(Received for publication, July 20, 1995)
From the
Apoprotein B100 (apoB) is a secretory protein that appears to be constitutively translated but inefficiently translocated into the lumen of the endoplasmic reticulum. Using several experimental approaches, we found that apoB is bound to the cytosolic chaperone protein, heat shock protein 72/73 (commonly referred to as Hsp70). Similar to other chaperone-protein interactions, this binding was transient and ATP-sensitive. The binding of apoB to Hsp70 in HepG2 cells was decreased by treatment with oleic acid, which increases both translocation and secretion of apoB, and was increased by N-acetyl-leucyl-leucyl-norleucinal, a protease inhibitor which efficiently protects apoB from cellular degradation without affecting translocation. The N-terminal 16% of apoB, which is efficiently translocated into the endoplasmic reticulum lumen in stably transfected Chinese hamster ovary (CHO) cells, showed minimal, if any, binding to Hsp70. The N-terminal 50% of apoB, which is very poorly translocated in CHO cells, was found to bind significantly to Hsp70. These results suggest that domains of nascent apoB localized on the C-terminal regions of the molecule are transiently exposed to the cytosol during translation and/or translocation, and that Hsp70 functions as a molecular chaperone to maintain apoB in a translocational competent conformation until translocation is completed.
Apoprotein B100 (apoB) ()is a very large (540 kDa),
extremely hydrophobic protein that is necessary for the assembly and
secretion of lipoproteins carrying the majority of plasma cholesterol
and triglyceride in man. Evidence from several laboratories indicates
that apoB secretion is regulated post-translationally, rather than at
the transcriptional or translational level(1) . The key step in
this post-translational regulation appears to be the translocation of
newly synthesized apoB across the endoplasmic reticulum (ER) membrane;
this step has been shown by most laboratories to be inefficient and
slow. Thus, unlike albumin, which is efficiently translocated into the
ER and quantitatively secreted into the medium, only a fraction of
nascent apoB is assembled with lipids and secreted as very low density
lipoprotein. Instead, the majority of newly synthesized apoB is
degraded intracellularly. Inefficient translocation allows some portion
of apoB to be exposed to the cytosol for a significant period of time (2, 3, 4, 5, 6) , and this
appears to render nascent apoB susceptible to degradation by a cysteine
protease(7) . An unaddressed issue related to the translocation
of apoB is how this hydrophobic molecule is able to retain its native
conformation during the period when it is, at least partly, exposed to
the cytosol.
Recent studies indicate that many newly synthesized proteins transiently interact with molecular chaperones during their folding, maturation, and transmembrane targeting(8, 9) . One class of these molecular chaperones is the 70-kDa heat shock protein family. Evidence indicates that both the constitutively expressed Hsp73 and the highly stress-inducible Hsp72 (subsequently referred to jointly as Hsp70) are cytoplasmic molecular chaperones which can target to the hydrophobic domains of substrate polypeptides. They not only assist various cytosolic proteins as they mature into their final conformation(10) , but also participate in post-translational transmembrane targeting of proteins to cellular organelles(11, 12) . For example, genetic manipulation in yeast showed that depletion of cytosolic Hsp70 protein in vivo caused the accumulation of presecretory proteins in the cytosol(13) . In vitro studies indicated that Hsp70 could stimulate protein translocation into microsomes(14, 15) . It is believed that Hsp70 participates in maintaining the translocation competence of proteins that are post-translationally targeted for transport across organelle membranes. Hsp70 also appears to participate in the insertion into the ER of transmembrane proteins destined for transport to the plasma membrane(15) . A role for Hsp70 in the translocation of mammalian secretory proteins is ill defined at present; it is commonly believed that the transport of such proteins into the ER occurs co-translationally through a pathway involving signal sequences, signal recognition particles and docking proteins(16, 17) . Whether Hsp70 interacts with any secretory protein in mammalian cells in vivo is still unknown. In this study, we demonstrate that nascent apoB is associated with cytosolic Hsp70 in HepG2 cells, and that this association is related to apoB translocational status. This may be how apoB, a hydrophobic protein, is able to maintain its translocational competence while exposed to the aqueous environment of the cytosol.
For denaturing immunoprecipitation, the labeled cells were lysed with PBS containing 1% Triton X-100, 1% deoxycholate, 1% SDS, and protease inhibitors. The samples were boiled for 5 min, diluted with 10 volumes of PBS containing 1% Triton X-100 and proteinase inhibitors, and centrifuged. The supernatants were used for immunoprecipitation followed by protein A-Sepharose collection of the immunocomplexes.
For sequential immunoprecipitation, radiolabeled cells were first immunoprecipitated under the nondenaturing conditions described above, and the immunocomplexes were mixed with an equal volume of 2% SDS and boiled for 5 min. The supernatants were diluted with 1% Triton X-100 to a final concentration of 0.1% SDS and used for a second, denaturing immunoprecipitation.
