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Endocytic uptake and intracellular transport of acidic fibroblast growth factor (aFGF) was studied in cells transfected with FGF receptor 4 with mutations in the cytoplasmic part. Endocytic uptake in HeLa cells was reduced but not abolished when the tyrosine kinase of the receptor was inactivated by mutations or deletions. The tyrosine kinase-dependent endocytosis of aFGF was prevented by the expression of a dominant negative dynamin mutant that blocks endocytosis from coated pits and caveolae. However, more than half of the total endocytic uptake of aFGF was not affected under these conditions, indicating an endocytic uptake mechanism not involving coated pits or caveolae. Mutation or deletion of a putative caveolin-binding sequence did not prevent the localization of part of the receptors to a low density, caveolin-containing subcellular fraction. Whereas wild-type receptor transfers the growth factor from early endosomes to the recycling compartment, kinase negative, full length receptors were inefficient in this respect and the growth factor instead accumulated in lysosomes. By contrast, when most of the intracellular part of the receptor, including the kinase domain, was removed, aFGF was transported to the recycling compartment, as in cells that express wild-type receptors, suggesting the presence of a kinase-regulated targeting signal in the cytoplasmic tail.

Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution.

Diphtheria toxin A-fragment enters the cytosol of target cells, where it inhibits protein synthesis by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). We have here analyzed toxin-induced protein synthesis inhibition in single cells by autoradiography and compared it with inhibition of protein synthesis in the whole cell culture. The data show that half-maximal protein synthesis inhibition in the whole cell population after a short incubation time is achieved by partially inhibiting protein synthesis in basically all the cells, while half-maximal protein synthesis inhibition after a long incubation time is due to a complete protein synthesis block in about half the cells in the population. We have also compared stable and unstable A-fragment mutants with respect to the kinetics of cell intoxication. While the toxicity of the stable mutants increased with time, the unstable mutants showed a similar toxicity at early and late time points. When studying the kinetics of cell intoxication by toxins with short cytosolic half-life, we could not detect any recovery of protein synthesis at late time points when all the mutant A-fragments should be degraded. This indicates that the ADP-ribosylation of EF-2 cannot be reversed by an endogenous activity in the cells. The data indicate that entry of toxin into a cell is not associated with an immediate block in protein synthesis, and that prolonged action of single A-fragment molecules in the cytosol is sufficient to obtain complete protein synthesis inhibition at low toxin concentrations.

Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61alpha precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.

Acidic fibroblast growth factor (aFGF) contains a phosphorylation site recognized by protein kinase C. A non-mitogenic mutant growth factor is devoid of this phosphorylation site. We have changed amino acids in and close to the phosphorylation site and studied the consequences of this for binding of the growth factor to high affinity receptors as well as to heparin. We have also studied the ability of the mutants to stimulate DNA synthesis and cell proliferation as well as phosphorylation of mitogen-activated protein kinase and the ability of the growth factor mutants to be transported to the nucleus. The results indicate that while the mutations strongly affect the ability of the growth factor to bind to heparin, they do not affect much the binding to the specific FGF receptors, activation of mitogen-activated protein kinase or transport of the growth factor to the nucleus. The mutations affect to various extents the ability of the growth factor to stimulate DNA synthesis and to induce cell multiplication. We find that phosphorylation of aFGF is not required for mitogenic activity. The data suggest that altered interaction of the growth factor with a cellular component different from the receptor, possibly a component in the nucleus, is the reason for the different mitogenicity of the different growth factor mutants.

We have investigated the role of the transmembrane and cytoplasmic domains of the diphtheria toxin (DT) receptor [heparin‐binding epidermal growth factor (HB‐EGF) precursor] in the intoxication pathway. Two mutants were constructed in which these domains were replaced by either a 37 amino acid sequence signalling membrane attachment via a glycosylphosphatidylinositol (GPI) anchor (DTR‐GPI) or by the transmembrane and cytoplasmic domains of the human EGF receptor (DTR‐EGFR). Similar amounts of DTA fragment were translocated through the plasma membrane of NIH 3T3 cells transfected with the wild‐type receptor (DTR), DTR‐GPI and DTR‐EGFR, but translocation was about six times less efficient in the case of DTR‐GPI and DTR‐EGFR when taking into account the number of receptors expressed. Interestingly, DT‐induced 22Na+ influx was weak in DTR‐EGFR cells and not detectable in DTR‐GPI cells. Whole cell patch‐clamp analysis showed the DT at low pH induced depolarization and decreased input resistance in DTR cells (and to a lesser extent also in DTR‐EGFR cells) but not in DTR‐GPI cells. These results suggest that the transmembrane and cytoplasmic part of the receptor might be involved in channel activity and that translocation of the A fragment is independent of toxin‐induced cation channel activity.

Receptor‐dependent translocation of diphtheria toxin across the surface membrane of Vero cells was studied using patch clamp techniques. Translocation was induced by exposing cells with surface‐bound toxin to low pH. Whole cell current and voltage clamp recordings showed that toxin translocation was associated with membrane depolarization and increased membrane conductance. The conductance increase was voltage independent, with a reversal potential of approximately 15 mV. This value was unaffected by changing the Cl‐ gradient across the membrane and microfluorometric measurements showed that the cytosolic Ca2+ concentration was only marginally elevated by the translocation. The conductance increase is thus mainly due to monovalent cations. Exposing outside‐out and cell‐attached patches with bound toxin to low pH induced a new type of ion channel in the membrane. The channel current was inward at negative membrane potentials and the single channel conductance was approximately 30 pS. This value is about three times larger than for receptor‐independent channels induced by diphtheria toxin or toxin fragments in artificial lipid membranes.

