR and dextran in early endosomes. With increasing internalization time (3-13 min) at 37 degrees C, FITC-dextran-labeled compartments co-localized with late, ASOR-containing endosomes. Since localization of FITC-dextran in late transcytotic compartments was not observed upon FFE separation, it is concluded that the majority of internalized markers is directed to lysosomes. The FITC-label did not account for the predominant lysosomal targeting of the dextran, since [3H]dextran-labeled endosomes exhibited an identical FFE pattern. Taken together, these data indicate that the fluid-phase marker dextran is transported through intracellular compartments with identical characteristics as endosome subcompartments of the receptor-mediated lysosomal route.     

High-resolution density gradient electrophoresis of subcellular organelles and proteins under nondenaturing conditions

We have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high-resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis 1993, 14, 1011-1018). Isoforms of bovine beta-lactoglobulin, human apo-transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and proteins.     

Free-flow electrophoretic analysis of endosome subpopulations of rat hepatocytes

The separation of functional early and late endosomes from other cellular compartments by free-flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells. Here, using 125I-labeled anti-secretory component antibodies ([125I]SC Ab) and FITC-labeled asialoorosomucoid (FITC-ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16 degrees C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20 degrees C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37 degrees C, transcytotic endosomes carrying [125I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC-ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common (i.e. early endosomes) and two distinct endosome subpopulations (i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.     

Separation and characterization of humic acids and metal humates by electrophoretic methods

A modified buffer for the separation of humic acids (HA) by capillary zone electrophoresis (CZE) was studied. The addition of hydroxycarboxylic acids to borate buffer makes a more efficient separation of HA possible. Two sharp peaks are obtained. Preparative isolation of these two fractions can be reached by applying the conditions used in CZE to free-flow electrophoresis (FFE). The fractions obtained were characterized by repetition of the CZE procedure. Also metal complexes of HA were investigated by electrophoretic methods. In the presence of metal cations, HA form stable complexes of different electrophoretic mobility. These complexes can be separated by free-flow isotachophoresis (FF-ITP). The different fractions are analyzed by CZE, ultraviolet-visible and atomic absorption spectrometry. Significant changes in both the ITP-pattern of the FFE and the 'humic hump' in CZE demonstrate the influence of complexation on the macromolecules.     

Phosphorylation regulates the delivery of MHC class II invariant chain complexes to antigen processing compartments

Transport of newly synthesized MHC class II glycoproteins to endosomal Ag processing compartments is mediated by their association with the invariant chain (Ii). Targeting to these compartments is dependent upon recognition of leucine-based endo. somal/lysosomal targeting motifs in the Ii cytosolic domain. Ii, like many molecules that contain leucine-based endosomal targeting motifs, is phosphorylated in vivo. In this report we demonstrate that the cytosolic domain of the p35 Ii isoform is phosphorylated in class II Ii complexes isolated from human B lymphoblastoid cell lines or freshly obtained PBMC. Mutation of serine residue 6 or 8 prevents phosphorylation of Ii-p35 expressed in HeLa cells. Treatment of B lymphoblastoid cell lines with the serine/threonine kinase inhibitor staurosporine prevented Ii phosphorylation and significantly delayed trafficking of newly synthesized class II Ii complexes to endosomal Ag processing compartments. By contrast, staurosporine had no effect on the rate of transport of class I or class II glycoproteins through the Golgi apparatus and did not inhibit the delivery of the chimeric molecule Tac-DM, to endocytic compartments, suggesting that staurosporine does not nonspecifically inhibit protein transport to the endocytic pathway. These results demonstrate that phosphorylation regulates the efficient targeting of MHC class II Ii complexes to Ag processing compartments and strongly suggest that this effect is mediated by phosphorylation of the MHC class II-associated Ii chain.     

MHC class II transport from lysosomal compartments to the cell surface is determined by stable peptide binding, but not by the cytosolic domains of the alpha- and beta-chains

Inside APCs, MHC class II molecules associate with antigenic peptides before reaching the cell surface. This association takes place in compartments of the endocytic pathway, more related to endosomes or lysosomes depending on the cell type. Here, we compared MHC class II transport from endosomal vs lysosomal compartments to the plasma membrane. We show that transport of MHC class II molecules to the cell surface does not depend on the cytosolic domains of the alpha- and beta-chains. In contrast, the stability of the alphabeta-peptide complexes determined the efficiency of transport to the cell surface from lysosomal, but not from endosomal, compartments. In murine B lymphoma cells, SDS-unstable and -stable complexes were transported to the cell surface at almost similar rates, whereas after lysosomal relocalization or in a cell line in which MHC class II molecules normally accumulate in lysosomal compartments, stable complexes were preferentially addressed to the cell surface. Our results suggest that when peptide loading occurs in lysosomal compartments, selective retention and lysosomal degradation of unstable dimers result in the expression of highly stable MHC class II-peptide complexes at the APC surface.     

