Toxicol Rev 2003; 22 (1): 53-64

REVIEW ARTICLE 1176-2551/03/0001-0053/$30.00/0

© Adis Data Information BV 2003. All rights reserved.

Ricin
Mechanisms of Cytotoxicity
Michael J. Lord, Nicholas A. Jolliffe, Catherine J. Marsden, Cassandra S.C. Pateman, Daniel C. Smith,
Robert A. Spooner, Peter D. Watson and Lynne M. Roberts
Department of Biological Sciences, University of Warwick, Coventry, UK

Contents
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
1. Structure and Function of Ricin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
1.1 Ricin Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
1.2 The Substrate for Ricin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
1.3 Mode of Action of Ricin A Chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
1.4 Novel Enzymatic Activities of Ricin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
1.5 Ricin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2. Getting Into the Cytosol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2.1 Binding of Ricin to Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2.2 Endocytosis of Ricin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
2.3 Transport of Ricin from Endosomes to the Golgi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
2.4 Transport of Ricin from Golgi to Endoplasmic Reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.5 Membrane Translocation of Ricin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.6 Retro-Translocation of Ricin A Chain in Other Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Abstract Ricin is a heterodimeric protein produced in the seeds of the castor oil plant (Ricinus communis). It is
exquisitely potent to mammalian cells, being able to fatally disrupt protein synthesis by attacking the Achilles
heel of the ribosome. For this enzyme to reach its substrate, it must not only negotiate the endomembrane system
but it must also cross an internal membrane and avoid complete degradation without compromising its activity in
any way. Cell entry by ricin involves a series of steps: (i) binding, via the ricin B chain (RTB), to a range of cell
surface glycolipids or glycoproteins having β-1,4-linked galactose residues; (ii) uptake into the cell by
endocytosis; (iii) entry of the toxin into early endosomes; (iv) transfer, by vesicular transport, of ricin from early
endosomes to the trans-Golgi network; (v) retrograde vesicular transport through the Golgi complex to reach the
endoplasmic reticulum; (vi) reduction of the disulphide bond connecting the ricin A chain (RTA) and the RTB;
(vii) partial unfolding of the RTA to render it translocationally-competent to cross the endoplasmic reticulum
(ER) membrane via the Sec61p translocon in a manner similar to that followed by misfolded ER proteins that,
once recognised, are targeted to the ER-associated protein degradation (ERAD) machinery; (viii) avoiding, at
least in part, ubiquitination that would lead to rapid degradation by cytosolic proteasomes immediately after
membrane translocation when it is still partially unfolded; (ix) refolding into its protease-resistant, biologically
active conformation; and (x) interaction with the ribosome to catalyse the depurination reaction.
It is clear that ricin can take advantage of many target cell molecules, pathways and processes. It has been
reported that a single molecule of ricin reaching the cytosol can kill that cell as a consequence of protein
synthesis inhibition. The ready availability of ricin, coupled to its extreme potency when administered
intravenously or if inhaled, has identified this protein toxin as a potential biological warfare agent. Therapeuti-
cally, its cytotoxicity has encouraged the use of ricin in ‘magic bullets’ to specifically target and destroy cancer
54
cells, and the unusual intracellular trafficking properties of ricin potentially permit its development as a vaccine
vector.
Combining our understanding of the ricin structure with ways to cripple its unwanted properties (its
enzymatic activity and promotion of vascular leak whilst retaining protein stability and important immunodomi-
nant epitopes), will also be crucial in the development of a long awaited protective vaccine against this toxin.
Ricin is a heterodimeric protein produced in the seeds of the
castor oil plant (Ricinus communis). It is a potent and opportunistic
ribotoxin that can bind to the surface of many kinds of cell. What
is very striking is the way ricin can exploit intracellular transport
pathways to travel from the plasma membrane to its target ribo-
somes in the cytosol. This journey involves retrograde routing to
the endoplasmic reticulum lumen, and retro-translocation from
this site to the cytosol.[1] It has been reported that a single molecule
of ricin reaching the cytosol can kill that cell as a consequence of
protein synthesis inhibition.[2] Indeed, the lethal dose of ricin in
humans has been estimated at between 5–10 μg/kg bodyweight.[3]
The ready availability of ricin, coupled to its extreme potency
when administered intravenously[3] or if inhaled[4] has identified
this protein toxin as a potential biological warfare agent.[5] Thera-
peutically, its cytotoxicity has encouraged the use of ricin in
‘magic bullets’ to specifically target and destroy cancer cells,[6,7]
and the unusual intracellular trafficking properties of ricin poten-
tially permit its development as a vaccine vector.[8,9]
This review will focus on the structure, function and intracellu-
lar trafficking of ricin, highlighting the features that underlie its
cytotoxic potency.
1. Structure and Function of Ricin
1.1 Ricin Structure
Ricin is a prototype ribosome-inactivating protein. Such pro-
teins have been isolated from many sources including bacteria,
fungi and the leaves, seeds and roots of plants.[10-12] In general,
most ribosome-inactivating proteins can be classified as either
Type 1 or Type 2.[13] Type 1 ribosome-inactivating proteins are the
most common (~100 described to date), and consist of a single
enzymatic chain of ~30 kDa. Type 2 ribosome-inactivating pro-
teins, typified by ricin, are potent cytotoxins with an A chain that is
structurally and functionally equivalent to the Type 1 ribosome-
inactivating proteins, disulphide-linked to a lectin referred to as
the B chain. The ~30 kDa B chain is able to bind terminal
galactose residues on cell surface components. It follows that
although Type 1 and Type 2 ribosome-inactivating proteins are
active against ribosomes in vitro, only the Type 2 ribosome-
inactivating proteins, such as ricin, are cytotoxic due to the pres-
© Adis Data Information BV 2003. All rights reserved.
Lord et al.
ence of a B chain that mediates the binding and entry of holotoxin
(the disulphide-linked A- and B-chain complex) into susceptible
cells.
Amongst ribosome-inactivating proteins, the tertiary structure
of ricin was the first to be determined.[14-16] It was shown to be a
globular, glycosylated heterodimer in which ricin A chain and
ricin B chain are joined by a single disulphide bond (figure 1,
panel a). The 262 amino acyl residues of ricin B chain form a
bilobal structure lacking α-helices or β-sheets that probably arose
by gene duplication.[17] Each of the structurally homologous lobes
contains three related subdomains and at least one of these in each
lobe possesses a sugar-binding pocket. In contrast, ricin A chain,
comprising 267 amino acyl residues, has three structural domains
with approximately 50% of the polypeptide arranged into α-
helices or β-sheet [18-20] (figure 1, panel a).
1.2 The Substrate for Ricin
The way in which ricin inactivates ribosomes was first de-
scribed by Endo and Tsurugi[22] who showed that ricin A chain
acted by cleaving a specific glycosidic bond within the large
ribosomal RNA (rRNA) of the 60S subunit of eukaryotic ribo-
somes. Ricin A chain must be separated from ricin B chain to
display this activity.[23,24] Subsequently, Endo and Tsurugi[25] dem-
onstrated that ricin A chain was an RNA-specific N-glycosidase
that hydrolytically cleaved the N-glycosidic bond of the adenine
residue at position 4324 (A4324) within the 28S rRNA of rat liver
ribosomes leaving the phosphodiester backbone of the RNA at this
site intact. It is very striking that this single, specific depurination
completely and irreversibly inactivates the ribosome. Closer
examination revealed that the target adenine lay in a universally
conserved sequence of 12 nucleotides, 5′-AGUACGAGAGGA-3′
(target adenine shown in italics), known as the sarcin-ricin loop.[26]
The sarcin-ricin loop is associated with the binding of elongation
factors.[27] Modification of this region by ricin A chain thereby
prevents the binding of such factors and inhibits the elongation
cycle of protein synthesis.[28,29] However, the relevance of
ribosomal proteins in rendering ribosomes susceptible to attack by
ricin A chain is highlighted by the fact that the kcat (a measure of
the catalytic rate of an enzyme reaction) for depurination of naked
28S rRNA by ricin A chain is nearly 105-fold lower than the kcat
for rRNA within a ribosome.[25] Moreover, although ricin A chain
Toxicol Rev 2003; 22 (1)
Ricin Toxicity 55