We have developed a new tool for studying the role of rho in actin stress fibre formation. Clostridium botulinum exoenzyme C3 which affects actin microfilament assembly by ADP‐ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT). The resulting chimeric toxins were tested on Vero cells. Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT. Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification. In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin. The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol. DC3B inhibited the bacterially induced microfilament assembly indicating that L. monocytogenes utilizes a cellular rho dependent mechanism in this process.

We have developed a system to study the interactions of diphtheria toxin with the cell surface using non‐toxic mutant proteins synthesized in vitro. Proteins obtained by N‐terminal deletions containing the whole B fragment bound strongly to cells. Deletions extending into the B fragment did not yield an autonomous binding domain. Loss of only the N‐terminal 3 kd of the B fragment significantly impaired the ability to recognize the receptor. This, together with previous reports that the C‐terminal end of the B fragment is required for binding, suggests that both ends of the B fragment are necessary for receptor recognition. Receptor bound diphtheria toxin undergoes a conformational change at pH less than 5.3 that results in translocation of the A fragment to the cytosol and the appearance of a B fragment‐derived 25 kd polypeptide (P25) resistant to externally applied protease. Only the B fragment was required for generation of P25. N‐terminal deletions of 130 amino acids or more resulted in proteins that gave rise to P25 at higher pH than full length toxin. Furthermore, a second protease‐inaccessible polypeptide of 18 kd (P18) was observed.

Diphtheria toxin B‐fragment binds to cell‐surface receptors and facilitates translocation of the enzymatically active A‐fragment to the cytosol. In this process the B‐fragment inserts into the plasma membrane and induces formation of cation‐selective channels. We examined the ability of a number of diphtheria toxin‐derived molecules translated in vitro to permeabilize cells. Two proteins consisting of the whole B‐fragment and small parts of the A‐fragment, and one protein comprising most of the B‐fragment alone, were more efficient than full‐length toxin in permeabilizing the plasma membrane to monovalent cations. Two shorter B‐fragment‐derived proteins, with 3 and 10 kd N‐terminal deletions, permeabilized the cells to sulfate and sucrose in addition to monovalent cations. The relationship between channel formation and toxin translocation is discussed.

HeLa S3 cells were protected against infection by poliovirus type I by the presence of monensin and N,N'‐dicyclohexylcarbodiimide (DCCD), compounds elevating the pH of acidic intracellular compartments. The protection was fully overcome by exposing the cells to pH 5.5 and lower, and at approximately pH 6.1 it was reduced by half. Measurements of the ability of the virus to enter the detergent phase under conditions where Triton X‐114 was separated from water indicated that the virus is hydrophilic at neutral pH, and that it exposes hydrophobic regions at low pH. When the cells were pretreated with acetic acid, which reduces the intracellular pH, virus entry was inhibited, indicating that a pH gradient across the membrane is necessary for infection. Under all conditions which induced infection, the virus particles were altered to more slowly sedimenting material. Also, virus bound to aldehyde‐fixed cells was altered when exposed to low pH at 37 degrees C. The data indicate that poliovirus bound to receptors on cells exposes hydrophobic regions at low pH, and that at physiological temperature it undergoes alteration. This alteration may be a necessary, but not sufficient requirement for infection.

A fusion protein of acidic fibroblast growth factor and diphtheria toxin A‐fragment was disulfide‐linked to the toxin B‐fragment. The complex bound specifically to diphtheria toxin receptors, and subsequent exposure to low pH induced the fusion protein to translocate to the cytosol. Heparin, inositol hexaphosphate and inorganic sulfate strongly increased the trypsin resistance of the growth factor part of the fusion protein, indicating tight folding, and they prevented translocation of the fusion protein to the cytosol. The data indicate that only a more disordered form of the growth factor is translocation competent.

Active diphtheria toxin consists of two fragments, A and B, joined by a disulfide bond. The B fragment binds to cell surface receptors and aids in the translocation of the enzymatically active A fragment to the cytosol. Normally, the toxin A fragment enters the cytosol from acidic endosomes, but translocation can also be induced at the level of the plasma membrane by exposing cells with surface-bound toxin to low pH. Recently, we showed that disulfide bonds introduced into the A fragment by mutation are inhibitory for translocation. In the present work, we found that although the complete translocation of the A fragment is blocked, three mutant toxins underwent reduction of the interfragment disulfide bond upon low pH exposure, whereas the internal disulfide in the A fragment remained intact. In the case of two of these mutants, the A fragment was released into the extracellular medium upon exposure of cell-bound toxin to low pH. The pH profile for the release of the mutant A fragments was the same as for translocation of wild-type A fragment to the cytosol, and the release was inhibited by conditions that interfere with A fragment translocation. In the case of the third mutant, which remained cell-associated upon reduction of the interfragment disulfide bond, a translocation intermediate was detected. The results show that the reduction of the interfragment disulfide bond can occur in the absence of complete translocation of the A fragment to the cytosol, and they indicate that the reduction takes place at an early stage in the translocation process. Our findings suggest that the translocation of the A fragment across the membrane is initiated at the C terminus.