Characterization of a multicatalytic proteinase complex (20S proteasome) from Trypanosoma brucei brucei

African trypanosomes are tsetse-transmitted protozoan parasites that cause sleeping sickness in humans and 'Nagana' in animals. A high relative molecular mass multicatalytic proteinase complex (MCP) was purified and biochemically characterized from the cytosolic fraction of Trypanosoma brucei brucei. The isolation procedure consisted of fractionation of the lysate by high speed centrifugation, chromatography on Q-sepharose molecular sieve filtration on Sephacryl S-300, chromatography on HA-Ultrogel and glycerol density gradient centrifugation (10-40%). The final enzyme preparation yielded a single protein band corresponding to a relative molecular mass of 630 kDa on a non-denaturing polyacrylamide gel. The enzyme hydrolyses a wide range of peptide substrates characteristic of chymotrypsin-like, trypsin-like, peptidylglutamylpeptide-hydrolysing activities determined by fluorogenic peptides, Z-Gly-Gly-Leu-NHMec, Z-Arg-Arg-NHMec and Z-Leu-Leu-Glu-beta NA, respectively. The enzyme was found to have a wide variation in pH optimal activity profile, with optimum activity against Z-Gly-Gly-Leu-NHMec at 7.8, Z-Arg-Arg-NHMec at pH 10.5 and Z-Leu-Leu-Glu-beta NA at pH 8.0, showing that the different activities are distinct. The enzyme hydrolysed oxidized proteins. In addition, the chymotryptic and trypsin-like activities were susceptible to inhibition by peptide aldehyde inhibitors with variable inhibition effects. The study demonstrates the presence of a non-lysosomal proteasome pathway of intracellular protein degradation in the bloodstream form of T. b. brucei. Further, the ability of the enzyme to hydrolyse most oxidized proteins, and the high immunogenicity exhibited suggests a possible involvement of the enzyme in pathogenesis of the disease.     

High-resolution density gradient electrophoresis of proteins and subcellular organelles

Following a concept developed by Bier et al. (Electrophoresis 1993, 14, 1011-1018), binary mixtures of amphoteric buffers with low conductivity and a good buffering capacity permit rapid rate zonal separation of proteins on a density gradient electrophoresis apparatus (7 cm, x 2.2 cm). At pH 8.66 and 250 V, beta-lactoglobulin (Mr 36600) was separated into the A and B isoforms within 44 min; human transferrin (Mr 76000-81000) was separated into its sialylated glycoforms and carbonic anhydrase (Mr 30000) separated into its isoenzymes. From these results we arrive at the term high-performance density gradient electrophoresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicles, intermediate endosomes, late endosomes and lysomes became well-separated after 80 min at 10 mA using [125I]transferrin and horseradish peroxidase as reporter molecules in pulse-chase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short separation times (19 min at 10 mA) for the endosomal compartments. Concommittantly, endoplasmic reticulum and proteasomes were well resolved.     

Membrane transport in rat liver endocytic pathways: preparation, biochemical properties and functional roles of hepatic endosomes

The endocytic compartment has emerged as a major regulator of the uptake and processing of circulating ligands, and has been extensively studied during the last decade. In this work, the polypeptides of the three endosomal fractions: compartment of uncoupling receptors and ligands (CURL), multivesicular bodies (MVB) and receptor recycling compartment (RRC), isolated from livers of estradiol-treated rats, were analyzed by two-dimensional gel electrophoresis. Silver-stained gels revealed that although the three endosomal fractions shared a generally similar pattern of approximately 120 components, qualitative and quantitative differences between the three endocytic fractions could be demonstrated. The polypeptide composition of the bile was also studied and compared with ligands and proteins identified in the different endosomal fractions. One- and two-dimensional gel electrophoresis and Western blotting were used to investigate the protein composition of the three isolated endocytic fractions and 39 proteins were identified. The distribution of identified receptors, ligands and structural proteins among the three endosomal fractions was in agreement with their expected functionalities and with the different endocytic pathways in the hepatocyte.     

Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers

Intoxication of mammalian cells with the vacuolating toxin (VacA) released by Helicobacter pylori causes the formation of large acidic vacuoles containing the vacuolar ATPase proton pump and Rab7, a late endosome marker. Here, we describe a novel subcellular fractionation procedure, and we show that nanomolar concentrations of VacA induce a clear redistribution of lysosomal membrane glycoproteins among endocytic compartments. This redistribution is an early event in the process of cellular intoxication by VacA and precedes the formation of macroscopic vacuoles. The absence of the cation independent mannose 6-P receptor and the presence of Rab7 and of lysosomal membrane proteins in the newly formed compartment suggest that the vacuolating toxin induces the accumulation of a post-endosomal hybrid compartment presenting both late endosomal and lysosomal features.     

Intracellular pathway for the generation of functional MHC class II peptide complexes in immature human dendritic cells

Binding of antigenic peptides to MHC class II (MHC-II) molecules occurs in the endocytic pathway. From previous studies in B lymphocytes, it is believed that most but not all of the newly synthesized MHC-II molecules are directly targeted from the trans-Golgi network to endosomal compartments. By using pulse-chase metabolic labeling followed by cell surface biotinylation, we show here that in contrast to an EBV-transformed B cell line and human monocytes, the majority of newly synthesized MHC-II molecules (at least 55 +/- 13%) are first routed to the plasma membrane of dendritic cells derived from human monocytes. They reach the cell surface in association with the invariant chain (Ii), a polypeptide known to target MHC-II to the endosomal/lysosomal system. Following rapid internalization and degradation of Ii, these alphabeta Ii complexes are converted into alphabeta-peptide complexes as shown by their SDS stability. These SDS-stable dimers appear as soon as 15 to 30 min after internalization of the alphabeta Ii complexes. More than 80% of alphabeta dimers originating from internalized alphabeta Ii complexes are progressively delivered to the cell surface within the next 2 h. Depolymerization of microtubules, which delays the transport to late endosomal compartments, did not affect the kinetics of conversion of surface alphbeta Ii into SDS-stable and -unstable alphabeta dimers. Altogether, these data suggest that newly liberated class II alphabeta heterodimers may bind peptides in different compartments along the endocytic pathway in dendritic cells derived from human monocytes.     

A silicone centrifugation technique for the detection of low parasitaemias of salivarian trypanosomes

A method using silicone fluid of specific gravity 1.075 was employed to detect low numbers of salivarian trypanosomes in rats infected with T. brucei, T. gambiense, T. congolense or mouse-adapted T. vivax. This method compared favourably with other microsensitive techniques such as the miniature anion-exchange centrifugation and microhaematocrit buffy-coat microscopy methods. The silicone centrifugation technique is based on the density differences between the host's erythrocytes and the parasites. Under the conditions used, the red cells are pelleted by centrifugation through a layer of silicone fluid whereas the trypanosomes remain in the plasma supernatant.     

Late events in the intracellular sorting of major histocompatibility complex class II molecules are regulated by the 80-82 segment of the class II beta chain

The molecular mechanisms that regulate sorting of major histocompatibility complex (MHC) class II molecules into the endocytic pathway are poorly understood. For many proteins, access to endosomal compartments is regulated by cytosolically expressed sequences. We present evidence that a sequence in the lumenal domain of the MHC class II molecule regulates a very late event in class II biogenesis. Class II molecules containing single amino acid changes in the highly conserved 80-82 region of the beta chain were introduced into invariant chain (Ii)-negative fibroblasts with wild-type alpha chain, and the derived transfectants were analyzed biochemically. Using an endosomal isolation technique, we have quantified the level of class II molecules expressed in endocytic compartments and found that in the absence of Ii, approximately 15% of total cellular class II molecules can be isolated from endosomal compartments. Mutation at position 80 enhances this localization, while changes at positions 81 and 82 ablate class II expression in endosomal compartments. In addition, we have evaluated whether the induced changes in intracellular distribution of class II molecules were due to alterations in early biosynthetic events, indicative of misfolding of the molecules, or to modulation of later trafficking events more likely to be a consequence of the modulation of a specific transport event. Despite the dramatic effects on endosomal localization induced by the mutations, early biosynthetic events and maturation of class II were unaffected by the mutations. Collectively, our data argue that late trafficking events that control the ability of the class II molecule to access antigens is regulated by the 80-82 segment of the MHC class II beta chains.     

