1 Department of Biochemistry, University of WisconsinMadison, Madison,
Wisconsin 53706, USA
2 Department of Chemistry, University of WisconsinMadison, Madison,
Wisconsin 53706, USA
* Current address: Department of Biology, Massachusetts Institute of Technology,
77 Massachusetts Avenue, Cambridge, MA 02139, USA
Author for correspondence (e-mail:
raines{at}biochem.wisc.edu)
Accepted 10 October 2002
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Summary |
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Key words: Dynamin, Endocytosis, Endosome, Ribonuclease, Toxin
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Introduction |
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The characterization of alternative endocytic pathways has been due, in
large part, to the creation of dominant-negative mutants that disrupt
clathrin-mediated endocytosis. One such mutant involves dynamin, which is a
GTPase required for the release of clathrin-coated pits from the cell surface
(Schmid et al., 1998;
McNiven et al., 2000
). A
variant of dynamin in which Lys44 is replaced with an alanine residue (K44A
Dyn) is defective in GTP binding and hydrolysis
(van der Bliek et al., 1993
).
Previous studies have shown that the overproduction of K44A Dyn in HeLa cells
blocks clathrin-mediated endocytosis and inhibits the internalization of both
transferrin and epidermal growth factor
(van der Bliek et al., 1993
;
Damke et al., 1994
). Dynamin is
also required for the internalization of caveolae
(Henley et al., 1998
), as well
as for vesicle budding from the trans-Golgi network
(Jones et al., 1998
). A poorly
defined clathrin-independent endocytic pathway is upregulated when
dynamin-dependent endocytosis is blocked
(Damke et al., 1995
).
Ribonuclease (RNase) A and its homologs are secretory proteins that
manifest diverse biological activities following internalization
(D'Alessio and Riordan, 1997;
Raines, 1998
). For example,
bovine seminal ribonuclease demonstrates antitumor, antiviral and
immunosuppressive activity (D'Alessio et
al., 1997
; Matousek,
2001
). Onconase® (ONC) demonstrates both antitumor and
antiviral activity (Youle and D'Alessio,
1997
; Leland and Raines,
2001
) and is in Phase III clinical trials for the treatment of
malignant mesothelioma (Mikulski et al.,
2002
). RNase A itself, however, does not have marked antitumor,
antiviral or immunosuppressive activity.
Ribonuclease-mediated cell death consists of two major steps: (1) cytosolic
internalization and (2) RNA cleavage. Several studies have focused on the
contribution of the intracellular ribonucleolytic activity to cytotoxicity
(Leland and Raines, 2001). In
comparison, little is known about the internalization pathway of
ribonucleases. Ribonucleases must reach the cytosol to degrade cellular RNA.
Ribonucleases that are microinjected into the cytosol are more toxic than
those added to cells externally (Saxena et
al., 1991
), suggesting that internalization limits toxicity. The
potency of ONC can be enhanced by adding drugs that alter cellular routing
(Wu et al., 1995
). Likewise,
conjugating ribonucleases to delivery molecules can enhance the specificity
and potency of their cytotoxicity (Suzuki
et al., 1999
; Newton et al.,
2001
; De Lorenzo et al.,
2002
).
The pathway by which ribonucleases reach the cytosol is not known. The
internalization of ONC could rely on an endocytic pathway, as its cytotoxicity
is blocked by inhibitors of this energy-dependent pathway
(Wu et al., 1995). Still, the
endocytosis of ONC has not been observed directly. In addition, the type of
vesicles that mediate ONC internalization have not been identified. Even less
is known about the internalization of RNase A. Indeed, other workers have
reported that RNase A is not even bound by cells
(Leamon and Low, 1993
).
The toxicity of a molecule can be a powerful tool for assessing the
efficiency and delineating the pathway of its intracellular routing. As RNase
A is not toxic to cultured cells, this approach cannot be used to probe its
internalization. Variants of RNase A can, however, be toxic to cells. For
example, replacing the glycine residue at position 88 with arginine (G88R)
decreases the affinity of RNase A for the cytosolic ribonuclease inhibitor
protein (RI) and makes RNase A cytotoxic, even when added to cells externally
(Leland et al., 1998).
Here, we investigate the internalization of ONC and RNase A with tools from cell biology, pharmacology and somatic cell genetics. In addition, we take advantage of the toxicity of G88R RNase A to probe the mechanism of its cytosolic entry. Our data reveal a pathway by which secretory ribonucleases are internalized, as well as the first evidence for differences in trafficking between ONC and RNase A, two homologous proteins.
