(Received for publication, June 20, 1995; and in revised form, July 28, 1995)
From the
Translocation to the cytosol is an essential and rate-limiting step in the cytotoxicity of the potent plant toxin ricin. In an attempt to study the mechanism of ricin A-chain (RTA) translocation in a cell-free assay, we have partially purified Golgi and endoplasmic reticulum from Jurkat cells by discontinuous sucrose gradient fractionation. The membranes of the organelle fractions were solubilized by the addition of sodium cholate and reconstituted into proteoliposomes by dialyzing out the detergent. The resulting vesicles supported cell-free translocation of RTA (as assessed by an enzyme protection assay) at a rate which was linearly dependent on the concentration of the vesicle preparation. Ricin B-chain (RTB) neither translocated into the vesicles, nor increased the efficiency of RTA translocation. Liposomes prepared from purified phospholipids were not capable of supporting RTA translocation. Furthermore, protease treatment or concanavalin A adsorption of proteins from lysates prior to vesicle reconstitution resulted in abrogation of the translocation process, suggesting that the protein components of organelle membranes are required for RTA translocation. Reconstitution of translocation-competent proteoliposomes from detergent-solubilized membranes of endoplasmic reticulum- and Golgi-enriched fractions provides a convenient cell-free system to study the mechanism of RTA translocation.
Ricin is a potent plant toxin which is used in the preparation
of immunotoxins for the therapy of cancer and autoimmune and infectious
diseases(1, 2) . It is composed of two polypeptide
chains, the 32-kDa ribosome-inactivating chain, RTA, ()and
the 33-kDa cell binding chain, RTB, which are linked to each other with
a disulfide bond. Following endocytosis of ricin or immunotoxin by the
target cell, RTA is transported to a cell compartment where it
translocates to the cytosol and exerts its ribosome-inactivating effect (3) . RTA translocation is the rate-limiting step in the
cytotoxicity of ricin and immunotoxins(4) . However, the
intracellular site(s) and the mechanism of RTA translocation are poorly
understood. Electron microscopic studies have shown that ricin is
transported as far as the trans-Golgi network in the endocytic
pathway(5) . Treatment of cells with brefeldin A, which
disrupts the Golgi structure and blocks the connection between the
endocytic pathway and the secretory pathway, results in the protection
of cells from the toxic effects of ricin and other related plant and
bacterial toxins such as modeccin, abrin, volkensin, viscumin, and
Shiga toxin, but not of Diphtheria toxin, which is known to translocate
to the cytosol from endosomes(6, 7) . Furthermore,
brefeldin A protects cells from cholera toxin-induced elevation of
intracellular cAMP(8) . These studies suggest that the
retrograde transport of ricin and related toxins into proximal
compartments of the secretory pathway such as the Golgi stacks and ER
may be important for efficient cytotoxicity. Addition of a ER retrieval
sequence, KDEL, onto the C-terminal end of RTA significantly increased
the cytotoxicity of ricin (9) and RTA (10) lending
credence to the concept that RTA translocates to the cytosol most
efficiently from the ER. A recent study demonstrated that ricin
translocation starts in the endosomes, but that translocation
efficiency increases as the toxin is transported deeper into the
endocytic pathway(11) .
A major obstacle for the study of ricin translocation in organelles deep in the internalization pathway, such as the Golgi stacks and ER, is death of cells before they accumulate sufficient concentrations of ricin to permit detection and experimentation. To circumvent this obstacle and to study the mechanism of translocation of RTA in these organelles, we developed a cell-free system in which we reconstituted translocation-competent proteoliposomes from detergent-solubilized membranes of Golgi- and ER-enriched fractions of Jurkat cells. Using this system, we studied the kinetics and the membrane requirements of RTA translocation and the effect of RTB on the translocation of RTA.
Figure 1:
Characterization of fractions obtained
by discontinuous sucrose gradient centrifugation. Jurkat cells were
disrupted with a Dounce homogenizer and fractionated on discontinuous
sucrose gradients by ultracentrifugation. Harvested fractions were
analyzed for their activities for galactosyltransferase (), a
Golgi-specific enzyme;
-galactosidase (
), a
lysosome-specific enzyme; and glucose-6-phosphatase (
), an
ER-associated enzyme (upper panel), as well as for protein
concentrations (
) (bottom
panel).
Figure 2:
Topology of the reconstituted vesicles.
Vesicles reconstituted from Golgi- () and ER-enriched (
)
fractions were incubated with
I-IgG
,
I-anti-MPR, and
I-anti-calnexin for 45 min
at room temperature and then diluted with buffer G and pelleted. The
vesicle associated antibody was determined by
counting the
radioactivity of the pellet and expressed as the percentage of the
total. The total counts/min (bound + unbound) was 4,141 for
I-IgG
, 22,732 for
I-anti-MPR
and 9,782 for
I-anti-calnexin. One of two concordant
experiments is shown.
