Endosome to Golgi Transport of Ricin Is Independent of Clathrin and of the Rab9- and Rab11-GTPases

The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.     

Dependence of ricin toxicity on translocation of the toxin A-chain from the endoplasmic reticulum to the cytosol

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.     

Endosome-to-Golgi transport is regulated by protein kinase A type II alpha

Studies of RII alpha-deficient B lymphoid cells and stable transfectants expressing the type II alpha regulatory subunit (RII alpha) of cAMP-dependent protein kinase (PKA), which is targeted to the Golgi-centrosomal area, reveal that the presence of a Golgi-associated pool of PKA type II alpha mediates a change in intracellular transport of the plant toxin ricin. The transport of ricin from endosomes to the Golgi apparatus, measured as sulfation of a modified ricin (ricin sulf-1), increased in RII alpha-expressing cells when PKA was activated. However, not only endosome-to-Golgi transport, but also retrograde ricin transport to the endoplasmic reticulum (ER), measured as sulfation and N-glycosylation of another modified ricin (ricin sulf-2), seemed to be increased in cells expressing RII alpha in the presence of a cAMP analog, 8-(4-chlorophenylthio)-cAMP. Thus, PKA type II alpha seems to be involved in both endosome-to-Golgi and Golgi-to-ER transport. Because ricin, after being retrogradely transported to the ER, is translocated to the cytosol, where it inhibits protein synthesis, we also investigated the influence of RII alpha expression on ricin toxicity. In agreement with the other data obtained, 8-(4-chlorophenylthio)-cAMP and RII alpha were found to sensitize cells to ricin, indicating an increased transport of ricin to the cytosol. In conclusion, our results demonstrate that transport of ricin from endosomes to the Golgi apparatus and further to the ER is regulated by PKA type II alpha isozyme.     

Role of lipids in the retrograde pathway of ricin intoxication

The plant toxin ricin binds to both glycosphingolipids and glycoproteins with terminal galactose and is transported to the Golgi apparatus in a cholesterol-dependent manner. To explore the question of whether glycosphingolipid binding of ricin or glycosphingolipid synthesis is essential for transport of ricin from the plasma membrane to the Golgi apparatus, retrogradely to the endoplasmic reticulum or for translocation of the toxin to the cytosol, we have investigated the effect of ricin and the intracellular transport of this toxin in a glycosphingolipid-deficient mouse melanoma cell line (GM95), in the same cell line transfected with ceramide glucosyltransferase to restore glycosphingolipid synthesis (GM95-CGlcT-KKVK) and in the parental cell line (MEB4). Ricin transport to the Golgi apparatus was monitored by quantifying sulfation of a modified ricin molecule, and toxicity was studied by measuring protein synthesis. The data reveal that ricin is transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum and translocated to the cytosol equally well and apparently at the same rate in cells with and without glycosphingolipids. Importantly cholesterol depletion reduced endosome to Golgi transport of ricin even in cells without glycosphingolipids, demonstrating that cholesterol is required for Golgi transport of ricin bound to glycoproteins. The rate of retrograde transport of ricin was increased strongly by monensin and the lag time for intoxication was reduced both in cells with and in those without glycosphingolipids. In conclusion, neither glycosphingolipid synthesis nor binding of ricin to glycosphingolipids is essential for cholesterol-dependent retrograde transport of ricin. Binding of ricin to glycoproteins is sufficient for all transport steps required for ricin intoxication.     

Saporin and ricin A chain follow different intracellular routes to enter the cytosol of intoxicated cells

Several protein toxins, such as the potent plant toxin ricin, enter mammalian cells by endocytosis and undergo retrograde transport via the Golgi complex to reach the endoplasmic reticulum (ER). In this compartment the catalytic moieties exploit the ER-associated degradation (ERAD) pathway to reach their cytosolic targets. Bacterial toxins such as cholera toxin or Pseudomonas exotoxin A carry KDEL or KDEL-like C-terminal tetrapeptides for efficient delivery to the ER. Chimeric toxins containing monomeric plant ribosome-inactivating proteins linked to various targeting moieties are highly cytotoxic, but it remains unclear how these molecules travel within the target cell to reach cytosolic ribosomes. We investigated the intracellular pathways of saporin, a monomeric plant ribosome-inactivating protein that can enter cells by receptor-mediated endocytosis. Saporin toxicity was not affected by treatment with Brefeldin A or chloroquine, indicating that this toxin follows a Golgi-independent pathway to the cytosol and does not require a low pH for membrane translocation. In intoxicated Vero or HeLa cells, ricin but not saporin could be clearly visualized in the Golgi complex using immunofluorescence. The saporin signal was not evident in the Golgi, but was found to partially overlap with that of a late endosome/lysosome marker. Consistently, the toxicities of saporin or saporin-based targeted chimeric polypeptides were not enhanced by the addition of ER retrieval sequences. Thus, the intracellular movement of saporin differs from that followed by ricin and other protein toxins that rely on Golgi-mediated retrograde transport to reach their retrotranslocation site.     