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Materials and Methods |
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Wild-type RNase A, G88R RNase A, K7A/G88R RNase A and wild-type ONC were
purified using methods described previously
(delCardayré et al.,
1995; Leland et al.,
1998
). A19C RNase A and D16C ONC were purified as described for
the wild-type proteins but with the following modifications
(Kothandaraman et al., 1998
).
Refolding solutions were saturated with Ar(g) to remove O2(g).
Immediately after anion-exchange chromatography, the sulfhydryl group of
native Cys19 (A19C RNase A) or Cys16 (D16C ONC) was protected from oxidation
by reaction with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) at pH 9
(Messmore et al., 1995
).
DTNB-protected protein was isolated from unprotected protein using
anion-exchange chromatography. Protein concentrations were determined by UV
spectroscopy using
=0.72 ml mg-1 cm-1 at 277.5 nm
for RNase A (Sela et al.,
1957
) and its variants and
=0.87 ml mg-1
cm-1 at 280 nm for ONC (Leland
et al., 1998
). All ribonucleases were dialyzed exhaustively versus
phosphate-buffered saline (PBS), which contained (in 1 litre) KCl (0.20 g),
KH2PO4 (0.20 g), NaCl (8.0 g) and
Na2HPO4 (2.16 g).
Fluorescent labeling of ribonucleases
DTNB-protected A19C RNase A and D16C ONC were deprotected immediately
before fluorescent labeling. Protected protein was incubated with a threefold
molar excess of dithiothreitol or tris(2-carboxyethyl) phosphine (TCEP) and a
40-fold molar excess of label for 25 minutes at 22°C. The fluorescent
probes 5-iodoacetamido-fluorescein (5-IAF),
N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl)iodoacetamide
(BODIPY® 507/545 IA) and
2',7'-difluorofluorescein-iodoacetamide (Oregon GreenTM 488
IA) were from Molecular Probes (Eugene, OR). Excess, unreacted probe was
removed by gel filtration chromatography with a NICK column (Pharmacia
Biotech; Piscataway, NJ). Protein concentration was determined using Equation
1 below:
![]() | 1 |
![]() | 2 |
Labeled protein was purified away from unlabeled protein by using reverse-phase HPLC. The HPLC peak containing labeled protein was dialyzed exhaustively versus PBS for biological assays.
Assay of thermal stability
Conformational stability assays were performed as described previously
(Eberhardt et al., 1996), with
the following modifications. The conformational stability of RNase A, ONC, and
each labeled variant (in PBS) was determined by monitoring the change in
absorbance at 287 nm as the temperature was increased from 25 to 75°C in
1°C increments. The absorbance at 287 nm was recorded after a 7 minute
equilibration at each temperature. The value of Tm is the
temperature at the midpoint of the thermal denaturation. Data were analyzed
with the program THERMAL (Varian Analytical Instruments; Walnut Creek,
CA).
Assay of catalytic activity
Ribonucleolytic activity was measured using
6-FAMdArU(dA)2
6-TAMRA, a fluorogenic substrate
(Kelemen et al., 1999
). Assays
were performed at 23°C in 2.00 ml of 0.10 M MES-NaOH buffer (pH 6.0)
containing NaCl (0.10 M), substrate (50 nM) and enzyme (1.0-5.0 pM). Data were
obtained and values of Kcat/KM were
calculated as described previously
(Kelemen et al., 1999
).
Cell culture
K-562, JAR and HeLa cells were obtained from the American Type Culture
Collection (Manassas, VA). K-562 and JAR cells were grown in RPMI medium 1640.
HeLa cells were grown in DMEM medium. All culture medium contained FBS (10%
v/v), penicillin (100 units/ml) and streptomycin (100 µg/ml). Cell culture
medium and supplements were from Life Technologies (Gaithersburg, MD). Cells
were cultured at 37°C in a humidified incubator containing CO2
(g; 5% v/v). All studies were performed using asynchronous log-phase
cultures.
