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INTRODUCTION |
Exosomes are small membrane-bound vesicles secreted in the
extracellular medium by hematopoietic cells, such as reticulocytes (1),
B lymphocytes (2), or dendritic cells (3). These vesicles are formed by
membrane budding into the lumen of an endocytic compartment, leading to
the formation of multivesicular intracellular structures. Fusion of
these multivesicular structures with the plasma membrane leads to the
release of internal vesicles (exosomes) into the medium (4). The
protein composition of exosomes depends on the cell type. Some membrane
proteins become enriched in exosomes, whereas others are excluded. For
example, the transferrin receptor (TfR)1 is a major protein of
exosomes secreted by reticulocytes, leading to the complete loss of TfR
from maturing reticulocytes, whereas no anion transporter is lost
during maturation into erythrocytes. By contrast, TfR is absent from
the surface of exosomes secreted by B lymphocytes, which instead are
enriched with major histocompatibility complex class II
molecules (2). The molecular basis of the process of protein sorting
into exosomes is yet not known. However, it seems specifically related
to the particular cell involved because TfR is a major actor in
reticulocyte and prereticulocyte function and major histocompatibility
complex class II is essential in B lymphocyte physiology.
In reticulocyte exosomes, another protein is enriched in a
stoichiometric ratio with TfR. This protein has been identified as
clathrin uncoating ATPase, (also known as the heat shock cognate 70-kDa
protein (Hsc70) (5). This protein has been shown to interact with the
cytosolic domain of the TfR (6), and this interaction was suggested to
induce sorting of the receptors into exosomes. This conclusion is
strengthened by the fact that Hsc70 is also present in exosomes
secreted by dendritic cells (3) and thus may be a general marker of
exosome formation. Interestingly, however, clustering of TfRs on the
reticulocyte cell surface by using antibodies or lectins was shown to
induce receptor sorting into exosomes (7). These observations
demonstrated that protein sorting into exosomes is not necessarily
induced by cytosolic sorting machinery. The fact that
glycosylphosphatidylinositol-anchored proteins such as
acetylcholinesterase, CD55, CD58, and CD59 are enriched in reticulocyte
exosomes (1, 8), whereas they do not cross the plasma membrane,
supports the idea that protein sorting into exosomes may occur in the
absence of a cytoplasmic domain.
The proposal that protein aggregation may be the signal triggering
molecules toward the exosome pathway could account for these
observations. For example, Hsc70 could interact with proteins following
partial unfolding of the cytoplasmic domain. If TfR were to unfold,
Hsc70 would become associated with the TfR without participating
further in the sorting process. It is known that, besides its role in
clathrin uncoating of coated vesicles through interaction with auxilin
(9), Hsc70 has general chaperone properties and is involved in protein
folding and unfolding (10).
Previous studies have described the general characteristics of the
Hsp70 class of molecular chaperones in binding to peptides (11). Hsc70
binds preferentially to hydrophobic sequences containing basic amino
acids. The ADP-bound form of Hsp70 has a high affinity for peptides,
whereas the ATP form has a lower affinity, thus resulting in
dissociation (12). In the present study, we carried out an in
vitro binding assay to study characteristics of the interaction
between Hsc70 and TfR. Our results demonstrate that Hsc70 binds to
exosomal TfR with the characteristics expected of a chaperone/peptide
interaction. We used deoxyspergualin
(DSG) and LF15-0195, two immunosuppressive agents that interact with Hsc70 (13, 14) (Fig. 1). Both compounds diminished the
interaction between Hsc70 and TfR in our in vitro binding
assay. Both induced an increase in TfR release in exosomes when added
during in vitro maturation of reticulocytes. These data
demonstrate that TfR aggregation instead of Hsc70 binding may be the
signal targeting TfR molecules toward the exosome pathway.
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EXPERIMENTAL PROCEDURES |
Materials--
Rat monoclonal antibody raised against Hsc70
(SPA-815) and mouse monoclonal antibody raised against rat TfR
(CL-071AP) were from StressGen (Victoria, Canada) and Cedarlane
(Hornby, Canada), respectively. A mouse
anti-Na+/K+-ATPase was kindly provided by Dr.
