From the Molecular Medicine Unit, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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Several secretory proteins, including apolipoprotein B, have been shown to undergo degradation by proteasomes. We found that the rapid degradation of nascent apolipoprotein B in HepG2 cells was diminished but not abolished by the addition of any of three different inhibitors of proteasomes. Ubiquitin is conjugated to apolipoprotein B that is not assembled with sufficient lipids either during or soon after synthesis. In addition, we found that apolipoprotein B that has entered the endoplasmic reticulum sufficiently to become glycosylated can be degraded by proteasomes. Furthermore, we detected ubiquitin-apolipoprotein B that is associated with the Sec61 complex, the major constituent of the translocational channel. Treatment of cells with monomethylethanolamine or dithiothreitol decreased the translocation of apolipoprotein B and increased the proportion of ubiquitin-conjugated molecules associated with Sec61. Conversely, treatment of cells with oleic acid, which increased the proportion of translocated apolipoprotein B, decreased the amount of ubiquitin-apolipoprotein B in the Sec61 complex. Finally, we found that inhibition of the interaction between calnexin and apolipoprotein B decreases the translocation of apolipoprotein B, increases the ubiquitin-apolipoprotein B in the Sec61 complex, and increases the proteasomal degradation of glycosylated apolipoprotein B. Thus, ubiquitin can be attached to unassembled apolipoprotein B in the Sec61 complex, and this process is affected by factors including calnexin that alter the translocation of apolipoprotein B.
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INTRODUCTION |
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Apolipoprotein B (apoB)1 is the large protein (>500 kDa) assembled with cholesterol and triglycerides into lipoprotein particles in hepatic and intestinal cells (1-4). Full-length apoB100 is secreted on very low density lipoproteins from hepatic cells, and apoB serves as a ligand on low density lipoproteins for the low density lipoprotein receptor. Increased levels of apoB and low density lipoprotein-associated cholesterol in human plasma correlate with increased risks of coronary artery disease.
In HepG2 cells, a large amount of newly synthesized apoB is degraded
(5-8). Intracellular disposal appears to be the principal means of
regulating the secretion of apoB. Since an inhibitor of calpains and
cysteine proteases, N-acetyl-leucyl-leucyl-norleucinal (ALLN), can protect apoB from degradation, an uncharacterized cysteine
protease was proposed to be responsible for the apoB degradation (6,
7). ALLN also acts on proteasomes (9), and other chemicals that more
specifically inhibit proteasomes also decrease the degradation of apoB
(10-12). In addition, ubiquitin has been found on apoB (10, 11).
Several proteins that enter the endoplasmic reticulum (ER) are degraded
by cytosolic proteasomes (13-19). Recently, it has been shown that
most if not all of these proteins undergo retrograde transport from the
lumen of the ER back into the cytosol through a protein-conducting
channel containing the Sec61 complex (20-22). The Sec61 complex, which
consists of ,
, and
subunits, is the major constituent of the
channel used for translocation into the ER. However, apoB has not been shown to be transported retrograde prior to degradation.
Several experimental interventions have been shown to affect translocation, secretion, and degradation. Stimulating lipid synthesis by the addition of oleic acid (OA) increases the proportion of apoB that is fully translocated and secreted and decreases the amount that is degraded (7, 23). In contrast, treatment of hepatocytes with monomethylethanolamine (MME) (24), dithiothreitol (DTT) (25, 26), or inhibitors of microsomal triglyceride-transfer protein (MTP) (27) decreases translocation and secretion of apoB. The effect of calnexin, another chaperone in the ER, on the translocation of apoB has not been examined previously. Molecules of apoB that are not completely translocated into the ER can be degraded. Thrift et al. (6) first showed in a non-hepatic cell line that molecules of apoB that are not fully translocated into the ER are degraded by an ALLN-sensitive pathway. More recent data suggest that the ubiquitin-proteasome pathway could be responsible for degradation of incompletely translocated apoB (11, 12).