Immunohistochemistry
Wild-type RNase A, G88R RNase A or ONC was added to a culture of K-562
cells at 4°C. After 20 minutes, cells were washed twice with PBS and fixed
for 30 minutes at 4°C in PBS containing paraformaldehyde (2% w/v) and
Triton X-100 (0.1% w/v). After being fixed, the cells were rinsed three times
in PBS and incubated for 1 hour at 37°C with primary antibody. Rabbit IgG
raised against RNase A was from Biodesign International (Kennebunk, ME), and
was used at a concentration of 1 µg/ml in PBS containing Tween-20 (0.1%
v/v) (PBST). Chicken antiserum raised against ONC was the generous gift from
Alfacell Corporation (Bloomfield, NJ) and was used at a 1:100 dilution in
PBST. After incubation with primary antibodies, cells were washed three times
in PBST and incubated with the appropriate secondary antibodies (1:500 in
PBST) conjugated to fluorescein or rhodamine (Molecular Probes; Eugene, OR).
After a 1 hour incubation with secondary antibodies, cells were washed three
times with PBST and stained with propidium iodide (1 µg/ml in PBS) for 5
minutes. Then, cells were washed twice with PBST and mounted onto glass
microscope slides using Vectashield (Vector Laboratories; Burlingame, CA).
Fluorescence staining of cells was visualized on a Zeiss Axiovert 100 TV
microscope (Zeiss; Germany). Images were analyzed with the programs BioRad MRC
1024 Laser Scanning Confocal Imaging System (Hercules, CA) and Adobe Photoshop
(San Jose, CA).
Assays of internalization
The internalization of RNase A was visualized directly in living cells.
K-562 cells in PBS (1x2'106 cells/ml) were incubated
with either BODIPY-labeled A19C RNase A (BODIPYRNase A) or
fluorescein-labeled A19C RNase A (fluorescein
RNase A) (1 µM) at
4°C for 20-30 minutes. Cells were washed three times with ice-cold PBS to
removed unbound protein. Then, cells were incubated for 5 minutes at 37°C
in a humidified incubator containing CO2 (g; 5% v/v). After
incubation, living cells were washed three times in PBS and placed on a
microscope slide; fluorescence was monitored immediately.
To quantify the pH sensitivity of fluoresceinRNase A, K-562 cells were
incubated at 4°C for 20-30 minutes with fluorescein
RNase A or
OG
RNase A (10 µM). Cells were washed with ice-cold PBS, and the
fluorescence of each cell was measured after a 0-6 minutes of incubation at
37°C with a FACScan flow cytometer and a 530DF30 bandpass filter (Becton
Dickenson, San Jose, CA).
To probe the co-internalization of ONC and RNase A, JAR or HeLa cells were
grown on coverslips in the wells of a six-well plate for 24 hours before the
assay. Oregon-Green-labeled D16C ONC (OGONC) and BODIPY
RNase A (both
at a concentration of 1 µM and labeled to
10%) were incubated with
cells at 4°C for 20-30 minutes. Cells were washed three times with
ice-cold PBS to remove unbound protein and then incubated for 5 minutes at
37°C in a humidified incubator containing CO2 (g; 5% v/v).
Cells were washed three times with ice-cold PBS and fixed as described for
immunohistochemistry. After fixing, cells were mounted on microscope slides
and visualized immediately.
To study the dose dependence of RNase A and ONC, both proteins were labeled
with Oregon Green and used at a labeling efficiency of 30%. HeLa cells were
incubated with increasing concentrations of either OGRNase A or
OG
ONC (0.01, 0.1, 1 and 10 µM). Samples were pulsed, fixed and
visualized as described for the ribonuclease co-internalization studies. To
quantify the fluorescence in each cell, pulsed cells were detached with
trypsin and analyzed by flow cytometry.
To investigate the internalization of RNase A with endocytic markers, cells
were pulsed with BODIPYRNase A (1 µM), OG
RNase A (1 µM),
FMTM 1-43 (1 µg/ml; Molecular Probes; Eugene, OR),
BODIPY-FL-transferrin (50 µg/ml; Molecular Probes; Eugene, OR) or
tetramethylrhodamine-labeled transferrin (TAMRA
transferrin; 50 µg/ml;
Molecular Probes; Eugene, OR). Endocytosis assays were performed as described
previously (Lamaze et al.,
2001
).
Assays of cytotoxicity
The effect of ONC, RNase A and G88R RNase A on cell proliferation was
determined by measuring [methyl-3H]thymidine uptake into DNA.
Briefly, cells (95 µl of a solution of 5x104 cells/ml)
were incubated with PBS containing a known concentration of a ribonuclease (5
µl) in a 96-well plate for 20 hours at 37°C in a humidified incubator
containing CO2 (g; 5% v/v). Data on cell proliferation were
obtained and analyzed as described previously
(Haigis et al., 2002).
