Department of Biological Sciences, University of Warwick, Coventry CV4 7AL United Kingdom
THE synthesis, segregation, intracellular transport,
and exocytic export of secretory proteins in eukaryotic cells is now well understood. Synthesis begins
on free cytosolic ribosomes that subsequently attach to the
ER, resulting in the cotranslational discharge of the nascent proteins into the ER lumen. The targeting signals
and the cytosolic and ER membrane components that mediate the translocation step have been extensively characterized and reviewed (for example, see 29). The ER lumen
contains a range of resident proteins responsible for modifying newly synthesized polypeptides, and for ensuring
correct folding into the biologically active conformation
(9). Secretion entails transport of the proteins from the
ER, via the Golgi stack and the TGN, to secretory vesicles
that ultimately fuse with the plasma membrane to complete protein export. The secretory pathway was elucidated by Palade and his colleagues (24). Protein transport
between the various compartments of the secretory pathway is mediated by carrier vesicles that bud from one compartment and fuse with the next. Once again, many of the
cellular components that are required for, and that regulate, vesicular transport have been identified, and the
mechanisms by which cytoplasmic protein coats drive the
individual transport steps are now emerging (31). It has
been recognized for some time that the secretory pathway
is at least partially reversible. For example, retrograde vesicular transport from the Golgi to the ER is needed to retrieve resident ER proteins that have escaped from this
compartment (25), and endocytosis can transport proteins from the cell surface to the TGN (2), which is the cellular location where the secretory and endocytic pathways converge (31). ER resident proteins contain a retrieval signal, the COOH-terminal tetrapeptide Lys-Asp-Glu-Leu
(KDEL),1 which binds to a membrane receptor in the cis-Golgi region and returns them to the ER. Indeed the
KDEL receptor is capable of retrieving escaped ER resident proteins from as far along the secretory pathway as
the TGN; an exogenous synthetic peptide with a COOH-terminal KDEL sequence, which was introduced into the
TGN from the cell surface was subsequently transported
to the ER lumen (20). Recently, it has become apparent
that certain protein toxins follow this same route from the
surface to the ER lumen. To reach their targets in the cytosol of mammalian cells the toxins apparently go one step
further and cross the ER membrane. The emerging experimental evidence in support of this is reviewed here.
Toxin Structure and Mode of Action
Certain bacteria and plants produce proteins that are able
to enter and kill mammalian cells. These proteins all act by
catalytically modifying essential cellular components so
that their normal function is prevented. In this review, we
focus on a subset of these toxins that act by inhibiting cellular protein synthesis. Other catalytic toxins enter cells by
the same mechanisms, and reference will be made to them
(where appropriate). Bacterial toxins that inhibit protein
synthesis in eukaryotic cells include diphtheria toxin (DT),
Pseudomonas exotoxin A (PE), and Shiga toxin (ST),
whereas the best characterized of a group of related plant
toxins is ricin. DT, PE, and ricin are all synthesized as single polypeptide chains but in their functional form they exist as heterodimers covalently joined by a single disulfide
bond. DT and PE are cleaved into dimers after entering
mammalian cells by the ubiquitous protease furin (7),
whereas ricin is cleaved in the plant cells that produce it
(17). In the heterodimeric toxins, one polypeptide (often
designated the A chain or fragment) is catalytically active whereas the other (the B chain or fragment) is responsible
for binding the toxin to receptors on the surface of susceptible cells. ST differs in structure from the other toxins described here in that the catalytic A chain is associated with
a pentamer of B subunits, the so called AB5 structure possessed by other bacterial toxins such as cholera toxin and
Escherichia coli heat labile enterotoxin.
