Geldanamycin Enhances Retrograde Transport of Shiga Toxin in HEp-2 Cells

The heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) has been shown to alter endosomal sorting, diverting cargo destined for the recycling pathway into the lysosomal pathway. Here we investigated whether GA also affects the sorting of cargo into the retrograde pathway from endosomes to the Golgi apparatus. As a model cargo we used the bacterial toxin Shiga toxin, which exploits the retrograde pathway as an entry route to the cytosol. Indeed, GA treatment of HEp-2 cells strongly increased the Shiga toxin transport to the Golgi apparatus. The enhanced Golgi transport was not due to increased endocytic uptake of the toxin or perturbed recycling, suggesting that GA selectively enhances endosomal sorting into the retrograde pathway. Moreover, GA activated p38 and both inhibitors of p38 or its substrate MK2 partially counteracted the GA-induced increase in Shiga toxin transport. Thus, our data suggest that GA-induced p38 and MK2 activation participate in the increased Shiga toxin transport to the Golgi apparatus.     

Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking

The mammalian tumor susceptibility gene tsg101 encodes the homologue of Vps23p, a class E Vps protein essential for normal membrane trafficking in the late endosome/multivesicular body of yeast. Both proteins assemble into large (∼350 kDa) cytosolic protein complexes and we show that the yeast complex contains another class E Vps protein, Vps28p. tsg101 mutant cells exhibit defects in sorting and proteolytic maturation of the lysosomal hydrolase cathepsin D, as well as in the steady-state distribution of the mannose-6-phosphate receptor. Additionally, endocytosed EGF receptors that are normally sorted to the lysosome are instead rapidly recycled back to the cell surface in tsg101 mutant cells. We propose that tsg101 mutant cells are defective in the delivery of cargo proteins to late endosomal compartments. One consequence of this endosomal trafficking defect is the delayed down-regulation/degradation of activated cell surface receptors, resulting in prolonged signaling. This may contribute to the tumorigenic phenotype exhibited by the tsg101 mutant fibroblasts.     

A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides

The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the trans-Golgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8-10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins.     

Endosome disruption enhances the functional nuclear delivery of Tat-fusion proteins

Tat protein from human immunodeficiency virus can deliver biologically active proteins in vivo and is of considerable interest for protein therapeutics. The mechanism responsible for Tat-fusion protein internalization is still poorly understood and controversial. The punctuate distribution, timing, and temperature sensitivity observed in our experiments with Tat-fusion proteins are consistent with endocytosis. After a few hours, Tat-fusion proteins accumulated around the nucleus without any significant visible nuclear targeting. Using a Cre/Lox based functional assay, lysosomotropic agents known to disrupt endosome integrity, increased by up to 23-fold the nuclear delivery of functional Tat-Cre recombinase without increasing cell uptake in a similar fashion. This shows that endosome disruption can significantly increase Tat-fusion protein access to the cytosol and nucleus. In addition, we found that internalized Tat-fusion proteins persisted several hours and that inhibitors of lysosome acidification did not increase functional nuclear delivery of Tat-Cre. This suggests that Tat-fusion proteins enter via the endosomal pathway, circumvent lysosomal degradation, and are then sequestered in the periphery of the nucleus. Most importantly, our work indicates that an inadequate intracellular trafficking is the main factor limiting the efficiency of protein cargo delivery using Tat.     

Exchange of a single amino acid switches the substrate properties of RhoA and RhoD toward glucosylating and transglutaminating toxins

Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.     

Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates

Multi-domain bacterial protein toxins are being explored as potential carriers for targeted delivery of biomolecules. Previous approaches employing isolated receptor binding subunits disallow entry into the cytosol. Strategies in which catalytic domains are replaced with cargo molecules are presumably inefficient due to co-operation of domains during the endosomal translocation step. Here, we characterize a novel transport vehicle in which cargo proteins are attached to the amino terminus of the full-length botulinum neurotoxin type D (BoNT/D). The intrinsic enzymatic activity of the neurotoxin allowed quantification of the efficacy of cargo delivery to the cytosol. Dihydrofolate reductase and BoNT type A (BoNT/A) light chain (LC) were efficiently conveyed into the cytosol, whereas attachment of firefly luciferase or green fluorescent protein drastically reduced the toxicity. Luciferase and BoNT/A LC retained their catalytic activity as evidenced by luciferin conversion or SNAP-25 hydrolysis in the cytosol of synaptosomes, respectively. Conformationally stabilized dihydrofolate reductase as cargo considerably decreased the toxicity indicative for the requirement of partial unfolding of cargo protein and catalytic domain as prerequisite for efficient translocation across the endosomal membrane. Thus, enzymatically inactive clostridial neurotoxins may serve as effective, safe carriers for delivering proteins in functionally active form to the cytosol of neurones.     

Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.     

Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugate

Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments.     

Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides

A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways.     

Protein transduction in human cells is enhanced by cell-penetrating peptides fused with an endosomolytic HA2 sequence

Endocytosis has been proposed as one of the primary mechanisms for cellular entry of cell-penetrating peptides (CPPs) and their cargoes. However, a major limitation of endocytic pathway is entrapment of the CPP-cargo in intracellular vesicles from which the cargo must escape into the cytoplasm to exert its biological activity. Here we demonstrate that a CPP tagged with an endosomolytic fusion peptide derived from the influenza virus hemagglutinin-2 (HA2) remarkably enhances the cytosolic delivery of proteins in human A549 cells. To determine the endosome-disruptive effects, recombinant DNA plasmids containing coding sequences of HA2, CPPs and red fluorescent proteins (RFPs) were constructed. The fusion proteins were purified from plasmid-transformed Escherichia coli, and their effects on protein transduction were examined using live cell imaging and flow cytometry. Our data indicate that endocytosis is the major route for cellular internalization of CPP-HA2-tagged RFP. Mechanistic studies revealed that the fusogenic HA2 peptide dramatically facilitates CPP-mediated protein entry through the release of endocytosed RFPs from endosomes into the cytoplasm. Furthermore, incorporating the HA2 fusion peptide of the CPP-HA2 fusion protein improved cytosolic uptake without causing cytotoxicity. These findings strongly suggest that the CPP-HA2 tag could be an efficient and safe carrier that overcomes endosomal entrapment of delivered therapeutic drugs.     

Palmitoylation controls recycling in lysosomal sorting and trafficking

For the efficient trafficking of lysosomal proteins, the cationic-dependent and -independent mannose 6-phosphate receptors and sortilin must bind cargo in the Golgi apparatus, be packaged into clathrin-coated trafficking vesicles and traffic to the endosomes. Once in the endosomes, the receptors release their cargo into the endosomal lumen and recycle back to the Golgi for another round of trafficking, a process that requires retromer. In this study, we demonstrate that palmitoylation is required for the efficient retrograde trafficking of sortilin, and the cationic-independent mannose 6-phosphate as palmitoylation-deficient receptors remain trapped in the endosomes. Importantly, we also show that palmitoylation is required for receptor interaction with retromer as nonpalmitoylated receptor did not interact with retromer. In addition, we have identified DHHC-15 as the palmitoyltransferase responsible for this modification. In summary, we have shown the functional significance of palmitoylation in lysosomal receptor sorting and trafficking.     

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.     

Cholera toxin and Escherichia coli enterotoxin B-subunits inhibit macrophage-mediated antigen processing and presentation: evidence for antigen persistence in non-acidic recycling endosomal compartments

