vol. 265, no. 17, issue of june 15, pp. 10005-10011,199o...

THE .JOURNAL OF B~OGICAL CHEMISTRY Cc> 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 17, Issue of June 15, pp. 10005-10011,199O Printed m U.S. A.

Apolipoprotein B Is Both Integrated into and Translocated across the Endoplasmic Reticulum Membrane EVIDENCE FOR TWO FUNCTIONALLY DISTINCT POOLS*

(Received for publication, December 1, 1989)

Roger A. Davis$$, Richard N. Thrift& Christine C. Wu7l, and Kathryn E. HowellllII From the $Cell and Molecular Biology Unit, Atherosclerosis and Hepatobiliary Research Center and the (Department of Cellular and Structural Biology, University of Colorado Medical School, Denver, Colorado 80262

Apolipoprotein B (apoB), a protein containing several hydrophobic p-sheet structures, is essential for the assembly of triglyceride-rich lipoproteins. Previously, we found that only a fraction of de nova synthesized apoB is secreted; the remainder is retained in the endoplasmic reticulum where it is degraded. To under- stand the basis for these observations, translocation, the first step in the secretory pathway, was examined. Translocation of apoB was determined by its sensitiv- ity to degradation by the exogenous protease, trypsin. In rough microsomes, about half of the apoB was degraded by trypsin. In contrast, in Golgi fractions little (if any) apoB was accessible to trypsin. Essentially all of the apoB that was degraded was membrane bound. Monoclonal IgGs against either the N-terminal or C- terminal halves of apoB were bound to magnetic beads and used to immunoisolate microsomes. In contrast to the specific ability of the IgGs against apoB to isolate microsomes, little or no microsomes were isolated using nonimmune IgG and IgG against albumin. Since microsomes remained intact and oriented right-side out as demonstrated by the inability of trypsin both to degrade albumin and to affect the capacity of the intralumenal enzyme glucose-6-phosphatase to dephosphorylate mannose 6-phosphate, the data suggest that a pool of apoB is exposed on the cytoplasmic surface of the endoplasmic reticulum membrane. To determine if the trypsin-accessible pool of apoB is a transient form, pulse-chase experiments were performed. The results show that the percent of apoB that was trypsin accessible increased during the first 20 min of the chase, suggesting that during this time the trypsin-accessible pool of apoB is not translocated (it does not become trypsin insensitive). Thus, in two in uiuo models (cultured cells and rat liver) translocation of apoB is not quantitative. We propose that apoB translocation across the endoplasmic reticulum determines its entry into two functionally distinct pools. The intralumenal trypsin-insensitive pool participates in the assembly of very low density lipoprotein; the trypsin-accessible nontranslocated cytoplasmic pool is shunted into a degradative pathway. Regulated translocation of apoB

* This work was supported in part by National Institutes of Health Grants HL41624 (to R. A. D.) and HL25596 (to R. A. D.) from the Heart, Lung, and Blood Institute and DK34914 (to R. A. D. and K. E. H.) from the Digestive Disease institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adoertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be sent: Box B-158, UCHSC, 4200 E. Ninth Ave., Denver, CO 80262.

)I Supported by American Heart Association Grant-in-Aid AH89- 06.53.

may provide a unique mechanism with which to determine the rate of very low density lipoprotein assembly/ secretion.

Hydrophobicity appears to be one of the dominant structural motifs determining targeting and topography of proteins. Hydrophobic segments (signal peptides) usually present at the N terminus of nascent secretory proteins provide a signal facilitating translocation across the endoplasmic reticulum (ER)’ membrane into the lumen (Blobel and Dobberstein, 1975; Blobel, 1980; Walter et al., 1984). Hydrophobic segments at any site in a newly synthesized peptide act to “stop- transfer” the translocation step, resulting in the integration of the protein into the membrane of the ER (Gething and Sambrook, 1982; Rose and Bergmann, 1982; Boeke and Model, 1982; Yost et al., 1983; Davis and Model, 1985; Wick- ner and Lodish, 1985). In several experimental examples, signal sequences and stop-transfer sequences have functionally substituted for each other (Coleman et al., 1985; Mize et al., 1986).

Apolipoprotein B (apoB) is an amphipathic protein that is essential for the assembly of very low density lipoproteins (VLDL), a process that is thought to occur in the lumen of the ER (Alexander et al., 1976). There are two molecular mass forms of apoB. One has a molecular mass of approximately 550,000 daltons and is secreted by the rat liver, and in the rat a smaller form consisting of the N-terminal 48% of the larger form is secreted by both the intestine and rat liver (Krish- naiah et al., 1980). Since both forms can assemble VLDL, the N terminus 48% contains all of the essential features required. Although the unique molecular features that afford apoB the ability to assemble VLDL remain unknown, it is likely that the unusual affinity of apoB for lipids contributes to this function. ApoB has several small hydrophobic P-sheets situated throughout the molecule (Knott et al., 1986; Yang et al., 1986,1989; Cladaras et al., 1986). Although hydropathy algorithm analysis predicts these hydrophobic segments to be too small to allow stable integration as a transmembrane protein, apoB clearly integrates into the phospholipid monolayer surface of a lipoprotein particle and remains stably associated. Unlike other plasma apolipoproteins, apoB does not exchange among lipoprotein particles, does not exist naturally unasso- ciated with lipids, and cannot be extracted from lipoprotein

