Overexpression of the Tumor Autocrine Motility Factor Receptor Gp78, a Ubiquitin Protein Ligase, Results in Increased Ubiquitinylation and Decreased Secretion of Apolipoprotein B100 in HepG2 Cells*

Apolipoprotein B100 (apoB) is a large (520-kDa) complex secretory protein; its secretion is regulated posttranscriptionally by several degradation pathways. The best described of these degradative processes is co-translational ubiquitinylation and proteasomal degradation of nascent apoB, involving the 70- and 90-kDa heat shock proteins and the multiple components of the proteasomal pathway. Ubiquitinylation involves several proteins, including ligases called E3s, that coordinate the covalent binding of ubiquitin to target proteins. The recent discovery that tumor autocrine motility factor receptor, also known as gp78, is an endoplasmic reticulum (ER)-associated E3, raised the possibility that this E3 might be involved in the ER-associated degradation of nascent apoB. In a series of experiments in HepG2 cells, we demonstrated that overexpression of gp78 was sufficient for increased ubiquitinylation and proteasomal degradation of apoB, with reduced secretion of apoB-lipoproteins. This action of gp78 was specific: overexpression of the protein did not affect secretion of either albumin or apolipoprotein AI. Furthermore, overexpression of a cytosolic E3, Itch, had no effect on apoB secretion. Finally, using an in vitro translation system, we demonstrated that gp78 led to increased ubiquitinylation and proteasomal degradation of apoB48. Together, these results indicate that an ER-associated protein, gp78, is a bona fide E3 ligase in the apoB ER-associated degradation pathway.

Apolipoprotein B100 (apoB) 1 is the essential protein component of atherogenic very low density and low density lipoproteins (1), and overproduction of these lipoproteins is a common feature of human dyslipidemia (2). Extensive studies of cul-tured primary hepatocytes and hepatoma cells have established that significant control over apoB secretion can be achieved at the post-transcriptional level by degradation. One form of presecretory degradation, namely the ER-associated degradation (ERAD) of apoB, is regulated by the availability of newly synthesized core lipids (triglyceride, cholesterol ester) and microsomal triglyceride transfer protein activity (1,3,4). When lipid availability or microsomal triglyceride transfer protein activity is limited, translocation of apoB is incomplete, and apoB is ubiquitinylated co-translationally and degraded by the cytosolic ubiquitin-proteasome pathway (5)(6)(7)(8)(9).
Cytosolic and nuclear proteins are targeted for proteasomal degradation by the addition of multi-ubiquitin chains. The specificity of this process is largely conferred by ubiquitin (Ub) protein ligases (E3s) (10 -12). E3s interact directly or indirectly with substrate and mediate the transfer of Ub from Ub-conjugating enzymes to target proteins. Two major E3 classes have been identified: HECT domain E3s and RING finger E3s (10 -12). HECT domain E3s are homologous to E6-AP C-terminal domain E3s, which accept Ub from Ub-conjugating enzymes, thereby forming thiol-ester intermediates with Ub. The Ub is then transferred to the target protein. In contrast, RING finger E3s bind Ub-conjugating enzymes and apparently mediate the direct transfer of Ub from an Ub-conjugating enzyme to the target protein. Gp78 was originally isolated as a membrane glycoprotein from murine melanoma cells and was implicated in cell migration (13). Subsequently, gp78 was identified as the tumor autocrine motility factor receptor mediating tumor invasion and metastasis (14). Recently, we identified the tumor autocrine motility factor receptor gp78 as a RING finger-dependent Ub protein ligase, the first mammalian ER-resident E3 (15). This protein localizes primarily to the ER and targets itself, as well as a well-characterized ERAD substrate, the T cell antigen receptor CD3 ␦-subunit, for proteasomal degradation.
Because of the demonstrated role of gp78 in the ERAD for one target protein, we investigated whether it played a similar role for apoB, a secretory protein. When apoB's "lipid-ligands" are limited, it undergoes extensive degradation by the Ubproteasome system (1). We investigated this role by two approaches: transfection studies in HepG2 cells and co-expression of gp78 and apoB in a cell-free system. Our results with both approaches were consistent with each other and together indicate that gp78 is a bona fide E3 ligase in the apoB ERAD pathway.