a b

RTA

Glu177
Arg180

Tyr123

Tyr80

RTB
FMP

c
E177 NH2 E177 NH2 NH2

N N N E177 N
C C N N
− O −
O O O
N C
N N N − N N
O O O
H H
C O
O H H O H
C O H NH NH
H +
NH H OH
+ R180
+ R180 R180
H2N NH H2N NH
H2N NH

Fig. 1. Structure of ricin and catalytic mechanism of the A chain. (a) The α-carbon backbone of RTB is shown in green whilst that of RTA is coloured
according to its structure with α-helices in pink, β-strands in yellow and coil in white. The site of the single interchain disulphide bond is shown in blue. (b)
The active site of RTA bound with a substrate analogue, formycin-5′-monophospate (FMP), is shown with critical catalytic residues, Arg180, Glu177,
Tyr123 and Tyr80 coloured with carbon atoms in grey, oxygen atoms in red, nitrogen atoms in blue and FMP in green. (c) A schematic of the proposed
catalytic mechanism of RTA.[21] RTA = ricin A chain; RTB = ricin B chain.

is active against naked 23S prokaryotic rRNA, intact Escherichia
coli ribosomes are completely refractory to depurination by ricin
A chain,[30] and plant ribosomes are ~5000-fold less sensitive to
ricin A chain than mammalian ribosomes.[31] The ribosome is a
large, complex substrate that operates via structural transitions,
and it is likely that the ricin A chain polypeptide also interacts with
sites outside the immediate vicinity of the sarcin-ricin loop. The
presence of particular ribosomal proteins may therefore allow or
prevent access of ricin A chain to the sarcin-ricin loop and might
thereby contribute to substrate specificity.
1.3 Mode of Action of Ricin A Chain
The catalytic activity of ricin lies exclusively within the A
chain. Ricin A chain, along with other ribosome-inactivating
proteins, contains several invariant amino acid residues. These
include tyrosine residues at positions 80 and 123 (Y80 and Y123),
a glutamic acid residue at position 177 (E177) and an arginine
residue at position 180 (R180), all of which are located within the
active site (figure 1, panel b). Although the precise mechanism of
N-glycosidase action for ricin A chain remains unclear, Monzingo
and Robertus[21] have proposed a mechanistic model based on the
crystal structures of ricin A chain in complex with small molecules
and site-directed mutagenesis studies.[20,21,32-36] The kinetic ana-
lysis of ricin A chain variants with mutations at either Arg180 or
Glu177 showed little change in Km, the binding affinity of the
enzyme for its substrate. However, the kcat (a measure of the
catalytic rate of an enzyme reaction) of such mutants was affected
suggesting that these two residues are involved in the catalytic
mechanism of the enzyme rather than in substrate binding.
The Monzingo and Robertus[21] model (figure 1, panel c) took
these studies into account, and proposed that the rRNA substrate
becomes bound in the active site cleft with the target adenine
making stacking interactions with tyrosine residues Y80 and
Y123. Residue R180 is positioned such that it can protonate the
leaving group at N-3 of adenine, facilitating an electron flow that

