| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(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, ( 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 (
Figure 2:
Topology of the reconstituted vesicles.
Vesicles reconstituted from Golgi- (
Figure 3:
Translocation of RTA into vesicles
reconstituted from Golgi- and ER-enriched fractions. a,
Figure 4:
Effect of the concentration of vesicles on
the translocation of RTA.
Figure 5:
Effect of incubation time on the
translocation of RTA.
Figure 6:
Effect of protein depletion on
translocation of RTA.
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.
Figure 8:
Effect of RTB on the translocation of RTA
into reconstituted vesicles.
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) .
Volume 270,
Number 40,
Issue of October 06, pp. 23720-23725, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
)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) .
Reagents
RTA and RTB were purchased from Inland
Laboratories (Austin, TX); Triton X-100, proteinase K, papain, and
concanavalin A-Sepharose beads were from Sigma; ultrapure sodium
cholate and Nonidet P-40 were from Calbiochem; immobilized protease Sg
(a nonspecific protease) and immobilized papain were from Pierce; and
phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol
were from Avanti Polar Lipids Inc. (Alabaster, AL). A murine IgG antibody recognizing the luminal domain of calnexin (AF8)(12) was
a generous gift of Dr. Michael Brenner. A murine IgG antibody targeting the luminal domain of the MPR (86f7) was
kindly provided by Dr. Stuart Kornfeld. Murine IgG was
obtained by purification of monoclonal antibody DA 4.4 from ascitic
fluid by affinity chromatography on Sepharose-staphylococcal protein A
(Sigma). Jurkat cells were purchased from ATCC (Rockville, MD) and
maintained as described previously(13) .Radioiodination
RTA, RTB, mouse IgG, anti-MPR
antibody, and anti-calnexin antibody were radioiodinated as described
previously (13) . The specific activities of I-RTA and
I-RTB were 14 and 70 Ci
mmol
, respectively.
Discontinuous Sucrose Gradient
Fractionation
Fractionation methods were adapted from previous
studies(14, 15) . All procedures were carried out on
ice. 1 
10
Jurkat cells were suspended in buffer A
(50 mM TEA, 50 mM KOAc, 6 mM
Mg(OAc)
, 1 mM EDTA, 1 mM DTT, 0.5
mM phenylmethylsulfonyl fluoride) containing 0.25 M
sucrose. The cells were homogenized with 30 strokes of a Dounce
homogenizer (Kontes Inc., Moline, IL), and the nuclei and intact cells
were removed by centrifuging at 10,000 g for 5 min at
4 °C. The sucrose concentration of the supernatant was adjusted to
1.3 M by the addition of an appropriate amount of 2.5 M sucrose in buffer A and 4.5 ml of the resultant mixture placed in
each of two Beckman ultraclear tubes. The 1.3 M sucrose/homogenate mixture was overlaid with 5 ml of 1.2 M sucrose in buffer A and with 3 ml of 0.8 M sucrose in
buffer A. After ultracentrifugation for 2.5 h at 4 °C in a SW40
rotor at 94,000 g, 1-ml fractions were collected and
numbered from bottom to top. Fractions were then
analyzed by enzyme assays and by electron microscopy as described
previously(16) .Enzyme and Protein Assays
The Golgi-specific
enzyme, galactosyltransferase, was assayed by measuring the transfer of
UDP-[U-
C]galactose (Amersham Corp., Arlington
Heights, IL) to ovalbumin (Sigma) as described previously(17) .