Figure 3:
Translocation of RTA into vesicles
reconstituted from Golgi- and ER-enriched fractions. a,I-RTA was incubated with and without vesicles
reconstituted from fractions 1 (ER), 10 (Golgi), and 12 and then
digested with 1 unit/ml papain for 75 min on ice. Degradation was
analyzed by SDS-PAGE and autoradiography. b,
I-RTA was incubated with and without vesicles
reconstituted from Golgi- and ER-enriched fractions in the presence and
absence of 1% Nonidet P-40 and then subjected to papain digestion and
analyzed by SDS-PAGE and autoradiography as described
above.
Figure 4:
Effect of the concentration of vesicles on
the translocation of RTA. I-RTA was incubated with and
without vesicles reconstituted from ER-enriched fractions at the
indicated dilutions and then digested with 1 unit/ml papain for 75 min
on ice. The concentrations of vesicle suspensions (Dilution 1)
were standardized spectrophotometrically so that 250 µl of each
vesicle preparation possessed an absorbance of 0.2 at 405 nm.
Degradation was analyzed by SDS-PAGE and autoradiography (upper
panel). The amount of translocated RTA at each dilution was
determined by densitometric analysis of the lanes and plotted (lower panel).
Figure 5:
Effect of incubation time on the
translocation of RTA. I-RTA was incubated with and
without vesicles for the indicated times and then digested with 1
unit/ml papain for 75 min on ice. Degradation was analyzed by SDS-PAGE
and autoradiography. The autoradiographs of two separate experiments
are shown (upper panel). The amount of the translocated RTA at
each time point was determined by densitometric analysis of the lanes
and plotted (lower panel). (
, experiment 1;
,
experiment 2).
Figure 6:
Effect of protein depletion on
translocation of RTA. I-RTA was incubated with vesicles
reconstituted from protease-treated cholate extracts (upper three
panels) or ConA-adsorbed cholate extracts (lower panel)
of the Golgi-enriched fraction, digested with proteinase K, and
analyzed by SDS-PAGE and autoradiography. Similar results were obtained
in experiments using ER-enriched fractions for vesicle reconstitution
(data not shown).
An alternative protein depletion approach corroborated the conclusions derived from the protease experiments. Cholate extracts of Golgi- and ER-enriched fractions were passed through ConA columns to deplete glycoprotein constituents prior to vesicle reconstitution. This method of protein depletion also abrogated the translocation competence of the subsequently reconstituted vesicles as illustrated in Fig. 6(lower panel).
Figure 7:
Inability of RTB to translocate into
reconstituted vesicles. I-RTB was incubated with and
without vesicles reconstituted from Golgi-enriched fractions in the
presence and absence of 1% Nonidet P-40 and then digested with 1
unit/ml papain for 75 min. Degradation of
I-RTB was
analyzed by SDS-PAGE and autoradiography. Similar results were obtained
using proteoliposomes reconstituted from ER-enriched membranes (data
not shown).
Figure 8:
Effect of RTB on the translocation of RTA
into reconstituted vesicles. I-RTA was incubated with and
without vesicles reconstituted from ER-enriched fractions in the
presence and absence of 0.1 µg of unlabeled RTB and then digested
with 1 unit/ml proteinase K. Degradation of
I-RTA was
analyzed by SDS-PAGE, autoradiography, and densitometry. Similar
results were obtained using proteoliposomes reconstituted from
Golgi-enriched membranes (data not shown).
Several independent lines of investigation have suggested that most plant and bacterial toxins, including ricin, translocate to the cytosol most efficiently from organelles deep in the internalization pathway (e.g. the trans-Golgi region or endoplasmic reticulum). Ricin has been demonstrated in the trans-Golgi network by electron microscopic studies(5, 6) . Furthermore, brefeldin A treatment has protected the cells from the detrimental effects of most related bacterial and plant toxins, suggesting that the organelles of the secretory pathway (Golgi and ER) might be involved in the action of these toxins(6, 7, 8) . The increased cytotoxicity of the KDEL-modified RTA has provided additional evidence for the trafficking of RTA to Golgi and ER for efficient translocation (9, 10) . Unfortunately, it has been very difficult to study toxin translocation from the Golgi or ER, because the extreme potency of these reagents results in cell death before appreciable concentrations of toxin accumulate in these compartments. For example, it has been estimated that one ricin molecule can inactivate 1500 ribosomes per minute and that this rate is sufficient to kill a cell(28, 29) . Although the efficiency of the translocation process is not known with certainty, it appears plausible to assume that most standard assays are insufficiently sensitive to detect toxin concentrations capable of causing irreversible cytotoxicity. Consequently, we have developed an in vitro translocation assay based on proteoliposomes reconstituted from purified Golgi- or ER-enriched membranes by adapting methodology which has proven indispensable for the study of translocation of secretory proteins from ribosomes to the ER lumen(20, 21, 30) . As demonstrated in the present study, translocation-competent proteoliposomal vesicles from detergent solubilized membranes of Golgi- and ER-enriched fractions provide convenient cell-free systems for the study of RTA translocation.