Abstract.: Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel, and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin, Δg m , was initially determined. DT-AB induced a ∼10-fold lower Δg m than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Δg m , whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Δg m . Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches. No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as Δg m and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel.

The effects of retinoic acid and 12-O-tetradecanoylphorbol 13-acetate on the sensitivities of a number of cell lines to the toxins modeccin, abrin, ricin and diphtheria toxin were studied. Retinoic acid and some other retinoids were found to protect a number of the cell lines against the toxins. HeLa cells that were protected bound much more retinoic acid than L-cells that were not protected. The tumour promoter 12-O-tetradecanoylphorbol 13-acetate was found to increase the sensitivity of cells to abrin, ricin and modeccin in the absence as well as in the presence of retinoic acid. Neither retinoic acid nor 12-O-tetradecanoylphorbol 13-acetate affected the extent of binding and pinocytotic uptake of toxins by the cells. Apparently retinoic acid and 12-O-tetradecanoylphorbol 13-acetate interfere with the entry of the toxins through the cell membrane.

Radioimmunoassays for abrin and ricin are described. There is little cross-reactivity between the two toxins. The procedures described are capable of determining blood concentrations down to 50-100 pg/ml, permitting identification of abrin and ricin poisoning and monitoring of the blood concentrations in cancer patients treated with these agents.

Acidic fibroblast growth factor (aFGF) is a potent mitogen. It acts through activation of specific cell surface receptors leading to intracellular tyrosine phosphorylation cascades, but several reports also indicate that aFGF enters cells and that it has an intracellular function as well. The aFGF(K132E) mutant binds to and activates fibroblast growth factor receptors equally strongly as the wild-type, but it is a poor mitogen. We demonstrate that aFGF(K132E) enters NIH 3T3 cells and is transported to the nuclear fraction like wild-type aFGF. A fusion protein of aFGF(K132E) and diphtheria toxin A-fragment (aFGF(K132E)-DT-A) and a similar fusion protein containing wild-type aFGF (aFGF-DT-A) were reconstituted with diphtheria toxin B-fragment. Both fusion proteins were translocated to the cytosol by the diphtheria toxin pathway and subsequently recovered from the nuclear fraction. Whereas translocation of aFGF-DT-A stimulated DNA synthesis in U2OSDR1 cells lacking functional fibroblast growth factor receptors, aFGF(K132E)-DT-A did not. The mutation disrupts a protein kinase C phosphorylation site in the growth factor making it unable to be phosphorylated. The data indicate that a defect in the intracellular action of aFGF(K132E) is the reason for its strongly reduced mitogenicity, possibly due to inability to be phosphorylated.

Shiga toxin consists of an enzymatically active A-chain and a pentameric binding subunit. The A-chain has a trypsin-sensitive region, and upon cleavage two disulfide bonded fragments, A1 and A2, are generated. To study the role of the disulfide bond, it was eliminated by mutating cysteine 242 to serine. In T47D cells this mutated toxin was more toxic than wild type toxin after a short incubation, whereas after longer incubation times wild type toxin was most toxic. Cells cleaved not only wild type but also mutated A-chain into A1 and A2 fragments. The mutated A-chain was more sensitive than wild type toxin to Pronase, and it was degraded at a higher rate in T47D cells. Subcellular fractionation demonstrated transport of both wild type and mutated toxin to the Golgi apparatus. Brefeldin A, which disrupts the Golgi apparatus, protected not only against Shiga toxin but also against the mutated toxin, indicating involvement of the Golgi apparatus. After prebinding of Shiga(C242S) toxin to wells coated with the Shiga toxin receptor, Gb3, trypsin treatment induced dissociation of A1 from the toxin-receptor complex demonstrating that in addition to stabilizing the A-chain, the disulfide bond prevents dissociation of the A1 fragment from the toxin-receptor complex.

Ion fluxes associated with translocation of diphtheria toxin across the surface membrane of Vero cells were studied. When cells with surface-bound toxin were exposed to low pH to induce toxin entry, the cells became permeable to Na+, K+, H+, choline+, and glucosamine+. There was no increased permeability to Cl-, SO4(-2), glucose, or sucrose, whereas the uptake of 45Ca2+ was slightly increased. The influx of Ca2+, which appears to be different from that of monovalent cations, was reduced by several inhibitors of anion transport and by verapamil, Mn2+, Co2+, and Ca2+, but not by Mg2+. The toxin-induced fluxes of N+, K+, and protons were inhibited by Cd2+. Cd2+ also protected the cells against intoxication by diphtheria toxin, suggesting that the open cation-selective channel is required for toxin translocation. The involvement of the toxin receptor is discussed.

Preincubation of Vero cells with 1 microM phorbol 12-myristate 13-acetate (PMA) decreased the specific binding of diphtheria toxin by about 50%, whereas the toxic effect, endocytic uptake and membrane translocation were completely blocked. Toxin bound to PMA-treated cells was released upon incubation with heparinase. The effect of PMA was abrogated in the presence of EDTA or N-(DL-[2-(hydroxyaminocarbonyl)methyl]-4-methyl-pentanoyl)-L-3-(2′ - naphthyl)-alanyl-L-alanine 2-aminoethyl-amide (TAPI), a specific inhibitor of matrix metalloproteases. The results indicate that PMA induces proteolytic cleavage of the diphtheria-toxin receptor [heparin-binding EGF-like growth factor (HB-EGF)-precursor] outside the membrane anchor, and that about 50% of the growth-factor ecto-domain remains associated with the cells, due to binding to surface proteoglycans containing heparan sulphates. Although the cleaved cell-associated HB-EGF binds diphtheria toxin, it does not serve as a functional receptor, since neither toxin internalization nor translocation occurs. Thus the intact HB-EGF precursor is of crucial importance for its function as the diphtheria-toxin receptor.

