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Journal of Experimental Botany, Vol. 49, No. 326, pp. 1473–1480, September 1998 The enemy within: ricin and plant cells Lorenzo Frigerio1,2 and Lynne M. Roberts2,3 1 Istituto Biosintesi Vegetali, Consiglio Nazionale delle Ricerche, via Bassini 15, 20133 Milano, Italy 2Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK Received 30 March 1998; Accepted 29 May 1998 Abstract Ricin, a ribosome-inactivating protein from the seeds of the castor oil plant (Ricinus communis L.) is one of the most potent cell poisons known. It is able to bind and enter most mammalian cells where it exploits their fully reversible secretory pathway to reach the endoplasmic reticulum. Ricin is then able to exit the endoplasmic reticulum to access the cytosol where it inhibits protein synthesis, thus killing the cells. Castor bean ribosomes are sensitive to ricin, but the plant has developed strategies to protect its own cells from suicide. The intracellular routing of ricin has been traditionally studied by exogenously adding toxin to mammalian cells and by following its path through the cell. However, the extreme potency of this protein has prevented the final membrane transport step from being studied in detail. Now, the expression of ricin in heterologous plant cells is proving helpful in elucidating details of both toxin biosynthesis and vacuolar targeting, and in studying membrane translocation of the catalytic subunit from the endoplasmic reticulum to the cytosol. Key words: Ricin, ribosome-inactivating protein, castor oil plant, seeds, inhibitor, membrane transport. Introduction Certain plants produce proteins that are able to enter and kill mammalian cells. These proteins act by catalytically and irreversibly modifying essential cellular components. In this review the focus is on the cytotoxin ricin, a member of the large family of plant ribosome-inactivating proteins (RIPs). Ricin is a type II (dimeric) RIP able to bind to the surface of most mammalian cells. Once delivered to the cytosol, it can inactivate ribosomes, promoting cell death by the inhibition of protein synthesis. Cytosolic entry of a single toxin molecule may be sufficient to cause the death of a cell, making ricin one of the most potently cytotoxic biomolecules known. Interestingly, the cells of the Ricinus communis endosperm, which produce the ricin toxin, possess ribosomes that are sensitive to its action. The strategy the plant uses to avoid its own suicide during ricin biosynthesis forms a major part of this review. Occurrence, structure and mode of action There are several isoforms of ricin, including ricin D, ricin E and the closely related lectin Ricinus communis agglutinin (RCA), encoded by a small multigene family of approximately eight members, some of which are nonfunctional ( Tregear and Roberts, 1992). Ricin is produced in endosperm tissue during the post-testa maturation of the castor oil seed (Roberts and Lord, 1981a) and is stored within protein bodies of the endosperm cells along with storage albumins and crystalloid proteins ( Tulley and Beevers, 1976; Youle and Huang, 1976). Within these organelles, ricin accumulates to around 5% of total particulate protein and is degraded in the first few days of postgerminative growth. Structurally, all forms of ricin are heterodimeric glycoproteins composed of a catalytically active A-chain (or RTA, 32 kDa) disulphide bonded to a galactose- and Nacetylgalactosamine-specific lectin known as the B-chain (or RTB, 34 kDa). RCA, by contrast, is tetrameric, being composed of two ricin-like heterodimers (Roberts et al., 1985) that are disulphide bonded together. The threedimensional structure of ricin has been solved by X-ray crystallography (Montfort et al., 1987; Monzingo and Robertus, 1992) allowing a detailed description of both subunits ( Katzin et al., 1991; Rutenber and Robertus, 1991). RTA is a 267 residue globular polypeptide with a pronounced cleft that forms the active site. Since both 3 To whom correspondence should be addressed. Fax: +44 1203 523568. E-mail: [email protected] © Oxford University Press 1998 1474 Frigerio and Roberts ricin cDNAs and genes have been cloned (Lamb et al., 1985; Halling et al., 1985; Tregear and Roberts, 1992), the active site has been well characterized by mutational analyses (for review see Lord et al., 1994). In contrast to the globular structure of RTA, the 262 residue RTB is bilobal. Each lobe is comprised of four subdomains, three of which are homologous and believed to have arisen from the duplication of an ancient galactose binding peptide (Rutenber and Robertus, 1991). Two of these six subdomains have retained their capacity to bind galactose and the binding sites are seen as shallow pockets at opposite ends of the bilobal structure. Once again, the residues critical for sugar binding have been identified by mutational analyses ( Wales et al., 1991). RTA is able