Download The enemy within: ricin and plant cells

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
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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.
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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
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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.
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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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