The dependence of the endocytosis of the epidermal growth factor receptors on the degree of receptor occupancy

Two subpopulations of epidermal growth factor (EGF) receptor with different affinity to EGF have been demonstrated for many cell types. There are reasons to assume a key role of high-affinity receptors in stimulation of cell response to EGF. Nevertheless, characteristics of its action remain obscure. In the present work an attempt has been made to study internalization and intracellular compartmentalization of high-affinity receptors. For this purpose endocytosis of 125I-EGF in A431 cells was stimulated by low (less than 1 nM) EGF concentrations as well as after blocking of low-affinity binding sites with specific monoclonal antibodies 2E9. By subcellular fractionation in 17% Percoll gradient it was demonstrated that in both cases internalized 125I-EGF was found first in light, and then in heavy endosomes staying there for a long time. Effectiveness of 125I-EGF delivery to prelysosomal heavy endosomes as well as initial internalization rate is maximal at low EGF concentrations and is reduced dramatically with increasing of receptor occupancy. Monoclonal antibody Mab108 specifically recognizing high-affinity receptors were capable of stimulation of receptor internalization with initial rate higher than that of high EGF concentrations, but Mab108-receptor complexes were localized in light endosomes. Preincubation of the cells with low concentrations of EGF led to redistribution of considerable portion of 125I-Mab108 into heavy endosomes, which may be a result of high-affinity receptor tyrosine kinase activation. Our data confirm a hypothesis of TK involvement in regulation of both internalization and sorting of EGF-receptor complexes. Structural organization of high-affinity receptors is not sufficient for receptor targeting to degradation pathway.     

EEA1 links PI(3)K function to Rab5 regulation of endosome fusion

GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.     

Cytoskeletal protein ABP-280 directs the intracellular trafficking of furin and modulates proprotein processing in the endocytic pathway

Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.     

EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine "finger" motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.     

Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera

A chimera of the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP) has been engineered by fusing GFP to the carboxyl terminus of EGFR. Data are provided to demonstrate that the GFP moiety does not affect the expected functioning of EGFR. EGFR-GFP becomes phosphorylated at tyrosine residues in response to EGF and is capable of phosphorylating endogenous substrates and initiating signaling cascades. EGF-dependent association of the chimeric receptor with the clathrin adaptor protein AP-2, involved in endocytosis, and with Shc adaptor protein, which binds in close proximity to the fusion point, is not affected by the GFP moiety. Receptor down-regulation and internalization occur at rates similar to those in cells expressing wild-type EGFR. Western blot analysis reveals that lysosomal degradation of EGFR-GFP proceeds from the extracellular domain and that GFP is not preferentially cleaved. Time-dependent co-localization of EGFR-GFP and Texas Red-conjugated EGF in living cells using digital deconvolution microscopy demonstrates the trafficking of ligand-receptor complexes through the early and multivesicular endosomes followed by segregation of the ligand and receptor at the late stages of endocytosis. Time-lapse optical analysis of the early stages of endocytosis reveals localization of EGFR-GFP in the tubular-vesicular endosomal compartments. Rapid dynamics of membrane movement and fusion within these compartments were observed. This approach and the fidelity of the biochemical properties of the EGFR-GFP demonstrate that real-time visualization of trafficking and protein interactions of tyrosine kinase receptors in the presence or absence of the ligand are feasible.     

In vitro fusion of phagosomes with different endocytic organelles from J774 macrophages

We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in nonphagocytic cells.     

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.     