For metabolic inhibition studies, cells were incubated in the presence of NaN3 (10 mM) and 2-deoxyglucose (50 mM) before the addition of a ribonuclease. For pharmacological studies, cells were incubated in the presence of BFA (5.0 µg/ml), NH4Cl (20 mM) or monensin (10 µM) before the addition of a ribonuclease. Data were analyzed as the percentage of [methyl-3H]thymidine incorporated compared to cells incubated instead with PBS.
To investigate the role of dynamin in ribonuclease toxicity, the toxicity
of ONC and G88R RNase A was measured in a cell line overproducing K44A Dyn
(Damke et al., 1994). Stably
transformed tTA-HeLa cells with tightly regulated expression of dynamin and
K44A Dyn was a generous gift from S. L. Schmid (Scripps Research Institute;
LaJolla, CA). Briefly, transformed tTA-HeLa cells were cultured in DMEM
containing FBS (10% v/v), penicillin (100 units/ml), streptomycin (100
µg/ml), G418 (400 µg/ml) and tetracycline (2 µg/ml) as described
previously (Damke et al.,
1994
). To induce the overexpression of HA-tagged dynamin (or K44A
Dyn), subconfluent cultures (<50%) were washed several times in PBS,
detached with trypsin and plated onto 10 cm culture dishes in the absence of
tetracycline. After 48 hours, the overproduction of dynamin was monitored by
immunoblot analysis using antibodies to the HA epitope. Then, toxicity assays
were performed as described above using both uninduced and induced cells.
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Results |
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Conformational stability and ribonucleolytic activity
Data from experiments with incubations at 37°C can be interpreted
properly only if the ribonuclease is folded at that temperature.
Fluorescein-labeled A19C RNase A (fluoresceinRNase A) and DTNB-labeled
D16C ONC (DTNB
ONC) had Tm values of 61 and 88°C,
respectively (Table 1). These
values are similar to those reported for the wild-type ribonucleases
(Leland et al., 2000
).
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The cytotoxicity of ribonucleases relies on their ribonucleolytic activity.
Alkylation of ONC results in the loss of catalytic as well as cytotoxic
activity (Wu et al., 1993).
RNase A variants with altered active-site residues have lowered enzymatic
activity and cytotoxic activity (Bretscher
et al., 2000
). We measured the ribonucleolytic activity of labeled
ONC and RNase A variants using 6-FAM
dArU(dA)2
6-TAMRA as
the substrate (Table 1). The
values of kcat/KM for RNase A and ONC
were found to be 4.3x107 M-1 s-1 and
3.5x102 M-1 s-1, which are in good
agreement with the kcat/KM values
reported previously (Kelemen et al.,
1999
; Leland et al.,
2000
). The values of
kcat/KM for fluorescein
RNase A
and DTNB
ONC were found to be 0.54x107 M-1
s-1 and 1.7x102 M-1 s-1,
respectively. Thus, thermal stability and ribonucleolytic activity are
retained in the modified variants.
Binding and internalization of ribonucleases
First, we investigated whether ribonucleases interacted with the cell
surface. Previous studies have reported a high-affinity interaction
(Kd=62 nM) between ONC and the surface of 9L cells
(Wu et al., 1993). An
interaction between RNase A and the cell surface has not been reported
previously. We investigated the binding of RNase A, G88R RNase A and ONC to
the plasma membrane of human leukemia (K-562) cells. All three proteins were
bound to the cell surface after a 30 minute incubation at 4°C
(Fig. 2). The signal intensity
does not increase significantly after incubation for 2 hours at 4°C (data
not shown). Both RNase A and ONC accumulate at the cell surface at 4°C and
do not cross the plasma membrane, even after 2 hours. These results suggest
that the internalization of ribonucleases does not occur at 4°C and thus
probably relies on an endocytic process. A preincubation of metabolic
inhibitors protects K-562 cells from the toxicity of ONC and K7A/G88R RNase A
(Fig. 3A), which is the most
toxic known variant of RNase A (Haigis et
al., 2002
). In addition, metabolic inhibitors block the
internalization of RNase A (Fig.
3B).
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|
Next, we examined the initial events of internalization. A19C RNase A was
labeled with fluorescein (fluoresceinRNase A), which has pH-sensitive
fluorescence, or BODIPY (BODIPY
RNase A), which is a pH-insensitive probe.