© Adis Data Information BV 2003. All rights reserved. Toxicol Rev 2003; 22 (1)
56
results in the breaking of the bond between N-9 of the adenine ring
and the C-1′ of the ribose. This bond cleavage results in the
production of an oxycarbonium ion on the ribose, which is
stabilised by the presence of E177. The protonation of N-3 of
adenine by R180 increases the basic character of the R180 side
chain, allowing it to pull a proton from a nearby water molecule
thus activating it and allowing solvent attack on the ribose carboni-
um creating a neutral ribose. Through this action, it has been
reported that a single A chain molecule in the cytosol can
depurinate ~1500 ribosomes per minute, leading to a rapid inhibi-
tion of protein synthesis.
1.4 Novel Enzymatic Activities of Ricin
It has become evident that intact ricin can modify non-
ribosomal nucleic acid substrates in vitro, allowing it to be consid-
ered a polynucleotide:adenosine glycosidase.[37] Nuclear DNA
damage has also been reported in human endothelial cells,[38]
possibly as a prelude to the induction of apoptosis.[39,40] While
certain ribonuclease and deoxyribonuclease activities ascribed to
some ribosome-inactivating proteins are likely to be due to con-
tamination,[41,42] it is unclear whether additional enzyme activities
reported for ricin and other ribosome-inactivating proteins (e.g.
chitinase, phosphatase, phospholipase and superoxide dismutase
activities) are in any way physiologically relevant.[13,43,44]
Recently, it has been shown that ricin contains a neutral lipase
activity in vitro, correlating to the presence of a catalytic triad
between the two subunits.[45] Again, however, it remains unclear
whether this activity is functionally relevant since abolition of the
lipase activity had only a marginal effect on cytotoxicity.
1.5 Ricin Biosynthesis
Ricin is made in the fatty endosperm cells of maturing castor oil
seeds (Ricinis communis) where it accumulates in protein storage
vacuoles to 5% of total organelle protein.[46] Although ricin is a
heterodimer, its individual subunits are initially synthesised to-
gether in the form of a precursor.[47] The first 26 residues constitute
a signal peptide for translocation into the endoplasmic reticulum
lumen of the plant cell[48] followed by a nine-residue propeptide of
no known function. This combined 35 residue presequence is
followed by the mature ricin A chain sequence, which in turn is
joined to ricin B chain by a 12-residue intramolecular propep-
tide.[49] The signal peptide and the two propeptides are absent in
mature ricin, with the former being removed during synthesis and
segregation into the endoplasmic reticulum lumen, and the latter
being removed in vacuoles. Within the endoplasmic reticulum
lumen, the nascent protein becomes core-glycosylated at one site
in ricin A chain (unpublished data) and at two sites within ricin B
© Adis Data Information BV 2003. All rights reserved.
Lord et al.
chain.[50] Five disulphide bonds are also formed, four within ricin
B chain and one between the mature subunit domains.[51]
The resulting core-glycosylated, folded proricin is then trans-
ported to storage vacuoles via the Golgi complex.[50,52] This intra-
cellular transport is accompanied by several poorly characterised
modifications, including trimming of oligosaccharides and addi-
tion of fucose to the ricin toxin A glycan.[53] Upon deposition in
vacuoles, the mature disulphide-linked ricin A chain/ricin B chain
heterodimer is finally generated by the endoproteolytic removal of
the N-terminal and internal propeptides.[54] The intramolecular
propeptide has been shown to be both necessary[55] and suffi-
cient[56,57] for vacuolar targeting.
Recombinant proricin has been produced and its properties
examined.[58] It is an active prolectin containing functional sugar
binding sites. In contrast, proricin lacks N-glycosidase activity,
consistent with reports that in mature holotoxin, ricin A chain must
be reductively separated from ricin B chain in order to have
activity.[23,24] It appears that in both the dimeric ricin holotoxin and
in proricin, the ricin B chain moiety can sterically obstruct the
active site of ricin A chain. Manufacture as a precursor perhaps
explains why Ricinus cells are able to synthesise large amounts of
ricin even though endogenous ribosomes are susceptible (albeit
much less so than animal ribosomes[31,59,60]), to the action of ricin
A chain.[25] Since the endosperm cells of Ricinus show no signs of
ribosome damage whilst storing large amounts of ricin, it can
further be deduced that active ricin A chain does not escape from
low pH vacuoles to reach the cytosol. The generation of an active
holotoxin only within the safe haven of vacuoles therefore enables
Ricinus seeds to synthesise and store large quantities of a potent
toxin that can effectively function in plant defence against
predators without compromising its own survival.
2. Getting Into the Cytosol
2.1 Binding of Ricin to Mammalian Cells
When ricin is applied to the exterior of most animal cells, it will
bind to complex carbohydrates containing terminal N-acetyl
galactosamine or β-1,4-linked galactose residues.[3] The abun-
dance of cell surface galactolipids and glycoproteins provides an
explanation for the diversity of cell lines that are sensitive to ricin.
Indeed, HeLa cells have been shown to possess around 3 × 107
potential ricin binding sites,[61] although not all of these sites may
