Selective translocation of the A chain of diphtheria toxin across the membrane of purified endosomes.

Translocation is a necessary and rate-limiting step for diphtheria toxin (DT) cytotoxicity. We have reconstituted DT translocation in a cell-free system using endosomes purified from lymphocytes and have demonstrated this using two different probe/cell systems, which provided identical results: 125I-DT/human CEM cells and 125I-transferrin-DT/mouse BW cells. The cell-free DT translocation process was found to be dependent on the presence of the pH gradient endosome (pH 5.3)/cytosol (pH 7). Among the pH equilibrating agents, nigericin (5 microM) was found to be the most effective, inhibiting DT translocation by 88%. An optimum pH value of 7 on the cytosolic side of the membrane (pH gradient approximately 1.7) was determined. ATP per se is not required for DT translocation. 125I-DT translocation was 3-fold more active from late than from early endosomes, probably because of their slightly more acidic pH. Only the A chain of the toxin was found to escape from either 125I-DT/CEM or 125I-transferrin-DT/BW endosomes. Translocation of control endosome labels (125I-transferrin and 125I-horseradish peroxidase) was never observed. We also show that DT receptors present on resistant (mouse) cells block the translocation of the toxin and are responsible for the resistance of these cells to DT.     

Pathways for lipid antigen presentation by CD1 molecules: nowhere for intracellular pathogens to hide

A crucial feature of peptide antigen presentation by major histocompatibilty complex (MHC) class I and II molecules is their differential ability to sample cytosolic and extracellular antigens. Intracellular viral infections and bacteria that are taken up in phagosomes, but then escape from the endocytic compartment efficiently, enter the class I pathway via the cytosol. In contrast, phagosome-resident bacteria yield protein antigens that are sampled deep in the endocytic compartment and presented in a vacuolar acidification-dependent pathway mediated by MHC class II molecules. Despite this potential for antigen sampling, microbes have evolved a variety of evasive mechanisms that affect peptide transport in the MHC class I pathway or blockade of endosomal acidification and inhibition of phagosome–lysosome fusion that may compromise the MHC class II pathway of antigen presentation. Thus, besides MHC class I and II, a third lineage of antigen-presenting molecules that bind lipid and glycolipid antigens rather than peptides exists and is mediated by the family of CD1 proteins. CD1 isoforms (CD1a, b, c, and d) differentially sample both recycling endosomes of the early endocytic system and late endosomes and lysosomes to which lipid antigens are differentially delivered. These CD1 pathways include vacuolar acidification-independent pathways for lipid antigen presentation. These features of presenting lipid antigens, independently monitoring various antigen-containing intracellular compartments and avoiding certain evasive techniques employed by microbes, enable CD1 molecules to provide distinct opportunities to function in host defense against the microbial world.     

To be helped or not helped,that is the question

Diphtheria toxin (DT)* is the paradigm of the powerful A-B toxins. These bacterial poisons bind to cells, are endocytosed, and inject their catalytic domain into the cytosol causing the irreversible modification of a key component of the the host cellular machinery. The mechanism by which the hydrophilic enzymatic fragment of DT crosses the endosomal membrane and is released into the cytosol remains controversial. In this issue, Ratts et al. (2003) demonstrate that delivery of the DT catalytic domain from the lumen of purified early endosomes to the external medium requires the addition of a cytosolic translocation factor complex composed in part of Hsp90 and thioredoxin reductase.     

Free Ricin A Chain Reaches an Early Compartment of the Secretory Pathway before It Enters the Cytosol

During the intoxication of mammalian cells by ricin, the catalytically active A chain must cross the membrane of an intracellular compartment in order to reach its ribosomal substrates in the cytosol. The actual site of ricin A chain translocation is unclear, and conflicting views hold that it enters the cytosol from endosomes or from an early compartment of the secretory pathway, possibly the lumen of the endoplasmic reticulum. Here we show that treating cells with brefeldin A, or transiently overexpressing mutant GTPases known to inhibit biochemical complexes mediating anterograde and retrograde transport between the endoplasmic reticulum and the Golgi complex, protected cells from intoxication by free ricin A chain. These data indicate that ricin A chain, either free or as part of intact ricin, reaches an early compartment of the secretory pathway before translocation into the cytosol occurs.     