DT and PE inhibit protein synthesis by ADP-ribosylating elongation factor-2 (45), whereas ST and ricin act by
removing a specific adenine residue from a highly conserved loop present in 28S rRNA (5). Since the toxin substrates are in the cell cytosol, the A chain must enter the
target cell cytosol and retain its biologically active conformation. To enter mammalian cells, the toxins must first
bind to a surface component. Binding can be to a specific
protein such as the Cell Entry
Surface-bound toxin enters cells by endocytosis; the endocytic route depends on the nature of the receptor. DT,
for example, appears to enter exclusively via clathrin-coated pits and vesicles. As such, overexpression of mutant dynamin, which prevents clathrin-dependent endocytosis, protects cells from intoxication by DT (40). Such cells remain completely sensitive to ricin, however. Normally ricin uses both clathrin-dependent and -independent
endocytosis, so when the former route is effectively
blocked the latter remains available. The clathrin-independent route used by ricin does not appear to be mediated by caveolae since it is unaffected by treatment with
cholesterol-binding drugs such as nystatin (40). ST appears to enter cells exclusively via clathrin-dependent endocytosis (33), suggesting that its glycolipid receptor becomes
fixed in coated pits, possibly by interacting with a protein
already anchored there. Both the clathrin-dependent and
the clathrin-independent uptake routes converge in endosomes. At this stage the endocytosed toxins fall into two
different behavioral groups: those that translocate into the
cytosol from an endosomal compartment, and those that do not. Toxins whose A fragment crosses the endosomal
membrane include DT, which has been most thoroughly
studied, and others such as anthrax, tetanus, and botulinum toxins. DT translocation depends crucially on the low
pH of the endosomal compartment, which induces a conformational change in DT B causing it to insert into the
endosomal membrane and form, or contribute to the formation of, a proteinaceous pore through which the A fragment is able to pass. Further discussion of the DT translocation mechanism can be found elsewhere (37).
Since DT translocation requires low pH, treating cells
with reagents that increase endosomal pH protects them
from intoxication. Conversely, lowering the extracellular
pH to that typically found in endosomes causes surface-bound DT to directly cross the plasma membrane. Increasing the intracellular pH does not protect cells against
members of the second group of toxins; toxins that do not
translocate from endosomes including PE, ST, and ricin.
Members of this second toxin group must proceed beyond
endosomes to reach the compartment for A chain translocation.
Intracellular Transport Beyond Endosomes
The trafficking of endocytosed ricin beyond endosomes
was initially followed by electron microscopy (42). In addition to entering lysosomes or being recycled back to the
cell surface, a significant amount of toxin accumulated in
the TGN. Initially this was interpreted as suggesting that
ricin A chain translocated to the cytosol from the TGN
(43). With hindsight, a critical limitation of this experimental approach was its sensitivity. Whereas it clearly
defined the fate of the bulk of the endocytosed toxin, the
extreme potency of such proteins (entry of just a few molecules into the cytosol is believed sufficient to kill a cell) meant that the productive but inefficient trafficking to the
translocation compartment went undetected. One of the
first experimental indications that toxins such as PE, ST,
and ricin might not translocate from the TGN was the
demonstration that treating the cells with brefeldin A
(BFA) protected against these toxins but had no effect on
their susceptibility to DT (50). A careful morphological
study of ricin trafficking in BFA-treated cells showed that
in all lines where protection was seen, the typical BFA-
induced dissembly of the Golgi stack had occurred (32).
Transport of ricin to the TGN was unaffected by BFA
treatment. In contrast, treating MDCK cells with BFA did
not confer protection against ricin nor did it structurally
perturb the Golgi apparatus.
Experiments with PE mutants suggested that PE might
also undergo retrograde transport from the TGN to the
ER, and provided a likely explanation of how this occurred. The last five COOH-terminal residues of the translocated PE fragment are Arg-Glu-Asp-Leu-Lys (REDLK).
The effect of mutations in this pentapeptide on the catalytic activity and cytotoxicity of PE were determined. Deletions or substitutions predicted to prevent interaction
with the KDEL receptor dramatically reduced cytotoxicity
(3). Such changes had no effect on catalytic activity and
apparently cause reduced cytotoxicity by preventing the
mutant forms from reaching the cytosol. Replacing the
wild-type REDLK sequence with KDEL produced a mutant toxin even more cytotoxic than wild-type PE. (36).