to inactivate eukaryotic ribosomes irreversibly by acting as an rRNA-specific N-glycosidase ( Endo et al., 1987). The modification involves the removal of a specific adenine in the large rRNA of the 60S ribosomal subunit (A4324 in rat liver 28S rRNA). This conserved adenine is part of a region of 28S rRNA that is critical for the binding of the EF2 ternary complex in the translocation phase of a protein synthesis elongation cycle. Its removal, therefore, causes an immediate cessation of elongation cycles and quickly leads to a halt in all cytosolic protein synthesis. A mechanism of action has been proposed for RTA which incorporates the structural, mutational and kinetic analyses of RTA ( Ready et al., 1992). Although the sequence in the 28S rRNA immediately around the target adenine is totally conserved in all types of ribosomes, the sensitivity of ribosomes from different sources to RTA action varies considerably. Thus, while mammalian ribosomes are most sensitive to the action of RTA, yeast ribosomes are slightly less sensitive and prokaryotic ribosomes are resistant (Lord et al., 1991). The situation with plant ribosomes is rather more complex, for whilst some ribosomes appear resistant (e.g. wheat germ ribosomes), others are moderately sensitive ( Taylor et al., 1994). Ricin A-chain is known to be active on Ricinus communis ribosomes, but at concentrations much higher than those required to depurinate mammalian ribosomes (Harley and Beevers, 1982). Tobacco ribosomes are also particularly recalcitrant to the action of ricin A-chain: the concentration of RTA that causes depurination to 50% of tobacco rRNAs (DC ) is 12550 fold higher than the DC for yeast rRNAs ( Taylor et al., 50 1994). The reason for this reduced sensitivity is not completely clear at present, but it would seem that the precise conformation or accessibility of the target rRNA sequence in the intact ribosome is important in determining whether it becomes a good substrate for RTA, and this presumably varies in ribosomes from different sources. In the absence of any known natural inhibitors of RTA, the varying array of ribosomal proteins is presumably responsible for these conformational or steric differences. Indeed, E. coli ribosomes, which are normally resistant to ricin A-chain, become completely susceptible to depurination when their rRNA is deproteinized ( Endo and Tsurugi, 1988). Recent work on RTA-sensitive ribosomes in vitro seems to indicate that these can promote refolding, and thus recovery of toxicity, of heat-denatured RTA (Frigerio and Roberts, unpublished results). Ricin uptake by mammalian cells Ricin enters mammalian cells by endocytosis after opportunistic binding, via the B-chain, to surface components bearing exposed galactosides. Uptake occurs by both clathrin coated vesicles and by non-clathrin coated vesicles that form in a dynamin-independent process (Simpson et al., 1998). Both the clathrin-dependent and the clathrinindependent uptake routes converge in endosomes. However, for productive toxicity it is known that ricin must proceed beyond endosomes to reach a compartment compatible for the membrane translocation of its Achain. This intracellular trafficking of ricin was initially followed by electron microscopy (van Deurs et al., 1988). In addition to entering lysosomes or being recycled back to the cell surface, a small but significant amount of toxin was seen to accumulate in the trans Golgi network ( TGN ). A critical limitation of this experimental approach however, was its sensitivity. So, while it clearly defined the fate of the bulk of the endocytosed ricin, the extreme potency of ricin (whereby the entry of just a few molecules into the cytosol may be sufficient to kill a cell ) meant that the physiologically significant but inefficient trafficking to the translocation compartment could well go undetected. One of the first experimental indications that ricin might not translocate from the TGN was the demonstration that treating cells with the Golgi stackdisrupting agent brefeldin A (BFA) afforded protection ( Yoshida et al., 1991). This led to the hypothesis that toxins such as ricin may need to be transported to the endoplasmic reticulum of animal cells in order to exploit a route to the cytosol (Pelham et al., 1992). The idea of retrograde transport to the ER was supported by the finding that cells expressing trans dominant negative forms of various Rab mutants involved in regulating vesicular traffic in the early secretory pathway were significantly protected against ricin but not against diphtheria toxin, which is known to enter the cytosol from the endosomes (Simpson et al., 1995). However, the most direct evidence to date for the trafficking of ricin to the endoplasmic reticulum comes from the finding that endocytosed ricin containing a non-glycosylated A subunit, can become core glycosylated upon uptake, and that this glycosylated ricin could be