Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14733–14738, December 1998 Cell Biology Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence DEBORAH M. MITCHELL*†,MINGYUE ZHOU†‡,RAJALAKSHMI PARIYARATH*, HONGXING WANG*, JOHN D. AITCHISON§,HENRY N. GINSBERG‡, AND EDWARD A. FISHER*¶ *Laboratory of Lipoprotein Research, Cardiovascular Institute, Department of Medicine, and Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, 1 Gustave Levy Place, Box 1030, New York, NY 10029; ‡Department of Medicine, Columbia University College of Physicians and Surgeons, 630 West 168 Street, New York, NY 10032; and §Department of Cell Biology and Anatomy, University of Alberta, Edmonton, AB, Canada T6G 2H7 Communicated by Jan L. Breslow, The Rockefeller University, New York, NY, October 8, 1998 (received for review August 5, 1998) ABSTRACT When lipid synthesis is limited in HepG2 cells, tion. This presecretory degradation is increased when the avail- apoprotein B100 (apoB100) is not secreted but rapidly degraded ability of lipid ligands of apoB100 is limited by inadequate levels by the ubiquitin-proteasome pathway. To investigate apoB100 of either lipid synthesis or MTP-mediated lipid-transfer activity biosynthesis and secretion further, the physical and functional (7, 8). In recent studies with the human hepatocarcinoma cell line states of apoB100 destined for either degradation or lipoprotein HepG2, a standard model of lipoprotein metabolism, we and assembly were studied under conditions in which lipid synthesis, others have shown that most, if not all, of the degradation of proteasomal activity, and microsomal triglyceride transfer pro- apoB100 that occurs when lipid availability is limited is mediated tein (MTP) lipid-transfer activity were varied. Cells were pre- by the ubiquitin-proteasome pathway (9–11). treated with a proteasomal inhibitor (which remained with the Although the components of this pathway are cytosolic, a cells throughout the experiment) and radiolabeled for 15 min. number of recent reports have shown that a variety of membrane- During the chase period, labeled apoB100 remained associated associated and secretory proteins can be targeted to the protea- with the microsomes. Furthermore, by crosslinking sec61b to some for degradation in eukaryotic cells (reviewed in refs. 12 and apoB100, we showed that apoB100 remained close to the trans- 13). The precise mechanism by which these proteins become locon at the same time apoB100–ubiquitin conjugates could be substrates for a cytosolic protease has remained elusive. Based on detected. When lipid synthesis and lipoprotein assemblyy results for major histocompatibility complex class I molecules, it secretion were stimulated by adding oleic acid (OA) to the chase has been hypothesized that a protein translocated into the ER can medium, apoB100 was deubiquitinated, and its interaction with be fully ‘‘dislocated’’ back into the cytosol and subsequently sec61b was disrupted, signifying completion of translocation attacked by the proteasome (14). The need for complete dislo- concomitant with the formation of lipoprotein particles. MTP cation, which would require some form of reverse translocation, participates in apoB100 translocation and lipoprotein assembly. may not be general, given the finding that proteasomes can be In the presence of OA, when MTP lipid-transfer activity was found in association with the cytosolic face of the ER (15, 16). inhibited at the end of pulse labeling, apoB100 secretion was Thus, the initial attack of the proteasome could be directed abolished. In contrast, when the labeled apoB100 was allowed to against a protein domain that is or becomes exposed to the cytosol accumulate in the cell for 60 min before adding OA and the while other domains remain segregated by the ER membrane. inhibitor, apoB100 lipidation and secretion were no longer Consistent with this possibility is the finding that two yeast impaired. Overall, the data imply that during most of its asso- ubiquitin-conjugating enzymes essential for ER-associated pro- ciation with the endoplasmic reticulum, apoB100 is close to or teasomal degradation, Ubc6p and Ubc7p, can be localized to the within the translocon and is accessible to both the ubiquitin- ER. Ubc6p does so as an integral membrane protein (17), and proteasome and lipoprotein-assembly pathways. Furthermore, Ubc7p does so by docking onto the membrane protein Cue1p MTP lipid-transfer activity seems to be necessary only for early (18). translocation and lipidation events. The extremely large size of apoB100, its potential to pause or arrest during translocation (19, 20), as well as its multiple hydro- b Apolipoprotein B100 (apoB100) is a 4,536-aa