R. Blostein (Montreal General Hospital Research Institute,
Montreal, Canada). An antibody against a known peptide sequence
corresponding to the cytoplasmic domain of the human transferrin
receptor was kindly provided by Dr. I. S. Trowbridge (Salk
Institute, La Jolla, CA). Peroxidase-conjugated goat anti-rat IgG and
peroxidase-conjugated donkey anti-mouse IgG were both obtained from
Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). Protein
A-Sepharose beads were from Amersham Pharmacia Biotech. ATP-agarose gel
was from Sigma. Recombinant firefly Luciferase was purchased from
Promega France (Charbonnieres, France). Thin layer chromatography
plates (polyethylenimine-cellulose) were purchased from Macherey-Nagel
(Düren, Germany). [
-32P]ATP and
Na125I were obtained from ICN Pharmaceuticals (Orsay,
France). [3H]Nitrobenzylthioinosine was obtained from
Moravek Biochemicals (Brea, CA). DSG and methyl-DSG were provided by
Nippon Kayaku Co (Tokyo, Japan), and LF15-0195 was from Laboratoires
Fournier (Daix, France).
Reticulocyte and Exosome Preparation--
Reticulocyte-enriched
blood was obtained from phenylhydrazine-treated rats (7) or from
phlebotomized sheep (6). Exosomes were collected by sequential
centrifugations from the supernatant of reticulocyte subcultures as
previously described (1). Briefly, blood samples were centrifuged at
1000 × g for 10 min at 4 °C to remove plasma and
the buffy coat. After three washes with Hanks' buffer, the cells (3%
final packed cell volume) were cultured in RPMI 1640 supplemented with
5 mM adenosine, 10 mM inosine, 5 mM
glutamine (plus 3% (v/v) fetal calf serum in the case of rat
reticulocytes). To assess the effect of DSG and LF15-0195 on exosome
formation, the compounds were added during the in vitro maturation of reticulocytes. After the indicated times at 37 °C in a
O2/CO2 incubator, the cells were pelleted, and
the supernatant was centrifuged for 20 min at 20,000 × g to remove mitochondria and cellular fragments. The
supernatants were ultracentrifuged for 90 min at 100,000 × g, and the vesicle pellets were either analyzed for specific
protein content by Western blotting or for [3H]nitrobenzylthioinosine binding (1) or were
solubilized in phosphate-buffered saline containing 1% Triton X-100
and the cleared supernatants were used for TfR immunoprecipitation (see below).
Purification of Hsc70 from Rat Brain--
The protocol described
in Ref. 15 was adapted for the isolation of Hsc70 from rat brains. All
steps were carried out at 4 °C. Four rat brains were homogenized
with a Dounce homogenizer in 50 ml of buffer A (20 mM
Tris-HCl, pH 7.3, 15 mM KCl, 10% (w/v) sucrose, 0.01%
(v/v) Nonidet P-40, 1 mM dithiothreitol, 2 mM
EDTA, 1 mM PMSF, 10 mM sodium bisulfite, 1 µg/ml pepstatin, 1 µg/ml leupeptin), dialyzed against buffer B (50 mM Tris-HCl, pH 7.3, 10% (w/v) sucrose, 1 mM
EDTA, 5 mM
-mercaptoethanol, 0.2 mM PMSF) containing 50 mM KCl, and loaded onto a 25-ml Q-Sepharose
column equilibrated with the same buffer. The column was washed with 750 ml of buffer B containing 50 mM KCl. Elution was
performed with a 200-ml gradient from 50 to 600 mM KCl in
buffer B. Fractions containing Hsc70 (detected by Western blotting)
were pooled, dialyzed against buffer C (10 mM imidazole
HCl, pH 7, 10 mM MgCl2, 10% (w/v) sucrose, 1 mM EDTA, 5 mM
-mercaptoethanol, 0.2 mM PMSF), and loaded onto a 5 ml ATP-agarose column
equilibrated with buffer C. The column was sequentially washed with 25 ml of buffer C, 25 ml of buffer C containing 500 mM KCl, 25 ml of buffer C, 25 ml of buffer C containing 1 mM GTP (to
elute GTP binding proteins), and 25 ml of buffer C. Finally, the
Hsc70 protein was eluted with 50 ml of buffer C containing 4 mM ATP. Fractions containing Hsc70 protein were pooled and
dialyzed against buffer D (50 mM Tris-HCl, pH 7.3, 10%
(v/v) glycerol, 1 mM EDTA, 1 mM dithiothreitol,
and 0.2 mM PMSF). Purified Hsc70 was freed from ATP using a
Sephadex-G25 spin column equilibrated in buffer E (25 mM
HEPES, pH 7.4, 50 mM KCl) containing 0.1 mM
EDTA and then dialyzed for 48 h against the same buffer. The
concentration of Hsc70 purified was 0.3 mg of protein/ml.
Radioiodination of Hsc70 was carried out using Iodo-Gen reagent
(16).