Unlike typical secretory proteins, not all of the apoB molecules are completely translocated into the ER. Several investigators have found newly synthesized apoB with large domains exposed to the cytosol. These domains are accessible to proteolytic cleavage (26, 28, 29) and binding by antibodies (30) or cytosolic proteins such as Hsp70 (31). The cytosolic domains of such molecules of apoB would be accessible to ubiquitin-conjugating enzymes and proteasomes. The location of this incompletely translocated apoB that could be bound by ubiquitin is not known. Such molecules could reside entirely on the cytosolic side of the ER membrane, whether due to aborted forward translocation or retrograde transfer. Alternatively, these ubiquitin-conjugated apoB proteins (ub-apoB) might be spanning the ER membrane either in the lipid bilayer itself or within the translocational apparatus.
In this study, we examine the kinetics and action of ubiquitin and proteasomes in the degradation of different subsets of apoB. We also demonstrate a role for calnexin in the proteasomal degradation of glycosylated apoB. Furthermore, we investigate the intertwined roles of translocation and proteasomal degradation in regulating the secretion of apoB.
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EXPERIMENTAL PROCEDURES |
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Reagents--
modification of Eagle's medium (
MEM) and
Dulbecco's modified Eagle's medium (DMEM) were purchased from
Mediatech. Minimum essential medium (MEM) without methionine/cysteine,
water-soluble oleic acid, 2-(methylamino)ethanol
(monomethylethanolamine, MME), ALLN, and brefeldin A (BFA) were
obtained from Sigma. Castanospermine (CST) was purchased from
Calbiochem. DTT was obtained from Fisher. Protein A- and protein
G-agarose beads were purchased from Life Technologies, Inc. Trans-label
[35S]methionine/ cysteine and
[3H]D-mannose were obtained from ICN
Biochemicals. Carboxylbenzyl-leucyl-leucyl-leucinal (ZL3H,
which is the same compound as MG135) was the generous gift of Hidde
Ploegh (15). Lactacystin was obtained from E. J. Corey (32). The
antibody that was used for immunoprecipitation of ubiquitin was the
kind gift of Arthur L. Haas (33). Antibodies against Sec61
and
Sec61
, and the peptide that was used to raise the antibody against
Sec61
, were generous gifts from Walther Mothes and Tom Rapoport (34,
35). Antibody against ubiquitin that was used for Western blotting was
purchased from Boehringer Mannheim, antisera against human apoB were
purchased from Calbiochem and Boehringer Mannheim, and antiserum
against calnexin was obtained from StressGen.
Cell Culture and Pulse-Chase Labeling--
HepG2 cells (obtained
from American Type Culture Collection) were maintained at 37 °C with
5% CO2 in MEM with 10% fetal bovine serum and 2 mM glutamine. Cells were used at about 90% confluence. In
some experiments, proteasomal inhibitors, BFA, MME, OA, CST, or DTT
were added to the preincubation, labeling, and chase media as indicated
in the figure legends. HepG2 cells were preincubated in cysteine-,
methionine-, and serum-free MEM for 1 h and then pulse-labeled
with 50-200 µCi/ml trans-label
[35S]methionine/cysteine for 10 min or 1 h. After
washing with phosphate-buffered saline (PBS) once, labeled cells were
chased for various times in serum-free
MEM. For labeling with
[3H]mannose, glucose- and serum-free DMEM were used for
the preincubation and pulse labeling. After 1 h preincubation,
HepG2 cells were pulse-labeled with 50 µCi/ml
[3H]mannose for up to 1 h. The cells were washed
with PBS once and then chased for different times in medium containing
a 1,000-fold excess of unlabeled mannose.
Immunoprecipitation--
After various times of chase,
pulse-labeled cells were lysed with TXSWB (1% Triton X-100, 100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride
(PMSF)) (36) for 30 min at 4 °C. For immunoprecipitation of
calnexin, cells were lysed in cholate buffer (2% sodium cholate, 200 mM NaCl, 50 mM Hepes, pH 7.4, 1 mM
EDTA, 20 mM N-ethylmaleimide, 1 mM
PMSF). For immunoprecipitation of Sec61, cells were lysed in
digitonin buffer (1% digitonin, 100 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1 mM PMSF) containing 100 mM NaCl for 1 h at room temperature.