The pH-dependent properties of fluorescein and BODIPY are retained when linked
to a specific site on RNase A (data not shown). We incubated JAR cells at
either 4°C or 37°C. The fluorescence of the former cells is located
uniformly on the plasma membrane (Figs
2,
3B) and that of the latter
cells is located internally (Fig.
4A,B). Next, we pulsed cells at 37°C with either
fluorescein
RNase A or BODIPY
RNase A and then analyzed their
fluorescence by microscopy. Cells pulsed with BODIPY
RNase A have vivid
internal fluorescence (Fig.
4A,B). By contrast, cells incubated with fluorescein
RNase A
lack strong internal fluorescence (Fig.
4C). To verify that unlabeled RNase A is likewise internalized by
vesicles, we pulsed K-562 cells with unlabeled RNase A and then analyzed them
by immunohistochemistry. RNase A appears in vesicles similar in morphology to
those seen in living cells (Fig.
4C). The addition of the fluorescent label does not hinder or
alter the mode of ribonuclease internalization.
|
To monitor the pH surrounding an internalized ribonuclease, K-562 cells
were pulsed with fluoresceinRNase A or RNase A labeled with Oregon
GreenTM (OG
RNase A), which has a lower pKa
(=4.7) than the pKa (=6.4) fluorescein and hence retains
much more of its fluorescence at low pH. Fluorescence was measured by using
flow cytometry after timed incubations at 37°C. The fluorescence of
fluorescein
RNase A decreases with longer incubations at 37°C,
providing evidence that ribonucleases are internalized through acidic
endosomes (Fig. 4D). By
contrast, the fluorescence of OG
RNase A does not decrease significantly,
indicating that it is in an environment with a pH>5
(Fig. 4D). All 37°C pulses
were for
6 minutes, so as to probe only the initial phase of ribonuclease
internalization. Cells incubated with unconjugated flourescein, BODIPY or
Oregon Green (10 µM) are not fluorescent (data not shown).
We then investigated whether ONC and RNase A are cointernalized. We labeled
D16C ONC at residue 16 with Oregon GreenTM (OGONC) to allow for
colocalization studies with BODIPY
RNase A. K-562 cells internalize
OG
ONC and BODIPY
RNase A using the same compartments
(Fig. 5). OG
ONC and
BODIPY
RNase A are also internalized in the same compartments in cervical
epithelioid carcinoma (HeLa) and choriocarcinoma (JAR) cells. Identical
results were seen in living cells that were viewed without fixation after
incubation with ribonucleases (data not shown). The colocalization was not due
to fluorescence bleed-through (data not shown).
|
To compare the dose dependence of ONC and RNase A internalization, we
pulsed HeLa cells with increasing concentrations of either OGONC or
OG
RNase A and analyzed the cells by microscopy or flow cytometry. The
amount of OG
ONC and OG
RNase A internalized by cells is identical and
increases with increasing protein concentration
(Fig. 6A,B). In addition, the
10 µM samples show a clear increase in the number of ribonuclease-filled
punctate vesicles (Fig. 6A). The data indicate that ribonuclease internalization is not saturable
(Fig. 6B); in addition, the
fluorescence intensity of OG
RNase A (1 µM) is not diminished by the
addition of a 100-fold excess of either unlabeled RNase A or ONC (data not
shown).
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|
We analyzed the clathrin-mediated endocytosis of K-562 cells by using
BODIPY FL-labeled transferrin. We find that transferrin, a marker for
clathrin-mediated endosomes and recycling endosomes, is readily internalized
by K-562 cells, but does not colocalize with BODIPYRNase A
(Fig. 7). In addition, RNase A
and transferrin do not colocalize in cells pulsed for 2, 5 or 15 minutes at
37°C, which eliminates a scenario of co-internalization and then
post-endocytic sorting (data not shown). Thus, BODIPY
RNase A is
internalized by a pathway distinct from that expected for clathrin-mediated
endocytosis.
Dynamin-independent toxicity and internalization of
ribonucleases
We investigated the toxicity of ONC and G88R RNase A using tTA HeLa cell
lines that overexpress the gene encoding wild-type dynamin or its K44A variant
(Damke et al., 1994). Removal
of tetracycline results in the overproduction of K44A Dyn, as shown by
immunoblotting (Fig. 8A), as
well as morphological changes in these cells that are similar to those
described previously (Damke et al.,
1994
). Specifically, cells overproducing K44A Dyn are flatter and
have rounder edges and distinct actin formation
(Fig. 8A).
|
We find that HeLa cells are not equally susceptible to ONC-and G88R RNase A-mediated toxicity (Fig. 8B,C). ONC and G88R RNase A kill HeLa cells with IC50 values of 1 and >50 µM, respectively (Table 2). The cytotoxicity of ONC and G88R RNase A is unaffected by the overproduction of wild-type dynamin (Fig. 8B; Table 2).