Hsc70 Binding to Immobilized TfR--
Immunoprecipitations were
performed with exosomes (0.4 mg of protein/ml) solubilized in 40 µl
of phosphate-buffered saline, 1% Triton X-100 and incubated for 2 h at room temperature with 3 µg of anti-TfR. The antigen-antibody
complex was precipitated with 40 µl of a 50% slurry of protein
A-Sepharose beads and incubated overnight at 4 °C. The beads were
recovered by centrifugation for 2 min at 400 × g and
washed five times with 1 ml of TG buffer (10 mM Tris-HCl,
pH 7.6, 50 mM NaCl, 10% (w/v) glycerol, 0.1% Nonidet
P-40). Determination of purified Hsc70 association with immobilized TfR
was carried out by adding 3 µg of Hsc70 with the beads. The samples
were then incubated at 30 °C for 1 h with gentle shaking every
5 min, followed by seven washes with TG buffer. Samples were denatured
for 5 min at 90 °C with Laemmli loading buffer and analyzed by
SDS-PAGE (17). Copelleting of Hsc70 with TfR was determined by Western
blot and quantified using ImageQuaNT software. Where indicated,
purified Hsc70 was incubated with 5 mM of different
nucleotides (ADP, ATP
S) or pretreated with 10 mM of DSG
or LF15-0195. In the case of Hsc70 pretreated with DSG or LF-15, all
washes were carried out using TG buffer without Nonidet-P40.
NEM Treatment of Purified Hsc70--
Purified nucleotide-free
Hsc70 was preincubated with or without MgATP (5 mM) at
4 °C for 30 min. NEM (2.5 mM) was then incubated for
2 h with ATP-bound and ATP-free Hsc70 at 30 °C before adding 5 mM dithiothreitol (10 min at 4 °C) to inactivate
residual NEM. Incubation at 4 °C was continued for 30 min with the
addition of MgATP (5 mM) to the ATP-free Hsc70 sample.
NEM-treated Hsc70 was then added to immobilized TfR, and Hsc70
copelleting was determined as described above.
Aggregation Assay by Centrifugation--
Recombinant firefly
luciferase (58 µM) was incubated in buffer E (25 mM Tris-HCl, pH 7.8, 50 mM KCl, 1 mM dithiothreitol) containing the indicated combinations of
proteins (2.6 µM Hsc70 and 2.6 µM
transferrin) for 10 min at 4 or 42 °C. Reaction mixtures were
centrifuged in an air-driven ultracentrifuge (Beckman Airfuge with
18° A-100 rotor) at 5 p.s.i. for 10 min. Supernatants were analyzed by SDS-PAGE and Coomassie staining.
ATPase Assay--
ATPase assay was performed as described (18,
19) with slight modifications. The assay was carried out in buffer F
(20 mM HEPES, pH 7, 25 mM KCl, 10 mM NH4SO4) at 30 °C for 1 h. [
-32P]ATP (10 µCi) was diluted in 25 µl of a 50 µM solution of ATP (8 µCi/µmol ATP) was aliquoted to
achieve 3.8 µCi/6 µg of purified Hsc70. Incubation was conducted
for 40 min at 30 °C. The sample was applied to a 1-ml Sephadex G-25
spin column. 32P-Labeled nucleotide·Hsc70 complex was
recovered by centrifugation and added to immobilized TfR for 2 h
at 30 °C. As control, 0.4 µCi of [
-32P]ATP was
added along with 100 µg of adenosine 5'-triphosphatase (Sigma), 9 µl of buffer F, and protein A-Sepharose beads incubated with 400 µg
of exosomal proteins and anti-TfR antibody. Aliquots were spotted on
thin layer chromatography plates (polyethylenimine-cellulose) and
developed using 0.4 M NH4HCO3
buffer. [
-32P]ADP and [
-32P]ATP were
visualized by autoradiography.
Western Blot Analysis--
Proteins were separated by 10%
SDS-PAGE according to Laemmli (17) and electrophoretically transferred
to PVDF membrane (Immobilon-P, Millipore), unless otherwise specified,
as described by Towbin et al. (20). Immunoblotting was
performed using peroxidase-conjugated antibodies and ECL (Amersham
Pharmacia Biotech) procedure.