Immunoprecipitation of Sec61
required lysis of cells in TX2 buffer
(2% Triton X-100, 500 mM potassium acetate, 50 mM Tris-HCl, pH 8.0, 1 mM PMSF) for 30 min at
4 °C. The cellular lysates were spun for 10 min in a microcentrifuge. No apoB was detected in the unsolubilized cellular debris. The supernatants were incubated at 4 °C with an excess amount of the appropriate antibodies for at least 1 h. Protein A-
or protein G-agarose beads were added, and the samples were rotated
overnight at 4 °C. The beads were washed three times with lysis
buffer and then twice with wash buffer (500 mM potassium acetate, 50 mM Tris-Cl, pH 8.0, for Sec 61
immunoprecipitates; 200 mM NaCl, 50 mM Hepes,
pH 7.4, for calnexin immunoprecipitates; 100 mM Tris-Cl, pH
8.0, 100 mM NaCl for all other immunoprecipitates).
Electrophoresis and Immunoblotting--
The immunoprecipitates
were solubilized in sample buffer (4% SDS, 125 mM
Tris-HCl, pH 6.8, 20% glycerol, 500 mM DTT) at 37 °C, boiled for 10 min, and separated by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). For labeling studies, the gel was treated
with ENHANCETM (DuPont), dried, and exposed to film. For Western
blotting, proteins were transferred overnight onto nitrocellulose
membranes, probed with antibody as indicated in the figure legends, and
revealed by peroxidase conjugated to anti-rabbit or anti-goat IgG
antibodies using the LumiGLO substrate kit (Kirkegaard & Perry
Laboratories) according to the manufacturer's instructions. Some blots
were stripped in buffer (2% SDS, 62.5 mM Tris-HCl, pH 6.8, 100 mM -mercaptoethanol) at 70 °C for 30 min prior to
a second immunodetection.
Subcellular Fractionation and Flotation-- HepG2 cells were suspended in homogenization buffer (10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 1 mM PMSF, 50 µM ALLN). Cell homogenization was carried out using a motorized Dounce homogenizer (30 strokes). The homogenate was centrifuged at 10,000 × g for 10 min to pellet cellular debris and nuclei. Microsomes were isolated from the supernatant by centrifugation at 170,000 × g for 2 h at 4 °C. The microsomes were extracted with sodium carbonate, pH 11.5, for 1 h at 4 °C (37), and the supernatant was subjected to sucrose gradient ultracentrifugation according to Boren et al. (38). (No apoB was detected in the pelleted membranes.) The gradient was unloaded into 12 fractions; the densities of each fraction were very similar to those previously reported with this method (38). ApoB was recovered from each fraction by immunoprecipitation and visualized by SDS-PAGE and Western blotting.
Sec61 Peptide Competition--
HepG2 cells were preincubated
for 2 h in cysteine-, methionine-, and serum-free MEM and then
pulse-labeled with 200 µCi/ml trans-label
[35S]methionine/cysteine for 10 min. ALLN was included in
the preincubation and labeling media. After washing with PBS, the
labeled cells were lysed in digitonin buffer containing 150 mM NaCl. The peptide used to raise antibody against
Sec61
was added to 1 aliquot of the cell lysate at a concentration
of 50 µg/ml. Immunoprecipitation with Sec61
antibody was carried
out as described above.
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RESULTS |
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ApoB Is Degraded Rapidly by Proteasomes-- To define the kinetics and extent to which proteasomes degrade apoB, we performed pulse-chase analyses using HepG2 cells treated with ALLN, ZL3H, or lactacystin. HepG2 cells were pulse-labeled with [35S]methionine/cysteine for 10 min in the presence or absence of different proteasome inhibitors and followed by various times of chase. ApoB was immunoprecipitated and analyzed by SDS-PAGE.
The three inhibitors had similar protective effects on apoB. Nascent apoB in untreated cells was dramatically degraded starting at a very early chase time; 40% of the apoB was degraded after 10 min of chase (Fig. 1). Since it has been estimated that 14-20 min are necessary to synthesize a full-length apoB molecule (12, 39), degradation must begin during or within a very short time after synthesis. Treatment of cells with lactacystin, ALLN, or ZL3H resulted in increased levels of apoB. After 30 min of chase, more than 75% of apoB molecules in the untreated cells were degraded, whereas about 70% of apoB molecules still remained intact in inhibitor-treated cells (Fig. 1). Analysis of the kinetics of degradation demonstrated a significant difference (p < 0.01) in rates of removal (slopes in Fig. 1B) in the presence or absence of an inhibitor. These data indicate that proteasomes degrade apoB molecules rapidly within a short time after synthesis.