Cholera toxin (Ctx) and the closely related Escherichia coli heat-labile enterotoxin (Etx) not only act as mediators of diarrhoeal disease but also exert potent immunomodulatory properties on mammalian immune systems. The toxins normally exert their diarrhoeagenic effects by initiating receptor-mediated uptake into vesicles that enter a retrograde trafficking pathway, circumventing degradative compartments and targeting them to the trans-Golgi network (TGN) and endoplasmic reticulum. Here, we examine whether receptor-mediated binding and cellular entry by the toxin B-subunits also lead to concomitant changes in uptake and trafficking of exogenous antigens that could contribute to the potent immunomodulatory properties of these toxins. Treatment of the macrophage (J774.2) cell line with Etx B-subunit (EtxB) resulted in EtxB transport to the TGN and also led to the formation of large, translucent, non-acidic, EtxB-devoid vacuoles. When exogenous antigens were added, EtxB-treated cells were found to be proficient in both internalization of ovalbumin (OVA) and phagocytosis of bacterial particles. However, the internalized OVA, instead of trafficking along a lysosome-directed endocytic pathway via acidified endosomes, persisted in a non-acidic, light-density compartment that was distinct from the translucent vacuoles. The rerouted OVA did not co-localize with the endosomal markers rab5 or rab11, nor with EtxB, but was retained in a transferrin receptor-positive compartment. The failure of OVA to enter the late endosomal/lysosomal compartments correlated with a striking inhibition of OVA peptide processing and presentation to OVA-responsive CD4+ T-cells. CtxB also modulated OVA trafficking and inhibited antigen presentation. These findings demonstrate that the B-subunits of Ctx and Etx alter the progression of exogenous antigens along the endocytic processing pathway, and prevent or delay efficient epitope presentation and T-cell stimulation. The formation of such 'antigen depots' could contribute to the immunomodulatory properties of these bacterial virulence determinants.     

WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion

Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) is an important regulator of vesicle trafficking. By generating actin on the surface of intracellular vesicles, WASH is able to directly regulate endosomal sorting and maturation. We report that, in Dictyostelium, WASH is also required for the lysosomal digestion of both phagocytic and autophagic cargo. Consequently, Dictyostelium cells lacking WASH are unable to grow on many bacteria or to digest their own cytoplasm to survive starvation. WASH is required for efficient phagosomal proteolysis, and proteomic analysis demonstrates that this is due to reduced delivery of lysosomal hydrolases. Both protease and lipase delivery are disrupted, and lipid catabolism is also perturbed. Starvation-induced autophagy therefore leads to phospholipid accumulation within WASH-null lysosomes. This causes the formation of multilamellar bodies typical of many lysosomal storage diseases. Mechanistically, we show that, in cells lacking WASH, cathepsin D becomes trapped in a late endosomal compartment, unable to be recycled to nascent phagosomes and autophagosomes. WASH is therefore required for the maturation of lysosomes to a stage at which hydrolases can be retrieved and reused.     

A Conserved Structural Motif Mediates Retrograde Trafficking of Shiga Toxin Types 1 and 2

Shiga toxin‐producing Escherichia coli (STEC) produce two types of Shiga toxin (STx): STx1 and STx2. The toxin A‐subunits block protein synthesis, while the B‐subunits mediate retrograde trafficking. STEC infections do not have definitive treatments, and there is growing interest in generating toxin transport inhibitors for therapy. However, a comprehensive understanding of the mechanisms of toxin trafficking is essential for drug development. While STx2 is more toxic in vivo, prior studies focused on STx1 B‐subunit (STx1B) trafficking. Here, we show that, compared with STx1B, trafficking of the B‐subunit of STx2 (STx2B) to the Golgi occurs with slower kinetics. Despite this difference, similar to STx1B, endosome‐to‐Golgi transport of STx2B does not involve transit through degradative late endosomes and is dependent on dynamin II, epsinR, retromer and syntaxin5. Importantly, additional experiments show that a surface‐exposed loop in STx2B (β4–β5 loop) is required for its endosome‐to‐Golgi trafficking. We previously demonstrated that residues in the corresponding β4–β5 loop of STx1B are required for interaction with GPP130, the STx1B‐specific endosomal receptor, and for endosome‐to‐Golgi transport. Overall, STx1B and STx2B share a common pathway and use a similar structural motif to traffic to the Golgi, suggesting that the underlying mechanisms of endosomal sorting may be evolutionarily conserved.      

Recognising the signals for endosomal trafficking

The endosomal compartment is a major sorting station controlling the balance between endocytic recycling and lysosomal degradation, and its homeostasis is emerging as a central factor in various neurodegenerative diseases such as Alzheimer's and Parkinson's. Membrane trafficking is generally coordinated by the recognition of specific signals in transmembrane protein cargos by different transport machineries. A number of different protein trafficking complexes are essential for sequence-specific recognition and retrieval of endosomal cargos, recycling them to other compartments and acting to counter-balance the default endosomal sorting complex required for transport–mediated degradation pathway. In this review, we provide a summary of the key endosomal transport machineries, and the molecular mechanisms by which different cargo sequences are specifically recognised.     