The lysosomal enzyme, 
-galactosidase, was assayed using a
colorimetric method (18) . The ER-associated enzyme,
glucose-6-phosphatase, was assayed by measuring the release of
inorganic phosphate from glucose 6-phosphate
(Sigma)(17, 19) . Protein concentrations of the
fractions were determined by bicinchoninic acid (BCA) assay (Pierce).Reconstitution of Proteoliposomes
The
reconstitution method was adapted from the procedure described by
Nichitta and Blobel(20, 21) . Fractions 12 and 10
(Golgi) of the gradient were diluted to 0.5 M sucrose
concentration by the addition of the appropriate amount of buffer B
(0.25 M sucrose, 50 mM TEA, 1 mM DTT), and
3-ml aliquots were placed in tubes on ice. The pellet (fraction 1) was
suspended in 1.1 ml of buffer B and supplemented by an equal amount of
buffer D (0.2 M EDTA, 0.25 M sucrose, 50 mM TEA, 1 mM DTT) to strip ribosomes. Following 15-min
incubation on ice, this sample was placed in tubes underlaid with 0.8
ml of buffer E (0.5 M sucrose, 50 mM TEA, 1 mM DTT). All the samples were then centrifuged in a Beckman TL 100.3
rotor at 140,000 g for 1 h at 4 °C followed by
resuspension of the pellets of each fraction in 230 µl of buffer F
(0.4 M sucrose, 0.4 M KCl, 20 mM Tris, pH
7.6, 1.5 mM MgCl, 1 mM EDTA). The
membranes were solubilized by the addition of sodium cholate and
reconstituted into vesicles by dialysis as described
previously(20, 21) . Vesicle concentrations were
standardized prior to the performance of assays so that 250 µl of
each preparation yielded an absorbance of 0.2 at 405 nm using a EL-310
model enzymelinked immunosorbent assay reader (Bio-Tek Instruments,
Inc., Winooski, VT).Topology Assays
Ten nanograms of I-IgG (DA 4.4),
I-anti-MPR, and
I-anti-calnexin were incubated with 30 µl of vesicles
reconstituted from Golgi- and ER-enriched fractions at room temperature
for 45 min. Each sample was then diluted with the addition of 100
µl of buffer G and centrifuged in an Airfuge A100/30 rotor
(Beckman) for 10 min at 18 p.s.i. (110,000
g). The
radioactivity in the supernatant, and the pellet was determined by
counting the samples with a Gamma 5500 (Beckman, Palo Alto, CA)
counter and the vesicle (pellet)-associated counts/min was expressed as
the percentage of the total.
ConA Chromatograpy
A 0.5-ml column of
ConA-Sepharose was equilibrated with 10 column volumes of extraction
buffer (0.8% sodium cholate in buffer F) and then loaded with 0.25 ml
of cholate extract. The column was eluted at a flow rate of 0.25 ml/h,
the eluate was concentrated to the original volume, and vesicles were
reconstituted as described above.Protease Digestion of Cholate Extract
125 µl
of cholate extract was incubated with a mixture of 100 µl of
immobilized papain and 100 µl of immobilized protease Sg at room
temperature for 2, 18, and 42 h. Another 125 µl of cholate extract
was incubated in the absence of protease beads for preparation of
control vesicles. The protease beads were then removed by
centrifugation at 1000 g for 30 s, and vesicles were
reconstituted from protease-digested and undigested cholate extracts as
described above.Preparation of Liposomes
Purified
phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol
were mixed to a total lipid concentration of 1 mM at a ratio
of 75, 15, and 10%, respectively, and dried under Argon gas. The lipids
were then resuspended in 1 ml of buffer G and sonicated for 10 min to
form liposomes.Translocation Assay
35 ng of I-RTA
or
I-RTB was incubated with or without reconstituted
vesicles in 20 µl of buffer G at 37 °C for 1 h. The samples
were then placed on ice and proteinase K, or papain was added to a
final concentration of 1 unit/ml. Following incubation on ice for 75
min, 20 µl of 2
Laemmli sample buffer (22) containing 10 mM phenylmethylsulfonyl fluoride
was added, and the samples were ana-lyzed by SDS-PAGE, autoradiography
and densitometry as described previously(13) .
Purification and Characterization of Golgi and ER
Jurkat cell homogenates were fractionated by centrifugation
on a discontinuous sucrose gradient composed of 1.3, 1.2, and 0.8 M layers. Twelve fractions were collected (numbered from the bottom)
and examined for their organelle content by electron microscopy and
enzymatic analysis (Fig. 1). Relatively pure ER was recovered in
fraction 1 as assessed by the presence of rough microsomes by electron
microscopy (data not shown) and by the recovery of
glucose-6-phosphatase activity. The microsome-bound ribosomes in this
fraction were removed by washing with EDTA. Fraction 5 (1.3 M/1.2 M interface) was composed of lysosomes,
mitochondria, and smooth ER by electron microscopic criteria (data not
shown). Most of the lysosomal -galactosidase activity was
contained in this fraction. Galactosyltransferase activity was
recovered in fractions 10 and 11, indicating the presence of Golgi in
these fractions. Fraction 10 (1.2 M/0.8 M interface)
also contained some contaminating endosomes and surface membranes (data
not shown). Although vesicular structures were abundant in fraction 12
by electron microscopy (data not shown), this fraction contained very
little -galactosidase, glucose-6-phosphatase, or
galactosyltransferase activity. A protein profile of the fractions is
presented in Fig. 1.