Our experiments show that RTA is able to translocate efficiently into reconstituted vesicles derived from either Golgi- or ER-enriched fractions of Jurkat cells in a manner linearly dependent on the vesicle concentration. The kinetics of the translocation process show that the amount of RTA that translocates increases linearly for 1 h and then plateaus. It appears likely that the cessation of translocation after 1 h in this reconstituted system is due to depletion of an endogenous (organelle membrane-derived) energy source that drives the translocation machinery. In their recent study of ricin translocation across endosomal membranes, Beaumelle et al.(11) showed that ricin translocation in endosomes is ATP-dependent and stops after 30 min unless exogenous ATP is added. The difference in the duration of translocation without exogenous ATP between the present study and that of Beaumelle et al.(11) may reflect inherent differences in the distribution of energy-generating systems between Golgi, ER and endosomes.
RTB is known to augment the toxicity of RTA(23, 24, 25, 26, 27) , although the mechanism underlying this potentiating effect is poorly understood. It has long been postulated that RTB facilitates translocation of RTA in a manner analagous to that proposed for diphtheria toxin B-chain which is believed to insert into endosomal membranes at acidic pH values, forming a channel for A-chain translocation to the cytosol(23, 24, 26, 31) . Alternatively, Lord and co-workers (32, 33) have suggested that RTB may subserve an intracellular shuttling function by binding to intracellular galactose residues expressed by molecules along the internalization pathway, thereby ``ratcheting'' RTA along the endocytic pathway to its translocation site. Finally, recent studies have shown that RTB protects RTA from degradation by endosomal and lysosomal proteolytic enzymes and may thereby enhance the cytotoxicity of RTA(13) .
In the present study, we tested the ability of RTB to translocate into reconstituted Golgi and ER vesicles and its effect on the translocation of RTA. RTB neither translocated by itself, nor augmented the amount of RTA that translocated into reconstituted vesicles. In contrast to our findings with reconstituted Golgi and ER proteoliposomes, Beaumelle et al.(11) found that RTB was able to translocate across the membranes of endosomes purified by gradient centrifugation. The discrepancy between our findings and those of Beaumelle et al.(11) may reflect methodologic differences or could result from structural differences in the organelles studied. The membrane component which facilitates translocation of RTB in endosomes may not exist in Golgi and ER, or alternatively, it could be lost or inactivated in the reconstitution process.
One of the most active areas of current research in cell biology concerns investigations into the mechanisms involved in protein transport across biological membranes. Studies of peptide (34, 35) and nascent chain (36) translocation across membranes of the endoplasmic reticulum, mitochondria(37) , and bacterial surface membranes (38) have demonstrated that protein channels exist which facilitate transmembrane protein transport (e.g. ``translocon'' protein channels for nascent secretory proteins (36, 39) and the ``TAP transporter'' channels (34, 35) for targeting cytosolic peptides to class I major histocompatibility complex molecules). Since our present studies indicate that liposomes prepared from purified phospholipids or from cellular fractions depleted in membrane glycoproteins are unable to support the translocation of RTA, it appears likely that proteinaceous constitutents of Golgi and ER membranes play an integral role in the translocation of ricin A-chain and other toxins. As suggested by Wales et al.(10) , it is conceivable that RTA utilizes previously identified translocation components of the ER (i.e. the translocon complex) in a retrograde direction, from lumen to the cytosol. Alternatively, heretofore unrecognized protein channels may be employed for toxin translocation.
Recent studies by Theuer et al.(40, 41) employing an in vitro translation/translocation system suggest that a truncated form of Pseudomonas exotoxin (PE37) can insert into the membrane of canine pancreatic microsomes if guided by the preprocecropin signal sequence, but that a ``stop-transfer'' sequence in domain II of the toxin arrests translocation and releases the toxin from the microsomes before it can transfer into the lumen of the microsomes. Our data suggest that a less complicated interaction occurs between ricin A-chain and Golgi/ER proteoliposomes and that full translocation of the molecule occurs in the absence of a signal sequence and without interruption by a stop transfer sequence. We believe that translocation-competent proteoliposomes such as those described in this report will prove to be as useful for the molecular dissection of the structural components involved in the translocation of plant and bacterial toxins as they have been in studying the translocation of secretory proteins(20, 21, 30) .