We next measured the toxicity of ONC and G88R RNase A for cells that overproduce K44A Dyn (Fig. 8C). The toxicity of ONC and G88R RNase A for cells not overproducing K44A Dyn does not differ significantly from that for cells containing wild-type dynamin (Table 2). Surprisingly, we find that the overproduction of K44A Dyn makes cells more susceptible to ribonuclease-mediated toxicity. The IC50 value for ONC decreases from 1.6 to 0.7 µM in uninduced versus induced cells, respectively (Table 2). We observed an even more dramatic enhancement in the cytotoxicity of G88R RNase A, as its IC50 value decreases from >50 to 17 µM, in uninduced versus induced cells, respectively.
The increase in cytotoxicity upon K44A Dyn overproduction suggests that ONC
and G88R RNase A are internalized via a dynamin-independent mechanism. To test
this hypothesis, we pulsed cells overproducing K44A Dyn with OGRNase A
and TAMRAtransferrin. OG
RNase A is internalized in cells
overproducing K44A Dyn (Fig.
9). The majority of transferrin remains at the surface of these
cells (Fig. 9). Control cells
not overproducing the dynamin variant internalize both OG
RNase A and
labeled transferrin by vesicles that do not overlap
(Fig. 9). These data show that
ribonucleases can be internalized in the absence of clathrin- and
dynamin-mediated endocytosis.
|
Routing of toxic ribonucleases to the cytosol
The pharmacological agents NH4Cl, monensin and brefeldin A (BFA)
have distinct and established effects on cellular compartments
(Pelham, 1991;
Sciaky et al., 1997
;
Dinter and Berger, 1998
). We
used these drugs to examine the intracellular pathway(s) by which
ribonucleases reach the cytosol. In all experiments, K-562 cells were
incubated with a drug for 2 hours prior to the addition of a ribonuclease.
NH4Cl is a weak base that increases the endosomal pH. We find that NH4Cl (20 mM) has no effect on the cytotoxicity of either ONC or G88R RNase A, indicating that routing to the cytosol is not perturbed in the presence of deacidified vesicles (Fig. 10; Table 3). An NH4Cl concentration of 30 mM also has no effect on ribonuclease cytotoxicity (data not shown).
|
|
Monensin is a carboxylate ionophore that also leads to the deacidification of endosomes and can disrupt Golgi trafficking. We find that monensin (10 µM) potentiates the cytotoxicity of ONC by 10-fold but has little effect on the cytotoxicity of G88R RNase A (Fig. 10). These data reveal that G88R RNase A is internalized via a monensin-insensitive pathway and verify that the internalization of ONC or G88R RNase A is not dependent on a low pH environment.
To investigate whether downstream events in the retrograde pathway are
important for cytotoxicity, we analyzed the toxicity of ONC and G88R RNase A
in the presence and absence of BFA. BFA disassembles the Golgi stack and
disrupts retrograde transport from the Golgi to the ER
(Pelham, 1991) and has been a
powerful tool for studying the internalization pathway of other protein toxins
(Hudson and Grillo, 1991
). We
find that BFA potentiates the cytotoxicity of both ONC and G88R RNase A by
10-fold (Fig. 10; Table 3). Hence, retrograde
transport from the Golgi to the ER is not an essential component of
ribonuclease translocation to the cytosol.
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Discussion |
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|
We were surprised to discover that secretory ribonucleases are more toxic
to cells in the absence of dynamin-mediated endocytosis
(Fig. 8; Table 2). These data can be
explained by invoking known mechanisms of dynamin action. The
dynamin-dependent endocytic pathways, such as clathrin- and caveolae-mediated
endocytosis, could represent an inefficient route for the cytosolic entry of
ribonucleases. By bypassing a dead-end pathway, ribonucleases could enter the
cytosol more efficiently, leading to enhanced toxicity. Thus, a block in
dynamin function could alter the translocation of ribonucleases, as well as
their initial uptake. Finally, some clathrin- and dynamin-independent
pathways, such as pinocytosis, are upregulated in cells that lack functional
dynamin (Damke et al., 1995).