We have developed a system that makes it possible to study the fate of a foreign polypeptide that is inserted in the plasma membrane. Diphtheria toxin is a bacterial protein toxin that, upon acidification, has the ability to insert into the plasma membrane from the outside of eukaryotic cells. We present results that indicate endocytic uptake and degradation of the diphtheria toxin B-fragment after insertion into the membrane of Vero cells. The degradation rate of the fragment was found to be very high (t1/2 = 6 min) and dependent on cleavage of the extracellular part of the polypeptide with protease. Degradation was strongly inhibited in ATP-depleted cells, as well as at temperatures below 18 degrees C, and it was partially inhibited when the cytosol was acidified to block endocytosis from clathrin-coated pits. Degradation was also reduced in the presence of NH4Cl. The results indicate that the inserted and cleaved B-fragment is degraded by a process requiring endocytosis and transport to late endosomes or to lysosomes.

Four nonsteroidal anti-inflammatory analgesic drugs acetylsalicylic acid (aspirin), salicylic acid, indomethacin and piroxicam were tested for their action on chloride/bicarbonate exchange by anion antiport and on the regulation of cytosolic pH (pHi) in Vero cells. The Na+-independent Cl-/HCO3- exchange regulates pHi back to normal after alkalinization and is therefore in a state of high activity at alkaline pHi, whereas it is in a state of low activity when pHi is below neutrality. Preincubation with the drugs at low pH strongly increased the activity of the Na+-independent Cl-/HCO3- exchange, whereas at high pH the antiport was inhibited. The Na+-dependent Cl-/HCO3- exchange, which is most active at low pHi and normalize pHi after acidification, was inhibited after preincubation with the drugs. In cells that had been incubated with the drugs, pHi was 0.1 to 0.4 pH units lower than in cells that had been incubated in the absence of drugs. The prostaglandins E2, F2 and I2 had little effect on anion antiport, and did not counteract the effects of the anti-inflammatory drugs on Cl-/HCO3- exchange, indicating that these actions of the drugs are not due to the inhibition of prostaglandin synthesis. The relevance of our findings to the clinical effects of these drugs is discussed.

Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protected only when it was added within a short period of time after modeccin. Compounds that increase the pH of intracellular vesicles, protected even when added after 2 h, indicating that modeccin remains inside vesicles for a considerable period of time before it enters the cytosol. After addition of diphtheria toxin to the cells, compounds that increase the pH of intracellular vesicles protected only approximately to the same extent as antitoxin. This indicates that after endocytosis diphtheria toxin rapidly enters the cytosol. At 20 degrees C, the cells were more strongly protected against modeccin than against diphtheria toxin. The residual toxic effect of diphtheria toxin at 20 degrees C could be blocked with NH4Cl whereas this was not the case with modeccin. This indicates that at 20 degrees C the uptake of diphtheria toxin occurs by the normal route, whereas the uptake of modeccin occurs by a less efficient route than that dominating at 37 degrees C. The results indicate that after endocytosis diphtheria toxin rapidly enters the cytosol from early endosomes with low pH (receptosomes). Modeccin enters the cytosol much more slowly, possibly after fusion of the endocytic vesicles with another compartment.

Volkensin, a highly toxic protein from the roots of Adenia volkensii (kilyambiti, kinoria), was purified by affinity chromatography on acid-treated Sepharose 6B. The toxin is a glycoprotein (Mr 62,000, neutral sugar content 5.74%) consisting of an A subunit (Mr 29,000) and of a B subunit (Mr 36,000) linked by disulfide and noncovalent bond(s). The amino acid, amino sugar, and neutral sugar composition of the protein were determined. Volkensin is a galactose-specific lectin and is a potent inhibitor of eukaryotic protein synthesis in whole cells as well as in a cell-free system (a rabbit reticulocyte lysate). The inhibitory and the lectin activities are functions of the A and B subunits, respectively. Volkensin can be included amongst the ricin-like toxins and resembles most closely modeccin, the toxin of Adenia digitata.

Diphtheria toxin contains a trypsin-sensitive region with 3 closely spaced arginines in the sequence (Asn189, Arg190, Val191, Arg192, Arg193, Ser194). Cleavage of the toxin to yield A- and B-fragments (“nicking”) appears to occur in a stochastic manner after either of these arginine residues. Isoelectric focusing of A-fragment prepared in vitroshowed four bands of varying intensity with pI between 4.5 and 5.0, three of which could be accounted for by the three different cleavage sites. Exposure of cells with surface-bound toxin to pH < 5.3 induces translocation of A-fragment to a position where it is shielded from external Pronase, presumably in the cytosol. A-fragment translocated in this manner had the same pI as the most acidic A-fragments, indicating that only A-fragments lacking both Arg192and Arg193are translocation-competent. This was confirmed by amino acid sequencing. Treatment of Afragment with carboxypeptidase B eliminated the two bands with the highest pI while there was a concomitant increase in the bands corresponding to the two most acidic A-fragments. Such treatment of nicked diphtheria toxin increased the amount of translocated A-fragment and the ability of toxin to form cationselective pores in the cell membrane. The site of trypsin cleavage therefore appears to be one of the factors limiting toxin entry to the cytosol.