), 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).
Topology of Reconstituted Proteoliposomes
The proteoliposome reconstitution system we have employed is
identical to the system employed by Nicchitta and Blobel (20, 21) to study translocation of preprolactin. Using
this methodology, the reconstituted vesicles produced are topologically
restructured so that vectorially oriented membrane proteins are
redistributed variably either in their native membrane orientations or
in orientations opposite their native configurations (i.e. some luminal domains become abluminal). Such
``reconstituted'' proteoliposomes should permit
``retrograde'' translocation from the exterior to the
interior of the vesicles. To confirm that the expected topological
reorganization of the proteoliposome membranes had occurred, we tested
the ability of I-labeled monoclonal antibodies specific
for the lumenal domains of integral Golgi and ER membrane proteins, the
mannose 6-phosphate receptor and calnexin, respectively, to bind to the
exterior of the reconstituted vesicles. As shown in Fig. 2,
specific binding of
I-anti-MPR antibody confirmed the
presence of the luminal domain of the mannose 6-phosphate receptor on
the exterior surface of the vesicles reconstituted from Golgi-enriched
fractions. Similarly, luminal determinants of calnexin were detected on
the exterior surface of the vesicles reconstituted from ER-enriched
fractions using
I-anti-calnexin antibody (Fig. 2).
A nonspecific mouse IgG
used as a control reagent bound
minimally to the vesicles (Fig. 2).
) 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.
Translocation of RTA into Vesicles Reconstituted from
Golgi- and ER-enriched Fractions
RTA Preincubated with Vesicles Reconstituted from
Golgi- and ER-enriched Fractions Is Protected from Subsequent Papain
Digestion
Vesicles were reconstituted identically from fractions
1 (ER-enriched), 10 (Golgi-enriched), and 12 and were standardized to
the same concentration spectrophotometrically before utilizing in
translocation assays. I-RTA was incubated with and
without vesicles reconstituted from fractions 1 (ER), 10 (Golgi), and
12 for 1 h at 37 °C. Samples were then subjected to papain
digestion on ice, and the degradation of
I-RTA was
assessed by SDS-PAGE and autoradiography. As shown in Fig. 3a,
I-RTA was completely digested in
the samples preincubated without vesicles or with vesicles
reconstituted from fraction 12; however a quantity of
I-RTA was protected from digestion in samples
preincubated with vesicles reconstituted from Golgi- and ER-enriched
fractions. Similar results were obtained with proteinase K digestion
(data not shown).
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.
Protection from Papain Digestion Is Abrogated by
Detergent Disruption of the Vesicles
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
for 1 h at 37 °C. The samples were then subjected to papain
digestion and analyzed by SDS-PAGE and autoradiography. As shown in Fig. 3b, the integrity of the vesicles was required for
protection from papain digestion, since
I-RTA was
completely digested when the vesicles were disrupted by detergents.
Similar results were obtained when Nonidet P-40 was added after the
incubation of the vesicles with RTA (data not shown). These data
suggest that a quantity of
I-RTA is protected from papain
digestion by translocating into the vesicles reconstituted from Golgi-
and ER-enriched fractions and that the vesicles reconstituted from
fraction 12 are not competent for RTA translocation.
Linear Relationship between the Amount of
Translocated RTA and the Concentration of the Vesicle Preparation
I-RTA was incubated with and without vesicles
at the dilutions indicated in Fig. 4in the presence and absence
of 1% Triton X-100 for 1 h at 37 °C. The samples were then
subjected to papain digestion and analyzed by SDS-PAGE and
autoradiography. As shown in Fig. 4, the amount of
I-RTA that translocated was dependent on the
concentration of the vesicle preparation. Dilution of the vesicles
resulted in lesser protection from papain digestion (upper
panel). Densitometric analysis of the lanes revealed that there
was a linear relationship between the amount of translocated RTA and
the concentration of the vesicles (lower panel).