I The ahhreviations used are: ER. endoplasmic reticulum; apoBL, large molecular weight apolipoprotein i; apoBs, small molecular weight auoliaoDrotein B: aooB. aoolioourotein B: VLDL. very low density Iipobroiein; SDS, sodium hod&l sulfate; PAGE, polyacryl- amide gel electrophoresis; Tricine, N-[2-hydroxy-l,l-bis(hydroxy- methyl)ethyl]glycine.

10005

Integration and Translocation of ApoB

particles without disruption by detergents. Although apoB is secreted with the VLDL particle, a number of groups have reported that it is associated with the ER membrane (Bor- chardt and Davis, 1985; Bostrom et al., 1986; Wong and Pino, 1987; Bamberger and Lane, 1988; Davis et al., 1989A). One interpretation of these data is that integration into the membrane of the ER provides a mechanism through which apoB can “extract” membrane lipids and assemble them into a VLDL particle.

In previous studies, we found that the rate of movement of apoB out of the ER was slower than that of albumin and that this transport step was rate limiting for secretion (Borchardt and Davis, 1987). Moreover, unlike albumin, which is quantitatively secreted, only a fraction of the apoB that is synthesized is secreted; the remainder is degraded intracellularly (Borchardt and Davis, 1987). Additional data show that proteolytic fragments of apoB can be identified in the ER but not in the Golgi (Davis et al., 1989B), suggesting that degradation occurs prior to the Golgi. Intracellular degradation of apoB is consistent with the findings that under several different circumstances, the secretion of apoB varies whereas the content of apoB mRNA remains constant (Davidson et al., 1988; Davis et al., 1989A; Pullinger et al., 1989).

To define the manner through which apoB is shunted into a degradative pathway, we examined the first committed step in the secretory pathway, translocation across the ER membrane. Using different but complementary experimental ap- proaches, we show that two functionally distinct pools of apoB exist in the ER. One pool of apoB is not translocated; it remains exposed on the cytoplasmic surface of the ER, does not move onward into the Golgi, and apparently is eventually degraded. The other pool of apoB is translocated across the ER and is secreted as a component of VLDL. The data support a novel mechanism in which the secretion of apoB is determined by the proportion is translocated across the ER.

MATERIALS AND METHODS

Preparation of Microsomal Fractions-Rat livers (Davis et al., 1989B) and cultured rat hepatocytes (Borchardt and Davis, 1987) were homogenized in the presence of proteolytic inhibitors (100 pM leupeptin, 100 pM phenylmethylsulfonyl fluoride, and 0.1% aprotinin) and separated into the designated microsomal fractions using the procedures described in detail. Characterization of the purity (marker enzyme recovery and enrichment) of the preparations has been re- norted in detail (Davis et al., 1989A. 1989B; Borchardt and Davis, i987).

Accessibility of Microsomal Membrane Proteins to Trypsin-Im- mediately upon isolation, the membrane fractions were sedimented through a 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, solution to remove the proteolytic inhibitors. The washed microsomal pellet was resuspended in 0.25 M sucrose, 10 mM Tris-HCl buffer. Each microsomal sample (rough and smooth microsomes contained 0.4 mg of protein; the Golgi fraction contained 0.1 mg of protein) was incubated with 240 pg of trypsin (Sigma, type XIII) for 30 min at room temperature. Preliminary studies showed that 30 min was sufficient to cause maximal degradation of proteins (analyzed by SDS-PAGE) without any loss of the latency of mannose-6-phosphatase; see data below). After the incubation, 2.4 mg of soybean trypsin inhibitor (Sigma, Bowman-Birk) and phenylmethylsulfonyl fluoride (final concentration 100 fiM) was added, the microsomes were sedimented, and the supernatant was discarded. The microsomal pellet was resuspended in 0.25 M sucrose, 10 mM Tris-HCI buffer. Aliquots were analyzed by Western blotting (see below) and were used to determine the latency for mannose-6-phosphatase s described (Arion et al., 1976).

Sodium Carbonate Treatm nt of Rough Microsomes-Rough microsomes were prepared and i I cubated with and without trypsin, as described above. Following the addition of inhibitors, the microsomes were pelleted as described above. The microsomal pellets were then resuspended in sodium carQonate, pH 11.3, incubated on ice for 30 min, and then pelleted as described in detail (Howell and Palade,

1982). The supernatant and pellet fractions were then subjected to Western blotting.