be functionally involved in the productive internalisation of the
toxin. The relative contribution of glycoproteins and/or
glycolipids in the uptake of ricin by target cells remains uncertain,
although a role for glycosphingolipids as receptors has recently
been ruled out.[62] Native ricin also possesses the ability to bind
Toxicol Rev 2003; 22 (1)
Ricin Toxicity
Recycling
endosome
Caveolae
Clathrin-dependent
pH
Rab9
EE
Clathrin-independent
LE
Lysosome
Fig. 2. Schematic representation of the pathways that may be used by ricin to enter eukaryotic cells. COP1 = coatomer protein 1; EE = early endosomes;
ER = endoplasmic reticulum; LE = late endosomes; TGN = trans-Golgi network.
and enter some cell types by virtue of its own carbohydrate side
chains. A limited number of cell lines express mannose receptors
on their surface, to which ricin, with its high mannose-type gly-
cans typical of plant glycoproteins, can bind. Mannose receptor-
mediated uptake of ricin has been described in both macro-
phages[63] and rat liver endothelial cells.[64]
2.2 Endocytosis of Ricin
Due to its promiscuous binding, ricin becomes localised to all
types of membrane invaginations[65] and as such, is a valuable tool
in the study of different endocytic mechanisms[66,67] (figure 2). The
binding and internalisation of ricin into Vero cells (African Green
Monkey kidney cells) was first visualised in clathrin-coated pits
using a ricin-gold conjugate.[68] However, evidence emerged to
suggest that clathrin-independent mechanisms were also involved
in toxin uptake. For example, Hep-2 cells, in which coated pit
formation was blocked by hypotonic shock and potassium ion
depletion, remained sensitive to ricin.[69] Furthermore, in HeLa
cells, over-expression of a trans-dominant negative mutant of the
protein dynamin (a guanosine triphosphate [GTP]-binding mecha-
noenzyme that constricts lipids to aid vesicle formation) failed to
protect cells against ricin, although it did abolish clathrin-depen-
dent endocytosis.[70] In a contradictory study, ricin uptake was
shown to depend on dynamin.[71]
Clathrin-independent endocytosis includes uptake by caveolae
(specialised, invaginated lipid rafts) and macropinocytosis.[72]
When both caveolae and clathrin-dependent endocytosis were
perturbed by the removal of membrane cholesterol, ricin uptake
still occurred.[70,73] The molecular machinery controlling this
clathrin-independent endocytosis has yet to be elucidated, but
uptake may occur by more than one mechanism.[65,71,74-79] In cells
lacking functional clathrin-dependent uptake, the rate of ricin
© Adis Data Information BV 2003. All rights reserved.
57
ER
TGN Golgi
COP1-independent?
Cytosol
COP1-dependent?
internalisation was reduced to half of that seen in control cells,
suggesting that ~50% of surface membrane uptake occurs by a
clathrin-independent mechanism.[65,66,80] Although ricin uptake
has been used to probe different endocytic pathways, careful
interpretation is needed since the area of the plasma membrane
occupied by, for example, clathrin-coated pits and caveolae invag-
inations, can differ dramatically from one cell type to another.[81,82]
Furthermore, the perturbation of one route of uptake might actual-
ly up-regulate entry by a different pathway.[71,77]
2.3 Transport of Ricin from Endosomes to the Golgi
The majority of ricin that initially enters the cell is delivered to
early endosomes from where most appears to be either recycled to
the cell surface or delivered via late endosomes to lysosomes for
degradation (figure 2). Raising the pH of endosomes/lysosomes
does not protect against ricin. This, taken together with an effect of
the ionophore monensin that influences Golgi structure and func-
tion, and a protection against ricin when endosome-to-Golgi trans-
port is blocked by low temperature,[65] suggested a requirement for
the Golgi apparatus in ricin intoxication. A proportion (~5%) of
internalised ricin was also visualised within the perinuclear Golgi
with 70–80% of this small fraction being located in the trans-
Golgi network.[83]
To date, the most direct evidence for the transport of ricin to the
Golgi complex comes from biochemical data. A recombinant form
of ricin A chain was engineered to contain an internal tyrosine
sulphation site.[84] Internalisation of the unlabelled holotoxin into
cells metabolically labelled with [35S]-sulphate led to the radio-
labelling of a proportion of toxin. This outcome demonstrated that
transport to the trans-Golgi network, the location of tyrosyl
sulphotransferase, had occurred.
Toxicol Rev 2003; 22 (1)
58
The question arises as to how ricin is trafficked from en-
dosomes to the Golgi apparatus? Studies have revealed the exis-
tence of more than one route,[67,85-88] one of which is utilised by the
mannose-6-phosphate receptor[89,90] in a ‘classical’ pathway in-
volving transport from early to late endosomes to the trans-Golgi
network. Early to late endosome transport requires the low pH-
dependent formation of carrier vesicles.[91] However, ricin trans-
port to the Golgi is not affected by reagents that perturb the low pH
of endosomes.[65,92] Additionally, over-expression of a dominant-
negative mutant of Rab9, a small GTPase involved in regulating
vesicular transport between late endosomes and the trans-Golgi
network,[89,90] failed to protect cells from intoxication by ricin[70]
and did not abrogate sulphation of internalised toxin.[67]
Collectively, these studies indicate that ricin may be transport-