polypeptide and is phobic -sheet domains (21), which would favor membrane the major structural protein of the liver-derived very low density interactions, led us to consider whether full translocation fol- and low density lipoproteins. Hepatic lipoprotein assembly begins lowed by complete dislocation was a likely path from the ER to when apoB100 is cotranslationally translocated across the endo- the proteasome. Key elements of the degradation pathway can be plasmic reticulum (ER) membrane (1) and interacts with the on the cytosolic side of the ER membrane; this finding suggested luminal microsomal triglyceride transfer protein (MTP; refs. that apoB100 that was incompletely translocated or partially 2–4). MTP catalyzes the initial transfer of lipid to nascent dislocated could be the proximal substrate of the ubiquitin- apoB100, with subsequent lipoprotein maturation steps occurring proteasome machinery. in the ER and possibly the Golgi apparatus (5, 6). Studies of We hypothesized, therefore, that even after translation, the cultured primary hepatocytes and transformed liver cells of apoB100 polypeptide could remain in close proximity to the human and nonhuman origin have established that significant translocon. While in close proximity, it could be either targeted control over apoB100 secretion can be exerted at the posttrans- for degradation or assembled as a lipoprotein, depending on the lational level by the targeting of the nascent protein to degrada- availability of lipid ligands. This hypothesis was tested primarily The publication costs of this article were defrayed in part by page charge Abbreviations: apoAI, apoprotein AI; apoB100, apoprotein B100; ER, endoplasmic reticulum; MTP, microsomal triglyceride transfer pro- payment. This article must therefore be hereby marked ‘‘advertisement’’ in tein; OA, oleic acid. accordance with 18 U.S.C. §1734 solely to indicate this fact. †D.M.M. and M.Z. contributed equally to this work. © 1998 by The National Academy of Sciences 0027-8424y98y9514733-6$2.00y0 ¶To whom reprint requests should be addressed. e-mail: edmd-phdo PNAS is available online at www.pnas.org. [email protected]. 14733 Downloaded by guest on September 26, 2021 14734 Cell Biology: Mitchell et al. Proc. Natl. Acad. Sci. USA 95 (1998) by examining the cellular localization and functional state of medium for 0, 20, 40, or 60 min. Cells were then disrupted by apoB100 protected from degradation by inhibition of the pro- sonication. After low-speed centrifugation (10,000 3 g, 10 min, teasome. As described below, the results not only support the 4°C) to remove nuclei and unbroken cells, the supernatant was hypothesis but further suggest that apoB100 destined to become fractionated by centrifugation at 100,000 3 g for1hat4°C. part of a lipoprotein has a prolonged interaction with the ER ApoB100 was then immunoprecipitated from the microsomal membrane; during this time, apoB100 becomes associated with its (pellet) and cytosolic (supernatant) fractions. lipid ligands in a process that depends on MTP only at an early Crosslinking of ApoB100 and sec61b. HepG2 cells were stage of the assembly process. pretreated with a proteasome inhibitor (either MG132 or lacta- cystin at a final concentration of 10 mM) for 1 h and then labeled EXPERIMENTAL PROCEDURES for 15 min by adding 100 mCiyml of [35S]methionineycysteine. Materials. Rabbit anti-human apoB100 and anti-human apo- The medium was removed, and the cells were washed twice with protein AI (apoAI) antisera were from Calbiochem; mouse chase medium and then incubated in chase medium containing anti-human apoB100 antiserum was from Caltag (South San the proteasome inhibitor for 0, 20, 40, or 60 min in the presence Francisco, CA). Rabbit anti-sec61b and anti-sec61a antisera were or absence of OA (0.8 mM). At the end of each chase period, cells gifts from Tom A. Rapoport (Harvard University, Cambridge, were washed with ice-cold PBS and harvested in crosslinking MA; ref. 22) and Arthur E. Johnson (TexasA&M,College buffer (PBSy0.4% digitoniny0.5 mM EDTA). The permeable cell Station, TX). Rabbit anti-ubiquitin antiserum was from Stress- suspension was incubated on ice for1hwiththehomobifunc- Gen Biotechnologies (Victoria, Canada). Antisera and associated tional chemical crosslinking reagent dithiobis(succinimidyl pro- reagents for immunofluorescence studies are described below. pionate) (final concentration of 0.25 mM added as a concentrated [35S]methionineycysteine was from DuPontyNEN. Lactacystin solution in dimethyl sulfoxide). The reaction was quenched by was from the laboratory of E. J. Corey (Harvard University; ref. incubation in 20 mM Tris (pH 7.5) for 15 min. ApoB100–sec61b 23) and MG132