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RESULTS |
Hsc70 Binding to Immobilized Exosomal TfR--
Association of
Hsc70 with exosomal TfR has been demonstrated by chemical
cross-linking, coimmunoprecipitation, and immobilization experiments
(6). To further characterize this association, we developed a binding
assay using Hsc70 purified from rat brain and TfR immunoprecipitated
from rat exosomes. As shown in Fig. 2A, a single band of ~70 kDa
was obtained by Coomassie staining (lane 2), and recognized
by an anti-Hsc70 antibody (lane 3). TfR was
immunoprecipitated from rat reticulocyte exosomes using a monoclonal
antibody against the extracellular domain of the receptor. As shown in
Fig. 2A (lane 5), a band with a molecular
mass (about 94 kDa) corresponding to the TfR monomer, was
detected by Western blot after immunoprecipitation. Note that a band
with a lower molecular mass (approximately 85 kDa) was also detected on
the membrane, corresponding to the soluble fragment of a cleaved
receptor already described (21, 22). TfR immobilized on protein
A-Sepharose was then used to characterize binding of purified Hsc70. As
highlighted by the Western blot in Fig. 2A (lane
7), after incubation of the purified chaperone with immobilized
TfR and several washes, Hsc70 was found to be associated with the
TfR-Sepharose beads. Protein A-Sepharose incubated with Hsc70 without
immobilized TfR did not result in any chaperone copelleting (Fig.
2A, lanes 4 and 6). To confirm the
specificity of the association between TfR and Hsc70, we examined
whether increasing amounts of TfR would bring down an increasing amount
of Hsc70. As shown in Fig. 2B, there was an increase in the
amount of Hsc70 brought down by the TfR immobilized on Sepharose beads.
Quantification of TfR and Hsc70 Western blots was done for each lane,
and the ratio (Hsc70/TfR) gave similar values.

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Fig. 2.
Purified Hsc70 binds to immobilized exosomal
TfR. A, Hsc70 was purified from rat brain. Lane
1, Coomassie Blue staining of SDS-PAGE from brain extract;
lane 2, purified Hsc70; lane 3, Western blot of
purified Hsc70. For immobilization of exosomal TfR, protein A-Sepharose
beads were incubated with solubilized exosomal proteins, in the absence
(lanes 4 and 6) or presence (lanes 5 and 7) of anti-TfR antibody. Purified Hsc70 was then added
for 1 h at 30 °C before washing the Sepharose beads as
described under "Experimental Procedures." Bead-associated proteins
were separated by SDS-PAGE and transferred on PVDF membrane. Western
blot of TfR (lanes 4 and 5) and Hsc70
(lanes 6 and 7) was then carried out on the
membrane. Molecular mass standards are indicated to the
left. B, increasing amounts of TfR were
immunoprecipitated using the same quantities of anti-TfR antibody and
protein A-Sepharose beads and increasing amounts of solubilized
exosomes (1, 4, 8, and 16 µg of protein, respectively, in lanes
1, 2, 3, and 4). Purified Hsc70
was then added, and the beads were treated as described in
A. Membrane was blotted for TfR (upper part) and
Hsc70 (lower part).
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Purified Hsc70 Binds to Exosomal TfR through Its Chaperone
Activity--
Contrary to other members of the Hsp70 protein family,
Hsc70 is constitutively expressed in almost all living cells. One of its best known roles in cell physiology is to remove clathrin triskelions from coated vesicles and to provide recycled components for
the formation of nascent coated pits (23). Hsc70 is also involved in
other cellular functions that require binding to partially unfolded
proteins during synthesis or en route to degradation. The fate of the
proteins can then vary. For example, the chaperone can prevent
aggregation of a misfolded protein as well as assist its refolding with
cochaperones (24), or it may target some cytosolic proteins to
lysosomes (25). The chaperone activity of purified Hsc70 was assessed
by measuring its binding to heat-denatured firefly luciferase (26).
Luciferase unfolding was obtained by 10 min of incubation at 42 °C.
This led to protein aggregation and was followed by pelleting during
centrifugation (Fig. 3A, lanes 1 versus 2). However, when Hsc70
was present during incubation at 42 °C, luciferase aggregation and
pelleting was prevented. The addition of transferrin instead of Hsc70
had no effect (Fig. 3A, lanes 3 and
4), and luciferase was removed from the supernatant. If
Hsc70 is limiting and binds to exosomal TfR via its chaperone capacity,
the addition of denatured luciferase could compete with the Hsc70-bound
TfR and lower the amount of Hsc70 associated with Sepharose-immobilized
TfR. As shown in Fig. 3B, incubation in the presence of
heat-denatured luciferase significantly decreased Hsc70 binding to TfR,
whereas the addition of native luciferase did not significantly affect
Hsc70/TfR binding. The minor effect of native luciferase could be the
result of a partial unfolding of luciferase during the 1-h incubation
at 30 °C, as suggested by the lower luciferase activity (not shown).