Induction of direct endosome to endoplasmic reticulum transport in Chinese hamster ovary (CHO) cells (LdlF) with a temperature-sensitive defect in epsilon-coatomer protein (epsilon-COP)

In the present study we demonstrate that ricin, apparently without passing through the Golgi apparatus, reaches the endoplasmic reticulum (ER) and intoxicates cells in which the Golgi apparatus has been vesiculated by depletion of epsilon-COP, a subunit of COPI. LdlF cells contain a temperature-sensitive mutation in epsilon-COP. At the nonpermissive temperature epsilon-COP is degraded, and the Golgi apparatus undergoes a morphological change. To study ricin transport in these cells we used ricin sulf-2, a modified ricin molecule containing glycosylation and sulfation sites. Measurements of the incorporation of radioactive mannose into ricin sulf-2 showed that ricin reached the ER in cells depleted of epsilon-COP. Importantly, by investigating the glycosylation of ricin sulf-2 that was modified with radioactive sulfate in the trans-Golgi network, it was demonstrated that transport of ricin to the ER via the Golgi apparatus was severely inhibited. Moreover, we found that ricin was able to intoxicate ldlF cells depleted of epsilon-COP in the presence of brefeldin A. In contrast, control cells were completely protected against ricin by brefeldin A. In conclusion, our results suggest that in ldlF cells depleted of epsilon-COP ricin might be transported to the ER by an induced brefeldin A-resistant pathway that circumvents the Golgi apparatus.     

Retrograde transport of pertussis toxin in the mammalian cell

Pertussis toxin (PT), an AB5 exotoxin and important virulence factor of Bordetella pertussis , is hypothesized to traffic along a retrograde transport pathway in mammalian cells. This pathway includes endosomal uptake, transport to the Golgi complex and endoplasmic reticulum (ER), dissociation of the holotoxin in the ER and translocation of the A subunit (S1) to the cytosol, where it ADP-ribosylates its G protein targets. In this study, PT was visualized in the Golgi complex by immunofluorescence microscopy, but transport beyond the Golgi could not be detected by this method. To gain evidence for the retrograde pathway, peptide tags with target sites for tyrosine sulfation (a trans -Golgi network-specific activity) and N-glycosylation (an ER-specific activity) were added to either S1 or a B subunit (S4) of PT. Modified PT retained in vitro enzymatic and cellular activity as assessed by ADP-ribosylation assays. Peptide-tagged PT subunits were found to be modified by tyrosine sulfation, and, at later time points, by N-glycosylation. Appearance of sulfated PT subunits was inhibited by pretreatment of cells with brefeldin A. In some cell types, much of the S4 glycosylation, but not that of S1, was resistant to endoglycosidase H, suggesting that, subsequent to core N-glycosylation in the ER, S4 was transported anterograde to the Golgi, where further glycosylation occurred. When cells were pretreated with methyl-β-cyclodextrin, sulfation of PT subunits and PT cytotoxicity were reduced, suggesting that PT transport is dependent on cellular cholesterol content. These data support a retrograde pathway for PT intracellular transport.     

Transport of ricin from endosomes to the Golgi apparatus is regulated by Rab6A and Rab6A'

Ricin is transported from early endosomes and/or the recycling compartment to the trans-Golgi network (TGN) and subsequently to the endoplasmic recticulum (ER) before it enters the cytosol and intoxicates cells. We have investigated the role of the Rab6 isoforms in retrograde transport of ricin using both oligo- and vector-based RNAi assays. Ricin transport to the TGN was inhibited by the depletion of Rab6A when the Rab6A messenger RNA (mRNA) levels were reduced by more than 40% and less than 75%. However, when Rab6A mRNA was reduced by more than 75% and Rab6A' mRNA was simultaneously up-regulated, the inhibition of ricin sulfation was abolished, indicating that the up-regulation of Rab6A' may compensate for the loss of Rab6A function. In addition, we found that a near complete depletion of Rab6A' gave approximately 40% reduction in ricin sulfation. The up-regulation of Rab6A mRNA levels did not seem to compensate for the loss of Rab6A' function. The depletion of both Rab6A and Rab6A' gave a stronger inhibition of ricin sulfation than what was observed knocking down the two isoforms separately. In conclusion, both Rab6A and Rab6A' seem to be involved in the transport of ricin from endosomes to the Golgi apparatus.     