Receptor-mediated endocytosis and intracellular routing of native ricin, and of ricin conjugated to colloidal gold (Ri-Au) and to horseradish peroxidase (Ri-HRP), have been studied in cultured MCF-7 and Vero cells by electron microscopical techniques including serial section analysis. Both native ricin, as demonstrated by immunoperoxidase cytochemistry, and the ricin conjugates were internalized via a common coated pit-coated vesicle pathway to reach vacuolar and tubulo-vesicular portions of the endosomal system. In addition, native ricin and a purified monovalent fraction of Ri-HRP reached distinct Golgi cisterns, whereas Ri-Au and polyvalent Ri-HRP did not. The results delineate intracellular routing of native ricin and compare it with the routing of different ricin conjugates. Moreover, our study shows that conjugates of a particular ligand (ricin) and various probes (e.g., gold and peroxidase), may be handled differently by cells. Sorting apparently takes place in the endosomal system, allowing some but not other molecules to reach Golgi elements. This sorting seems to depend on the valency of the ricin conjugate.

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.

When 125I-labeled nicked diphtheria toxin bound to Vero cells was exposed to pH less than 5.0, a small fraction was reduced to yield A- and B-fragments. The pH required for reduction correlates well with that required to induce intoxication, and the amount of A-fragment released was of the same order as that required to intoxicate the cells. Conditions that protect cells against intoxication, such as acidification of the cytosol, treatment with anion transport inhibitors, or treatment with anti-diphtheria toxin antibodies, prevented the reduction of the interfragment disulfide in cell-bound toxin. In vitro, thioredoxin reduced nicked diphtheria toxin only at pH 5.0 and lower, and the reduction was inhibited by anti-toxin antibodies. This indicates that a conformational change in the toxin, necessary for reduction by the thioredoxin system, is prevented by the antibodies. Reduction by glutathione and cysteine was most efficient at neutral pH and was not inhibited by anti-toxin. The results are consistent with the possibility that cell-mediated reduction of the interfragment disulfide is a measure of the entry of fragment A into the cytosol.

A toxic protein, viscumin, was isolated from extracts of mistletoe by affinity chromatography on acid-treated Sepharose 4B. Viscumin was selectively bound to the column and could be eluted with lactose. It migrated in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate corresponding to Mr = 60,000. In addition, two bands migrating corresponding to Mr = 29,000 and 32,000 were found. After treatment with 2-mercaptoethanol, only 2 bands (Mr = 29,000 and 34,000) were found. Apparently, viscumin consists of two chains which, in some of the molecules, are disulfide-linked. Protection experiments with antiserum against viscumin indicated that the major part of the cytotoxic activity in mistletoe extracts is due to viscumin. Gel filtration experiments on Sephacryl 200 indicated that, at low concentrations, viscumin occurs as a monomer and at higher concentrations as a dimer. Viscumin was found to inhibit protein synthesis in cell-free systems. When the two constituent peptide chains of viscumin were eluted from polyacrylamide gels and tested for ability to inhibit cell-free protein synthesis, this property was found to be associated with the fastest migrating chain, here denoted the A chain. The heavier chain was denoted the B chain. The A chain was found to inhibit protein synthesis by inactivating the ribosomes catalytically. Reconstitution experiments with isolated ribosomal subunits from untreated and A chain-treated ribosomes showed that the 60 S ribosomal subunit was selectively inactivated.

A toxin from mistletoe, viscumin, inhibited the incorporation of leucine in cells more rapidly than the incorporation of uridine and thymidine, indicating that the toxins act by inhibiting cellular protein synthesis. The presence of galactose, lactose, and melibiose in the medium protected cells against viscumin. The sensitivity to viscumin of different cell lines differed considerably. The cytotoxic properties of viscumin were compared to those of the related toxins abrin, ricin, and modeccin. The profile of the sensitivity of a panel of cell lines to viscumin was dissimilar from those obtained with abrin and modeccin. Cell lines selected for resistance to modeccin and ricin were fully sensitive to viscumin. Ca2+ was required for viscumin to express its toxic effect. In contrast, the Ca2+ ionophore A23187 protected the cells against viscumin. Whereas the sensitivity of cells to viscumin did not vary much between pH 7 and 9, the cells were much less sensitive at pH 6. Also, cells treated with the two metabolic inhibitors, 2-deoxyglucose and NaN3, were insensitive to the toxins. Viscumin taken up by cells in the absence of Ca2+ or at pH 6 was able to intoxicate the cells when Ca2+ was added or when the pH was adjusted to neutrality. In contrast, cells exposed to viscumin in the presence of 2-deoxyglucose and NaN3, which inhibit endocytosis, were not intoxicated when the cells were treated with antiviscumin and then transferred to normal medium. The results indicate that endocytosis is involved in the entry of viscumin.