Note that although aggregation and pellet formation of heat-denatured
luciferase were prevented by the addition of Hsc70, the latter could
not bring about protein refolding, as measured by restoration of its enzymatic activity (not shown).

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Fig. 3.
Purified Hsc70 binds to immobilized exosomal
TfR through its chaperone activity. A, purified Hsc70
has chaperone activity. Recombinant luciferase (Lucif.) was
treated at 4 °C (lane 1) or 42 °C (lanes
2-4) for 10 min in the presence (lane 3) or absence
(lanes 1, 2, and 4) of purified Hsc70,
plus transferrin (Tf, lane 4). Mixtures were
ultracentrifuged, and supernatants containing nonaggregated proteins
were analyzed by SDS-PAGE and Coomassie Blue staining. B,
heat-denatured luciferase competes with Hsc70 binding to exosomal TfR.
Binding of Hsc70 to immobilized exosomal TfR was carried out as
described in Fig. 2, except that purified Hsc70 was first loaded with
ADP and preincubated (lanes 2 and 3) or not
(lane 1), with recombinant luciferase for 10 min at 4 °C
(lanes 1 and 2) or 42 °C (lane 3).
Washed beads were resuspended with Laemmli buffer and loaded on
SDS-PAGE. Proteins were transferred to PVDF membrane and immunoblotted
using an anti-Hsc70 antibody.
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Effect of Nucleotides on Hsc70 Binding to Exosomal TfR--
The
Hsp70 family binds peptides under regulation by ATP binding and
hydrolysis. The ADP-bound form of Hsc70 binds peptides with high
affinity and has a slow rate of substrate release, whereas the
ATP-bound form is more prone to peptide release. Moreover, the complex
between Hsc70 and the nucleotide (ATP or ADP) is very stable, and when
purified from a cell homogenate, special methods are required to obtain
nucleotide-free Hsc70 (27, 28). To study the effects of added
nucleotides, we thus generated nucleotide-free chaperone, loaded it
with ADP or ATP
S, and assessed binding of the two forms to exosomal
TfR. As shown in Fig. 4A, more
ADP-bound Hsc70 than ATP
S-bound Hsc70 was retained by TfR
immobilized on Sepharose beads. Western blot quantification indicated
that about three times more of the ADP form was associated with the
TfR-bead pellet than the ATP
S bound form. These data are all
consistent with chaperone behavior.

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Fig. 4.
Effects of nucleotides on Hsc70 binding to
exosomal TfR. A, ADP-loaded Hsc70 binds TfR with a
higher affinity than ATP S-loaded chaperone. Hsc70 was freed from
nucleotide and loaded with ADP or ATP S by incubation with 5 mM nucleotides, as described under "Experimental
Procedures." Hsc70 binding to Sepharose-immobilized exosomal TfR was
carried out as in Fig. 2. Hsc70 immunoblots (upper part)
were scanned and quantified using ImageQuaNT software (lower
part). (Image quantification: ADP·Hsc70 was set to 100%).
B, nucleotide-bound Hsc70 is protected from NEM treatment.
Purified nucleotide-free Hsc70 was loaded or not with ATP before NEM
treatment as described under "Experimental Procedures." Hsc70
binding to exosomal TfR was carried out as described above. TfR and
Hsc70 Western blots are presented in the upper part of the
panel. Data of Hsc70 immunoblot quantification are given in the
lower part. (Image quantification: NEM-untreated ATP·Hsc70
was set to 100%).
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Ssa1p, the predominant yeast cytosolic Hsp70, was demonstrated to be
inactivated by NEM when in the nucleotide-free form but not when bound
to nucleotides (28). It was suggested that NEM modification may disrupt
the conformation of Ssa1p or interfere with nucleotide binding. To
assess the effect of NEM in this system, nucleotide-free or ATP-bound
Hsc70 were treated with NEM followed by binding to exosomal TfR. As
shown in Fig. 4B, Hsc70 binding to TfR immobilized on
Sepharose beads was significantly decreased by NEM treatment of the
nucleotide-free Hsc70 (about 40% of control) (Fig. 4B,
lane 3), whereas the nucleotide loaded form (lane
2) was much less affected by NEM treatment (about 70% of
control). This suggests that, like Ssa1p, NEM treatment of
nucleotide-free Hsc70 affects its activity and that the
nucleotide-bound chaperone is protected from modification by NEM.
Binding to Exosomal TfR Activates Hsc70 ATPase Activity--
To
further assess the characteristics of Hsc70 association with exosomal
TfR, the ATPase activity of Hsc70 upon substrate binding was
investigated. It has been shown that binding of peptide substrates to
Hsp70 homologs such as DnaK from Escherichia coli (29) or
Ssa1p from Saccharomyces cerevisiae (30) stimulate ATPase
activity of chaperone proteins. As shown in Fig.