The COOH-terminus of the catalytic polypeptide of cholera toxin is KDEL, and that of E. coli heat labile enterotoxin is RDEL. Subsequent work has established that cholera and E. coli heat-labile toxin action are also blocked by
BFA treatment (4). The cytotonic activity of these G protein-modifying proteins is not absolutely dependent on
the KDEL/RDEL tetrapeptide, although it apparently enhances delivery into the cytosol (16). EM studies have visualized endocytosed ST (34) and cholera toxin (35) in the
ER lumen, and cholera toxin transport from the plasma
membrane to the ER has been described (18). For toxins
that contain KDEL or a KDEL analogue at the COOH-terminus of the translocated polypeptide, retrograde transport from the TGN to the ER is most probably achieved
by interaction with the KDEL receptor which is now
known to recycle between these compartments (8, 20). Presumably, after endocytosis, recycling cell surface receptor
delivers a proportion of the toxin to the TGN where receptor-toxin dissociation occurs leaving the free toxin able to
opportunistically interact with the KDEL receptor.
How do toxins such as ST and ricin, which do not possess KDEL or a related peptide, travel from the TGN to
the ER? That ST can reach the ER lumen is beyond dispute since, as noted above, it has been visualized there.
The effect of BFA treatment suggests that ricin must also
traverse the Golgi stack to reach the ER. This contention
was supported by the finding that addition of the KDEL
retrieval signal to the COOH-terminus of ricin A chain significantly enhances cytotoxicity (46). It seems likely that ricin undergoes retrograde transport from the TGN to the
ER by binding to a recycling galactosylated component. Ricin B chain contains two functional galactose-binding sites,
and is itself glycosylated. Because it has a high mannose
oligosaccharide sidechain, ricin is able to intoxicate cells
with mannose receptors, such as macrophages, even when
binding to galactosylated surface components is prevented
(38). The effect of abrogating one or other or both B chain
galactose-binding sites on the cytotoxicity of ricin entering
macrophages via the mannose receptor showed that toxin retaining either of the B chain sugar-binding sites remained cytotoxic, but when both sites were destroyed, toxicity was lost (22). The putative recycling galactosylated
component used by ricin for retrograde transport might
itself carry a KDEL retrieval signal, be a transmembrane
glycoprotein with a retrieval signal such as KKXX (12), or
may interact with another molecule that has a retrieval
signal.
The ER as the Site of Toxin Translocation
Recently the first direct biochemical evidence that endocytosed ricin reaches the ER before translocating to the
cytosol has been presented (28). Ricin A chain was modified to contain a tyrosine sulfation site and overlapping
N-glycosylation sites. Mutant A chain was reassociated
with B chain and incubated with cells in the presence of
Na235SO4 and the A chain became radiolabeled. This labeling identified that proportion of the endocytosed toxin
that had reached the Golgi, and labeling was prevented by
treatment with BFA or ilimaquinone. The labeled A chain
underwent retrograde transport to the ER where it became core glycosylated, and then translocated to the cytosol. Only free glycosylated A chain entered the cytosol, indicating that transport to the ER was a prerequisite for
translocation. At this stage it is unclear whether the A and
B chains dissociate in the ER before translocation, or in
the cytosol after translocation of the holotoxin. A similar
experimental approach has confirmed that ST B fragment
also reaches the ER via the Golgi apparatus (13).
Do Toxins Use the ER-associated Protein Degradation
Pathway to Enter the Cytosol?