subsequently detected within the cytosolic fraction (Rapak et al., 1997). To visualize only that proportion of ricin that had reached the TGN, the RTA subunit used in these experiments carried a Ricin and plant cells tyrosine sulphation site and overlapping N-glycosylation sites. Mutant A-chain was reassociated with B-chain and incubated with cells in the presence of Na 35SO so that 2 4 the A-chain became radiolabelled in vivo if it reached the TGN (the site of protein sulphation). That the labelled A-chain became core glycosylated clearly demonstrated its subsequent arrival in the endoplasmic reticulum (the site of oligosaccharyl transferase). It remains unclear whether the A- and B-chains dissociate in the ER before membrane translocation, or whether both subunits translocate and become reduced in the cytosol. Recent findings in mammalian cells and yeast that both glycosylated membrane proteins ( Wiertz et al., 1996a) and glycosylated lumenal proteins (Hiller et al., 1996) of the endoplasmic reticulum can be exported to the cytosol for degradation if they are detected to be aberrant in some way, has led to the speculation that ricin may exploit such a pathway to reach its cytosolic substrates (Lord and Roberts, 1998). It is supposed that ricin must masquerade as a misfolded protein, perhaps after reduction of the interchain disulphide bond which would expose a hydrophobic patch at the C-terminus of RTA that is normally shielded by RTB. This, however, remains to be experimentally established. Whatever the events, the fact that export of proteins from organelles of the secretory pathway to the cytosol appears to occur exclusively from the endoplasmic reticulum, would seem to account for the need of a non-pore-forming toxin, such a ricin, to reach the endoplasmic reticulum in the first place. The factors involved in this quality control system, that detect abnormal proteins and effect their membrane transport, are only just beginning to be identified. The process appears to require the proteinaceous Sec61p channels ( Wiertz et al., 1996b; Plemper et al., 1997; Pilon et al., 1997) that are associated with the import of nascent polypeptides form the cytosol, though the mechanism of export remains unclear at present. Once aberrant proteins are exported to the cytosol it appears they are targeted for destruction by ubiquitination and degraded by cytosolic proteasomes ( Wiertz et al., 1996a, b). Indeed, the process is so effective that it is normally only possible to visualize exported proteins in the mammalian cytosol by using specific proteasomal inhibitors such as lactacystin ( Wiertz et al., 1996a, b). Ricin, or more specifically RTA, must avoid degradation if it is to inactivate ribosomes. Interestingly, RTA is remarkably protease-resistant ( Walker et al., 1996), and it may avoid ubiquitination by its paucity of lysines ( Hazes and Read, 1997). In effect, there is probably a competition between degradation, refolding and ribosome inactivation, in highly sensitive mammalian and yeast cells. As already pointed out, the process of entry and retrograde transport of ricin into the cells, although physiologically effective, is quantitatively very inefficient. 1475 The direct observation of the late events that lead to toxin retrotranslocation is therefore technically very difficult. One way to avoid this problem would be to deliver large amounts of ricin A-chain to the endoplasmic reticulum by direct toxin gene expression in heterologous cells. So far, however, all efforts to successfully express wild-type ricin A-chain in eukaryotic cells, including Xenopus oocytes, yeast, insect and mammalian cells have failed, due to the extreme sensitivity of their ribosomes, and most of this work has remained unpublished. The observation that ricin A-chain is less active on plant ribosomes suggests that plants could be resistant enough to allow the expression of the toxin and of its subunits. Synthesis in Ricinus communis RIP-producing plants have developed strategies to protect themselves from the action of their own toxins. The biosynthesis of ricin in Ricinus communis cells has been studied in detail and constitutes an elegant example of one of these strategies. During seed development, ricin is synthesized from a single messenger RNA that encodes a preproprotein containing both RTA and RTB. Preproricin consists of 576 amino acid residues: the first 35 residues contain the signal sequence for translocation into the ER lumen and a propeptide of unknown length, that is absent in the mature protein. The following 267 residues constitute mature RTA, joined to mature RTB (262 residues) by a 12-residue linker peptide. Preproricin is co-translationally translocated into the ER lumen, where the signal peptide is removed (Roberts and Lord, 1981b). The nascent peptide is N-glycosylated (Lord, 1985b), and five disulphide bonds are formed (Lord, 1985a). Four disulphide bonds occur within the