A human melanoma cell line with unusually high growth potential was established from a xenograft growing in athymic mice. When xenograft fragments were cultured in vitro, melanoma cells grew out rapidly without any contamination of mouse stromal cells. An established cell line, FME, derived from this tumour, grew both in monolayer and in shaker suspension culture with doubling times of about 20 h. The cells grew easily at low serum concentrations and could even be cultured in serum-free medium supplemented with insulin and transferrin. The cultured cells were hyperdiploid, as were the cells of the xenograft. The cells grew easily in soft agar and formed tumours in athymic mice. When growing exponentially, the cells were almost unpigmented, but when grown to high density, their melanin content increased. Upon treatment with dimethyl sulphoxide (DMSO), retinoic acid and theophylline, as well as with the tumour promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), the cells showed growth inhibition and increased melanin synthesis.

When Vero cells with surface-bound 125I-labeled, nicked diphtheria toxin were exposed to pH 4.5, two polypeptides of Mr 20,000 and 25,000 became protected against externally applied Pronase E. The 20-kDa polypeptide appears to be the toxin A-fragment, whereas the 25-kDa polypeptide must be derived from the B-fragment. Permeabilization of the cells with saponin allowed efflux of the 20-kDa fragment to occur, whereas most of the 25-kDa polypeptide remained associated with the cells. A number of compounds and conditions which protect cells against diphtheria toxin prevented the protection against Pronase E. Protection of the 25-kDa polypeptide occurred even when the transmembrane proton gradient (delta pH) was dissipated by acidification of the cytosol, whereas protection and release of the A-fragment were prevented under these conditions. Electrical depolarization and ATP depletion of the cells did not inhibit protection and release of the A-fragment. The data indicate that delta pH is required for the transfer of the A-fragment to the cytosol, whereas the insertion of part of the B-fragment into the membrane occurs at low pH, even in the absence of a delta pH.

The rate of shigella toxin entry into the cytosol of HeLa S3 cells was estimated from the toxin-induced reduction in protein synthesis. Whereas high toxin concentrations strongly reduced protein synthesis within 30 min, lower concentrations required longer times. The major part of the cell-bound toxin entered only after several hours. Toxin entered cells after incubation at 25 degrees C but not at 20 degrees C, although toxin binding was the same at the two temperatures. Increasing the KCl concentration to 0.2 M protected against toxin. The toxin entry was strongly reduced when the level of ATP in the cells was reduced by incubation with metabolic inhibitors. Lysosomotrophic agents such as NH4Cl and chloroquine had little or no protective effect, but the protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone and carbonyl cyanide m-chlorophenylhydrazone and the ionophore monensin protected cells against the toxin. Cells were also protected when the pH was reduced to 6.4. The entry of shigella toxin is discussed in relation to that of other protein toxins with intracellular sites of action.

Previous work has shown that when Vero cells with surface-bound diphtheria toxin are exposed to low pH, toxin entry across the plasma membrane is induced and that this entry involves two steps, insertion of the B-fragment of the toxin into the membrane and translocation of the enzymatically active A-fragment to the cytosol. Here we have studied the role of permeant anions in this process. It was found that when the B-fragment was inserted into the membrane, part of it, a 25-kDa polypeptide, was shielded from externally added Pronase. This insertion did not require permeant anions. The translocation of the A-fragment was monitored by measuring either its ability to inhibit protein synthesis in the cells or the appearance of radioactively labeled 21-kDa fragment after treatment of the cells with externally applied Pronase. The translocation of the A-fragment was dependent on the presence of permeant anions in the medium. However, when the cells were depleted of Cl−by incubation in Cl−free buffer at high pH, translocation of the A-fragment did not require permeant anions in the medium. The possibility that translocation of the A-fragment is inhibited by an outward directed chloride gradient rather than by the absence of chloride is discussed.

The toxicity of abrin, modeccin, and ricin to Vero cells was maximal at neutral and slightly alkaline pH, and it was strongly reduced at pH 6.0 and below. Diphtheria toxin was most toxic at low pH. Binding and endocytosis of abrin, modeccin, and ricin did not vary much within the pH range tested. High concentrations of the carboxylic ionophore Br-X-537A, protected against all four toxins. Combined treatment of cells with an inhibitor of glycolysis and an uncoupler of oxidative phosphorylation strongly inhibited endocytosis of toxins and protected against intoxication. The protective effect of Ca2+ deprivation, of pH 6.0, and of metabolic inhibitors disappeared soon after transfer of the cells to normal medium, whereas the protective effect of Br-X-537A and of trifluoperazine disappeared slowly. The decay rate of the protection by NH4Cl and by the ionophore A23187 differed with the different toxins. Cells exposed to abrin, modeccin, and ricin under protective conditions which did not inhibit endocytosis of the toxins (Ca2+ deprivation, pH 6.0, Br-X-537A), and then treated with antitoxins to inactivate extracellular toxin, were intoxicated when the protection was released. In contrast, cells exposed to toxins while endocytosis was arrested by treatment with metabolic inhibitors were not intoxicated when antitoxins were added and the metabolic inhibitors removed. Modeccin and diphtheria toxin endocytosed in the presence of trifluoperazine and NH4Cl were unable to intoxicate cells. The possibility that endocytosis is a step in the normal entry route of the toxins is discussed.