Role of CypA and Hsp90 in membrane translocation mediated by anthrax protective antigen

Bacillus anthracis lethal toxin consists of the protective antigen (PA) and the metalloprotease lethal factor (LF). During cellular uptake PA forms pores in membranes of endosomes, and unfolded LF translocates through the pores into the cytosol. We have investigated whether host cell chaperones facilitate translocation of LF and the fusion protein LF(N)DTA. LF(N) mediates uptake of LF(N)DTA into the cytosol, where DTA, the catalytic domain of diphtheria toxin, ADP-ribosylates elongation factor-2, allowing for detection of small amounts of translocated LF(N)DTA. Cyclosporin A, which inhibits peptidyl-prolyl cis/trans isomerase activity of cyclophilins, and radicicol, which inhibits Hsp90 activity, prevented uptake of LF(N)DTA into the cytosol of CHO-K1 cells and protected cells from intoxication by LF(N)DTA/PA. Both inhibitors, as well as an antibody against cyclophilin A blocked the release of active LF(N)DTA from endosomal vesicles into the cytosol in vitro. In contrast, the inhibitors did not inhibit cellular uptake of LF. In vitro, cyclophilin A and Hsp90 bound to LF(N)DTA and DTA but not to LF, implying that DTA determines this interaction. In conclusion, cyclophilin A and Hsp90 facilitate translocation of LF(N)DTA, but not of LF, across endosomal membranes, and thus they function selectively in promoting translocation of certain proteins, but not of others.     

Involvement of ATP-dependent Pseudomonas Exotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal ¹²⁵I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect ¹²⁵I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when ¹²⁵I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired ¹²⁵I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.     

Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes

The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.     

Plasma membrane-derived vesicles containing receptor-ligand complexes are fusogenic with early endosomes in a cell-free system

Receptor-mediated endocytosis involves the transport of receptor-ligand complexes from the cell surface to an intracellular endocytic compartment. This study shows that plasma membrane-derived vesicles containing receptor-bound ligands (e.g. aggregated anti-dinitrophenol (DNP) IgG bound to Fc receptors) fuse with early endosomes containing DNP-beta-glucuronidase in a cell-free system. Plasma membrane vesicles were generated by homogenization of cells that had been allowed to bind ligands at 4 degrees C. Fusion between vesicles containing the two probes was assessed by (i) the formation of anti-DNP IgG-DNP-beta-glucuronidase complexes and (ii) the colocalization within closed vesicles of two different sizes of colloidal gold coated with ligands. Fusion required ATP, cytosol, and KCl. The requirements were similar to those described for endosome-endosome fusion in in vitro systems. Mild trypsinization of vesicles prior to their addition to the assay inhibited fusion. When DNP-beta-glucuronidase was chased into more mature endocytic compartments, fusion was not observed. The results indicate that cell surface regions involved in receptor-mediated endocytosis are capable of fusing to early endosomes. This fusion event may constitute the first step in the transport of ligands to an intracellular endocytic compartment.     

Functional early endosomes are required for maturation of major histocompatibility complex class II molecules in human B lymphoblastoid cells.

Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place.     

Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin

The Rho-GTPases-activating toxin CNF1 (cytotoxic necrotizing factor 1) delivers its catalytic activity into the cytosol of eukaryotic cells by a low pH membrane translocation mechanism reminiscent of that used by diphtheria toxin (DT). As DT, CNF1 exhibits a translocation domain (T) containing two predicted hydrophobic helices (H1-2) (aa 350-412) separated by a short peptidic loop (CNF1-TL) (aa 373-386) with acidic residues. In the DT loop, the loss of charge of acidic amino acids, as a result of protonation at low pH, is a critical step in the transfer of the DT catalytic activity into the cytosol. To determine whether the CNF1 T domain operates similarly to the DT T domain, we mutated several ionizable amino acids of CNF1-TL to lysine. Single substitutions such as D373K or D379K strongly decreased the cytotoxic effect of CNF1 on HEp-2 cells, whereas the double substitution D373K/D379K induced a nearly complete loss of cytotoxic activity. These single or double substitutions did not modify the cell-binding, enzymatic or endocytic activities of the mutant toxins. Unlike the wild-type toxin, single- or double-substituted CNF1 molecules bound to the HEp-2 plasma membrane could not translocate their enzymatic activity directly into the cytosol following a low pH pulse.     