sequence of RTB, while the remaining one links the C terminus of RTA with the N terminus of RTB. Glycosylated, folded proricin is then transported to the protein storage vacuoles through the Golgi complex (Lord, 1985b). In the vacuole, the N-terminal propeptide and the 12 amino acid linker peptide are proteolytically removed, thus releasing the mature, disulphide-linked RTA-RTB heterodimer. Recently, a vacuolar processing enzyme, related to cysteine proteases, has been isolated from castor bean endosperm. This enzyme is able to convert proricin into its mature form (Hiraiwa et al., 1997; Hara-Nishimura et al., 1991). The signal for sorting of preproricin to the protein storage vacuoles has not been determined precisely, but recent evidence suggests that it could reside in the linker peptide (Frigerio et al., 1998, and see below). The synthesis in precursor form is the principal mechanism by which castor bean cells protect themselves from the toxicity of ricin, since it has been shown that proricin is not active per se (Richardson et al., 1989). This is 1476 Frigerio and Roberts probably due to the fact that the presence of the linker peptide generates structural constraints that cause the active site cleft of RTA to be in close contact with RTB, thus rendering it unavailable for catalysis. Furthermore, maturation of the precursor into the active form takes place in the vacuole, which is a compartment from which mature ricin is evidently unable to retrotranslocate. It should be noted that sequestration of RIPs in the secretory pathway is also used by other dicotyledonous plants whose ribosomes are sensitive to their endogenous RIPs, and is presumably a strategy to prevent these toxins from accidentally reaching the cytosol (Bonness et al., 1994). Most of these RIPs are, in fact, targeted into the ER lumen and sorted to the protein storage vacuoles, like ricin, or destined for secretion into the cell wall, as occurs for pokeweed antiviral protein (PAP) (Ready et al., 1986). The situation is somewhat different for some of the RIPs of monocotyledonous plants. Cereal RIPs appear to be made without signal peptides and are therefore not delivered into the secretory pathway ( Walsh et al., 1991; Leah et al., 1991). Their occurrence in the cytosol requires that conspecific ribosomes be insensitive to depurination. Indeed, wheat and maize ribosomes are resistant to the action of their endogenous endosperm RIPs (Bass et al., 1992; Taylor and Irvin, 1990). In the case of wheat, however, a leaf isoform of the RIP, tritin, is active against wheat ribosomes (Massiah and Hartley, 1995). Although most cereal RIPs are made as mature proteins in the cytosol, some, such as the maize kernel RIP (also known as b–32), are made as part of a precursor protein that requires proteolytic activation ( Walsh et al., 1991). The RIP of the barley leaf, made in the cytosol upon jasmonate induction, is also made as part of a cytosolic precursor (Chaudhry et al., 1994). These two different mechanisms of protection against cell suicide, toxin sequestration or ribosome insensitivity, may also be relevant for the physiological role of RIPs in conferring both viral and fungal resistance. For dicotyledonous plants, it has been proposed that mechanical entry of a virus into a cell may cause the release of any vacuolar or extracellular RIP into the cytosol, thereby promoting local cell suicide to prevent replication and spread of the virus (Bonness et al., 1994). For those cereals where conspecific ribosomes are resistant to the endogenous RIP, it is assumed that the ribosomeinactivating action is exploited directly against fungal pathogens (Massiah and Hartley, 1995). There is some supporting experimental evidence that cereal RIPs do indeed have an antifungal role (Logemann et al., 1992; Leah et al., 1991; Jach et al., 1995). However, the physiological role of all the RIPs in their producer plants is far from clear. For example, some plants producing high levels of type I RIPs are themselves susceptible to viral infection (Shepherd et al., 1969). Other work with transgenic plants expressing catalytically-inactive poke- weed antiviral protein (PAP), has revealed the activation of multiple plant defence mechanisms concomitant with the acquisition of fungal resistance ( Zoubenko et al., 1997), suggesting that N-glycosidase activity need not necessarily be involved in conferring the resistance phenotype. There is also evidence from grafting experiments, that enzymatically active PAP expressed in the rootstocks of transgenic tobacco is needed to generate a signal that renders viral resistance in wild-type scions, where the PAP protein is undetectable (Smirnov et al., 1997). Clearly, much more work is now needed to examine, in the field, the selective advantage conferred by each type of RIP on the plant that produces it. Ricin biosynthesis in a heterologous plant Expression of preproricin Due to its extreme