Western Blotting-Western blotting was perform as described (Towbin et al., 1979). Samples were electrophoresed as described above and then electroblotted onto nitrocellulose paper followed by addition of primary antibody (rabbit anti-rat apoB or albumin) and then horseradish peroxidase-conjugated goat anti-rabbit IgG. When the relative amounts of immunoreactive proteins were compared, all samples were subjected to electroblotting onto the same sheet of nitrocellulose. In some experiments, lz51-labeled apoB (as part of a lz51-labeled d c 1.21 g/ml fraction prepared from rat serum) was added to the samples prior to the SDS-PAGE step. Autoradiography of the SDS-PAGE gel and the nitrocellulose paper afforded an estimation of recovery of apoB among the different samples; there was no difference (see Fig. 2).

Western blots were analyzed by densitometry using a Bio-Rad densitometer equipped with an integration program. Values are reported in densitometry units as determined by the integration program.

P&e-Chase Studies-Hepatocytes were obtained from male Spra- gue-Dawley rats (175-250 g) via collagenase perfusion as described in detail (Davis, 1986). Cells were first plated in Dulbecco’s modified Eagle’s medium with 20% calf serum. After plating the hepatocytes for 4 h, the medium was changed to serum-free, methionine-free Joklik’s minimum essential medium. Cells were incubated in the methionine-free medium for 1 h after which 100 rrCi of translation grade [36S]methionine (I130 Ci/mmol) in Tricine buffer was added/ ml of minimum essential culture medium. After 10 min, the dishes were immediately placed on ice, and the culture medium was drawn off by suction. The dishes were than washed six times with Dulbecco’s modified Eagle’s medium containing 10 mM methionine. The cells incubated with Dulbecco’s modified Eagle’s medium plus 10 mM methionine were then incubated for the designated time (see figure legends).

Medium was removed, and the culture dishes were placed on ice, and then cells were covered with 5 ml of phosphate-buffered saline and placed on ice. The cells were scraped from the dishes using a rubber policeman and transferred to a culture tube. Cells were pelleted via centrifugation at 2,000 rpm for 5 min. The phosphate-buffered saline supeinatant was removed by aspiration, and the cells were resusnended in homotzenization buffer (0.25 M sucrose, 10 mM Tris- HCl, PH 7.4,lOO PM leupeptin, 100 FM phenylmethylsulfonyl fluoride 0.01% aprotinin). The cell suspension was placed in a 4 “C minicell bomb disruption chamber (Kontes) for 15 min at 500 p.s.i. The cell suspension was then released to atmospheric pressure to disrupt the cells. Repeating this process a second time resulted in disruption of all the cells as determined by phase-contrast light microscopy. A total microsomal fraction was prepared from the homogenate as described (Borchardt and Davis, 1587). This involved centrifuging for 10 min at 10.000 X P., in a Beckman 52-20 centrifuge using a Beckman JA- 20 rotor. Thesupernatant from the 10,000 x & spin-was layered onto a 2 M sucrose cushion (2 M sucrose, 10 mM Tris-HCl, pH 7.4, with the protease inhibitors) and centrifuged at 105,000 X g., for 90 min to yield a total microsomal fraction at the 0.25 M/2 M sucrose interface.

Microsomes were then resuspended in 0.25 M sucrose, 10 mM Tris- HCl buffer (without inhibitors), pelleted to remove inhibitors, and then subjected to trypsin (25 pg/ml) for 30 min at room temperature. Inhibitors were then added and the microsomes pelleted to remove trypsin.

Zmmunoprecipitation, SDS-PAGE, and Fluorography-Immuno- precipitation was performed using SDS-containing buffers as described previously (Davis et al., 1984). Membrane fractions were suspended in 0.5 ml of boiling buffer (buffer B: 2% SDS, 50 mM Tris- HCl pH 7.4,5 mM dithiothreitol, 50 p leupeptin, 100 @M phenylmethylsulfonyl fluoride). One-ml aliquots of the solubilized samples were placed in 1.5-ml microcentrifuge tubes along with an amount of antiserum, determined to bind all of the desired antigen quantitatively, This was determined empirically by the repeated addition of known amounts of antibody. The samples were mixed overnight with the antiserum on a shaker at room temperature. Protein A-Sepharose beads in buffer D (10 mM NaHPO,-. DH 7.4, 0.15% SDS, 0.5% Zwittergent 3-14, 50‘m~ NaCl, 5 mg)mi bovine serum albumin, 20 KM leupeptin, 10 mM dithiothreitol) (Faust et al., 1982) were added to the samples in an amount determined to bind all the antibody. The samples were then shaken for an additional h. The tubes were centrifuged in a Fischer microcentrifuge (10,000 X g..) for 5 min to pellet the antigen-antibody-protein A-Sepharose bead complex. The

Integration and Translocation of ApoB

supernatant was drawn off using a vacuum aspirator, and the samples were then washed with 1 ml of buffer D. The tubes were mixed for 5 s, and centrifuged again for 5 min. The wash process was repeated once more with buffer D and finally once with phosphate-buffered saline. The final pellet was solubilized in sample buffer that contained 2% SDS, 10 mM EDTA, 5 mM dithiothreitol, 8 M urea, 2% glycerol, 0.01% pyronin Y all dissolved in electrophoresis running buffer (pH 8.3). The tubes were placed in a boiling water bath for 5 min. The samples were then subjected to electrophoresis as described (Davis et al., 1984).