Dislocation of ricin toxin A chains in human cells utilizes selective cellular factors

Ricin is a potent A-B toxin that is transported from the cell surface to the cytosol, where it inactivates ribosomes, leading to cell death. Ricin enters cells via endocytosis, where only a minute number of ricin molecules reach the endoplasmic reticulum (ER) lumen. Subsequently, the ricin A chain traverses the ER bilayer by a process referred to as dislocation or retrograde translocation to gain access to the cytosol. To study the molecular processes of ricin A chain dislocation, we have established, for the first time, a human cell system in which enzymatically attenuated ricin toxin A chains (RTA(E177D) and RTA(Δ177-181)) are expressed in the cell and directed to the ER. Using this human cell-based system, we found that ricin A chains underwent a rapid dislocation event that was quite distinct from the dislocation of a canonical ER soluble misfolded protein, null Hong Kong variant of α(1)-antitrypsin. Remarkably, ricin A chain dislocation occurred via a membrane-integrated intermediate and utilized the ER protein SEL1L for transport across the ER bilayer to inhibit protein synthesis. The data support a model in which ricin A chain dislocation occurs via a novel strategy of utilizing the hydrophobic nature of the ER membrane and selective ER components to gain access to the cytosol.     

Cytotoxic ribosome-inactivating lectins from plants

A class of heterodimeric plant proteins consisting of a carbohydrate-binding B-chain and an enzymatic A-chain which act on ribosomes to inhibit protein synthesis are amongst the most toxic substances known. The best known example of such a toxic lectin is ricin, produced by the seeds of the castor oil plant, Ricinnus communis. For ricin to reach its substrate in the cytosol, it must be endocytosed, transported through the endomembrane system to reach the compartment from which it is translocated into the cytosol, and there avoid degradation making it possible for a few molecules to inactivate a large proportion of the ribosomes and hence kill the cell. Cell entry by ricin involves the following steps: (i) binding to cell-surface glycolipids and glycoproteins bearing beta-1,4-linked galactose residues through the lectin activity of the B-chain (RTB); (ii) uptake by endocytosis and entry into early endosomes; (iii) transfer by vesicular transport to the trans-Golgi network; (iv) retrograde vesicular transport through the Golgi complex and into the endoplasmic reticulum (ER); (v) reduction of the disulfide bond connecting the A- and B-chains; (vi) a partial unfolding of the A-chain (RTA) to enable it to translocate across the ER membrane via the Sec61p translocon using the pathway normally followed by misfolded ER proteins for targeting to the ER-associated degradation (ERAD) machinery; (vi) refolding in the cytosol into a protease-resistant, enzymatically active structure; (vii) interaction with the sarcin-ricin domain (SRD) of the large ribosome subunit RNA followed by cleavage of a single N-glycosidic bond in the RNA to generate a depurinated, inactive ribosome. In addition to the highly specific action on ribosomes, ricin and related ribosome-inactivating proteins (RIPs) have a less specific action in vitro on DNA and RNA substrates releasing multiple adenine, and in some instances, guanine residues. This polynucleotide:adenosine glycosidase activity has been implicated in the general antiviral, and specifically, the anti HIV-1 activity of several single-chain RIPs which are homologous to the A-chains of the heterodimeric lectins. However, in the absence of clear cause and effect evidence in vivo, such claims should be regarded with caution.     

Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin

We have studied the cytotoxicity of ricin in cells treated with brefeldin A (BFA), which dramatically disrupts the structure of the Golgi apparatus causing Golgi content and membrane to redistribute to the ER. BFA inhibits the cytotoxicity of ricin in Chinese hamster ovary, normal rat kidney, and Vero cells and abolishes the enhancement of ricin cytotoxicity by NH4Cl, nigericin, swainsonine, and tunicamycin or by a mutation in endosomal acidification. BFA protects cells from the cytotoxicities of modeccin and Pseudomonas toxin, but has no effect on the intoxication by diphtheria toxin. Pretreatment of BFA does not protect cells from ricin treatment in the absence of BFA. Our results suggest that ricin, modeccin, and Pseudomonas toxin share a common pathway of intracellular transport from endosomes to the Golgi region where they are released into the cytosol. In contrast, the lack of protection of Vero cells from diphtheria toxin by BFA indicates that diphtheria toxin is released from acidified endosomes without involving the Golgi region.     