Ribonucleases are indeed endocytosed by K44A Dyn cells that do not internalize
transferrin, which is a marker for clathrin-mediated endocytosis
(Fig. 9). Hence, inactivating
dynamin-dependent pathways could increase the influx and cytotoxicity of
ribonucleases (Fig. 8).
We were also surprised to learn that monensin potentiates the cytotoxicity of ONC by 10-fold but does not alter significantly the cytotoxicity of G88R RNase A (Fig. 10). Monensin deacidifies vesicles by exchanging protons for other cations, but it can also disrupt cellular functions, such as trafficking through the Golgi apparatus. Our results provide evidence for a possible difference between ONC and G88R RNase A trafficking through the Golgi. For example, the Golgi could be an inefficient site for ONC translocation to the cytosol; monensin could prevent such inefficient routing of ONC to the Golgi, resulting in enhanced cytotoxicity.
Perturbing intracellular trafficking by using drugs may have indirect
consequences on cellular pathways and must be interpreted with caution. For
example, BFA has diverse effects on endocytosis and has been shown to
interfere with the sorting of the transferrin receptor
(Wang et al., 2001). Such a
block could lead to the decrease of a protein or lipid receptor at the cell
surface, which would result in a decrease of toxicity. The toxicity of G88R
RNase A and ONC is enhanced when cells are treated with BFA
(Fig. 10). These data provide
evidence that ribonuclease cytotoxicity is not diminished by this indirect
affect. Cells treated with BFA or monensin internalize the same amount of
ribonuclease as do control cells in flow cytometry assays (data not
shown).
Ribonucleases are internalized in a dose-dependent manner
(Fig. 6), which is consistent
with their dose-dependent cytotoxicity
(Fig. 8B,C, Fig. 10). We propose that as
the extracellular concentration of a ribonuclease increases, so does its
cytosolic concentration. The arrows in Fig.
6 indicate the protein concentrations that correspond to the
IC50 values of >50 and 1 µM for G88R RNase A and ONC,
respectively (Table 2). We were
surprised to find that ONC and G88R RNase A have a similar dose-dependent
influx into HeLa cells, despite having IC50 values that differ by
more than 50-fold (Fig. 7;
Table 2). These results imply
that the difference in the cytotoxicity of ONC and G88R RNase A is not due to
differences in cell-surface binding and internalization. Still,
ribonuclease-mediated toxicity also requires translocation to the cytosol.
Once in the cytosol, uninhibited ribonucleolytic activity is necessary for
toxicity. Thus, ribonucleases must evade binding and inhibition by cytosolic
RI. ONC has low affinity for RI (Wu et
al., 1993; Boix et al.,
1996
), while RNase A binds RI with fM affinity
(Lee et al., 1989
;
Vicentini et al., 1990
). Our
data suggests that either (or both) of these hurdles is responsible for the
difference in the toxicity of ONC and G88R RNase A for HeLa cells. Indeed,
much work has demonstrated that evasion of RI is a requirement for the
cytotoxicity of mammalian ribonucleases
(Cafaro et al., 1995
;
Kim et al., 1995b
;
Leland et al., 1998
;
Bretscher et al., 2000
;
Haigis et al., 2002
).
Is there a cell-surface receptor for secretory ribonucleases? We
hypothesize that ribonucleases bind to the cell surface without interacting
with a specific protein receptor on the cell surface. We find that
ribonucleases bind to the cell surface in a nonsaturable manner
(Fig. 6B). This nonspecific
binding is not perturbed by treatment of the cells with proteases (K. A.
Dickson and R.T.R., unpublished). In addition, homolog-scanning mutagenesis
results suggest that there is not a single protein receptor for bovine seminal
ribonuclease (Kim et al.,
1995a). Finally, the random cationization of RNase A increases
both its cellular uptake and its cytotoxicity
(Futami et al., 2001
;
Futami et al., 2002
).
A distinct internalization pathway for a protein toxin
Ribonucleases are internalized by a pathway that is distinct from that of
all toxins characterized previously (Fig.