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Ubiquitin Binds to ApoB within a Short Time after Its Synthesis-- Protein ubiquitination is a critical step in the degradation of most proteins by the proteasome pathway (40). Ubiquitin has been detected on apoB (10, 11), although it is unclear how soon after synthesis this conjugation occurs. To determine when ubiquitination of apoB takes place, HepG2 cells were pulse-labeled with [35S]methionine/cysteine and then chased for 0, 10, 30, and 60 min. Sequential immunoprecipitation was performed using antiserum against apoB followed by antiserum against ubiquitin. Ub-apoB appears at a very early time even in the sample without chase which suggests that ubiquitin is conjugated to apoB during or immediately after synthesis (Fig. 2).
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Only Non-floating ApoB Is Associated with Ubiquitin-- Association with lipids is a prerequisite for apoB secretion, and some early steps in particle formation can occur cotranslationally (38). We wondered whether apoB that is assembled with lipids into nascent particles would escape conjugation with ubiquitin. To investigate the correlation between ubiquitin binding and the amount of lipid bound to apoB, microsomes were prepared from HepG2 cells, extracted with sodium carbonate, and the proteins that were released were fractionated by sucrose gradient ultracentrifugation according to Boren et al. (38). ApoB molecules were recovered from each fraction of the gradient by immunoprecipitation and analyzed by Western blotting using antibodies either against apoB or ubiquitin.
As described previously (38), some of the apoB was found to float in the gradient with a density of high density lipoprotein (HDL)-like particles (Fig. 3A, fractions 3-9). When the blot was reprobed with anti-ubiquitin antiserum, ubiquitin was found only on the lipid-poor apoB recovered from the bottom of the gradient (Fig. 3B, fractions 1 and 2). The anti-ubiquitin immunoreactive smear with apparent higher molecular weight reflects the covalent attachment of multiple ubiquitin chains to apoB molecules. These results indicate that only apoB that is not assembled with sufficient lipids can be marked with ubiquitin.
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Glycosylated ApoB Is Degraded by Proteasomes-- It has been shown that incompletely translocated apoB53 molecules are degraded by an ALLN-sensitive pathway in non-hepatic cells (6). However, it is not known to what extent full-length apoB100 enters the ER of hepatic cells prior to its degradation by proteasomes. To determine whether apoB with at least some domains translocated is degraded by proteasomes, we investigated the degradation of glycosylated apoB. Furthermore, we tested whether N-linked high-mannose oligosaccharides protect apoB or become attached to those molecules of apoB that have escaped proteasomal degradation. We pulse-labeled HepG2 cells with [3H]mannose for 10 min, washed the cells with PBS, and then incubated the cells in medium with a 1,000-fold excess of unlabeled mannose for various times. Glycosylated apoB was degraded in a manner similar to 35S-labeled apoB (Fig. 4). When HepG2 cells were treated with 10 µM lactacystin, about three times as much glycosylated apoB remained after 240 min indicating that glycosylated apoB can be degraded by proteasomes.
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Calnexin Protects ApoB from Degradation-- Calnexin is a molecular chaperone that is localized in the ER (41). By associating transiently with nascent influenza hemagglutinin, calnexin can regulate protein folding, oligomerization, and degradation (42). Calnexin can bind to a number of glycoproteins including apoB, although the role of calnexin in apoB biogenesis is not defined (41, 43). When cells are preincubated with castanospermine (CST), the trimming of glucose residues is prevented and the binding of glycoproteins by calnexin is inhibited (42). Since glycosylated apoB is degraded by proteasomes, we investigated the role of calnexin in the degradation of apoB.
First, we confirmed the effect of CST on the binding of calnexin to apoB. HepG2 cells were labeled for 1 h with [35S]methionine/cysteine in the presence or absence of 1 mM CST, and the association of calnexin with apoB was measured by co-immunoprecipitation. As shown in Fig. 5A, treatment with CST decreased the co-immunoprecipitation of apoB by about 45%. However, CST had no effect on the immunoprecipitation of calnexin.