MATERIALS AND METHODS
Reagents-N-Acetyl-leucinyl-leucinyl-norleucinal (ALLN), oleic acid, Triton X-100 and protein A-Sepharose CL 4B were purchased from Sigma. ALLN was used at a concentration of 100 M (40 g/ml). Sheep anti-human apoB polyclonal antibody was purchased from Roche Applied Science. [ 35 S]Methionine/cysteine was from PerkinElmer Life Sciences. LipofectAMINE was purchased from Invitrogen.
Growth of Cells-HepG2 cells were obtained from the American Type Culture Collection. Briefly, cells were maintained at 37°C in 5% CO 2 in 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum. The medium was changed every 3 days, and experiments were started after the cells were 70 -90% confluent. During the experiments, cells were maintained at 37°C in 5% CO 2 in serum-free minimum essential medium containing 1.5% bovine serum albumin, with the indicated additions or treatments.
In transfection experiments, HepG2 cells were treated with Lipo-fectAMINE with or without human gp78 cDNA (15) or murine Itch cDNA (15). Itch is a cytosolic E3 that is not involved in ERAD (16,17). After 36 h, cells were labeled with [ 35 S]Methionine/cysteine in the presence or absence of ALLN. The cell lysates and conditioned medium were analyzed by immunoprecipitation as described below.
Immunoprecipitation-Briefly, after samples were incubated on a shaker for 10 h at 4°C with an excess amount of anti-apoB antiserum, the immune complexes were precipitated by an additional 3-h incubation with protein A-Sepharose CL-4B. The samples were analyzed on 4% SDS-polyacrylamide gels. For analysis of ubiquitinylated apoB, cellular immunoprecipitates produced by the addition of anti-ubiquitin antibodies were resolubilized by boiling 4 min in 2% SDS and subjected to a second immunoprecipitation with anti-apoB antibody after adjusting the SDS concentration to 0.1%.
Cell-free System Studies-The cell-free expression of human apoB48 cDNA in the Promega TNT system was described previously (18). Briefly, human apoB48 cDNA (1.5 g of circular plasmid) was transcribed/translated in the presence of rabbit reticulocyte lysate, canine pancreatic microsomes, and 35 S protein labeling mix (1000 Ci/mmol; PerkinElmer Life Sciences); hereafter, this is referred to as the "reaction." ApoB48 was studied either as translated alone or co-translated with gp78 cDNA (0.5 g) and either with or without 50 M benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI; proteasome inhibitor obtained from S. Wilk, Mount Sinai School of Medicine) (18,19) dissolved in Me 2 SO. In the reactions in which PSI was absent, an equivalent volume of Me 2 SO was substituted. Incubation of the coupled transcription/translation reactions (total volume, 50 l) was carried out at 30°C for 1.5 h. Aliquots (25% of the transcription/translation reaction mixture) were taken to isolate microsomes by centrifugation at 100,000 ϫ g for 30 min at 4°C. The microsomes were then solubilized in gel loading buffer and proteins separated by SDS-PAGE (3-17% acrylamide gradient). Fluorography was used to detect the radioactive apoB48 protein bands.
The remaining 75% of the reaction mixture was used to study apoB48 ubiquitinylation. A 100ϫ excess of unlabeled Met/Cys was added, and the reaction mixture was composed of 2 mM ATP, 10 mM creatinine phosphate, and 100 g/ml creatine kinase. Then 5 l of 1 g/l hemagglutinin-tagged ubiquitin (provided by Z. Ronai, Mount Sinai School of Medicine) was added. After incubation at 37°C for 30 min, the microsomes were isolated as above. To recover ubiquitinylated ApoB48, the microsomes were then subjected to a two-step immunoprecipitation procedure (first step, 5 l of monoclonal anti-hemagglutinin antibody (Covance, Princeton, NJ); second step, 5 l of anti-human apoB antibody (Roche Diagnostics)). The final immunoprecipitates were then analyzed by SDS-PAGE and fluorography as above.