5, two radioactive spots appeared
(lane 8) when a purified [
-32P]ATP·Hsc70
complex was added to TfR immobilized on Sepharose beads followed by
2 h of incubation at 30 °C and polyethylenimine-cellulose chromatography. The reaction mixture at t0 gave only one
radioactive spot (lane 7). Migration comparison with
unlabeled nucleotides identified the spots as ADP and ATP, as
indicated. Spots corresponding to AMP, ADP, and ATP were also obtained
by incubation of [
-32P]ATP with excess porcine brain
ATPase (lanes 3 and 4). Neither spontaneous
(lanes 1 and 2) nor immobilized-TfR induced
(lanes 5 and 6) [
-32P]ATP
hydrolysis was found during the incubation period. The nucleotide hydrolysis induced by binding of ATP·Hsc70 to exosomal TfR thus confirmed that the interaction between the two proteins was that expected of an interaction with a chaperone.

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Fig. 5.
Stimulation of the Hsc70 ATPase by
immunoprecipitated TfR. [32P]ATP·Hsc70 complex was
obtained as described under "Experimental Procedures."
[32P]ATP (lanes 5 and 6) or
[32P] ATP·Hsc70 (lanes 7-10) were added to
immobilized TfR (lanes 7 and 8) or to protein
A-Sepharose beads (lanes 9 and 10) for 2 h
at 30 °C. Aliquots of the reaction mixtures were spotted on
polyethylenimine-cellulose plate at the beginning of incubation
(lanes 5, 7, and 9) or 2 h later
(lanes 6, 8, and 10). Nucleotides were
visualized by autoradiography after migration, as described under
"Experimental Procedures." Spontaneous hydrolysis of
[32P]ATP during the incubation period at 30 °C was
visualized by incubation of the nucleotide alone and spotting at
t0 (lane 1) and after 2 h
(lane 2). Partial and total [32P]ATP
hydrolysis was obtained by [32P]ATP incubation in the
presence of adenosine 5'-triphosphatase and spotting at
t0 (lane 3) and after 2 h
(lane 4). Nucleotide species are indicated to the
left.
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DSG and LF15-0195 Decrease in Vitro Hsc70/TfR Interaction but
Increase TfR Sorting in Exosomes during Reticulocyte
Maturation--
One way to interfere with the interaction of Hsc70/TfR
in vitro is, as described above, to add a competitor for the
interaction using an unfolded target protein. Another possibility is to
use agents capable of interfering with normal functioning of the
chaperone, such as the immunosuppressant DSG (31) or analogs like
methyl-DSG and LF15-0195 (14) (Fig. 1). Although the mechanism of
action is still poorly understood, DSG binds Hsc70 with a
Kd in the micromolar range (13), suggesting that
Hsc70 may be a target for the immunosuppressive action of DSG in
vivo.
First we assessed the effect of the compounds on in vitro
interaction between Hsc70 and exosomal TfR. Purified Hsc70 was
preincubated with or without DSG or LF15-0195 before using it for
in vitro binding to TfR. As shown in Fig.
6, preincubation of both
immunosuppressive agents with the chaperone decreased its association
with immobilized TfR. Radiolabeled Hsc70 was used in the binding assay
to improve the quantification. As shown in Fig. 6, the data in both
assays gave similar values.

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Fig. 6.
DSG and LF15 decrease Hsc70 binding to
exosomal TfR. Binding of Hsc70 to immobilized exosomal TfR was
carried out as described in Fig. 2, except that purified Hsc70 was
first preincubated for 1 h at 30 °C with buffer alone or in the
presence of DSG (10 mM) or LF15-0195 (10 mM).
Washed beads were then resuspended with Laemmli buffer and loaded on
SDS-PAGE. Proteins were transferred to PVDF membrane and immunoblotted
using an anti-Hsc70 antibody. Hsc70 immunoblot was scanned and
quantified using ImageQuaNT software. (Image quantification: untreated
Hsc70 was set to 100%). 125I-Hsc70 was added to unlabeled
chaperone to obtain a specific activity of ~20 cpm/ng. Radiolabeled
Hsc70 was then used in the binding assay as described for unlabeled
chaperone. Hsc70 association with Sepharose bead pellet was determined
by radioactivity counting. (The 100% binding obtained with CTRL
corresponded to ~10000 cpm associated with Sepharose beads).
CTRL, control.