Protein recycling from the Golgi apparatus to the endoplasmic reticulum in plants and its minor contribution to calreticulin retention

Using pulse-chase experiments combined with immunoprecipitation and N-glycan structural analysis, we showed that the retrieval mechanism of proteins from post-endoplasmic reticulum (post-ER) compartments is active in plant cells at levels similar to those described previously for animal cells. For instance, recycling from the Golgi apparatus back to the ER is sufficient to block the secretion of as much as 90% of an extracellular protein such as the cell wall invertase fused with an HDEL C-terminal tetrapeptide. Likewise, recycling can sustain fast retrograde transport of Golgi enzymes into the ER in the presence of brefeldin A. However, on the basis of our data, we propose that this retrieval mechanism in plants has little impact on the ER retention of a soluble ER protein such as calreticulin. Indeed, the latter is retained in the ER without any N-glycan-related evidence for a recycling through the Golgi apparatus. Taken together, these results indicate that calreticulin and perhaps other plant reticuloplasmins are possibly largely excluded from vesicles exported from the ER. Instead, they are probably retained in the ER by mechanisms that rely primarily on signals other than H/KDEL motifs.     

Retrograde transport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP

Endocytosed Shiga toxin is transported from the Golgi complex to the endoplasmic reticulum in butyric acid-treated A431 cells. We here examine the extent of this retrograde transport and its regulation. The short B fragment of Shiga toxin is sufficient for transport to the ER. The B fragment of cholera toxin, which also binds to glycolipids, is transported to all the Golgi cisterns, but cannot be localized in the ER even after butyric acid treatment. Under all conditions the toxic protein ricin was found predominantly in the trans-Golgi network. There is no transport of endocytosed fluid to the Golgi apparatus or to the ER even after butyric acid treatment and in the presence of Shiga toxin, indicating that transport to the ER, through the trans-Golgi network and the cisterns of the Golgi apparatus, involves several sorting stations. Since Shiga toxin receptors (Gb3) in butyric acid- treated A431 cells seem to have a ceramide moiety with longer fatty acids than in untreated cells, the possibility exists that fatty acid composition of the receptor is important for sorting to the ER. Both retrograde transport and intoxication with Shiga toxin can also be induced by cAMP, supporting the idea that retrograde transport from the Golgi to the ER is required for intoxication. The data suggest that transport to the ER in cells in situ may depend on fatty acid composition and is regulated by physiological signals.     

Relationship between Golgi architecture and glycoprotein biosynthesis and transport in Chinese hamster ovary cells

We have investigated the effect of colcemid-induced disassembly of microtubules, which is accompanied by retraction of the endoplasmic reticulum and fragmentation of the Golgi apparatus, on glycoprotein biosynthesis and transport in Chinese hamster ovary (CHO) cells. CHO cells were metabolically radiolabeled with [6- 3H]galactose or [2- 3H]mannose in the presence of either 0.1% dimethyl sulfoxide or 10 microM colcemid in dimethyl sulfoxide. The fine structure of glycoprotein asparagine-linked oligosaccharide structures synthesized in the presence or absence of colcemid was analyzed by lectin affinity chromatography, ion exchange chromatography, and methylation analysis using radiolabeled glycopeptides prepared by Pronase digestion. The fractionation patterns of [3H]mannose- and [3H]galactose-labeled glycopeptides on immobilized lectins indicated that processing to complex N-linked chains and poly-N-acetyllactosamine modification were similar in control and colcemid-treated cells. In addition, colcemid treatment did not alter the extent of sialylation or the linkage position of sialic acid residues to galactose. Using a trypsin release protocol, it was also found that the transport of newly synthesized glycoproteins to the cell surface was not affected by colcemid. These results demonstrate that the morphologically altered ER and Golgi apparatus in colcemid-treated CHO cells are completely functional with respect to the rate and fidelity of protein asparagine-linked glycosylation. Furthermore, movement of newly synthesized glycoproteins to and through the ER and Golgi apparatus and their transport to the cell surface in nonpolarized cells appears to be microtubule-independent.     