In the absence of Ca2+ cells were not sensitive to the toxic proteins abrin and modeccin and the sensitivity to ricin and diphtheria toxin was reduced. Calcium deprivation had little effect on the binding and endocytosis of abrin, modeccin, and ricin. The binding of diphtheria toxin to cells was, however, reduced, Verapamil and Co2+ inhibited 45Ca2+ uptake and protected cells against abrin and modeccin at low concentrations of Ca2+. At higher Ca2+ concentrations the protection was overcome. La3+ inhibited strongly 45Ca2+ uptake and protected well against all four toxins, even at high Ca2+ concentrations. Fe3+ also afforded protection although it did not inhibit Ca2+ uptake. The Ca2+ ionophore, A23187, which strongly increases the uptake of 45Ca2+, protected cells well against abrin and modeccin, slightly against diphtheria toxin, but not against ricin. Both Ca2+ deprivation and treatment with A23187 protected well against the hybrid toxin abrin A-chain/ricin B-chain. Such treatment afforded little protection against the hybrid ricin A-chain/abrin B-chain. Apparently the protection against abrin is associated with its A-chain. The calmodulin inhibitor, trifluoperazine, protected strongly against modeccin and diphtheria toxin. The data indicate that Ca2+ is involved in the entry mechanism for abrin, modeccin, and ricin, possibly as a Ca2+ flux together with the toxins.

A highly toxic conjugate of ricin A-chain and diphtheria toxin fragment B was prepared by disulfide exchange reaction. A similar conjugate between diphtheria toxin fragment A and ricin B-chain was nontoxic. Like native diphtheria toxin, the conjugate ricin A/diphtheria toxin B was much more toxic to Vero than to HeLa cells. Ricin was equally toxic to these cell lines. Lactose, which inhibits ricin binding, did not protect against the conjugate. Cells resistant to ricin, partly due to a reduced number of ricin-binding sites, were fully sensitive to the conjugate, indicating that the conjugate binds to diphtheria toxin receptors. The conjugate was fully toxic to two Vero cell mutants, resistant to diphtheria toxin because the elongation factor 2 could not be ADP-ribosylated by the diphtheria toxin A-fragment. Therefore, the inhibition of protein synthesis by the conjugate must be caused by the ricin A-chain. Ammonium chloride which prevents entry of diphtheria toxin, but not of ricin, also protected against the conjugate. Like diphtheria toxin, the conjugate was most toxic at low pH, whereas ricin is most active at pH above neutrality and inactive at low pH. The results indicate that the conjugate ricin A/diphtheria toxin B binds to diphtheria toxin receptors and inhibits cellular protein synthesis due to the action of ricin A-chain which appears to enter the cell by the diphtheria toxin pathway.

The binding domain (R domain) of diphtheria toxin as defined from the recently published crystal structure [Choe, Bennett, Fujii, Curmi, Kantardjieff, Collier and Eisenberg (1992) Nature (London) 357, 216-222] was subcloned. The 17 kDa peptide containing amino acids 378-535 from fragment B of diphtheria toxin preceded by the tripeptide Met-His-Gly bound specifically and with high affinity to diphtheria-toxin receptors. It efficiently inhibited the toxicity of full-length toxin. The binding domain entered the detergent phase of Triton X-114 at pH values below 6, indicating that it exposed hydrophobic regions at acidic pH.

Natural diphtheria toxin is synthesized as a single polypeptide chain that is activated by cleavage into an A- and a B-fragment, which are linked by a disulphide bond. In the present work the ability of independently translated A- and B-fragments to associate was investigated. Low amounts of A- and B-fragments synthesized in vitro were mixed under conditions that allowed formation of a disulphide bridge between the fragments. Under these conditions toxin was reconstituted in close to 100% yield and found to be as toxic to Vero cells as natural diphtheria toxin. Efficient association between the A- and B-fragment was dependent on the formation of a disulphide bridge. Reconstituted toxin obtained from one [35S]methionine-labelled fragment and one unlabelled fragment proved useful in translocation studies. Addition of a number of different polypeptides to the N- and C-termini of either fragment did not, in most cases, prevent reconstitution. The ready reconstitution allows easy manipulations with the toxin to form targeted molecules and to develop diphtheria toxin as a vector for translocation of peptides to the cytosol. The fact that the reconstituted toxin does not need to be nicked with proteinases to be active allows experimentation with proteinase-sensitive constructs.

The effect of dimethyl sulphoxide and other cryoprotective compounds on the sensitivity of cells to polypeptide toxins and to poliovirus was tested. In the presence of these compounds, which all affect membrane fluidity, the cells were protected against the toxic proteins and against poliovirus. The large protection obtained was not due to reduced binding and endocytosis of the toxins. Apparently, the cryoprotective compounds interfere with the entry of toxins and of the poliovirus genome across the cell membrane.

The effect of a cytotoxin isolated from Shigella shigae has been tested on different cell lines. HeLa S3 cells, as well as some other human carcinoma cells, were killed by picomolar to femtomolar concentrations of the pure toxin, whereas certain other human carcinoma cells and a variety of non-epithelial cells from human tissue and from various animal tissues were resistant to nanomolar concentrations of the toxin. Binding studies with 125I-labelled Shigella shigae cytotoxin showed that the sensitive HeLa S3 cells contain 1.3 × 10 binding sites per cell, whereas in an insensitive HeLa cell line 2.6 × 10 sites per cell were measured. In all cases the apparent association constant, ka, was found to be about 1010 M-1. The binding occurred fairly rapidly, whereas dissociation of bound toxin occurred at a very slow rate, even in the presence of excess unlabelled toxin. All toxin sensitive cell lines bound similar amounts of toxin as HeLa S3 cells, whereas some of the resistant cell lines did not contain measurable amounts of toxin receptors.