ed to the Golgi apparatus by a pathway that is independent of late
endosomes (figure 2). Existence of a direct pathway from early/
recycling endosomes to the Golgi apparatus has been more thor-
oughly investigated using a related bacterial ribotoxin, Shiga
toxin.[72,86,93] Recently, specific proteins, e.g. Rab6a′ and cognate
SNAREs,1 have been implicated in the direct early endosome-to-
trans-Golgi network transport step utilised by Shiga toxin.[94] The
Rab9-independent transport of ricin suggests that this protein can
also exploit the direct route from early endosomes to the Golgi,
although further work is required to correlate such transport with
other markers of this pathway. It is indeed conceivable that ricin
can utilise multiple transport pathways from the endosomal com-
partments to the Golgi apparatus due to the multiplicity of its
binding partners.
Other molecules have a lesser or uncertain relevance. Retro-
grade routing to the Golgi apparatus of ricin and Shiga toxin was
only marginally affected by mutations in the recycling endosome-
localised GTPase, Rab11.[67,93] This would suggest that a
Rab11-dependent route from the recycling endosome to the trans-
Golgi network is of minor significance. The importance of lipids
such as cholesterol, and cholesterol-binding proteins, in regulating
the intracellular transport of ricin is unclear at present. Indeed,
only marginal effects on ricin sulphation have been reported when
cholesterol homeostasis was perturbed,[88,95] or when signalling
via protein kinase A was inhibited.[96]
2.4 Transport of Ricin from Golgi to
Endoplasmic Reticulum
Ricin has been visualised in the Golgi.[97] However, as moni-
tored by immunogold labelling, only ~5% of toxin was accumulat-
ed in this compartment after a 1-hour internalisation.[83] Indeed,
1 SNARE is the SNAP receptor, SNAP is the soluble NSF attachment protein, and NSF is the N-ethyl-maleimide-sensitive fusion protein.
2 KDEL is tetrapeptide lysine-aspartic acid-glutamic acid-leucine.
© Adis Data Information BV 2003. All rights reserved.
Lord et al.
using electron microscopy, it has not been possible to visually
detect this toxin within either the Golgi cisternae or the endoplas-
mic reticulum. Such studies do not allow us to determine whether
ricin can be translocated from this location or whether an even
smaller fraction, too low to allow detection, is sorted and trans-
ported in a retrograde manner to the endoplasmic reticulum.
The question as to whether ricin escapes to the cytosol from the
Golgi or whether it needs to traffic elsewhere for the membrane
transport step has been more successfully addressed using bio-
chemical assays. An early study demonstrated that ricin cytotoxic-
ity required a brefeldin A-sensitive step.[98-100] In most cells,
brefeldin A interferes with a membrane bound GTP exchange
factor,[101] leading to a reorganisation of internal membranes and a
redistribution of Golgi contents to the endoplasmic reticu-
lum.[102,103] Ilimaquinone, a drug that causes Golgi vesiculation in
some cell types, can also protect against ricin.[104] The simplest
interpretation of these protective phenotypes is that ricin must
travel through the Golgi before it gains access to its ribosomal
substrates in the cytosol. Furthermore, transient expression of
mutant GTPases that regulate vesicle transport steps in the early
secretory pathway partially protected cells from ricin,[105] Intro-
duction of a C-terminal endoplasmic reticulum retrieval signal
(KDEL2) into ricin A chain increased the cytotoxicity of reconsti-
tuted holotoxin,[106,107] and of free ricin A chain,[108,109] suggesting
an encounter of incoming toxin with Golgi-to-endoplasmic reticu-
lum cycling KDEL receptors. Under these conditions, free ricin A
chain would enter the cells by fluid phase uptake.[110] Ricin
holotoxin has been shown to interact in vivo with the highly
abundant endoplasmic reticulum chaperone calreticulin.[111]
The most direct evidence for Golgi-to-endoplasmic reticulum
transport of ricin, however, has been shown by the brefeldin A-
sensitive uptake and subsequent core-glycosylation of a non-
glycosylated holotoxin.[84] Post-translational core-glycosylation of
an endocytosed protein is atypical, but it provides compelling
evidence for complete retrograde transport of ricin from the cell
surface to the endoplasmic reticulum where oligosaccharyl trans-
ferase is exclusively located. The presence of this glycosylated A-
chain in a cytosolic fraction further indicated that membrane
translocation takes place once transport of the toxin to endoplas-
mic reticulum is accomplished.[84]
How might ricin traffic through the Golgi apparatus? The
classic pathway of retrograde transport involves movement via
coatomer protein 1-coated vesicles.[112,113] A second, less well
characterised pathway is coatomer protein 1-independent[114,115]
(figure 2). This pathway mediates the transport of some cycling
Toxicol Rev 2003; 22 (1)
Ricin Toxicity
Golgi glycosylation enzymes and possibly lipids, in a manner
controlled by Rab6[114] (a small GTPase that modulates intra-Golgi
transport). It should be noted, however, that the overall signifi-
cance of coatomer protein 1-independent transport and the nature
of any carriers involved remain unclear. Both pathways are
utilised by a number of bacterial toxins. For example, cholera
toxin and Pseudomonas exotoxin A exploit KDEL receptors that
in turn are sequestered into coatomer protein 1 coated vesi-