Involvement of the endosomal autoantigen EEA1 in homotypic fusion of early endosomes

In mammalian cells, fusion between early endocytic vesicles has been shown to require the ubiquitous intracellular fusion factors N-ethylmaleimide-sensitive factor (NSF) and alpha-SNAP, as well as a factor specific for early endosomes, the small GTPase Rab5 [1-3]. We have previously demonstrated an additional requirement for phosphatidylinositol 3-kinase (PI 3-kinase) activity [4]. The membrane association of early endosomal antigen 1 (EEA1), a specific marker of early endosomes [5,6], has recently been shown to be similarly dependent on PI 3-kinase activity [7], and we therefore postulated that it might be involved in endosome fusion. Here, we present evidence that EEA1 has an important role in determining the efficiency of endosome fusion in vitro. Both the carboxy-terminal domain of EEA1 (residues 1098-1411) and specific antibodies against EEA1 inhibited endosome fusion when included in an in vitro assay. Furthermore, depletion of EEA1, both from the membrane fraction used in the assay by washing with salt and from the cytosol using an EEA1-specific antibody, resulted in inhibition of endosome fusion. The involvement of EEA1 in endosome fusion accounts for the sensitivity of the endosome fusion assay to inhibitors of PI 3-kinase.     

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.     

Children''s emerging regulation of conduct: restraint, compliance, and internalization from infancy to the second year

Receptor-mediated endocytosis of circulating collagen is a major physiological scavenger function of the liver endothelial cell and an important catabolic event in the complete turnover of this abundant connective tissue protein. In the present study, transport of collagen through the endocytic pathway was investigated in cultured liver endothelial cells. Collagen conjugated to fluorescein isothiocyanate, to allow detection of the ligand by fluorescence and immunoelectron microscopy, was found sequentially in three different organelles that compose the basic degradative endocytic pathway of eukaryotic cells: early endosomes, late endosomes, and terminal lysosomes. Early endosomes were identified as vesicles positive for early endosome antigen 1 (EEA1). Late endosomes were distinguished as structures positive for the late endosomal/lysosomal marker rat lysosomal membrane glycoprotein 120, but negative for EEA1 and lysosomally targeted BSA-gold. Lysosomes were defined by their content of BSA-gold, injected 24 hours before isolation of cells. Coated pits and coated vesicles mediated an extremely rapid internalization. Shortly after internalization and during the first 20 minutes, ligand was found in early endosomes. From 20 minutes on, ligand started to appear in late endosomes (23%), and by 2 hours the transfer was largely complete (82.5%). Only 2.5% of ligand was transferred to the lysosomes after 2 hours, and this number slowly increased to 21% and 53% after 6 and 16 hours, respectively. We conclude that 1) EEA1 is a useful marker for tracing early events of endocytosis in liver endothelial cells; 2) in contrast to the rapid internalization, transit of internalized ligand through early sorting endosomes generally takes from 20 minutes to 2 hours; and 3) exit from the late endosomes is very slow, requiring several hours.     

Evidence for a symmetrical requirement for Rab5-GTP in in vitro endosome-endosome fusion

Early endosome fusion, which has been extensively characterized using an in vitro reconstitution assay, is Rab5-dependent. To examine the requirement for Rab5 on both fusion partners, we prepared cytosol and endosomes depleted of Rab5. Unlike control cytosol, Rab5-depleted cytosol was only marginally active in the in vitro endosome fusion. However, fusion could be restored by the addition of wild-type Rab5 or Rab5 D136N, a mutant whose nucleotide specificity favors xanthine over guanine. The addition of Rab5 D136N restored fusion only in the presence of XTP. In the absence of XTP or in the presence of XDP, Rab5 D136N failed to restore fusion. When fusion was carried out with endosomal vesicles depleted of Rab GTPases (by preincubation of vesicles with GDP dissociation inhibitor), together with cytosol immunodepleted of Rab5, fusion was virtually absent. We then used immunodepleted cytosol and GDP dissociation inhibitor-treated vesicles to determine whether Rab5 is required by both fusion partners. Using separate sets of endosomal vesicles, we found that priming both sets of Rab5-depleted vesicles with Rab5 Q79L, a GTPase-defective mutant, substantially stimulated endosome fusion. Priming one set of vesicles with Rab5 Q79L and a second set of vesicles with Rab5 S34N failed to activate fusion. When both sets of Rab5-depleted vesicles were primed with Rab5 D136N supplemented with XTP, endosome fusion was stimulated, similar to that observed with Rab5 Q79L. However, when one set of vesicles was preincubated with Rab5 D136N plus XTP and the second set with Rab5 D136N and XDP, no stimulation of fusion was observed. We conclude that Rab5-GTP is required on both fusion partners for docking and fusion of early endosomes. To confirm the fusion of Rab5-GTP-positive vesicles in vivo, we expressed GFP-Rab5 Q79L in fibroblasts and observed fusion of Rab5-positive vesicles. We failed to record fusion of Rab5-positive vesicles with Rab5-negative vesicles. We conclude that Rab5-GTP is required on both sets of endosomes for fusion in vitro and in living cells.     