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To improve our understanding of the nature and role of the Hsc70/TfR
interaction during reticulocyte maturation, we took advantage of the
inhibition of Hsc70 binding to TfR by DSG. DSG is transported across
the plasma membrane and accumulates in the cell cytoplasm (32). We were
thus able to assess the consequences of decreasing Hsc70/TfR
interaction during exosome formation. The immunosuppressive agents were
added during in vitro maturation of reticulocytes, and the
time course of exosome release was monitored after the addition of the
immunosuppressive compounds.
Reticulocytes from phlebotomized sheep that were cultured in
vitro for 3-4 h or for up to 2 days, in the presence of DSG or methyl-DSG, showed an increase in the
amount of TfR released via exosomes (Table I and Fig.
7). Although the extent of increase was variable, the direction of the response was consistent.
Immunoblotting for Hsc70 from exosomes did not show a comparable
quantitative increase in the presence of DSG. Similar results with DSG
and LF15-0195 were obtained using rat reticulocytes from animals
treated with phenylhydrazine (not shown). This increase of TfR release in exosomes in response to the immunosuppressive agents in two different species argues against a possible fortuitous event.
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Table I
DSG increases release of exosomal TfR
Sheep reticulocytes enriched to contain at least 80% reticulocytes
were incubated as described for the times indicated. The exosomes were
separated from the medium by ultracentrifugation of the cell-free
supernatant. An aliquot of exosomes corresponding to the number of
packed cells from which the exosomes were obtained were then subjected
to SDS gel electrophoresis, transferred to nitrocellulose membranes,
and blotted with a monoclonal mouse antibody to the cytoplasmic domain
of the human TfR (obtained from Trowbridge). A sheep anti-mouse
horseradish peroxidase-antibody was used as secondary antibody. The
blots were visualized with chemiluminescence reagents according to the
manufacturer's instructions and analyzed with a Bioimage system. The
arbitrary OD units (AOD) are given. Three separate experiments are
shown, two of which were done in triplicate. An immunoblot
corresponding to experiment 1 is shown in Fig. 7.
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Other membrane proteins have been demonstrated to be selectively sorted
into exosomes, together with TfR (1). If TfR release were part of a
general sorting mechanism, the presence of DSG during reticulocyte
maturation would also increase Na+/K+-ATPase
and nucleoside transporter release. The presence of DSG during
reticulocyte maturation increased the amount of
[3H]nitrobenzylthioinosine binding in the released
exosomes (Table II).
[3H]Nitrobenzylthioinosine is a nucleotide analog frequently
used to assess the level of equilibrating nucleoside transporter
(1). In addition, in a single experiment, there was also evidence of enhanced externalization of the
-subunit of the
Na+/K+-ATPase.
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Table II
DSG increases release of exosomes bearing the nucleoside transporter
Exosomes from sheep reticulocytes were collected after overnight
incubation in the presence or absence of the drugs as indicated.
[3H]Nitrobenzylthioinosine at a concentration of 1.0 nM was incubated with the recovered exosomes as described
using a sample of exosomes obtained from 150 µl of sheep
reticulocytes. To obtain specific binding, a control containing 1000×
excess of unlabeled [3H]nitrobenzylthioinosine was carried
out with each sample. The nonspecific uptake, which represented no more
than 5% of the total uptake, has been subtracted from the values
given.
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DISCUSSION |
Release of exosomes during reticulocyte maturation was first
described about 20 years ago (33) and is now a well known process (34).
More recently, this process has been demonstrated to occur in other
hematopoietic cells (2, 3, 35). Depending on the cell type, the
occurrence of the phenomenon of vesicle release may have different but
important implications. In the case of reticulocytes, it likely
represents a mechanism to remove obsolete proteins from the cell
surface. It is known that the plasma membrane of reticulocytes has a
greater variety and a higher amount of proteins than that of the mature
erythrocyte. In the case of antigen presenting cells, exosome release
could be a way to increase the cellular immunologic response (36).
However, the molecular basis of protein sorting into exosomes is still
not known. It was suggested that Hsc70, the only known cytoplasmic
protein enriched in reticulocyte exosomes, could be involved in TfR
sorting. In fact, Hsc70 was demonstrated to interact better with the
TfR present in exosomes than with those on the plasma membrane (6).
Moreover, the interaction in exosomes appeared stoichiometric.