Figure 1:
Identification of Hsp70apoB
complexes by immunoprecipitation of radiolabeled proteins. A,
monolayers of HepG2 cells were radiolabeled for 30 min with
[
H]leucine (200 µCi/ml) in leucine-free
medium. The labeled cells were lysed and immunoprecipitated either
under nondenaturing (lanes 1 and 2) or denaturing
conditions (lanes 3 and 4). as described under
``Experimental procedures'' and analyzed on SDS-PAGE gradient
gels followed by fluorography. Molecular mass markers mixed with human
LDL (as a source of apoB) were run in the end lane of the gel and
stained with Coomassie Blue. The fluorograph indicates that
nondenaturing immunoprecipitation with anti-Hsp70 resulted in
coprecipitation of Hsp70 and a protein of molecular mass similar to
that of apoB. B, sequential immunoprecipitation. After
immunoprecipitation with either preimmune mouse serum IgG as a control
or anti-Hsp70 antibody under nondenaturing conditions, PBS containing
2% SDS was added to the immunocomplexes, and the mixture was boiled for
5 min. The supernatants were diluted with 1% Triton X-100 to a final
concentration of 0.1% SDS and used for a second immunoprecipitation
with anti-apoB antibody. In these sequential immunoprecipitations,
anti-apoB antibody precipitated apoB from the complex that was first
precipitated with anti-Hsp70 antibody (lane 2) but not from
the complex that was initially precipitated with mouse IgG (lane
1).
To confirm the coimmunoprecipitation of Hsp70 and apoB, the immunocomplexes from the first nondenaturing precipitations with either anti-Hsp70 or preimmune mouse IgG (control) were subjected to a second (sequential) immunoprecipitation with anti-apoB antibody under denaturing conditions(20) . ApoB was isolated from the anti-Hsp70/anti-apoB sequential immunoprecipitate (Fig. 1B, lane 2) but not from the IgG/anti-apoB control precipitate (Fig. 1B, lane 1). If the labeled cells were chased for 3 h, we observed much less coimmunoprecipitation of newly synthesized, radiolabeled apoB with Hsp70 (data not shown). These results indicated that newly synthesized apoB is transiently associated with Hsp70 in HepG2 cells.
Additional confirmation of the findings in labeled HepG2 cells was obtained using immunoblotting techniques. After subjecting anti-Hsp70 immunoprecipitates obtained under nondenaturing conditions to immunoblot analysis using anti-apoB antibody as the probe, we could demonstrate the presence of apoB (Fig. 2A, lane 4). Similarly, if we subjected anti-apoB immunoprecipitates obtained under nondenaturing conditions to immunoblotting with anti-Hsp70 antibody as the probe, we could demonstrate a Hsp70 band (Fig. 2B, lane 3). In an attempt to determine the specificity of Hsp70 binding to apoB, we investigated the binding of Hsp70 to albumin, a typical secretory protein which is cotranslationally translocated across ER membranes and should not be exposed to the cytosol. Although we could demonstrate the presence of albumin when we immunoblotted the whole cell lysate with an anti-human albumin antibody (Fig. 2C, lane 4), no albumin could be detected in the anti-Hsp70 nondenaturing immunoprecipitate (Fig. 2C, lane 3). This result not only demonstrated that Hsp70 does not bind to typical secretory proteins such as albumin, but also suggested that Hsp70 does not bind nonspecifically to apoB after cell lysis occurs.
Figure 2:
Identification of Hsp70apoB
complexes by immunoblotting. A, nondenatured lysates from
nonradiolabeled HepG2 cell were immunoprecipated with anti-Hsp70
antibody (lane 4) or preimmune mouse IgG (lane 3).
The immunocomplexes were resolved with SDS-PAGE and then transferred to
a nitrocellulose membrane. After blocking, the membranes were probed
with anti-apoB antibody. Whole cell lysate (without
immunoprecipitation) (lane 5), BSA (lane 2), and LDL
plus molecular mass markers (lane 1) were loaded as positive
and negative controls. The results indicate that apoB can be identified
by immunoblotting in the complex immunoprecipitated by anti-Hsp70
antibody under nondenaturing conditions (lane 4). B,
when the same procedures were used, except that anti-apoB antibody was
used for lysate immunoprecipitation and anti-Hsp70 antibody was used
for immunoblotting, the results demonstrated that Hsp70 could be
identified in the complex immunoprecipitated by anti-apoB antibody
under nondenaturing conditions (lane 3). C, HepG2
cells were lysed and immunoprecipitated with either mouse IgG (lane
2) or anti-Hsp70 antibody (lane 3) under nondenaturing
conditions as above. The samples were immunoblotted with goat
anti-human albumin antibody. Although albumin was identified in the
whole cell lysate (lane 4), it was not present in either the
preimmune mouse IgG or the anti-Hsp70
immunocomplexes.