Coomassie Blue-stained bands corresponding to authentic apoB standards were cut horn the gel, transferred to a scintillation vial, dissolved in 0.5 ml of 30% hydrogen peroxide, and then solubilized with 0.2 ml of tissue solubilizer (NCS, Amersham Corp.). The samples were counted and quantitated for radioactivity on a scintillation counter (Beckman).

Several lines of evidence show that the [““Slmethionine-labeled apoB produced during the pulse-chase experiment was quantitatively immunoprecipitated. First, when the microsomal pellet was subjected to two rounds of immunoprecipitation, there was no detectable ‘jaS- apoB in the second immunoprecipitate (radioactivity for each apoB moiety equaled background 155-173 cpm). Second, when the supernatant obtained from a single round of immunoprecipitation was concentrated, and an unlabeled standard containing both forms of apoB was added, and the entire sample was separated by SDS-PAGE, there was no detectable “‘S radioactivity in either apoB band. These data show that immature ““S-apoB is quantitatively isolated by the immunoprecipitation procedure.

Immunoisolation of Microsomes-Immunoisolation was performed using the magnetic immunoabsorbent technique described in detail (Howell et al., 1989). The linker bound directly to the magnetic beads was a sheep IgG against the Fc portion of mouse IgG. The designated IgG was added to the magnetic beads containing the sheep antibody in a molar ratio of one sheep IgG to two mouse IgGs. The beads were then washed with 0.5% bovine serum albumin in phosphate-buffered saline, pH 7.2. In the immunoisolation experiments the indicated amount of microsomes were incubated with in 0.5% bovine serum albumin in 25 mM sodium acetate buffer (pH 6.5). The mixtures were incubated for 4 h at 4 “C. The magnetic beads were then isolated by placing a magnet on the side of the incubation vessel. The isolated beads were gently washed with buffer until the supernatant was clear. The beads were then subjected to assay for glucose-6-phosphatase (Arion et al., 1976). From the previously determined specific activity of glucose-6-phosphatase in the total microsomal fraction, the recovery was calculated.

Statistical Analysis-All values presented represent the mean f standard deviation. Student’s t test was used to determine statistical differences.

RESULTS

Accessibility of Microsomal Proteins to Trypsin Degrada- tion-Translocation was determined by the ability of exogenous trypsin to degrade apoB in sealed microsomes. Immedi- ately upon isolation, hepatic rough, smooth, and Golgi fractions were incubated with trypsin. There was a specific a decrease in the relative amount of immunoreactive apoBL and apoBs in the rough microsomes (Fig. 1). The decrease in immunoreactive apoB in the rough microsomes caused by trypsin was observed using both a polyclonal antibody and a monoclonal antibody that recognizes the C-terminal half of apoBL (Davis et al., 1989B). Estimation of the amount of immunoreactive apoB by densitometry showed that using the polyclonal antibody there was a significant 56% decrease in apoBI. and a 70% decrease in apoBs (see legend, Fig. 1, for statistics). Using the monoclonal antibody specific for apoBL, there was a 95% decrease in the amount apoBL. In marked contrast, incubation of Golgi fractions with trypsin resulted in no significant decrease in apoB. Smooth microsomes dis- played a small amount of trypsin accessible apoB. These data suggest that a significant proportion apoB is not translocated across the ER membrane; this form does not enter the Golgi, hence secretory pathway.

To verify this interpretation, a series of control experiments

123 123 123 FIG. 1. Effect of incubation of hepatic rough, smooth, and

Golgi fractions with trypsin on immunoreactive apoB and albumin. Rough, smooth, and Golgi microsomal fractions were prepared from rat liver. Aliquots of each fraction (400 rg of protein of rough and smooth and 100 rg of protein of Golgi) were incubated with trypsin (240 pg) for 30 min at room temperature after which soybean trypsin inhibitor and phenylmethylsulfonyl fluoride were added, and the samples were centrifuged to remove the trypsin. The microsomes were then subjected to Western blotting using rabbit antiserum against apoB (upper panel, A), albumin (lower panel, A), and a monoElona1 IgG found to be specific for the C-terminal half of aaoB, (Fig. 1B). The Western blots shown were all obtained from tie s&e experiment and the same sheet of nitrocellulose paper. Lane I, untreated samples; lane 2, inhibitors added before trypsin incubation; lane 3, samples incubated with trypsin. BL and By designate the migration of authentic standards of apoB. The relative amounts of immunoreactive apoB were determined by densitometry. Trypsin significantly decreased the amount of both apoB,. (in densitometric units: 170 f 60, nontrypsinized; 75 f 40, trypsin-treated = 56% decrease; n = 5 in each group, p c 0.05) and apoBs (390 f 40, nontrypsinized, 120 f 40, trypsin-treated = 70% decrease; n = 5 in each group, p < 0.01).