RESULTS AND DISCUSSION
We first investigated the effect of overexpression of gp78 in HepG2 cells on the synthesis and secretion of apoB. Itch (16,17), a cytosolic E3, was used as a control. Thirty-six hours after transient transfection with cDNAs for gp78 or myc-tagged Itch, FIG. 1. A, specific expression of gp78 in Hep G2 cells. Thirty-six hours after transfection (with mock reagents (c) or with cDNA encoding either gp78 (ER-E3) or myc-tagged Itch (cytosolic E3), the cells were lysed and cell lysates were analyzed by Western blotting analysis with anti-gp78 antibodies (left) or anti-myc antibodies (right). The arrows indicate the expected positions of gp78 and Itch. Both were efficiently expressed after transfection. B, overexpression of gp78 reduces secretion of apoB from HepG2 cells. After being transiently transfected with gp78 or Itch cDNAs, HepG2 cells were labeled for 2 h with [ 35 S]methionine. After labeling, cellular and medium apoB were analyzed by immunoprecipitation with anti-human apoB antibodies. The results shown are representative of five separate experiments. C, overexpression of gp78 has no effect on synthesis and secretion of albumin or apolipoprotein AI. After being transiently transfected with mock reagents (c) or with gp78 or Itch cDNAs for 36 h, HepG2 cells were labeled for 2 h with [ 35 S]methionine. After labeling, cellular and medium were analyzed by immunoprecipitation with anti-human albumin or apolipoprotein AI antibody. The data shown are representative of three experiments. D, ALLN reverses the decreased secretion of apoB associated with overexpression of gp78 in HepG2 cells. After being transiently transfected with mock reagents (c) or with gp78 or Itch for 36 h, HepG2 cells were labeled for 2 h with [ 35 S]methionine in the presence or absence of ALLN (40 g/ml). After labeling, cellular and medium apoB were analyzed by immunoprecipitation with anti-human apoB antibody. The data are representative of four experiments.
HepG2 cells were labeled for 2 h with [ 35 S]methionine. After labeling, cellular and medium were analyzed by immunoprecipitation with anti-human apoB, anti-human albumin, or antihuman apolipoprotein AI antibodies. The latter two proteins are not known to be ERAD substrates and served as controls. Specific expression of gp78 was confirmed by Western blotting analysis with an anti-gp78 polyclonal antibody (Fig. 1, left). Itch was also highly expressed after transfection, as demonstrated with anti-myc antibody (Fig. 1, right). Note that the lack of signal in control cells implies a low level of expression of each endogenous protein.
Overexpression of gp78 had a minimal effect on the level of labeled apoB in the cells (Fig. 1B, left). By contrast, overexpression of gp78 in HepG2 cells resulted in a marked reduction in the secretion of apoB (Fig. 1B, right). When the results of five separate experiments (with two to five wells per transfection per experiment) were pooled, overexpression of gp78 reduced secretion of apoB to 26.0 Ϯ 10.3% of mock transfected cells. Overexpression of Itch cDNA had no overall effect on secreted apoB (94.5 Ϯ 36.5% of mock-transfected cells). Importantly, overexpression of gp78 had no effect on either the synthesis or secretion of either apolipoprotein AI or human albumin, proteins that do not undergo intracellular degradation by the proteasome (Fig. 1C). As shown in Fig. 1D, the decreased secretion of apoB associated with overexpression of gp78 was completely reversed by ALLN, an inhibitor of proteasomal degradation that blocks apoB ERAD in HepG2 cells as effectively as lactacystin (6). The results shown in Fig. 1, C and D, indicate that the decrease in apoB secretion that resulted from overexpression of gp78 (Fig. 1B) was specific and was most probably caused by increased proteasomal degradation.
To extend these findings, we next immunoprecipitated ubiquitinylated apoB molecules in a two-step protocol while the proteasome was inhibited by ALLN. After labeling, cellular lysates were first immunoprecipitated by anti-ubiquitin antiserum; these immunoprecipitates were then denatured and subjected to a second immunoprecipitation with the anti-apoB antiserum. Fig. 2 shows that overexpression of gp78 resulted in a significant increase of ubiquitinylated apoB molecules in the cells. Because of the high molecular mass of apoB (ϳ520 kDa), we typically observe either minimally ubiquitinylated apoB running where native apoB is found on a 4% gel (Fig. 2, Ub-apoB100) or polyubiquitinylated apoB at the very top of the gel (Fig. 2, Ub-apoB). In this experiment, we demonstrated the presence of increased ubiquitinylated apoB in both of these regions of the gel as well as some intermediate sizes of ubiquitinylated apoB represented by the smear between the two clearer bands. Overexpression of Itch (Fig. 2, middle lane) may have resulted in a slight increase in ubiquitinylated apoB compared with control cells. Together, the results depicted in Figs. 1D and 2 indicate that overexpression of gp78 resulted in decreased secretion of apoB via increased ubiquitinylation and targeting of apoB for proteasomal degradation.