Intracellular trafficking of lysosomal membrane proteins

Lysosomes are the site of degradation of obsolete intracellular material during autophagy and of extracellular macromolecules following endocytosis and phagocytosis. The membrane of lysosomes and late endosomes is enriched in highly glycosylated transmembrane proteins of largely unknown function. Significant progress has been made in recent years towards elucidating the pathways by which these lysosomal membrane proteins are delivered to late endosomes and lysosomes. While some lysosomal membrane proteins follow the constitutive secretory pathway and reach lysosomes indirectly via the cell surface and endocytosis, others exit the trans-Golgi network in clathrin-coated vesicles for direct delivery to endosomes and lysosomes. Sorting from the Golgi or the plasma membrane into the endosomal system is mediated by signals encoded by the short cytosolic domain of these proteins. This review will discuss the role of lysosomal membrane proteins in the biogenesis of the late endosomal and lysosomal membranes, with particular emphasis on the structural features and molecular mechanisms underlying the intracellular trafficking of these proteins.     

Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1.

Certain uropathogenic and neonatal meningitis-causing strains of Escherichia coli express a 114 kDa protein toxin called cytotoxic necrotizing factor 1 (CNF1). The toxin causes alteration of the host cell actin cytoskeleton and promotes bacterial invasion of blood-brain barrier endothelial cells. CNF1 belongs to a unique group of large cytotoxins that cause constitutive activation of Rho guanosine triphosphatases (GTPases), which are key regulators of the actin cytoskeleton. This group also includes E. coli cytotoxic necrotizing factor 2 (CNF2, 114 kDa) and dermonecrotic toxins (DNT, 159 kDa) of Bordetella spp. with related sequences occurring in Yersinia spp. Here we show that the catalytic region of CNF1 exhibits a novel protein fold as determined by its 1.83 A resolution crystal structure. The structure reveals that CNF1 has a Cys-His-main chain oxygen catalytic triad reminiscent of enzymes belonging to the catalytic triad superfamily. The position of the catalytic Cys residue at the base of a deep pocket restricts access to potential substrates and helps explain the high specificity of this and related toxins.     

Mycobacterium tuberculosis phagosome maturation arrest: mycobacterial phosphatidylinositol analog phosphatidylinositol mannoside stimulates early endosomal fusion

Mycobacterium tuberculosis is a facultative intracellular pathogen that parasitizes macrophages by modulating properties of the Mycobacterium-containing phagosome. Mycobacterial phagosomes do not fuse with late endosomal/lysosomal organelles but retain access to early endosomal contents by an unknown mechanism. We have previously reported that mycobacterial phosphatidylinositol analog lipoarabinomannan (LAM) blocks a trans-Golgi network-to-phagosome phosphatidylinositol 3-kinase-dependent pathway. In this work, we extend our investigations of the effects of mycobacterial phosphoinositides on host membrane trafficking. We present data demonstrating that phosphatidylinositol mannoside (PIM) specifically stimulated homotypic fusion of early endosomes in an ATP-, cytosol-, and N-ethylmaleimide sensitive factor-dependent manner. The fusion showed absolute requirement for small Rab GTPases, and the stimulatory effect of PIM increased upon partial depletion of membrane Rabs with RabGDI. We found that stimulation of early endosomal fusion by PIM was higher when phosphatidylinositol 3-kinase was inhibited by wortmannin. PIM also stimulated in vitro fusion between model phagosomes and early endosomes. Finally, PIM displayed in vivo effects in macrophages by increasing accumulation of plasma membrane-endosomal syntaxin 4 and transferrin receptor on PIM-coated latex bead phagosomes. In addition, inhibition of phagosomal acidification was detected with PIM-coated beads. The effects of PIM, along with the previously reported action of LAM, suggest that M. tuberculosis has evolved a two-prong strategy to modify its intracellular niche: its products block acquisition of late endosomal/lysosomal constituents, while facilitating fusion with early endosomal compartments.     

Cytosolic delivery of granzyme B by bacterial toxins: evidence that endosomal disruption, in addition to transmembrane pore formation, is an important function of perforin

Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10% (51)Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal (51)Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin's ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.