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Ub-ApoB Is Associated with the Sec61 Complex-- Not all molecules of apoB are fully translocated into the ER upon completion of translation; many have large domains exposed to the cytosol (26, 28-31) where they could become bound by ubiquitin. We wondered if ubiquitin binds to this partially translocated apoB while it is still in the Sec61-containing translocation channel.
First, we established the specificity of co-immunoprecipitating apoB with antibody against Sec61
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Lipids That Alter the Translocation of ApoB Affect the Amount of
Ub-ApoB Found with the Sec61 Complex--
Two lipids have been found
to affect the proportion of newly synthesized apoB that is translocated
fully into the ER: MME inhibits whereas OA increases the translocation
and secretion of apoB (7, 23, 24). We examined the effect of OA and MME on ub-apoB associated with Sec61. HepG2 cells were incubated with OA
(0.8 mM, 2 h) or MME (0.4 mM, 16 h)
in the presence of ALLN (50 µg/ml, 2 h). Western blotting using
antibodies against ubiquitin or apoB were performed after sequentially
immunoprecipitating Sec61
and apoB (Fig.
7). More apoB is found with Sec61
in
cells treated with MME (Fig. 7A, compare lanes 1 and 2). Treatment with MME also increased the amount of
ub-apoB associated with Sec61 (Fig. 7B, compare lanes
1 and 2). In contrast, treatment with OA increased apoB
translocation so that fewer molecules of apoB were associated with
Sec61 (Fig. 7A, compare lanes 1 and
3). This effect on translocation corresponded with a
decrease in ub-apoB in the Sec61 complex (Fig. 7B, compare
lanes 1 and 3). The effects of these two lipids
suggest that alterations of apoB translocation affect the amount of
ub-apoB associated with Sec61.
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Inhibition of Calnexin Binding Increases the Amount of ApoB and
Ub-ApoB That Co-precipitates with the Sec61 Complex--
We next
investigated the effect of CST on apoB translocation and ub-apoB
associated with the Sec61 complex. The Sec61-apoB immunoprecipitates
from CST- and ALLN-treated HepG2 cells were compared with those from
HepG2 cells treated with ALLN only. CST increased the amount of apoB in
the Sec61 complex (Fig. 8A).
This effect correlated with an increase in ub-apoB associated with Sec61 (Fig. 8B). Thus, inhibition of the interaction between
calnexin and apoB increased the ub-apoB in the Sec61 complex.
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DTT, Which Results in Misfolded ApoB, Increases Ub-ApoB Associated
with the Sec61 Complex--
Degradation of apoB is greatly stimulated
if the proper formation of disulfide bonds is disrupted by DTT.
Treatment of HepG2 cells with DTT also has been correlated with a
decrease in the translocation of apoB into the ER (25, 26). We examined
the effect of DTT on the amount of apoB and ub-apoB associated with Sec61. ALLN-treated HepG2 cells were incubated in 2 mM
DTT for 0, 2.5, or 5 min. Increasing the length of incubation in DTT
caused a reduction in the total amount of intracellular apoB (Fig.
9A). In addition, increasing
exposure to DTT was associated with little change in the quantity of
apoB associated with the Sec61 complex (Fig. 9B). In view of
the overall decrease in intracellular apoB, however, the proportion of
intracellular apoB associated with Sec61 increased with increasing
exposure to DTT. Even more notable was the dramatic increase in ub-apoB
associated with Sec61
seen with longer exposure to DTT (Fig.
9C). Hence, the disruption of proper disulfide bond
formation and folding caused by DTT increases the ub-apoB associated
with the Sec61 complex.
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DISCUSSION |
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Yeung et al. (10) first demonstrated that apoB is conjugated to ubiquitin and degraded by proteasomes in an ATP-dependent manner. Two other groups have provided additional evidence for the role of the ubiquitin-proteasome pathway in the degradation of apoB (11, 12). Likewise, we have found that proteasomes rapidly degrade a significant proportion of newly synthesized apoB. The kinetics of this proteasomal degradation is rapid, proteolysis begins during or within a very short time after synthesis and continues for over 1 h. This early degradation is consistent with recent evidence that proteolysis of incompletely elongated apoB can occur (12). We found that apoB is bound by ubiquitin either during or very soon after translation and disappears rapidly during the chase period. Thus, degradation by the proteasome, rather than ubiquitin conjugation, is the rate-limiting step.