Because apoB degradation is regulated by lipid availability (1, 3, 4), we next investigated the effect of oleic acid treatment on the secretion of apoB in gp78-transfected HepG2 cells. Thirty-six hours after being transiently transfected with gp78, HepG2 cells were labeled for 30 min with [ 35 S]methionine in the presence of ALLN. The labeling medium was then washed out and cells were first incubated for an additional 15 min in new medium without ALLN. After the first 15-min chase, medium was changed again to fresh medium with or without oleic acid (0.4 mM), and the cells were incubated for 15, 30, or 60 min. Chase medium was collected at each time point and analyzed by immunoprecipitation with anti-apoB antibody. Fig. 3 shows that oleic acid significantly increased apoB secretion in HepG2 cells overexpressing gp78 compared with gp78-transfected cells incubated without OA (Fig. 3, compare lanes 12-14 (ϩOA) with lanes 9 -11 (ϪOA)). This is consistent with our previous observations that oleic acid, either by stimulating core lipid synthesis (20,21) or acting via some other pathway (22), can target apoB away from ERAD and toward secretion. Furthermore, these results are concordant with those of our previous study, in which oleic acid blocked the inhibition of apoB secretion by herbimycin, which increases expression of the 70-kDa heat shock protein and facilitates ubiquitinylation and proteasomal degradation of apoB (6). Oleic acid treatment also increased apoB secretion in HepG2 cells transfected with Itch (lanes 6 -8, ϩOA) but apoB secretion in cells overexpressing Itch incubated without OA was greater at all time points compared with gp78-expressing cells incubated without OA (Fig. 3, compare  lanes 3-5 with lanes 9 -11). The pulse-chase design used in this experiment also provided data demonstrating directly that the decrease in apoB recovery from media shown in Fig. 1 was a consequence of increased degradation of apoB, not effects of gp78 overexpression on apoB synthesis (Fig. 3, lanes 1 and 2; cell lysates obtained 15 min after labeling).
The second approach we took to establish the role of gp78 in apoB ERAD was to co-express gp78 cDNA and apoB48 cDNA in a cell-free system that we had previously demonstrated could recapitulate the major features of apoB100 ERAD (18). As shown in Fig. 4, the co-expression of gp78 led to decreased recovery of microsome-associated apoB48 (Fig. 4A, compare  lanes 1 and 3; for each sample, microsomes from 12.5 l of the transcription/translation reaction mixture were solubilized directly in gel buffer and subjected to SDS-PAGE). Concomitantly, overexpresssion of gp78 was associated with an increase in Ub-apoB48 (Fig. 4A, compare lanes 2 and 4; for each sample, microsomes from 37.5 l of the transcription/translation reaction mixture were subjected to a two-step immunoprecipitation to isolate ubiquitinylated apoB48). Nonspecific effects of coexpression could not explain these results, because the coexpression of luciferase and apoB48 had no impact on apoB48 recovery or ubiquitinylation relative to results when only apoB48 was expressed (data not shown). After labeling, cellular lysates were first immunoprecipitated by antiubiquitin antibodies. These immunoprecipitates were denatured by boiling for 4 min in 2% SDS and subjected to second immunoprecipitation with anti-apoB antibody after adjusting the SDS concentration to 0.1%. Equal quantities of newly synthesized protein, based on trichloroacetic acid-precipitable counts, were loaded onto each lane. Ub-apoB100 denotes where native apoB would normally run on the gel; all of the bands, however, represent only ubiquinylated apoB. These results are representative of two experiments.
That the decreased recovery of apoB48 observed when gp78 was co-expressed was caused by proteasomal degradation is supported clearly by the data in Fig. 4B, which employed PSI (19), an effective inhibitor of the proteasome in the cell-free system (18). As shown in Fig. 4B, PSI did not alter the recovery of microsome-associated apoB48 when it was expressed alone (Fig. 4, compare lanes A1 and B1), but did significantly increase recovery of translated apoB48 from reaction mixtures where gp78 was co-expressed (Fig. 4, compare lanes A3 and  B3). We have previously shown that the most highly ubiquitinylated apoB species are stable and do not undergo de-ubiquitinylation (23). Thus, the appearance of a wider molecular mass range of Ub-apoB48 species when PSI is added to inhibit proteasomal degradation is not surprising (Fig. 4, lanes A4 and B4, compare the regions above the expected migration of unmodified apoB48). The more labile forms of moderately ubiquitinylated apB48 that run between the top of the gel and where minimally ubiquitinylated apoB48 runs were now able to accumulate.