was carried out. Two criteria were used to show that the microsomes remained intact: protection of albumin, and glu- case-6-phosphatase from trypsin degradation. Albumin is a protein quantitatively secreted by cultured rat hepatocytes (Borchardt and Davis, 1987). It should therefore be quantitatively translocated and completely protected from trypsin degradation. As shown in Fig. 1, in the same samples of microsomes that showed significant degradation of apoB, there was essentially no detectable loss of albumin. There was sufficient trypsin present in the incubation to cleave all of the proteins as demonstrated by the complete loss of immunoreactive apoB,,, apoBs, and albumin when deoxycholate (0.02%) was added (to disrupt the membranes) prior to the addition of trypsin (data not shown). Thus, the degradation of apoB by trypsin was selective.

Glucose-6-phosphatase is an integral membrane protein of the ER having its active site directed into the lumen (Arion et al., 1976). It has the ability to dephosphorylate both its usual substrate, glucose 6-phosphate, as well as mannose 6- phosphate. Intact microsomes are impermeable to both sub- strates. However, there is a specific translocator that imports glucose 6-phosphate but not mannose 6-phosphate into the lumen of the ER. The capacity of microsomes to dephosphor-

10008 Integration and Translocatlon of ApoB

ylate mannose 6-phosphate is a reflection of the degree to which the membranes have sealed right-side out and have remain sealed (Arion et al., 1976). The parameter, percent latency, is determined as the rate of dephosphorylation of mannose 6-phosphate by microsomes in the presence and absence of detergents. The percent latency provides an accu- rate index of the relative amount of microsomes that are permeable (Arion et al., 1976). As shown in Table I, without deoxycholate disruption, rough microsomes were almost inca- pable of dephosphorylating mannose 6-phosphate. In contrast, after the addition of deoxycholate there was a lo-fold stimulation. The resulting 90% latency, which agrees with that obtained by others (Coleman and Bell, 1978), indicates that trypsin did not affect the percent latency or detergent- stimulated activity. However, when deoxycholate was added to disrupt the microsomes prior to adding the trypsin, essentially all of the activity was lost, showing that this assay is sensitive to trypsin degradation (data not shown). The combined data indicate that the rough microsomes were sealed right-side out and remained this way throughout the trypsin incubation.

Two criteria were used to show that trypsin was completely inhibited before disrupting the microsomes for the Western blot analysis. Adding the inhibitors prior to the trypsin pre- vented the degradation of endogenous apoB, as shown in the second lane of each group of experiments (see Figs 1 and 3). To show that trypsin was not active during either the SDS- PAGE or electroblotting steps, a standard amount of ‘*‘I- labeled apoB was added to each incubation mixture right after the trypsin inhibitors were added. Preliminary experiments showed that the amount of “l-labeled apoB added was not sufficient to be detected by the enzyme-linked immunosorbent assay method used in the Western blotting technique. Neither trypsin nor inhibitors had any effect on the recovery of lz51- labeled apoB, as shown by autoradiography of the SDS-PAGE gel (Fig. 2A) and the nitrocellulose following electroblotting (Fig. 2B). On the same nitrocellulose, Western blotting analysis using the apoB antibody showed that endogenous apoB was significantly decreased when this rough microsomal sample was incubated with trypsin. With the proviso that plasma ““I-labeled apoB behaves in a manner similar to the apoB contained in microsomes, the results indicate that the degradation of endogenous apoB occurred prior to the either the SDS-PAGE or the electroblotting steps.

Trypsin Specifically Degrades Membrane-associated ApoB- We examined the possibility that the trypsin-accessible pool of apoB, which is presumably located on the cytoplasmic surface of the ER membrane, is membrane associated. Sealed hepatic microsomal vesicles can be opened to release content proteins by treatment with sodium carbonate (pH 11.3). The

TABLE I Ability of microsomes to dephosphotylate mannose 6-phosphate

Rough microsomes were isolated and incubated under the designated conditions. After reisolation of microsomes by ultracentrifugation, the ability of glucose-6-phosphatase to dephosphorylate mannose 6-phosphate was determined. Results are the mean of three individual determinations. Variations within the same group were -a%.