Localization of phospholipase Cbeta isozymes in the mouse cerebellum

Recent studies demonstrate that processing of N-linked glycans plays an important role in the quality control of major histocompatibility complex (MHC) class I transport from the endoplasmic reticulum (ER) to the Golgi complex and beyond. Here, we investigated the importance of oligosaccharide chain length on the association of MHC class I proteins with molecular chaperones and their intracellular transport from the ER to the Golgi. These data show that calnexin interaction with class I proteins having truncated N-glycans was reduced compared to normal class I molecules, whereas the assembly of class I with calreticulin and TAP was unperturbed by N-glycan chain length. Additionally, these results demonstrate that class I proteins containing truncated N-glycans showed decreased detachment from calreticulin and TAP relative to class I proteins bearing typical oligosaccharides. Taken together, these studies show that N-glycan chain length is an important determinant for the quality control of newly synthesized MHC class I proteins in the ER.     

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.     

Endosome to Golgi transport of ricin is regulated by cholesterol

We have here studied the role of cholesterol in transport of ricin from endosomes to the Golgi apparatus. Ricin is endocytosed even when cells are depleted for cholesterol by using methyl-beta-cyclodextrin (m beta CD). However, as here shown, the intracellular transport of ricin from endosomes to the Golgi apparatus, measured by quantifying sulfation of a modified ricin molecule, is strongly inhibited when the cholesterol content of the cell is reduced. On the other hand, increasing the level of cholesterol by treating cells with mbetaCD saturated with cholesterol (m beta CD/chol) reduced the intracellular transport of ricin to the Golgi apparatus even more strongly. The intracellular transport routes affected include both Rab9-independent and Rab9-dependent pathways to the Golgi apparatus, since both sulfation of ricin after induced expression of mutant Rab9 (mRab9) to inhibit late endosome to Golgi transport and sulfation of a modified mannose 6-phosphate receptor (M6PR) were inhibited after removal or addition of cholesterol. Furthermore, the structure of the Golgi apparatus was affected by increased levels of cholesterol, as visualized by pronounced vesiculation and formation of smaller stacks. Thus, our results indicate that transport of ricin from endosomes to the Golgi apparatus is influenced by the cholesterol content of the cell.     

Expression of Mutant Dynamin Inhibits Toxicity and Transport of Endocytosed Ricin to the Golgi Apparatus

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dyn^G273D at the nonpermissive temperature or the dyn^K44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69–80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915–934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949–966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dyn^WT than in cells expressing dyn^K44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dyn^K44A. In contrast, ricin transport to lysosomes as measured by degradation of ¹²⁵I-ricin was essentially unchanged in cells expressing dyn^K44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.     

Uncoupling of chondroitin sulfate glycosaminoglycan synthesis by brefeldin A

Brefeldin A has dramatic, well-documented, effects on the structural and functional organization of the Golgi complex. We have examined the effects of brefeldin A (BFA) on the Golgi-localized synthesis and addition of chondroitin sulfate glycosaminoglycan carbohydrate side chains. BFA caused a dose-dependent inhibition of chondroitin sulfate glycosaminoglycan elongation and sulfation onto the core proteins of the melanoma-associated proteoglycan and the major histocompatibility complex class II-associated invariant chain. In the presence of BFA, the melanoma proteoglycan core protein was retained in the ER but still acquired complex, sialylated, N-linked oligosaccharides, as measured by digestion with endoglycosidase H and neuraminidase. The initiation of glycosaminoglycan synthesis was not affected by BFA, as shown by the incorporation of [6-3H]galactose into a protein-carbohydrate linkage region that was sensitive to beta-elimination. The ability of cells to use an exogenous acceptor, p-nitrophenyl-beta-D-xyloside, to elongate and sulfate core protein-free glycosaminoglycans, was completely inhibited by BFA. The effects of BFA were completely reversible in the absence of new protein synthesis. These experiments indicate that BFA effectively uncouples chondroitin sulfate glycosaminoglycan synthesis by segregating initiation reactions from elongation and sulfation events. Our findings support the proposal that glycosaminoglycan elongation and sulfation reactions are associated with the trans-Golgi network, a BFA-resistant, Golgi subcompartment.     

Retrograde transport of protein toxins through the Golgi apparatus

A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through the Golgi apparatus.