11). Protein toxins, characterized to date, that act within the
cytosol cross the plasma membrane from two subcellular locations: endosomal or
post-endosomal compartments (Lord and
Roberts, 1998). The best characterized toxin that translocates
from an endosomal compartment is diphtheria toxin
(Collier, 2001
)
(Fig. 11). Ricin is among the
best characterized toxins that translocates from a post-endosomal compartment
(Olsnes and Kozlov, 2001
)
(Fig. 11). Diphtheria- and
ricin-like toxins have evolved special mechanisms for entering and killing
mammalian cells. Such toxins comprise distinct domains one to provide
catalytic activity and another to facilitate cytosolic entry. During toxin
action, these domains physically dissociate. In a striking contrast, secretory
ribonucleases have a single globular domain that is responsible for
cell-surface binding, internalization and catalytic activity
(Fig. 1).
The internalization pathways of ribonucleases and diphtheria toxin differ
by their modes of uptake and translocation. The internalization of diphtheria
toxin is contingent on clathrin-dependent endocytosis
(Simpson et al., 1998).
Blocking this pathway by overexpressing a dominant-negative variant of dynamin
protects cells from diphtheria toxin
(Simpson et al., 1998
). By
contrast, ribonucleases are more toxic in the absence of dynamin-dependent
endocytosis (Fig. 8). The
translocation of diphtheria toxin is highly dependent on the low pH of the
endosome, as this acidic environment induces a conformational change in the B
domain that forms a pore through the membrane and allows the A domain to cross
(Draper and Simon, 1980
;
Sandvig and Olsnes, 1980
;
Sandvig and Olsnes, 1981
;
Sandvig and Olsnes, 1982
).
Agents such as NH4Cl and monensin, which neutralize acidic
environments within the cell, block cytosolic entry and thereby protect cells
from diphtheria toxin. These drugs do not protect cells from the toxicity of
ribonucleases (Fig. 10).
The internalization pathway of ribonucleases has similarities to and
differences from that of ricin (Sandvig
and van Deurs, 2000; Olsnes
and Kozlov, 2001
). Like some RNase A homologs
(Irie et al., 1998
), ricin is
a lectin and binds carbohydrates at the cell surface. ONC, RNase A and ricin
can be internalized and cytotoxic in the absence of dynamin-mediated
endocytosis (Llorente et al.,
1998
) (Fig. 9). In
addition, a low pH is not required for the cytotoxicity of ONC, G88R RNase A
or ricin. Unlike ribonucleases, ricin translocates to the cytosol from the ER
after retrograde transport from the Golgi network. Cells with a BFA-disrupted
Golgi stack are protected from ricin
(Yoshida et al., 1991
) but not
from ribonucleases (Fig. 10).
These data are consistent with the finding that BFA does not block the
toxicity of ONC for 9L cells (Wu et al.,
1995
). Olsnes and coworkers demonstrated that transport of the
ricin A chain from the Golgi to the ER was a prerequisite for translocation
(Rapak et al., 1997
).
Moreover, introduction of a KDEL tail to the ricin A chain dramatically
enhances its cytotoxicity (Wales et al.,
1993
; Tagge et al.,
1996
), but the presence of a KDEL tail on G88R RNase A has no
effect on its cytotoxicity (P. A. Leland and R.T.R., unpublished). Hence, an
increased ribonuclease concentration in the ER does not enhance its
cytotoxicity. Combined, these results indicate that ONC and G88R RNase A do
not translocate to the cytosol from the ER.
The study of toxin internalization has facilitated important discoveries in
the field of intracellular transport (Lord
and Roberts, 1998). Studies of Shiga toxin showed for the first
time that the secretory pathway is completely reversible
(Sandvig et al., 1992
). In
addition, Shiga toxin and ricin are models for dissecting retrograde transport
and as well as the molecular details of endocytosis
(Mallard et al., 1998
;
Sandvig and van Deurs, 2000
).
Studies using cholera toxin binding and entry have clarified pathways of lipid
trafficking (Orlandi and Fishman,
1998
; Radhakrishnan et al.,
2000
). Information about protein translocation from the ER to the
cytosol can also be gained using Shiga toxin and ricin. Likewise, secretory
ribonucleases can be a tool for studying clathrin- and dynamin-independent
endocytosis, as well as for investigating how cationic proteins enter
cells.
RNase A and its homologs are secreted proteins. Yet, the cytosol of every
cell contains RI (Hofsteenge,
1997; Shapiro,
2001
), indicating an imperative to protect cells from internalized
ribonucleases. The prevalence of RI suggests that ribonuclease internalization
is a widespread and important occurrence. Our work has revealed aspects of a
distinct pathway by which these common proteins secretory
ribonucleases can enter the cytosol
(Fig. 11).
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