Rough microsomes (nonincubated)

+Inhibitors, then trypsin

+Trypsin

Mannose 6-phosphatase activity Latency

-Deoxycholate +Deoxycholate nmol PO,/mgprotein/min %

6.8 75.6 91

7.2 64.0 89

5.6 73.2 92

I3

BS --

a 1 2 3

FIG. 2. Autoradiograms of exogenous ‘261-labeled apoB. A total rat serum lipoprotein fraction (d < 1.21 g/ml) iodinated with “‘ICl was added to microsomal samples after trypsin inhibitors were added (see legend of Fig. 1 for experimental protocol). The amount added was shown not to be sufficient to be detectable by the Western blot. A, autoradiogram of SDS-PAGE gel developed for 24 h. Lanes 1 and 2, duplicate samples of untreated microsomes; lanes 3 and 4, inhibitors added before trypsin; lanes 5 and 6, microsomes incubated with trypsin. B, autoradiogram of the nitrocellulose used for Western blotting developed for 24 h. Lane I, untreated microsomes; lane 2, inhibitors added before trypsin; lane 3, microsomes incubated with trypsin.

BL

Bs

PELLET SUPERNATANT

123 46 8 1 23 466

FIG. 3. Sodium carbonate pellets and supernatants from rough microsomes. Rough microsomes were incubated as described in Fig. 1. After the trypsin inhibitors were added, microsomes were reisolated and incubated with sodium carbonate and separated into membrane pellets and intracisternal contents (supernatants) as described (Howell and Palade, 1982). Pellets and supernatants repre- senting the same fraction of the total were subjected to Western blotting using a rabbit antiserum against rat apoB. Lanes 1 and 4, unincubated microsomes; lanes 2 and 5, inhibitors added before trypsin; lanes 3 and 6, microsomes incubated with trypsin. Lanes I- 3 represent microsomes from one rat, and lanes 4-6 represent microsomes from a different rat. All blots were performed at the same time using the same sheet of nitrocellulose paper.

preparations are centrifuged; intralumenal soluble proteins remain in the supernatant, whereas membrane-integrated proteins are pelleted (Howell and Palade, 1982; Fujiki et al., 1982). Following incubation with trypsin and addition of the inhibitors, rough microsomes were dissociated with sodium carbonate, and the resulting pellet and supernatant fractions were subjected to Western blot analysis. Most (X35%) of the immunoreactive albumin was in the supernatant (data not shown), showing that sodium carbonate treatment effectively released the intracisternal contents. Trypsin selectively decreased apoB in the pellet but not in the supernatant (Fig. 3). This is most clearly illustrated by an almost complete loss of apoBL in the sodium carbonate pellet (Fig. 3, lanes 3 and 6). In contrast, trypsin caused no detectable loss of apoB in the supernatant. In the supernatant fraction, several proteins

Integration and Translocation of ApoB 10009

having molecular weights smaller than apoB reacted with the antibody against apoB. Since these proteins are present in microsomes not incubated with trypsin, it is likely that the proteins are proteolytic fragments of apoB produced by endogenous proteases, not by trypsin. The combined data show that trypsin selectively degrades membrane-associated apoB.

Newly Synthesized Trypsin-accessible ApoB Is Retained in the Endoplasmic Reticulum: Evidence for a Nontranslocated Pool of ApoB-Pulse-chase experiments were performed to examine if the trypsin-accessible pool of apoB is a transient form that eventually enters the trypsin-insensitive translocated pool. Cultured rat hepatocytes were pulse labeled with [%]methionine for 10 min after which they were chased with 1000-fold excess cold methionine. Cells were harvested at designated times during the chase period, a total microsomal fraction prepared, and the amount of “5S-labeled apoB that was trypsin accessible was determined.

Both forms of apoB reached peak labeling within 10 min of the chase after which the intracellular 35S-labeled apoB de- clined rapidly, reflecting a completion of translation and subsequent secretion out of the cell (Fig. 4, A and B). If trypsin-accessible apoB were to enter the translocated pool after translation was complete, there would be a decrease in the proportion of apoB that is trypsin accessible. To the contrary, the proportion of both molecular weight forms of apoB which were degraded by trypsin increased for the first 20 min of the chase period after which it remained fairly constant (Fig. 5). This observation is in marked contrast to the rapid loss of %-labeled apoB from the cells which occurred after 10 min of the chase (Fig. 4). These findings

10 20 30 40 50 TIME OF CHASE (Inin)

“I

I 10 20 30 40 50

TIME OF CHASE bin)

Frc. 4. Pulse-chase analysis of trypsin-accessible apoB. Cultured rat hepatocytes were pulsed for 10 min with [35S]methionine and then chased with 1000-fold excess unlabeled methionine. At the times indicated, cells were harvested and disrupted with nitrogen decavitation, and a total microsomal fraction was obtained by ultracentrifugation. Each fraction was then incubated with trypsin (25 rg/ ml) for 30 min at room temperature after which the trypsin was inhibited and microsomes reisolated as described in Fig. 1. Micro- somes were then solubilized, subjected to immunoprecipitation using a rabbit antibody against rat apoB, and the immunoprecipitates were separated on an SDS-PAGE gel. The portions of the gel corresponding to authentic standards of apoBL and apoBs were cut out and their radioactivity quantitated. Each value represents the mean of two different samples. Closed symbol represent microsomes not treated with trypsin; open symbols represent microsomes incubated with trypsin.