It has been known for some time that small glycopeptides
can enter the cytosol directly from the ER (30), and more
recently it has become apparent that proteins and glycoproteins also do so as part of the ER quality control function (14). Nascent secretory proteins enter the ER through
a proteinaceous channel whose central component is
Sec61p, the Sec61p translocon. Only if secretory proteins
are folded correctly, and if appropriate, correctly assembled into oligomers, are they packaged into ER-Golgi transport vesicles. Proteins that fail to fold or assemble
properly are instead exported across the ER membrane to
the cytosol, where they are degraded by the proteasomes
(10, 47). Human cytomegalovirus (CMV) takes advantage
of this export machinery to downregulate antigen presentation at the cell surface by the class I major histocompatibility complex (MHC). CMV proteins in the ER cause dislocation of the transmembrane MHC class 1 heavy chain
from the ER membrane to the cytosol, and its subsequent
degradation by the proteasomes (48, 49). Genetic and biochemical experiments in Saccharomyces cerevisiae demonstrated that soluble, misfolded proteins are also exported
across the ER membrane to the cytosol for degradation
(10, 19). Coimmunoprecipitation studies indicated that the
MHC class 1 heavy chain in CMV-infected cells might be exported through the Sec61p translocon (49). Direct evidence for the involvement of Sec61p in the export of misfolded proteins from the ER to the cytosol has recently
been demonstrated in yeast (26, 27). It is likely that when
toxins such as PE, ST, or ricin reach the ER lumen, thiol
exchange catalyzed by protein disulfide isomerase causes
dissociation of the disulfide-linked A and B subunits. Reductive dissociation in the oxidizing environment of the
ER lumen has been demonstrated for disulfide-linked
cholera toxin A fragments (23). Dissociation of the toxin
subunits exposes a hydrophobic region present in the A
polypeptides that may promote unfolding or membrane
interaction. In the case of ricin A chain, point mutations in
this region do not affect catalytic activity but dramatically
reduce ricin cytotoxicity, which may indicate that the hydrophobic stretch plays some role in the membrane translocation step (39). The free A subunits may be perceived
as unassembled polypeptides or Summary and Future Prospects
Bacterial and plant toxins that kill mammalian cells by
modifying intracellular targets are unusual in that they
both fold and traverse a membrane twice. They cross a
membrane and fold for the first time as they are secreted
from the producing bacteria or enter the ER lumen of the
eukaryotic plant cells. During the intoxication process, the
toxin A subunits partially unfold and again cross a membrane before refolding in the cytosol. Whereas some toxins, including DT, take advantage of the low pH to cross the endosomal membrane, others such as PE, ST, and ricin
are apparently unable to form or become part of a translocation channel. Therefore, these toxins undergo retrograde
transport through the secretory pathway to reach a cellular compartment where protein-conducting channels are
already present
ARTICLE
Top
Article
References
2-macroglobulin receptor (used by
PE) (15) or a heparin-binding epidermal growth factor (EGF)-like growth factor precursor (used by DT) (21), to
glycolipids such as globotriaosyl ceramide (used by ST)
(11), or ganglioside GM1 (used by cholera toxin) (44), or to
any appropriate glycoprotein or glycolipid (as in the case
of ricin whose B chain is a galactose-specific lectin) (17).
if ER resident chaperones cause partial unfolding
as misfolded polypeptides.
Evidence that both DT A and ricin A chain partially unfold during cell entry has been presented (1, 6). This may
result in export to the cytosol by the ER quality control system. If this is the case, some of the exported toxin must
avoid proteasomal degradation and assume its biologically
active conformation in order to kill the cell. An in vitro
study has reported that the NH2-terminal region of the
translocated PE fragment promotes its own export after
microsomal membrane insertion (41).
the ER. This is achieved by parasitizing normal cellular components that recycle between the plasma
membrane and the TGN, and between the TGN and the
ER. From the ER, the toxins enter the cytosol, possibly via
the Sec61p translocon, and perhaps by masquerading as
substrates for the ER export machinery. It may be possible
to take advantage of the ability of these toxins to reach the
ER. For example, toxins may be able to directly deliver antigenic peptides to MHC class 1 molecules. Finally, toxin
entry demonstrates that the eukaryotic secretory pathway
is completely reversible. This is physiologically significant
in that it provides a route by which these exogenous proteins reach their cytosolic targets.
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Footnotes |
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Received for publication Received for publication 15 August 1997 and in revised form 29 December 1997..
We thank Dr. K. Romisch for reading this article and for helpful suggestions that led to its improvement. ![]() |
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