In this study, we set up an in vitro binding assay using
purified Hsc70 and immobilized exosomal TfR to characterize the nature of the interaction between these two molecules. We found that binding
of Hsc70 to exosomal TfR followed the pattern expected of an
interaction with a chaperone. Thus we found that (i) heat-denatured luciferase was able to compete with TfR for Hsc70 binding, (ii) binding
of Hsc70 to TfR was dependent upon the nucleotide bound to the
chaperone, (iii) binding to exosomal TfR activated Hsc70 ATPase
activity, and (iv) compounds known to interact and modify the chaperone
characteristics of Hsc70 decreased binding of Hsc70 to TfR. Thus,
during reticulocyte maturation, it is possible that the cytoplasmic
domain of the TfR becomes partially unfolded, which would then trigger
Hsc70 binding. It is noteworthy that a peptide sequence:
23FSLARQV29 in the TfR cytoplasmic domain has
the characteristic motif recognized by Hsc70. Hsc70 binds to peptides
enriched in large hydrophobic and aromatic residues and containing
basic amino acids. For example, it has been shown that the peptide
sequences FSGLWKL and LSRTLSV were responsible for high affinity
binding (<200 µM) of peptides P17G and P10K,
respectively, to Hsc70 (11). Moreover, the peptide sequence
23FSLARQV29 of TfR is also part of the protein
kinase C phosphorylation motif 22RFSLAR27.
Thus, binding of the chaperone with its target domain should impair
further phosphorylation of the serine residue by PKC. If Hsc70 remained
associated with the TfR upon immunoprecipitation of the latter, this
could explain why immunoprecipitates of the TfR from the plasma
membrane were phosphorylated by PKC in vitro (37), whereas
the immunoprecipitates of exosomal TfR, which are more likely
associated with Hsc70, could not be phosphorylated under identical conditions.
A common explanation of the role of Hsc70 in TfR externalization is
that after many rounds of endocytic cycles, along with changes in the
cytoplasm during cell maturation, partial unfolding of the TfR
cytoplasmic domain would expose the hydrophobic sequence that would
bind Hsc70. This would be analogous to the appearance of a "new"
signal during the lifespan of the receptor in red cells, leading to a
change in its intracellular routing. This type of rerouting signal
could also occur in other cell types where lysosomal degradation of
proteins takes place at the end of their lifespan.
Although this model has attractive features, data obtained using DSG
are inconsistent with this model and suggest that Hsc70 binding is not
the real signal for a change in TfR sorting. On the contrary, DSG data
suggest that TfR segregation into exosomes is inhibited by Hsc70 binding.
DSG is a synthetic analog of spergualin, a natural product isolated
from Bacillus laterosporus that has been shown to interact specifically with Hsc70 and Hsp90, with Kd values of 4 and 5 µM, respectively (13). Although its exact
mechanism of action is still poorly understood, DSG was demonstrated to be a potent immunosuppressive agent (38). The ability of DSG to bind
Hsc70 and modify the chaperone characteristics of Hsc70 in other
nonimmunologic areas has recently been shown. It was demonstrated that
DSG can rescue the
F508-CFTR trafficking defect (the most common
mutant of the transmembrane conductance regulator encountered in cystic
fibrosis).
F508-CFTR is purportedly retained and degraded in the ER
through interaction with Hsp70. However, it was targeted to the plasma
membrane when cells expressing the mutant protein were exposed to DSG
(39). In the present case, the addition of DSG or analogs during
in vitro reticulocyte maturation increased the amount of TfR
secreted via exosomes. If Hsc70 binding were required for
externalization along with the prevention of binding by DSG, a decrease
in exosome formation would be expected. This is in apparent
contradiction with the data on in vitro binding, which
demonstrated a decrease in Hsc70 binding to exosomal TfR in the
presence of DSG (Fig. 6). The data suggest that Hsc70 binding may
prevent or diminish sorting into exosomes. Thus, it is unlikely that
binding of Hsc70 itself leads to TfR externalization. In this context,
it is important to remember that one of the chaperone functions is to
fight against protein unfolding and aggregation. Aggregation is thus
probably the real signal triggering protein sorting into exosomes.
Hsc70 would bind to TfR as a consequence of receptor unfolding but
would not be the major factor of its segregation. Moreover, Hsc70 could
delay TfR aggregation by acting as a chaperone, and this reaction would
be impaired by DSG. The result would also be consistent with a greater
effect of DSG at short maturation times. In this perspective, it is
noteworthy that TfR has a marked tendency to interact strongly upon
reconstitution into lipid bilayers (40). This characteristic probably
favors the aggregation of TfR in the exosomal membrane and could
account for the finding that TfR is often detected as a dimer on
SDS-PAGE under reducing conditions. This is also consistent with our
previous work demonstrating that experimental aggregation of TfR
induces its secretion through exosomes (7) and that aggregation of furin in post-Golgi compartments triggers its targeting to lysosomes (41).