TlME OF CHASE (mid

FIG. 5. Change accessibility of apoB to trypsin during the pulse-chase experiment. The percent apoB accessible to trypsin was determined as the ratio of the amount of radioactive apoB degraded by trypsin to the total amount of apoB in the untreated microsomes. Each value represents the mean of two separate samples. Open symbols represent apoBL; closed symbols represent apoBs.

or . , 7 0 100 200 300 400 INPUT MICROSOMES (Kg)

FIG. 6. Immunoisolation of Microsomes using monoclonal antibodies against rat apoB. A fixed amount of magnetic beads coupled to the same amount of the designated IgG was incubated with the indicated amount of microsomal protein. The recovery of glucose&phosphatase was used to estimate the recovery of microsomes. Each point represents the mean of three individual determinations; variations were

Integration and Tramlocation of ApoB

were effective in isolating microsomes; as the concentration of microsomes increased, the total amount of microsomes bound to the beads increased (Fig. 6). In marked contrast, the amount of microsomes recovered by the magnetic beads with linked nonimmune IgG or IgG prepared against albumin was much less and not different from background. These data show a selective ability of the anti-apoB monoclonal IgGs to isolate microsomes assayed by glucose-6-phosphatase activity. In additional experiments, we examined the portion of microsomal glucose-6-phosphatase which could be recovered with the antibodies against apoB. In three separate experiments at bead-linked antibody excess, 67 + 8% of the total glucose-6- phosphatase activity was recovered by magnetic beads that bound monoclonal antibody DBll, which recognizes both molecular weight forms of apoB. The selective ability of antibodies against apoB to recover microsomes by the immunoisolation method provides compelling data showing the presence of apoB integrated into the cytoplasmic surface of the ER membrane.

DISCUSSION

Our results are consistent with the concept that membrane integration obfuscates translocation. As a result of inefficient translocation, there are two functionally distinct pools of apoB in the ER. One pool is located in the lumen, as demonstrated by protection from exogenous trypsin. This pool of apoB participates in the assembly of VLDL. The other pool is not translocated into the lumen; it is integrated into the cytoplasmic surface of the ER, as shown by the ability of exogenous trypsin to degrade it. The pool of nontranslocated apoB does not enter the Golgi, is shunted from the secretory pathway, and does not participate in the assembly of VLDL. Nontranslocated apoB probably accounts for the apoB that is synthesized but is not secreted by cultured rat hepatocytes (Borchardt and Davis, 1987).

These conclusions are supported by four different experimental protocols: 1) selective degradation by trypsin of endogenous apoB in rough microsomes as shown by Western blotting using specific polyclonal (Fig. IA) and monoclonal (Fig. 1B) antibodies against apoB; 2) demonstration that the apoB that is accessible to trypsin is derived from the membrane-associated pool of apoB (Fig. 3); 3) demonstration that the de novo synthesized 3”S-labeled apoB degraded by trypsin does not enter the trypsin-insensitive pool (Figs. 4A and B, and 5); and 4) demonstration that apoB epitopes recognized by monoclonal antibodies are present on the surface of microsomes and are effective antigens to immunoisolate hepatic microsomes (Fig. 6).

The alternative explanation that trypsin-accessible apoB was originally intracisternal but that during the homogenization step apoB leaked out of the microsomes and became stuck to the cytoplasmic surface was ruled out by a series of mixing experiments. ““I-Labeled apoB was added to microsomes (both in the presence and absence of sodium carbonate, pH 11.3). When microsomal membranes were recovered by ultracentrifugation, no detectable ““I-labeled apoB was recovered in the pellet (data not shown), showing that apoB does not stick to microsomes. Similar findings have been reported by others (Wong and Pino, 1987). With the proviso that plasma apoB reflects accurately the behavior of nascent apoB, these data argue strongly against the proposal that the cytoplasmic apoB was produced during the homogenization step.

ApoB could be translocated arrested by a stop-transfer sequence, resulting in its integration into the ER as a transmembrane protein. Although several different algorithm anal-

yses of the amino acid sequence of apoB predict that no hydrophobic domains in apoB are sufficiently long to act as a-helical transmembrane-spanning domains, it is possible that other motifs (e.g. P-sheets) may span the membrane bilayer (Knott et al., 1986; Yang et al., 1986; Cladaras et al., 1986). Our data do not address the issue of whether or not apoB is a transmembrane protein in the ER. The trypsin- sensitive nontranslocated pool of apoB could be all cytoplasmic or the major part of a protein that is transmembrane. The manner in which apoB is integrated into the ER membrane may be similar to the way porin is integrated into the outer membrane of Escherichia coli. Porin does not contain any transmembrane-spanning domains but does contain hydrophobic P-sheets (Paul and Rosenbusch, 1985). Like porin, apoB contains several hydrophobic P-sheet structures situated throughout the molecule (Yang et al., 1989). ApoB associates with lipoproteins mainly via these hydrophobic P-sheets. It is reasonable to expect that similar structures are responsible for the association of apoB with the ER membrane.