Figure 3:
ATP availability determines apoB binding
to Hsp70. A, radiolabeled HepG2 cells were pretreated with 50
units/ml apyrase (Apyrase), or 2.5 mM Mg-ATP
(+ATP), or 2.5 mM of a nonhydrolyzable ATP
analog (ATPS) in 0.1% Triton X-100-PBS, followed
by lysis, sequential immunoprecipitation, and analysis on SDS-PAGE (see
legend of Fig. 1B). Radioactivity (cpm) was also
measured in aliquots of the immunoprecipitates by liquid scintillation
counting. A background of 85 cpm was substracted from all values. The
results show that the association of Hsp70 with apoB is physiologically
regulated by the availability of ATP. B, HepG2 cells were
pretreated with BSA alone or with BSA plus ALLN (40 µg/ml) for 2 h
prior to labeling with [
H]leucine for 30 min
with/without ALLN. Cells were then placed on ice and treated for 15 min
with 0.1% Triton X-100-PBS with either no addition (No
Treatment), with apyrase (100 units/ml) (Apyrase), or
with ATP (2.5 mM) followed by treatment with apyrase for an
additional 15 min (ATP/Apyrase). All of the cells were then
lysed, and sequential immunoprecipitation was carried out as mentioned
above. The results are presented as counts/min from aliquots of
immunoprecipitates. The inset presents the fluorograph of
separate aliquots of immunoprecipitates from the ALLN-treated cells.
The results indicate that minimal binding of Hsp70 to apoB occurred
under conditions (ATP/apyrase) where maximal artifactual binding was
favored.
In all of the previous experiments,
use of detergents raised the possibility that the association between
apoB and Hsp70 could have occurred during lysis, e.g. the
association could have been artifactual. Therefore, we carried out
additional experiments in which cells incubated with either BSA or BSA
plus ALLN (40 µg/ml) were treated with PBS containing 0.1% Triton
X-100 and 5 mM MgCl for 15 min on ice with either (a) no addition, (b) apyrase alone (100 units/ml), or (c) Mg-ATP (2.5 mM) followed by treatment with
apyrase for an additional 15 min on ice. All cells were then lysed, and
sequential immunoprecipitation was carried out. The cells treated with
Mg-ATP should have had maximal dissociation of Hsp70 and bound
proteins; treatment of these cells with apyrase, which would hydrolyze
all ATP, should have allowed for maximal reassociation during
subsequent procedures (lysis, immunoprecipitation). As shown in Fig. 3B, in cells exposed to only 0.1% Triton X-100
with no addition, about 1000 cpm of apoB were precipitated by
anti-Hsp70 antibody; ALLN treatment increased this by about 50%. These
results are consistent with protection of apoB by ALLN (see below). In
cells exposed to 0.1% Triton X-100 and apyrase, both the control and
ALLN-treated cells showed greater immunoprecipitation of apoB by
anti-Hsp70 antibody; these results are consistent with the in vivo effects of apyrase on Hsp70 binding to cytosolic proteins (see Fig. 3A). Finally, in the cells exposed to 0.1% Triton
X-100 and Mg-ATP, followed by exposure to apyrase, there was very
litter Hsp70-bound apoB, particularly in ALLN-treated cells which had
much more apoB present. These results are incompatible with significant
artifactual association of apoB with Hsp70 during incubation in 0.1%
Triton X-100-PBS, during cell lysis, or during immunoprecipitation. The
results are, however, compatible with a physiologically regulated
interaction of apoB and Hsp70 in vivo. The inset in Fig. 3B depicts the results of sequential
immunoprecipitation with anti-Hsp70 followed by anti-apoB antibodies
under each of the conditions in the ALLN-treated cells.
Figure 4:
ApoB binding to Hsp70 is related to the
translocation status of apoB in ER membranes, and not to the total
cellular apoB content. For oleate treatment (OA), HepG cells were preincubated with serum-free MEM for 60 min. The cells
were labeled for 30 min with [
H]leucine (200
µCi/ml) in leucine-free and serum-free medium and then chased for
either 25 min (for cell apoB and Hsp70-bound apoB), or 60 min (for
medium apoB). Preincubation, labeling, and chase medium each contained
1.5% BSA alone (control, white bars) or BSA plus 0.4 mM oleate (oleate treatment, diagonal-striped bars). The
cells were lysed and immunoprecipitated with either anti-apoB antibody
under denaturing conditions (Cell ApoB100), or anti-Hsp70
antibody under nondenaturing condition (Hsp70-ApoB100) as
described in Fig. 1. Medium-apoB100 was immunoprecipitated with
anti-apoB antibody under denaturing conditions (Medium ApoB).