The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles

Interleukin 1beta (IL-1beta), a secretory protein lacking a signal peptide, does not follow the classical endoplasmic reticulum-to-Golgi pathway of secretion. Here we provide the evidence for a "leaderless" secretory route that uses regulated exocytosis of preterminal endocytic vesicles to transport cytosolic IL-1beta out of the cell. Indeed, although most of the IL-1beta precursor (proIL-1beta) localizes in the cytosol of activated human monocytes, a fraction is contained within vesicles that cofractionate with late endosomes and early lysosomes on Percoll density gradients and display ultrastructural features and markers typical of these organelles. The observation of organelles positive for both IL-1beta and the endolysosomal hydrolase cathepsin D or for both IL-1beta and the lysosomal marker Lamp-1 further suggests that they belong to the preterminal endocytic compartment. In addition, similarly to lysosomal hydrolases, secretion of IL-1beta is induced by acidotropic drugs. Treatment of monocytes with the sulfonylurea glibenclamide inhibits both IL-1beta secretion and vesicular accumulation, suggesting that this drug prevents the translocation of proIL-1beta from the cytosol into the vesicles. A high concentration of extracellular ATP and hypotonic medium increase secretion of IL-1beta but deplete the vesicular proIL-1beta content, indicating that exocytosis of proIL-1beta-containing vesicles is regulated by ATP and osmotic conditions.     

Rab7: a key to lysosome biogenesis

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.     

Rab15 effector protein: a novel protein for receptor recycling from the endocytic recycling compartment

Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways.     

The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.     

Cell-free systems to study vesicular transport along the secretory and endocytic pathways

Proteins bound for the cell surface, lysosomes, and secretory storage granules share a common pathway of intracellular transport. After their synthesis and translocation into the endoplasmic reticulum, these proteins traverse the secretory pathway by a series of vesicular transfers. Similarly, nutrient and signaling molecules enter cells by endocytosis, and move through the endocytic pathway by passage from one membrane-bound compartment to another. Little is known about the mechanisms by which proteins are collected into transport vesicles, or how these vesicles form, identify their targets, and subsequently fuse with their target membranes. An important advance toward our understanding these processes has come from the establishment of cell-free systems that reconstitute vesicular transfers in vitro. It is now possible to measure, in vitro, the transport of proteins from the endoplasmic reticulum to the Golgi, between Golgi cisternae, and the formation of transport vesicles en route from the trans Golgi network to the cell surface. Along the endocytic pathway, cell-free systems are available to study clathrin-coated vesicle formation, early endosome fusion, and the fusion of late endosomes with lysosomes. Moreover, the selective movement of receptors between late endosomes and the trans Golgi network has also been reconstituted. The molecular mechanisms of vesicular transport are now amenable to elucidation.     

RICIN-BINDING PROTEINS ALONG THE ENDOCYTIC PATHWAY: THE MAJOR ENDOSOMAL RICIN-BINDING PROTEIN IS ENDOSOME-SPECIFIC

Ricin is internalized after binding at the cell surface via lectin activity of the B-chain recognizing terminal galactose residues. Ricin-A chain is then translocated to the cytosol from various endocytic structures. Cell death is the result of catalytic inactivation of protein synthesis. Using ¹²⁵I-ricin overlays, we examined the distribution of ricin binding-proteins within highly purified preparations of plasma membrane vesicles, endosomes and lysosomes from lymphocytes. All compartments of the endocytic pathway had distinct profiles; some ricin-binding proteins were present throughout the pathway; others were restricted to the plasma membrane and endosomes. The major endosomal protein recognized by ¹²⁵I-ricin, a 166kDa glycoprotein, was endosome-specific. When endosomal proteins were solubilized before chromatography onto ricin-agarose this protein was also by far the major specifically-bound glycoprotein. This 166 kDa glycoprotein might be involved in ricin translocation from this compartment.     

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.     

EGF triggers caveolin redistribution from the plasma membrane to the early/sorting endocytic compartment of hepatocytes

In this study, we demonstrate that, in rat liver, epidermal growth factor (EGF) is responsible for the partial redistribution of caveolin-1 from the plasma membrane into the early/sorting endocytic compartment. Highly purified endosomes and plasma membrane fractions were isolated from control rat liver and from rats injected with EGF or pIgA for different times. Whereas in subcellular fractions from control hepatocytes most of caveolin was concentrated in the plasma membrane and the receptor-recycling fractions, after EGF injection there was a significant redistribution of caveolin toward the early/sorting (CURL) endocytic fractions. The recruitment of caveolin into the endocytic compartment was not induced by pIgA.