cles.[116-119] In contrast, Shiga toxin utilises the coatomer protein
1-independent route.[114,115] Only one study has been made with
ricin, where a block in coatomer protein 1 function did not protect
cells from intoxication.[120]
2.5 Membrane Translocation of Ricin
Although ricin translocation has been reported from fractionat-
ed endosomes,[121] this may represent a minor transport step or one
that occurred from contaminating microsomes, since a large body
of evidence (reviewed in section 2.4) has since accumulated to
support the idea that ricin is routed to the endoplasmic reticulum.
Why might ricin need to traffic to this organelle to exert a toxic
effect? It is likely that ricin’s complex intracellular journey is
connected to the membrane translocation step. Many bacterial
toxins form pores in biological membranes, either directly or upon
exposure to low pH. Ricin, however, does not form pores in
membranes. One appealing possibility is that ricin must be endo-
cytosed to a compartment that contains in its membrane pre-
existing protein-conducting channels through which the toxin can
exit.[122] In the endomembrane system, the only organelle that is
known to meet this criterion is the endoplasmic reticulum, whose
membrane contains abundant protein translocases (known as
Sec61 translocons)[123] and peptide transporters.[124] While peptide
transporter-independent cells have been shown to be as sensitive
to ricin as parental peptide transporter-positive cells,[8] effort has
focused on showing that protein translocon complexes provided
the conduits for toxin export. However, the tightly gated Sec61
channels were originally thought to be involved only in the import
of nascent proteins, making it difficult to explain how this machin-
ery might be used in the reverse direction.
The possible mechanism for this step remained elusive until
recognition of a quality control system in the endoplasmic reticu-
lum whereby newly made proteins detected as aberrant or orphan
become dislocated to the cytosol for degradation by proteasomes.
This quality control pathway is termed endoplasmic reticulum-
associated protein degradation (ERAD).[125,126] A schematic to
outline the fate of a typical misfolded soluble protein in the yeast
endoplasmic reticulum is shown in figure 3. In mammalian cells,
ERAD is more complex, with an elaborate system of molecular
© Adis Data Information BV 2003. All rights reserved.
59
chaperones and associated proteins that assist in the folding of
nascent proteins. Some proteins serve to ‘time-out’ interactions
with chaperones, whilst others somehow couple recognition of a
defective protein with the retrotranslocation machinery for extrac-
tion and destruction in the cytosol. Genetic and biochemical
evidence support a role for the Sec61 protein translocons in the
retro-translocation of endoplasmic reticulum-associated protein
degradation substrates.[127] Tightly coupled to this process is the
ubiquitination of internal lysyl residues of endoplasmic reticulum
degradation substrates. However, whether this modification is
required only for subsequent targeting to proteasomes or whether
it is needed for the actual retro-translocation process remains
unclear. Although understanding of retro-translocation is incom-
plete, there is a sufficient framework of implicated components to
test their involvement in ricin dislocation.
A pre-requisite for the activity of ricin A chain is that it is
reductively separated from ricin B chain. At present, it is unclear
whether this occurs in the endoplasmic reticulum as it does for
cholera toxin[129-131] and Pseudomonas exotoxin A[132] or whether
it occurs in the reducing environment of the cytosol. An early
study implicated translocation of a non-reducible ricin holotoxin
that was as cytotoxic as native ricin[133] although this has never
been further characterised and, as yet, the precise fate of ricin B
chain is unknown.
To date, no chaperone interactions other than holotoxin with
calreticulin have been detected[111] and, as such, it remains unclear
how ricin/ricin A chain is selected and targeted to the retro-
translocation apparatus. It remains possible that reduction of the
holotoxin in the endoplasmic reticulum exposes region(s) within
ricin A chain normally masked in ricin that allow this subunit to
interact with the membrane. Significant structural changes have
been shown to occur in ricin A chain, but not in ricin B chain or
holotoxin, when added to artificial liposomes containing negative-
ly charged lipids.[134] The physiological significance of this in
relation to the endoplasmic reticulum membrane requires clarifica-
tion. This situation contrasts with knowledge of cholera toxin
interactions where protein disulphide isomerase serves as an un-
foldase to present toxin to the retro-translocation machinery.[135]
The involvement of protein translocase complexes in toxin
retro-translocation has been shown by co-immunoprecipitation of
the protein translocase alpha subunit with the post-translationally
core-glycosylated ricin A chain variant that was endocytosed to
the endoplasmic reticulum as part of a holotoxin.[107] Interestingly,
Chinese hamster ovary cell lines with genetic defects in endoplas-
mic reticulum-associated protein degradation also exhibited in-
creased resistance to ricin intoxication.[136]
One problem faced by toxins that hijack the retro-translocation
machinery is that to be toxic they must avoid the ultimate fate of
Toxicol Rev 2003; 22 (1)
60 Lord et al.