Diversion of apoB from the VLDL assembly pathway may provide an explanation for the observation that only a fraction of apoB that is synthesized is secreted; the remainder is degraded intracellularly (Borchardt and Davis, 1987). The recent finding that proteolytic fragments of apoB exist in the rough and smooth microsomal fractions but not in Golgi fractions led to the hypothesis that the degradation of apoB occurs in the ER (Davis et al., 1989B). Excess subunits of the T-cell receptor are degraded in the ER (Lippencott-Schwartz et al., 1988).

In a recent publication, a transmembrane-spanning domain of the T-cell receptor subunit consisting of 21 amino acids was identified as a signal for targeting to the ER degradation pathway (Bonifacino et al., 1990). We have scanned the sequence of human apoB-100 and found two domains that are homologous with portions of this putative degradation targeting sequence. One 7-amino acid segment lies on the N-terminal side of the apoB-48 junction, whereas the other 6- amino acid segment lies on the C-terminal side. Since the minimal sequence necessary for signaling ER degradation has not yet been identified, the functional significance of the apoB sequence homology remains to be determined.

There are data showing that intracellular degradation of at least one secretory protein may be regulatable. In the absence of serum and hormones, the majority of de nouo synthesized fibrinogen is degraded by cultured hepatocytes (Grieninger et al., 1984). Addition of serum completely reversed the intracellular degradation of fibrinogen, suggesting a regulatable process. If the translocation of apoB can be varied in response to metabolic signals, this may serve as a mechanism with which to regulate apoB secretion. The secretion of apoB is sensitive to metabolic state; carbohydrate overload increases (Boogaerts et al., 1984) and fasting selectively decreases (Davis et al., 1985) apoB and VLDL secretion. Quantitation of apoB mRNA in these three different metabolic states using both 5’ and 3’ cDNA probes shows no change in mRNA in any of these different states, yet the secretion of apoB is clearly different (Davis et al., 1989A). A similar discordance between apoB synthesis and mRNA has been reported for rats having different thyroid hormone status (Davidson et al., 1988) and in HepG2 cells treated with various agents that alter lipoprotein secretion (Pullinger et al., 1989. The combined data clearly show that post-transcriptional mechanisms play a role in regulating the secretion of apoB by the liver.

Translocation efficiency may determine whether a protein is secreted, inserted into a membrane, or shunted into degradative pathways. Chaoptin, like apoB and porin, does not

Integration and Translocation of ApoB 10011

contain a transmembrane-spanning domain, yet it is integrated into the extracellular leaflet of the plasma membrane of Drosophila photoreceptor cells (Reinke et al., 1988). In ER membranes, in vitro translation/translocation assays produce two separate pools of chaoptin: a cytoplasmic (trypsin-accessible) pool that is membrane associated, and an intralumenal (trypsin-inaccessible) pool that is also membrane associated. Only a small proportion of chaoptin was translocated in vitro (i.e. becomes intralumenal and glycosylated, see Fig. 9 of Reinke et al., 1988). The combined data are consistent with the proposal that proteins that integrate into the ER membrane are inefficiently translocated. It is possible that these proteins require a unique translocation process capable of translocating hydrophobic membrane-integrating sequences.

Genetic evidence suggests that at least one step in trans- porting apoB out of the ER is unique from other secretory proteins; this may be the translocation step. Abetalipopro- teinemia is a recessive disorder expressed as an almost complete absence of plasma apoB (Herbert et al., 1982). Two lines of evidence show that the defective responsible for abetalipoproteinemia is not the apoB gene. First, there is one gene for apoB (Knott et al., 1985; Huang et al., 1985; Law et al., 1985; Knott et al., 1986; Yang et al., 1986). It has two co- dominantly expressed alleles (Young et al., 1987), which is inconsistent with recessive inheritance. Second, recent studies show clearly that restriction map polymorphisms of the apoB gene do not segregate with the inheritance of abetalipoproteinemia (Talmud et al., 1988). There is an accumulation of both apoB and its mRNA in the livers of abetalipoproteinem- its, suggesting that apoB is made, but cannot be transported out of, the hepatocyte (Lackner et al., 1986). However, abe- talipoproteinemics express no general impairment of the secretion of proteins other than apoB. The product of the defective gene responsible for abetalipoproteinemia probably interacts specifically with apoB. Whether or not the gene product responsible for abetalipoproteinemia is involved in translocation remains to be determined.

Although all of the trypsin-degraded apoB was membrane associated, clearly not all of the membrane-associated apoB was degraded by trypsin. This suggests that apoB is also integrated into the lumenal side of the ER membrane, which is probably the initial step in the assembly of VLDL. Physi- ologic variation in translocation of apoB may provide a unique mechanism accounting for the post-translational regulation of VLDL assembly.

Acknowledgments-We thank Tom Sand for expert technical as- sistance and Patricia Salaver for preparing the manuscript.

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