cytotoxic potency, ricin has been exploited in the therapy of cancer where ricin-based immunotoxins have been developed by chemically coupling RTA or whole ricin to monoclonal antibodies (Blythman et al., 1981; Lambert et al., 1991). A more efficient and cost-effective approach would be the direct expression of genetically engineered, recombinant ricin protein fusions in heterologous systems. The possibility of using plants to obtain large quantities of ricin holotoxin has been explored by Sehnke and coworkers (Sehnke et al., 1994). They produced transgenic tobacco plants expressing the preproricin gene under the control of the CaMV 35S promoter. Transformed plants were healthy and expressed mature ricin at levels of about 1 mg g−1 FW of leaves. RTA and RTB from leaves of transgenic tobacco were indistinguishable from their native, Ricinus-produced forms. Furthermore, ricin from transgenic plants retained full sugar binding and cytotoxic activity, suggesting correct folding and assembly of both chains (Sehnke et al., 1994). Recently, similar results, but with more reproducible yields, have been obtained by expressing preproricin in cultured tobacco cells ( Tagge et al., 1996). This system allows the production of very high amounts of mature, processed ricin at low purification and culture costs. The intracellular route of proricin and the final location of the mature toxin were not determined in these studies. However, such trafficking events have been the focus of a separate study ( Frigerio et al., 1998), as discussed below. When preproricin was transiently expressed in tobacco protoplasts, the precursor was shown to enter the lumen of the ER by virtue of its signal peptide and become Nglycosylated and disulphide bonded. Proricin (containing an N-terminal and a central propeptide) was then transported to the vacuole in a brefeldin A-sensitive manner. Brefeldin A is known to induce disassembly of Golgi stacks and to inhibit Golgi-mediated transport to the Ricin and plant cells vacuole in plant cells (Pedrazzini et al., 1997; Gomez and Chrispeels, 1993). Thus, proricin was being transported through the Golgi to reach the vacuole. Within the tobacco cell vacuole, proricin was proteolytically cleaved and processed to generate mature, disulphide-linked RTA and RTB ( Fig. 1). The fate of proricin in tobacco cells is thus similar to that of proricin in the cells of the castor bean endosperm (Lord, 1985b). Likewise, proricin was not toxic to tobacco cells. Expression of single chains RTA has been successfully expressed only in the cytosol of E. coli, since prokaryotic ribosomes are insensitive to its action (O’Hare et al., 1987). Such recombinant RTA is not glycosylated, but it possesses full biological activity. Recently, the RTA polypeptide has been transiently expressed in tobacco protoplasts (Frigerio et al., 1998). 1477 Analysis revealed that RTA was correctly translocated into the lumen of the ER and that it underwent N-glycosylation. However, as shown by pulse-chase experiments in the presence or absence of brefeldin A, glycosylated RTA does not move through the Golgi but is instead degraded (Fig. 1). This is similar to what is observed for an assembly-defective form of the storage protein phaseolin (Pedrazzini et al., 1997). By monitoring protein synthesis inhibition, it became clear that this glycosylated RTA was toxic for the cells: its toxicity was equivalent to that of a cytosolic RTA made in the absence of a signal peptide. In order to achieve such toxicity levels, glycosylated RTA must have been exported from the ER to reach the cytosol with high efficiency where it could inactivate ribosomes before being degraded. The existence of the cytosolic ubiquitin-proteasome protein degradation pathway has been well established in plants Fig. 1. A model for the cellular fates of proricin and separately expressed ricin chains in tobacco protoplasts. (1) A single polypeptide precursor (proricin), consisting of RTA and RTB joined by a short linker peptide (L), is initially translocated into the lumen of the ER, where N-glycosylation and disulphide bond formation occur. The precursor is then transported to the vacuole through the Golgi complex. In the vacuole, proricin is proteolytically processed to yield the mature toxin, in which the two subunits are held together by a single disulphide bond. (2) Ricin A-chain is translocated into the ER lumen, glycosylated and then degraded in a process that does not involve transport through the Golgi stack. RTA is toxic for the cell, raising the possibility that it is dislocated (by an unknown mechanism) from the ER lumen to the cytosol, where it inactivates ribosomes in a process that possibly competes with degradation. (3) Ricin B-chain is translocated into the ER lumen, glycosylated, and efficiently secreted. The co-expression of separate A and B-chains leads to the formation of disulphide-linked heterodimers, which are then secreted rather than targeted to vacuoles. 