ApoB is an atypical secretory protein that, after undergoing early translational targeting to the ER and initiation of cotranslational translocation into the ER lumen, assumes a bitopic orientation in the ER membrane. Thus, concomitant with a prolonged interaction between apoB and the Sec61 proteins of the translocon (24), partially translocated apoB has domains in both the ER lumen and in the cytosol (25). Because of this atypical orientation relative to the ER membrane, sequences of apoB are exposed to the cytosol, leading to an interaction with the 70-and 90-kDa heat shock proteins and the 26 S proteasome (18,26). These associations can lead to cotranslational ubiquitinylation (27) and degradation (7). The proportion of newly synthesized apoB that undergoes degradation by the proteasome is critically dependent on the availability of the lipid-ligands for apoB, especially triglycerides (4,28,29), and varies by cell type. However, ubiquitinylation and proteasomal degradation of apoB has been shown to occur in all systems, including primary hepatocytes (1).
Ubiquitinylation is dependent on several proteins that activate and transfer ubiquitin to the targeted protein substrate (10 -12). The fact that ubiquitinylation and proteasomal degradation of apoB occurs cotranslationally raised the question as to whether any of the proteins involved in those processes were associated with the ER. It has been demonstrated in yeast, for example, that mutant carboxypeptidase Y (30) or misfolded hydroxymethyl glutaryl-CoA reductase (31) interacts with E3 ligases that are members of the DER3/HRD complex, which contains multiple integral ER membrane proteins (32). The recent identification of a mammalian transmembrane E3 associated with the ER (15) allowed us to address that question. The results of the present studies provide clear evidence that the ER-associated E3, gp78, can participate in the ubiquitinylation and proteasomal degradation of apoB. This finding adds another important piece to a model of post-transcriptional reg-  figure) containing rabbit reticulocyte lysate and canine pancreatic microsomes (total volume, 50 l). ApoB48 was expressed either alone (lanes 1 and 2) or co-expressed with gp78 (lanes 3 and 4). After 1.5 h at 30°C, an aliquot (25% of total volume) was removed and microsomes isolated. Hemagglutinin-ubiquitin was added to the remaining mixture (75% of total volume), the incubation was continued (30 min at 37°C), and microsomes were isolated. Microsome-associated total apoB48 (lanes 1 and 3) was recovered by solubilizing microsomes in gel loading buffer. Ubiquitinylated (Ub)-apoB48 species (lanes 2 and 4) were recovered by a two-step immunoprecipitation of isolated microsomes using anti-hemagglutinin followed by anti-apoB antibodies as described under "Materials and Methods." Analysis of the solubilized microsomes or the immunoprecipitates was by SDS-PAGE/fluorography; in each case, the total volume available was loaded onto the gels. A and B comprise data from a representative experiment (three total experiments) conducted in the absence (A) or presence (B) of the proteasome inhibitor PSI. ApoB48 denotes the site on the gel where native, non-ubiquitinylated apoB48 (lanes 1 and 3) or minimally ubiquitinylated apoB48 (lanes 2 and 4) would be found; Ub-apoB denotes the site on the gel where polyubiquitinylated apoB48 would be found. Co-expression of gp78 significantly reduced the recovery of apoB48 in microsomes; this was caused by increased ubiquitinylation and proteasomal degradation. ulation of apoB secretion in which, after initial insertion of the amino terminus of apoB into the ER lumen, degradation can be initiated in the original translocon (33) after only 50 -60% (7,27) of the protein has been translated. Considering the size of apoB (4536 amino acids) and the complexity of its secondary and tertiary structure (34), the presence of all essential components of a major system for degradation at the site of synthesis of this protein provides the most efficient system for regulation. In this regard, the results of the present study are consistent with our previous data, which demonstrated that other cytosolic components involved in the targeting and degradation of apoB, such as the 70-and 90-kDa heat shock proteins and proteasomes, are all associated with microsomal apoB in lipid-deprived HepG2 cells (1,24). Whether other ERassociated E3s, or cytosolic E3s other than Itch, can participate significantly in apoB degradation remains to be determined. Because of the large size of apoB, it would not be surprising if its different domains are processed by distinct E3s, given the selectivity of these ligases.