Deglycosylation Proteasomal
degradation

PNGase

Import via
Cytosol Sec61 translocon
Hrd1p Ubiquitination

Sec61p
Sec61p
Ubc1p/7p

Hrd3 Cue1p
ER

Export via
Kar2p
Sec61 translocon
Glycosylation

Misfolded protein Protein folded

targeted for degradation correctly for activity
Fig. 3. Schematic representation of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway in the yeast Saccharomyces
cerevisiae. Newly made proteins are delivered through the translocon (comprising the Sec61p complex and accessory proteins) into the ER lumen. After
scrutiny by the quality control system, many persistently misfolded proteins are targeted via the translocon back to the cytosol for destruction (for a recent
review see Jarosch et al.[128]). The chaperone Kar2p, protein disulphide isomerase family members and mannose-binding lectins have been implicated in
the early events relating to the turnover of misfolded proteins. Membrane proteins (Hrd3p and Der1p) serve as potential receptors for these substrate
recognition components, whilst ubiquitin-conjugating enzymes (e.g. Ubc1p, Ubc6p and Ubc7p), and membrane bound ubiqutin ligases (e.g. Hrd1p) are
responsible for the polyubiquitination of internal lysyl residues of ERAD substrates. Note that the protein Cue1p recruits soluble Ubc7p to the surface of the
ER membrane. Ubiquitination of ERAD substrates on the cytosolic surface of the membrane appears to be crucial for the retrotranslocation process,
serving to provide a ratcheting mechanism or to recruit other complexes such as proteasomes or the Cdc48p complex that in turn leads to eventual
degradation by proteasomes. Peptide N-glycanase (PNGase) is a cytosolic peptide that removes glycans from glycoproteins at some point prior to their
degradation. For clarity, not all proteins are shown in the schematic.