1478 Frigerio and Roberts and may be responsible for the degradation of RTA. Indeed, several genes encoding ubiquitin-conjugating enzymes and proteasomal subunits have been characterized, and shown to be homologous to their yeast and mammalian counterparts (for review see Vierstra, 1996). So far however, very few plant proteins have been directly shown to be substrates for ubiquitin-mediated degradation, the best described being that of phytochrome A (Shanklin et al., 1987). The existence of an ER-associated degradation pathway in plants and the sensitivity of plant proteasomes to inhibiting drugs like lactacystin, which have allowed the study of this pathway in animal cells, still remain to be proved. RTB, being a non-toxic lectin, has been successfully expressed in Xenopus oocytes (Richardson et al., 1988a), and in mammalian and yeast cells (Richardson et al., 1988b; Chang et al., 1987). When ricin B-chain with a phaseolin signal peptide was transiently expressed in a tobacco protoplast system, it was shown to be secreted ( Frigerio et al., 1998) ( Fig. 1), strongly indicating that the vacuolar sorting information for ricin does not reside within the B-chain sequence. Interestingly, when RTA and RTB were co-expressed as separate subunits in tobacco protoplasts, they formed disulphide-linked heterodimers in the ER lumen (Frigerio et al., 1998), which, like RTB, were secreted to the extracellular medium ( Fig. 1). This would suggest that the short 12 amino acid linker sequence between the two subunits in proricin, that is absent in the reconstituted heterodimers, represents a good candidate for a vacuolar sorting signal. Direct experimental proof for this, however, remains to be provided. The existence of an intramolecular signal for sorting to the vacuole has been previously suggested for two plant storage proteins. In the first example, two long segments (one at the N terminus and one near the C-terminus) of the mature bean legumin have been shown to be effective in vacuolar targeting (Saalbach et al., 1991). Secondly, an internal region of the bean seed lectin phytohaemagglutinin was demonstrated to be both necessary and sufficient to sort yeast invertase to the vacuoles of transgenic plants (von Schaewen and Chrispeels, 1993). In neither case, however, were the putative sorting sequences removed upon deposition in the vacuole, contrary to what is observed for preproricin. An interesting observation was that the presence of RTB and RTA within the ER lumen permitted postsynthesis assembly of ricin heterodimers within this compartment, thus preventing export of a significant portion of free RTA to the cytosol. The reduction in the amount of RTA being degraded was clearly accompanied by a concomitant reduction in cytotoxicity, as measured by monitoring the synthesis of a reporter protein ( Frigerio et al., 1998). By analogy to mammalian and yeast cells, it is likely that an ER-associated degradation (ERAD) pathway exists in plant cells. Such a pathway would normally be used by secretory proteins detected as aberrant and condemned for export to the cytosol by proteins functioning in ER quality control. RTA, but clearly not RTB or proricin, would appear to behave as an abnormal protein in the ER of plant cells. This appears to lead to its ejection and subsequent degradation in the cytosol. The fact that the plant cell can survive at all under these circumstances is perhaps tied up with the fact that its ribosomes are more recalcitrant to the toxin than those of mammalian or yeast cells. Nevertheless, such is the potency of RTA that a detectable reduction in protein synthesis is observed even though the ultimate fate of this polypeptide in the plant cytosol is rapid degradation. It remains to be seen whether this degradation is mediated by plant proteasomes or whether it occurs through a different pathway. Conclusions Plant cells have been invaluable for studying the biosynthesis and cellular fate of ricin and its subunits. In fact, because of the reduced sensitivity of tobacco ribosomes to ricin, plant cells represent the only eukaryotic system permitting the expression of wild-type RTA. Clearly, different forms of ricin have different cellular fates and understanding the signals and mechanisms involved in these targeting and degradative processes will be the next big challenge. Such knowledge will be important for understanding not only RIP biosyntheses, but also how the plant cell handles both endogenous proteins and biotechnologically valuable foreign proteins that are delivered into the secretory pathway. Acknowledgements We thank Aldo Ceriotti and Alessandro Vitale for helpful suggestions and critical reading of the manuscript. Work in our laboratories was supported in part by the European Community Human and Capital Mobility Programme (grant no. CHRXCT94-0590). 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