For ALLN treatment (ALLN), the preincubation and labeling were
identical to that described above except that the medium contained
either BSA alone (control, white bars) or BSA plus 40
µg/ml ALLN (black bars), and the labeling was for 90 min
with no chase prior to cell lysis. Immunoprecipitation was identical to
those carried out in oleate-treated cells. The immunocomplexes were
separated with 3-10% gradient SDS-PAGE. After fluorography, the
apoB100 bands were scanned with a Molecular Dynamic Densinometer. Data
were presented as the mean of duplicate dishes, except for studies of
Hsp70-bound apoB in oleate-treated cells, which we present as the mean
of three independent experiments.
Figure 5:
apoB50, but not apoB16, is bound to Hsp70.
CHO cells were stably transfected with expression plasmids containing
either 16% (A) or 50% (B) of the N-terminal portion
of apoB cDNA, and these cells were grown up to 90% confluence in MEM
containing 5% fetal bovine serum and 300 µg/ml G418. The medium was
changed to serum-free MEM containing ALLN (60 µg/ml) and incubated
for 2 h at 37 °C. Cells were then labeled with
[H]leucine (200 µCi/ml) for 2 h and lysed
under nondenaturing conditions as described in Fig. 1. Aliquots
of cells lysates were immunoprecipitated either with anti-apoB (lanes A1 and B1), or anti-Hsp70 antibody (lanes
A2 and B2). A, apoB16 (molecular mass, 84 kDa),
which is rapidly and efficiently translocated was not
coimmunoprecipitated with Hsp70. B, in contrast, apoB50
(molecular mass, 250 kDa), which is ineffectively translocated across
the ER membrane, was found to coimmunoprecipitate with Hsp70. Each gel (A and B) represents a separate experiment. The lanes (1 and 2) on each gel were originally run adjacently;
we sliced the gel vertically and separated the lanes for purposes of
autoradiography. Because of different labeling efficiency in the two
experiments, the film in A was exposed for 5 days while the
film in B was exposed for 2 days.
We believe that this is the first demonstration of an association between Hsp70 and a secretory protein in a mammalian cell. Since Hsp70 is present in the cell cytoplasm under normal conditions and is not found within the lumen of the ER(25) , our results indicate that domains other than the N-terminal 16% of newly synthesized apoB must be exposed, at least transiently, to the cytosol(2, 3, 4, 5, 6, 24) . The exposure of hydrophobic domains in apoB to the aqueous cytosol would trigger transient associations with Hsp70. Through such interactions, those hydrophobic domains would be shielded from the polar environment of cytosol, and the nascent apoB would be maintained in a translocation-competent conformation and protected from aggregation or misfolding until its translocation was completed.
The fact that apoB has a signal peptide and has no typical membrane spanning domains (26) suggests that it should be cotranslationally translocated by the signal recognition particle pathway(16, 17) . Chuck and Lingappa (27, 28) have, however, suggested that apoB has sequences that cause it to pause during translocation. Several groups, including ours, have presented evidence, based on protease sensitivity or immunologic methods(2, 3, 4, 5, 6, 24) , that apoB domains are exposed to the cytosolic surface of isolated microsomes or ER. There are other investigators(29, 30) , however, who have reported contrasting findings using similar methods. We believe that our new data confirm and extend the previous studies that indicated that apoB is not fully translocated cotranslationally. Our finding of an association of Hsp70 with apoB is compatible with a pathway in which this very large, extremely hydrophobic secretory protein is maintained in a transmembrane position until adequate core lipid is available in the ER lumen to allow assembly of a nascent lipoprotein(1, 31) . It is in this transmembrane position that apoB appears to be very sensitive to proteolysis; the association with Hsp70 may protect cytosolic domains of apoB from degradation. Since the association of Hsp70 with proteins appears to be an ``on and off'' phenomenon, dependent on its ATP/ADP state, the likelihood that apoB will be degraded could depend on the length of time that it remains exposed in a transmembrane position. Of note in this regard is the recent demonstration that the human disorder abetalipoproteinemia, in which apoB is synthesized normally but appears to be degraded rather than secreted, is associated with mutations in an ER protein, microsomal triglyceride transfer protein (MTP)(32, 33) . It is likely that MTP mediates the transfer of newly synthesized triglyceride from the ER membrane to a luminal domain of nascent apoB, a step that is necessary before translocation of apoB can be completed (34, 35) . In the absence of normal MTP activity, apoB would remain in a transmembrane position and be degraded. It will be interesting to determine if Hsp70 also plays an active role in apoB translocation and intracellular degradation, and if apoB secretion from HepG2 cells can be affected by manipulation of Hsp70 levels.