Targeting a polyepitope protein incorporating multiple class II-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by CD4+ cytotoxic T lymphocytes: a novel approach to vaccine design

The role of CD4+ and CD8+ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4+- and CD8+-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8+- or CD4+-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4+ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4+ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4+-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4+ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.     

Analysis of subcellular organelles involved in major histocompatibility complex (MHC) class II-restricted antigen presentation by electrophoresis

Presentation of material derived from pathogenic organisms to the immune system requires uptake of antigens into antigen presenting cells, processing into peptide fragments and loading of the resulting fragments onto major histocompatibility complex (MHC) class II molecules. MHC class II-restricted antigen presentation involves both the biosynthetic as well as the endocytic pathway of antigen-presenting cells. In recent years, the general mechanisms that govern these processes have been delineated, and specialized organelles have been characterized in which processing and loading of antigens takes place. Here, we review the work that has led to the characterization of these MHC class II compartments, and describe the use of organelle electrophoresis and two-dimensional gel electrophoresis to analyze the molecular composition of the different subcellular organelles involved in MHC class II-restricted antigen presentation as well as in antigen uptake.     

Hemoglobin endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket

Four lines of evidence indicate that a specific high affinity binding site on the surface of Leishmania donovani promastigotes mediates rapid internalization and degradation of hemoglobin. 1) Binding and uptake of 125I-hemoglobin by Leishmania followed saturation kinetics and were competed by unlabeled hemoglobin but not by globin or hemin or other heme- or iron-containing proteins. 2) Immunogold labeling studies revealed that, at 4 degreesC, hemoglobin binding was localized in the flagellar pocket of the promastigotes. Indirect immunofluorescence assays showed that, at 37 degreesC, the bound hemoglobin in such cells entered an endocytic compartment within 2 min and dispersed throughout the cell body by 15 min. 3) After incubation with hemoglobin-gold conjugates at 25 degreesC or 37 degreesC, the particles accumulated in discrete intracellular vesicles. 4) A single biotinylated protein of 46 kDa was revealed when solubilized membranes from surface biotinylated intact Leishmania adsorbed by hemoglobin-agarose beads were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting with avidin-horseradish peroxidase. Considered together, these data indicate that this 46-kDa protein on the cell surface of L. donovani promastigotes mediates the binding of hemoglobin and its rapid internalization through a vesicular pathway characteristic of receptor-mediated endocytosis.     

Characterization of a novel, stage-specific, invariant surface protein in Trypanosoma brucei containing an internal, serine-rich, repetitive motif

A new surface membrane protein, invariant surface glycoprotein termed ISG100, was identified in Trypanosoma brucei, using catalyzed surface, radioiodination of intact cells. This integral membrane glycoprotein was purified by a combination of detergent extraction, lectin-affinity, and ion-exchange chromatography followed by preparative SDS-polyacrylamide gel electrophoresis. The protein was expressed only in bloodstream forms of the parasite, was heavily N-glycosylated, and was present in different clonal variants of the same serodeme as well as in different serodemes. The gene for this protein was isolated by screening a cDNA expression library with antibodies against the purified protein followed by screening of a genomic library. The nucleotide sequence of the gene (4050 base pairs) predicted a highly reiterative polypeptide containing three distinct domains, a unique N-terminal domain of about 10 kDa containing three potential N-glycosylation sites, which was followed by a large internal domain consisting entirely of 72 consecutive copies of a serine-rich, 17-amino acid motif (approximately 113 kDa) and terminated with an apparent transmembrane spanning region of about 3.3 kDa. The internal repeat region of this gene (3672 base pairs) represents the largest reiterative coding sequence to be fully characterized in any species of trypanosome. There was no significant homology with other known proteins, and overall the predicted protein was extremely hydrophobic. Unlike the genes for other surface proteins, the gene encoding ISG100 was present as a single copy. Although present in the flagellar pocket, ISG100 was predominantly associated with components of the pathways for endo/exocytosis, such as intracellular vesicles located in the proximity of the pocket as well a large, electron-lucent perinuclear digestive vacuole.     

A dominant-negative clathrin mutant differentially affects trafficking of molecules with distinct sorting motifs in the class II major histocompatibility complex (MHC) pathway

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 CD3gamma, 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.