The diphtheria toxin receptor (DTR) has been identified as the precursor of heparin-binding epidermal growth factor-like growth factor, which may interact with other membrane proteins to form the functional receptor. To test if mammalian DTR is able to confer toxin sensitivity onto phylogenetically distant cells, we expressed monkey DTR in the baculovirus system and tested infected insect cells for toxin sensitivity. cDNA encoding an epitope-tagged heparin-binding epidermal growth factor-like growth factor precursor (DTRB3) was inserted into the virus genome by allelic replacement to construct the recombinant virus vAc-DTRB3. SF9 cells infected with vAc-DTRB3 expressed functional DTR, which could be precipitated from the solubilized membrane fraction of infected cells with Sepharose-immobilized diphtheria toxin. The highest level of expression (about 5 x 10(6) receptors/cell) was observed 48 h after infection, at which time the infected cells were highly sensitive to diphtheria toxin. Uninfected SF9 cells and cells infected with the wild type virus were resistant to the toxin. The presence of heparin increased both the binding and the toxin sensitivity of vAc-DTRB3-infected SF9 cells. Translocation of toxin A fragment was induced when cells with surface-bound toxin were exposed to low pH, and the translocation was optimal at pH < or = 5.5. It was approximately 100 times more efficient at 24 degrees C than at 4 degrees C. The data indicate that monkey DTR is fully functional when expressed in insect cells.

For diphtheria toxin to be cytotoxic, the enzymatically active part (fragment A) must be translocated to the cytosol. We here demonstrate that additional proteins linked as N-terminal extensions can be translocated along with fragment A across the plasma membrane of toxin-sensitive cells. Thus, an extra fragment A of diphtheria toxin and some of apolipoprotein AI were translocated as passenger proteins along with mutant diphtheria toxin fragment A. Translocation was monitored by the cytotoxic effect of the additional fragment A as well as by the translocation of [35S]methionine-labelled protein to a compartment protected from externally added pronase. Cytotoxicity experiments indicated that double A fragments can also be translocated across the membrane of intracellular vesicles. The results demonstrate that the translocation apparatus used for toxin translocation is not limited to a single A fragment but can accommodate additional proteins as well. The fact that proteins as large as 20 kDa can be brought into cells by way of diphtheria toxin under both in vitro and in vivo conditions opens up the possibility of using diphtheria toxin mutants for introducing molecules with biological activity into cells.

The effects of acidification of the cytosol and of electrical depolarization on the entry of diphtheria toxin were studied. Entry of the toxin from the cell surface was induced by low pH, and the presence of the toxin in the cytosol was monitored as toxin-induced inhibition of protein synthesis. To reduce the membrane potential the cells were incubated in a buffer containing a high concentration of potassium. The cytosol was acidified either by incubating the cells with acetic acid, by incubating them with ammonium chloride which was subsequently removed in the presence of amiloride to prevent pH regulation by the Na+/H+ exchanger, or by incubating the cells in isotonic KCl in the presence of nigericin and valinomycin. The results showed that when the cytosol was acidified by either method toxin entry was inhibited, while a reduction in the membrane potential did not strongly interfere with the entry. A pH gradient across the membrane of at least 1 pH unit was required for entry. Possibly this gradient acts as a driving force for diphtheria toxin entry.

U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.

Sodium-linked and sodium-independent HCO3-/Cl- antiport was measured under different conditions in a number of cell lines. Transport of HCO3- was estimated from its effect on intracellular pH (pHi) measured with the fluorescent probe 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein. The associated ion fluxes were estimated from the transport of 36Cl- and 22Na+. Na+-dependent and Na+-independent HCO3-/Cl- antiport were found in many, but not in all cell lines tested. The Na+-independent HCO3-/Cl- antiport was found to be highly pHi-dependent in a number of cell lines, whereas in others this was not the case. Some cell lines were found to have both Na+-dependent and Na+-independent HCO3-/Cl- antiport, whereas in others we could detect only one of these mechanisms. Na+/H+ antiport, which is quantitatively the most important H+-extruding mechanism, was found in all cell lines tested, but the activity varied strongly. Possible reasons for the qualitative and quantitative differences in antiport activity are discussed.

The role of specific receptors in the translocation of diphtheria toxin A fragment to the cytosol and for the insertion of the B fragment into the cell membrane was studied. To induce nonspecific binding to cells, toxin was either added at low pH, or biotinylated toxin was added at neutral pH to cells that had been treated with avidin. In both cases large amounts of diphtheria toxin became associated with the cells, but there was no increase in the toxic effect. There was also no increase in the amount of A fragment that was translocated to the cytosol, as estimated from protection against externally added Pronase E. In cells where specific binding was abolished by treatment with 12-O-tetradecanoyl-phorbol 13-acetate, trypsin, or 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, unspecific binding did not induce intoxication or protection against protease. This was also the case in untreated L cells, which showed no specific binding of the toxin. When Vero cells with diphtheria toxin bound to specific receptors were exposed to low pH, the cells were permeabilized to K+, whereas this was not the case when the toxin was bound nonspecifically at low pH or via avidin-biotin. The data indicate that the cell-surface receptor for diphtheria toxin facilitates both insertion of the B fragment into the cell membrane and translocation of the A fragment to the cytosol.