In this study, the rapid intracellular degradation of apoB is
incompletely inhibited by any of three inhibitors of proteasomes. The
protection of apoB by ALLN in this study is similar to previous studies
in intact HepG2 cells (44, 45). Possibly these inhibitors cannot
sufficiently block the activity of proteasomes. However, no greater
protection was detected even when 10 times the concentration of
lactacystin was used (data not shown). In contrast, the degradation of
T-cell receptor chains by proteasomes is nearly completely inhibited by concentrations of ALLN or lactacystin similar to those
used in this study (19). An alternative explanation is that a
non-proteasomal pathway might proteolyze apoB in the presence of these
inhibitors. In particular, a pathway that is insensitive to ALLN has
been shown to degrade apoB (45, 46).
In HepG2 cells, assembly of lipoprotein particles of density similar to HDL appears to begin cotranslationally (38). Therefore, those molecules of apoB that are not assembled with sufficient lipids early in their biogenesis could be targeted for degradation. Indeed, we did not find ubiquitin associated with apoB assembled into HDL-like particles; instead, ubiquitin was found only on lipid-poor apoB. It is unknown what proportion of the lipid-deficient apoB is conjugated to ubiquitin since the Western blots cannot be compared quantitatively.
Glycosylated apoB proteins (that is molecules of apoB with some or all of their domains translocated into the lumen of the ER) also are degraded by proteasomes. Glycosylation of apoB in the ER does not correlate with assembly with sufficient lipid for secretion since glycosylated but not floating apoB can be degraded by the ubiquitin-proteasome pathway. Furthermore, our data demonstrate that apoB that has entered the ER far enough to be recognized by the glycosylation machinery still can undergo proteasomal degradation. This finding raises new questions such as whether the glycosylated region of apoB is degraded in the ER lumen or transferred into the cytosol and where the sugar residues are removed. Glycosylated regions of apoB might be deglycosylated prior to retrograde transfer just as major histocompatibility complex class I heavy chain molecules (20).
What is the topology of the ubiquitin-conjugated apoB in the bottom
fraction of the sucrose gradient? Since these proteins were isolated by
carbonate extraction of microsomes, the apoB could reside in the
following: 1) the lumen, 2) the cytoplasmic side of the ER membrane, or
3) spanning the membrane in a proteinaceous channel. In this study, we
demonstrate that ubiquitin can be conjugated to apoB while it still is
associated with the Sec61 complex. It is not known whether the ub-apoB
that co-immunoprecipitates with the Sec61 complex resides within a
translocation-competent channel. Although the ub-apoB is physically in
contact with Sec61 and Sec61
, other channel proteins that might
be necessary for anterograde or retrograde transport of apoB could be
missing. In addition, our data do not indicate directly whether ub-apoB
associated with Sec61 is on its way into or out of the ER. Proteins
such as major histocompatibility complex class I heavy chains (15, 20)
and carboxypeptidase Y (16, 22) that completely enter the ER also are
degraded by proteasomes. These proteins undergo retrograde transport
back into the cytosol via a channel of which the Sec61 complex is a
constituent. However, apoB is an atypical secretory protein since not
all newly synthesized molecules are fully translocated into the ER (26,
28-31). Those chains of apoB that are not translocated completely into
the ER have cytosolic domains that can be bound by ubiquitin. In the
presence of an inhibitor of MTP, apoB can be degraded before it is
completely synthesized (12). Hence, ub-apoB probably forms while apoB
is being translocated into the ER. This mechanism of degradation would
circumvent the need to unfold full-length apoB in the ER for retrograde
transport and prevent the accumulation of poorly lipidated apoB that
might be insoluble in the aqueous environment of the ER. Thus, apoB
might act more like a membrane-spanning protein such as the cystic
fibrosis transmembrane conductance regulator (13, 14) rather than a secretory protein en route to degradation by the ubiquitin-proteasome pathway.