endoplasmic reticulum-associated protein degradation substrates,
namely degradation by proteasomes. It was noticed that the cata-
lytic subunits of a number of toxins shown to reach the endoplas-
mic reticulum contained very few, if any, lysine residues.[137] This
intriguing observation led to a hypothesis that a paucity of lysyl
residues may serve to assist the uncoupling of toxin retro-translo-
cation from downstream degradation by avoidance of the ubiqui-
tination process.[137] This hypothesis has been tested by modulat-
ing the lysine content in ricin, abrin[138] and cholera toxin.[139] In all
cases, the lysine content altered cytotoxicity in the predicted way,
such that the greater the lysine content the lower the cytotoxicity
of the toxin unless proteasomes were inhibited. It would appear
that ricin A chain is not a natural substrate for ubiquitination and
that its low lysine content attenuates what might otherwise be a
very efficient process for removing and thereby disarming this
toxin in the cytosol. Interestingly, when proteasomes are inhibited,
cells are typically sensitised 2- to 3-fold to ricin.[8,111,138] This
suggests that ricin A chain can be targeted to proteasomes in a
ubiquitin-independent manner, but that uncoupling from the ubi-
quitination process provides an opportunity for refolding, and
hence degradation avoidance, of at least a fraction of the toxin.
Partially unfolded ricin A chain has been shown to refold in the
presence of ribosomes to regain full catalytic activity.[140] This
work has suggested that ribosomes may act as suicidal chaperones
since, by the very act of refolding ricin A chain, the ribosome
becomes irreversibly modified.
2.6 Retro-Translocation of Ricin A Chain in Other Systems
The genetically tractable budding yeast, Saccharomyces cerevi-
siae, has routinely been utilised to investigate eukaryotic cellular
processes that are more complex (and hence more difficult to
study) in mammalian systems. However, exogenously adminis-
tered ricin is not lethal to yeast due to the absence of an appropriate
galactosyl transferase. Yeast ribosomes are sensitive to ricin A

© Adis Data Information BV 2003. All rights reserved. Toxicol Rev 2003; 22 (1)
Ricin Toxicity
chaintoxicity, however,[141] and ricin A chaincan be synthesised de
novo and delivered into the yeast endoplasmic reticulum, render-
ing this system appropriate for the study of retro-translocation.
Quality control is less stringent in this organism than it is in
mammalian cells and there are some differences in the required
components, including the absence of a calreticulin homo-
logue.[126] Nevertheless, many of the players involved in the pro-
cess of ERAD in yeast have been identified (figure 3), although the
relevance and sequential involvement of most of these in the retro-
translocation of ricin A chain is unknown. As in mammalian cells,
the Sec61 translocon is known to be the channel through which
most misfolded yeast ERAD substrates are exported from the
endoplasmic reticulum.[142,143] Simpson et al.[144] investigated the
degradation kinetics of ricin A chain in a number of yeast endo-
plasmic reticulum-associated protein degradation mutants. Using
protein translocation mutants that were competent for import into
the endoplasmic reticulum while being cold sensitive for endo-
plasmic reticulum to cytosol export,[142] it was shown that ricin A
chain mutants could be stabilised relative to their rapid degrada-
tion in wild-type strains.[144] As in mammalian cells, it was also
evident that the ricin A chain was degraded in a ubiquitin-indepen-
dent, proteasome-dependent manner. The need for a recycling of
ricin A chain between Golgi and endoplasmic reticulum, as re-
quired for some soluble yeast endoplasmic reticulum-associated
protein degradation substrates,[145,146] is not known.
3. Conclusions
Ricin is exquisitely potent to mammalian cells, being able to
fatally disrupt protein synthesis by attacking the Achilles heel of
the ribosome. For this enzyme to reach its substrate, it must not
only negotiate the endomembrane system but it must also cross an
internal membrane and avoid complete degradation without com-
promising its activity in any way. It is clear that ricin can take
advantage of many target cell molecules, pathways and processes.
Future studies will attempt to clarify more exactly the range and
regulation of endocytic pathways used by ricin, the nature of intra-
molecular carriers and transport intermediates, the interactions
that lead to subversion of endoplasmic reticulum-associated pro-
tein degradation and toxin refolding, and the precise downstream
steps leading to apoptosis. In parallel with such fundamental
studies, ricin will continue to be used therapeutically for the
selective killing of cancer cells.[6,7] Where delivery of material to
the endoplasmic reticulum is required, the unusual intracellular
trafficking of this protein (as well as other bacterial toxins) can
also be exploited. In this way, disarmed (catalytically inactive)
versions will be developed as molecular carriers to trigger an
immune response against certain tumours and viruses.[8] Combin-
ing our understanding of the ricin structure with ways to cripple its
© Adis Data Information BV 2003. All rights reserved.
61
unwanted properties (its enzymatic activity and promotion of
vascular leak[147] whilst retaining protein stability and important
immunodominant epitopes[148]), will also be crucial in the develop-
ment of a long awaited protective vaccine against this toxin.[149]
Acknowledgements
Work cited from the Warwick laboratory was supported by the UK
BBSRC and The Wellcome Trust. The authors have no conflicts of interest
that are directly relevant to the content of this manuscript.
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Sciences, University of Warwick, Coventry, CV4 7AL, UK.
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