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).
Effect of Incubation Time on the Translocation of RTA
into Reconstituted Vesicles
I-RTA was incubated with and without vesicles
for 0.5, 1, 2, and 4 h time points at 37 °C. The samples were then
subjected to papain digestion and analyzed by SDS-PAGE and
autoradiography (Fig. 5, upper panel). The lanes from
the autoradiographs of two experiments were analyzed by densitometry
and plotted as shown in the lower panel of Fig. 5. The
results indicate that the amount of RTA that translocates increases
with the incubation time, reaching a plateau of approximately 25% of
total RTA in 1 h.
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).
Liposomes Prepared from Purified Phospholipids Do Not
Support RTA Translocation
Liposomes were prepared from purified phosphatidylcholine,
phosphatidylethanolamine, and phosphatidylinositol and then tested for
RTA translocation competence. I-RTA was incubated with
and without vesicles at the indicated dilutions for 1 h at 37 °C.
The samples were then subjected to papain digestion and analyzed by
SDS-PAGE and autoradiography.
I-RTA was completely
digested even in the presence of vesicles, suggesting that the protein
component of the Golgi or ER fractions is required for the
translocation of RTA (data not shown).
Removal of the Protein Components of Golgi and ER
Vesicles Abrogates RTA Translocation
To further investigate the role of the protein constitutents
of Golgi and ER proteoliposomes in the translocation process, we
treated cholate extracts with immobilized papain and protease Sg (a
nonspecific protease) for 2, 18 and 42 h prior to vesicle
reconstitution. After removal of the immobilized proteases, vesicles
were reconstituted and tested for their competence for RTA
translocation. As shown in Fig. 6(upper three panels),
vesicles reconstituted from Golgi-enriched fractions after 2 h of
protease treatment retained translocation competence, whereas longer
protease treatments completely abrogated the ability of reconstituted
vesicles to support translocation. Similar conclusions were reached
using vesicles reconstituted from ER-enriched fractions, although
translocation was only reduced by 47% after 42 h of incubation with
immobilized proteases (data not shown).
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).
RTB Does Not Translocate into Reconstituted Vesicles
RTB, the cell binding domain of ricin, is a potentiator of
RTA immunotoxins which is postulated to facilitate RTA translocation (23, 24, 25, 26, 27) . We
tested the ability 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 for 1 h at 37 °C. The samples were then
subjected to papain digestion and analyzed by SDS-PAGE and
autoradiography. RTB did not translocate into the reconstituted
vesicles, but was completely digested by papain (Fig. 7). A
similar result was obtained from an experiment in which vesicles
reconstituted from the ER-enriched fraction were used (data not shown).
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).
RTB Protects RTA from Enzyme Digestion
We tested the effect of RTB on the translocation of RTA into
reconstituted vesicles. I-RTA was incubated with and
without vesicles reconstituted from the ER-enriched fraction in the
presence and absence of unlabeled RTB for 1 h at 37 °C. The samples
were then subjected to proteinase K digestion and analyzed by SDS-PAGE,
autoradiography, and densitometry. The results are shown in Fig. 8. In the presence of vesicles,
I-RTA was
protected from digestion by proteinase K (lane C). Addition of
RTB in the absence of vesicles also resulted in the protection of
I-RTA from proteolysis (lane F). The presence of
RTB together with vesicles resulted in a protection that was not
significantly greater than the sum of the protection obtained in the
presence of either RTB or vesicles alone (lane G) as assessed
by densitometric analysis of the lanes in six separate experiments
(data not shown). Nonidet P-40 abrogated the protective effect of
vesicles but not of RTB on the digestion of RTA. (lanes D and
H). These data suggest that RTB is not essential for effective
translocation of RTA.
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).
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Morlon-Guyot, M. Helmy, S. Lombard-Frasca, D. Pignol, G. Pieroni, and B. Beaumelle Identification of the Ricin Lipase Site and Implication in Cytotoxicity J. Biol. Chem., May 2, 2003; 278(19): 17006 - 17011. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
ABSTRACT