Effect of prolonged staining with hematoxylin on detecting Helicobacter pylori in hematoxylin-eosin-stained gastric mucosa

Rab proteins are small GTP-ases localized to distinct membrane compartments in eukaryotic cells and regulating specific steps of intracellular vesicular membrane traffic. The Rab7 protein is localized to the late endosomal compartment and controls late steps of endocytosis. We have isolated, by library screening, the 5' region, including the promoter, of the mouse Rab7 gene and a Rab7 pseudogene. We have mapped, by genetic linkage analysis, the mouse Rab7 gene on Chromosome (Chr) 6 and the Rab7-ps1 pseudogene on Chr 9, where the Rab7 gene has been previously reported to map. By radiation hybrid mapping, we have located the human RAB7 gene on Chr 3, in a region homologous to the mouse Chr 6, where the Rab7 gene maps.     

Removal of the membrane-anchoring domain of epidermal growth factor leads to intracrine signaling and disruption of mammary epithelial cell organization

Autocrine EGF-receptor (EGFR) ligands are normally made as membrane-anchored precursors that are proteolytically processed to yield mature, soluble peptides. To explore the function of the membrane-anchoring domain of EGF, we expressed artificial EGF genes either with or without this structure in human mammary epithelial cells (HMEC). These cells require activation of the EGFR for cell proliferation. We found that HMEC expressing high levels of membrane- anchored EGF grew at a maximal rate that was not increased by exogenous EGF, but could be inhibited by anti-EGFR antibodies. In contrast, when cells expressed EGF lacking the membrane-anchoring domain (sEGF), their proliferation rate, growth at clonal densities, and receptor substrate phosphorylation were not affected by anti-EGFR antibodies. The sEGF was found to be colocalized with the EGFR within small cytoplasmic vesicles. It thus appears that removal of the membrane-anchoring domain converts autocrine to intracrine signaling. Significantly, sEGF inhibited the organization of HMEC on Matrigel, suggesting that spatial restriction of EGF access to its receptor is necessary for organization. Our results indicate that an important role of the membrane-anchoring domain of EGFR ligands is to restrict the cellular compartments in which the receptor is activated.     

A balance of opposing signals within the cytoplasmic tail controls the lysosomal targeting of P-selectin

The 35-amino acid cytoplasmic tail of the adhesion receptor P-selectin is subdivided into stop transfer, C1 and C2 domains. It contains structural signals needed for targeting this protein to specialized secretory organelles and to lysosomes. Recently, using site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras, we have uncovered a novel sequence within the C1 domain, KCPL, that mediates sorting from early, transferrin-positive endosomes to lysosomes and therefore operates as a positive lysosomal targeting signal (Blagoveshchenskaya, A. D., Norcott, J. P. , and Cutler, D. F. (1998) J. Biol. Chem. 273, 2729-2737). In the current study, we examined lysosomal targeting by both subcellular fractionation and an intracellular proteolysis assay and found that a balance of positive and negative signals is required for proper lysosomal sorting of P-selectin. First, we have found that within the sequence KCPL, Cys-766 plays a major role along with Pro-767, whereas Lys-765 and Leu-768 make no contribution to promoting lysosomal targeting. In addition, horseradish peroxidase-P-selectin chimeras were capable of acylation in vivo with [3H]palmitic acid at Cys-766, since no labeling of a chimera in which Cys-766 was replaced with Ala was detected. Second, analysis of mutations within the C2 domain revealed that substitution of two sequences, YGVF and DPSP, causes an increase in both lysosomal targeting and intracellular proteolysis suggesting the presence of lysosomal avoidance signals. The inhibition or promotion of lysosomal targeting resulted from alterations in endosomal sorting since internalization was not changed in parallel with lysosomal delivery. Analysis of the double mutants KCPL/YGVF or KCPL/DPSP revealed that although the positive lysosomal targeting signal operates in the early/sorting transferrin-positive endosomes, the negative lysosomal targeting (lysosomal avoidance) signals act at later stages of the endocytic pathway, most likely in late endosomal compartments.     

Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes

Endocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 we examined its intracellular distribution in nonpolarized cells. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 revealed that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of betaSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.     

Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.     

Major and minor receptor group human rhinoviruses penetrate from endosomes by different mechanisms

Intercellular adhesion molecule 1 and the low-density lipoprotein receptor are used for cell entry by major and minor receptor group human rhinoviruses (HRVs), respectively. Whereas minor-group viruses, exemplified by HRV2, transfer their genomic RNA to the cytoplasm through a pore in the endosomal membrane (E. Prchla, C. Plank, E. Wagner, D. Blaas, and R. Fuchs, J. Cell Biol. 131:111-123, 1995), the mechanism of in vivo uncoating of major-group HRVs has not been elucidated so far. Using free-flow electrophoresis, we performed a comparative analysis of cell entry by HRV2 and the major group rhinovirus HRV14. Here we demonstrate that this technique allows the separation of free viral particles from those associated with early endosomes, late endosomes, and plasma membranes. Upon free-flow electrophoretic separation of microsomes, HRV14 was recovered from endosomes under conditions which prevent uncoating, whereas the proportion of free viral particles increased with time under conditions which promote uncoating. The remaining virus eluted within numerous fractions corresponding to membraneous material, with no clear endosomal peaks being discernible. This suggests that uncoating of HRV14 results in lysis of the endosomal membrane and release of subviral 135S and 80S particles into the cytoplasm.     

Proper sorting of the cation-dependent mannose 6-phosphate receptor in endosomes depends on a pair of aromatic amino acids in its cytoplasmic tail

The 67-amino acid cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains a signal(s) that prevents the receptor from entering lysosomes where it would be degraded. To identify the key residues required for proper endosomal sorting, we analyzed the intracellular distribution of mutant forms of the receptor by Percoll density gradients. A receptor with a Trp19 --> Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either Phe18 --> Ala or Phe13 --> Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of Trp19 for sorting of the CD-MPR in endosomes, with Phe18, Phe13, and several neighboring residues contributing to this function. The addition of the Phe18-Trp19 motif of the CD-MPR to the cytoplasmic tail of the lysosomal membrane protein Lamp1 was sufficient to partially impair its delivery to lysosomes. Replacing Phe18 and Trp19 with other aromatic amino acids did not impair endosomal sorting of the CD-MPR, indicating that two aromatic residues located at these positions are sufficient to prevent the receptor from trafficking to lysosomes. However, alterations in the spacing of the diaromatic amino acid sequence relative to the transmembrane domain resulted in receptor accumulation in lysosomes. These findings indicate that the endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also present in the cytoplasmic tail of other receptors known to be internalized from the plasma membrane, this feature may prove to be a general determinant for endosomal sorting.     

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.     

Uptake of poliovirus into the endosomal system of HeLa cells

To understand the topology and mechanism of poliovirus uncoating, the question of whether intact virions can be endocytosed by the host cell was studied by a combination of various techniques. In order to prevent alteration of the virus to subviral particles, Hela cells were infected at 26 degrees C. At this temperature the majority of cell-associated virions remained at the plasma membrane, whereas a smaller amount accumulated in vesicles having the same mobility (upon free-flow electrophoresis) and migration behaviour on Nycodenz density gradients as early and late endosomes. Co-localization of native poliovirions with endosomal markers was verified by peroxidase-induced diaminobenzidine density-shift of endosomal vesicles. Internalization of poliovirions into endosomes makes it likely, but does not prove that viral RNA can be released into the cytoplasm from the vesicular compartment.