In this study, factors that altered the translocation of apoB into the
ER had inverse effects on ub-apoB associated with Sec61 that
parallel their effects on degradation. Treatment of HepG2 cells with
MME decreased the translocation of apoB and increased the ub-apoB
associated with Sec61. In contrast, OA increased the translocation of
apoB and decreased the ub-apoB that co-precipitates with the Sec61
complex. Finally, DTT caused an increase in apoB with the Sec61 complex
that was especially significant in view of the concomitant decrease in
total apoB. This effect of DTT on the translocation of apoB was
accompanied by a marked increase in ub-apoB associated with Sec61.
Thus, our data indicate that alterations in the translocation of apoB
lead to inverse changes in ub-apoB in the Sec61 complex. Other studies
can be viewed as supporting this relationship between translocation and
degradation. MTP is necessary for the translocation and secretion of
apoB, and chemical inhibitors of MTP induce cotranslational degradation of apoB (12). Similarly, Hsp70 has been shown to increase the proteasomal degradation of apoB (11) perhaps by retarding the translocation of cytosolic domains of apoB.
Calnexin interacts with newly synthesized glycoproteins including apoB to facilitate proper trimming of glucose residues, folding, and assembly (41). By using castanospermine, we found that interfering with trimming of glucose residues, and thereby inhibiting binding to calnexin, was associated with over twice as much degradation of glycosylated apoB by proteasomes. Inhibition of the interaction between calnexin and apoB also increased the apoB and ub-apoB associated with Sec61. Since calnexin only binds glycosylated apoB, the effect of CST on total apoB is less dramatic than the effects of OA, MME, and DTT. Thus, although core glycosylation by itself does not protect apoB from degradation, interacting with calnexin to ensure proper trimming of glucose residues and, presumably, to promote correct folding and complete translocation, does safeguard glycosylated apoB from degradation. A similar role of calnexin in maturation and protection from degradation has been reported for other nascent proteins including subunits of T-cell receptors (47), major histocompatibility complex class I and class II complexes (48, 49), influenza hemagglutinin (42), and nicotinic acetylcholine receptors (50).
Calnexin probably acts in concert with other molecular chaperones to facilitate the translocation, folding, and assembly of nascent apoB. Since apoB appears to be cotranslationally assembled into lipoprotein particles and the apoB that is unassembled with significant lipids can be bound by ubiquitin, the glycosylated chains of apoB that interact with calnexin might achieve a conformation that promotes their assembly with lipids catalyzed by microsomal triglyceride transfer protein (MTP) (27). Furthermore, calnexin may directly facilitate the forward translocation of apoB or prevent its retrograde transport into the cytosol. Calnexin could act cooperatively or successively with MTP, protein disulfide isomerase, BiP, and other molecular chaperones to promote forward translocation and assembly of secretion-competent apoB.
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ACKNOWLEDGEMENTS |
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We thank Nadine Earley McCall and Sarah Hevi
for excellent technical assistance. In addition, we thank Alan
Stuart-Tilley for assistance with mathematical analyses. We also thank
Arthur L. Haas for the gift of antibodies against ubiquitin; Hidde
Ploegh for the gift of ZL3H; and Walther Mothes and Tom
Rapoport for their gifts of antibodies against Sec61 and Sec61
and peptide used to raise the Sec61
antisera.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These two authors contributed equally to this work.
§ Supported by the Massachusetts Affiliate of the American Heart Association and the Harcourt General Charitable Foundation. To whom correspondence should be addressed: Molecular Medicine Unit, RW 663, Beth Israel Deaconess Medical Center-East, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-1625; Fax: 617-667-2913.
1
The abbreviations used are: apoB, apolipoprotein
B; ALLN, N-acetyl-leucyl-leucyl-norleucinal; ER, endoplasmic
reticulum; OA, oleic acid; MME, 2-(methylamino)ethanol; BFA, brefeldin
A; DTT, dithiothreitol; MTP, microsomal triglyceride-transfer protein; ub-apoB, ubiquitin-conjugated apolipoprotein B; MEM,
modification of Eagle's medium; DMEM, Dulbecco's modified Eagle's
medium; MEM, minimum essential medium; CST, castanospermine;
ZL3H, carboxylbenzyl-leucyl-leucyl-leucinal; PMSF,
phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; PAGE,
polyacrylamide gel electrophoresis; WB, Western blot; HDL, high density
lipoprotein.
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REFERENCES |
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