Microsomal Lumen Research Articles

SLC35B1 significantly contributes to the uptake of UDPGA into the endoplasmic reticulum for glucuronidation catalyzed by UDP-glucuronosyltransferases

The transport of UDP-glucuronic acid (UDPGA), a co-substrate of UDP-glucuronosyltransferase (UGT), to the intraluminal side of the endoplasmic reticulum (ER) is an essential step in the glucuronidation of exogenous and endogenous compounds. According to a previous study, the expression of recombinant SLC35B1, SLC35B4, or SLC35D1, nucleotide sugar transporters, in V79 cells has the potential to transport UDPGA into the lumen of microsomes. The purpose of this study is to examine whether the transport of UDPGA by these transporters substantially affects UGT activity. Since the knockdown of UDP-glucose 6-dehydrogenase, a synthetase of UDPGA, in HEK293 cells stably expressing UGT1A1 (HEK/UGT1A1 cells) resulted in a significant decrease in 4-methylumbelliferone (4-MU) glucuronosyltransferase activity, supplementation of a sufficient amount of UDPGA is required for UGT activity. By performing qRT-PCR using cDNA samples from 21 human liver samples, we observed levels of the SLC35B1 and SLC35D1 mRNAs that were 15- and 14-fold higher, respectively, than the levels of the SLC35B4 mRNA, and SLC35B1 showed the largest (37-fold) interindividual variability. Interestingly, 4-MU glucuronosyltransferase activity was significantly decreased upon the knockdown of SLC35B1 in HEK/UGT1A1 cells, and this phenomenon was also observed in HepaRG cells. Using siRNAs targeting 23 different SLC35 subfamilies, the knockdown of SLC35B1 and SLC35E3 decreased 4-MU glucuronosyltransferase activity in HEK/UGT1A1 cells. However, the 4-MU glucuronosyltransferase activity was not altered by SLC35E3 knockdown in HepaRG cells, suggesting that SLC35B1 was the main transporter of UDPGA into the ER in the human liver. In conclusion, SLC35B1 is a key modulator of UGT activity by transporting UDPGA to the intraluminal side of the ER.

Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates

Antibodies are indispensable tools for basic research as well as diagnostic and therapeutic applications. Consequently, the development of alternative manufacturing strategies which circumvent the hurdles connected to conventional antibody production technologies is of enormous interest. To address this issue, we demonstrate the synthesis of complex antibody formats, in particular immunoglobulin G (IgG) and single-chain variable fragment Fc fusion (scFv-Fc), in a microsome-containing cell-free system based on translationally active chinese hamster ovary (CHO) cell lysates. To mimic the environment for antibody folding and assembly present in living cells, antibody genes were fused to an endoplasmic reticulum (ER)-specific signal sequence. Signal-peptide induced translocation of antibody polypeptide chains into the lumen of ER microsomes was found to be the prerequisite for antibody chain assembly and functionality. In this context, we show the rapid synthesis of antibody molecules in different reaction formats, including batch and continuous-exchange cell-free (CECF) reactions, depending on the amount of protein needed for further analysis. In addition, we demonstrate site-specific and residue-specific labeling of antibodies with fluorescent non-canonical amino acids. In summary, our study describes a novel antibody production platform which combines the highly efficient mammalian protein folding machinery of CHO cells with the benefits of cell-free protein synthesis.

Ribonuclease-neutralized pancreatic microsomal membranes from livestock for in vitro co-translational protein translocation

Here, we demonstrate that pancreatic microsomal membranes from pigs, sheep, or cattle destined for human consumption can be used as a valuable and ethically correct alternative to dog microsomes for cell-free protein translocation. By adding adequate ribonuclease (RNase) inhibitors to the membrane fraction, successful in vitro co-translational translocation of wild-type and chimeric pre-prolactin into the lumen of rough microsomes was obtained. In addition, the human type I integral membrane proteins CD4 and VCAM-1 were efficiently glycosylated in RNase-treated microsomes. Thus, RNase-neutralized pancreatic membrane fractions from pig, cow, or sheep are a cheap, easily accessible, and fulfilling alternative to canine microsomes.

Misfolded Proinsulin Presenting an Unpaired Thiol is Recognized and Targeted for ERAD by Specific Lumenal Components in the ER

Objective The accumulation of misfolded proinsulin within the ER of pancreatic β-cells is toxic to the cell, leading to ER stress and cell death. Little is known about how misfolded proteins without glycosylation sites (i.e. proinsulin) are targeted for degradation. Mutant proinsulin, C96Y, (lacks a disulfide bond and has a reactive thiol) has been shown to accumulate and aggregate within the ER, leading to augmented cell apoptosis. We examined whether it is possible for an unpaired thiol on C96Y to act as an endoplasmic reticulum associated degradation (ERAD) recognition signal. Methods: Wild type and mutant (C96Y) proinsulins, labeled with a fluorescent (BODIPY) or radioactive probe (35S-Met), were made using an in vitro transcription/translation system and targeted to the ER lumen of canine pancreatic microsomes. ER microsomes containing proinsulin and lumenal proteins were resuspended in the cystolic medium containing rabbit reticulocyte lysate, which possesses all the required cystolic components for retrotranslocation ERAD. Rates of retrotranslocation and degradation were observed. Results: Our studies, utilizing 35S-Met, show that the ERAD of proinsulin increases when C96Y is present. The rate of retrotranslocation of BODIPY labeled C96Y proinsulin was found to be greater than that of wild type proinsulin. In retrotranslocation studies utilizing reconstituted microsomes, the rate of retrotranslocation was found to be linearly related to lumenal protein concentration. Conclusion Misfolded proinsulin presenting an unpaired thiol is recognized and targeted for ERAD by specific lumenal components in the ER. Funded by: Western University of Health Sciences New Faculty Startup Fund

Microsome-associated lumenal lipid droplets in the regulation of lipoprotein secretion

Liver is the major organ in mammals that possesses the capacity to release triglyceride within VLDL. VLDL assembly requires apolipoprotein (apo) B-100 with the assistance of microsomal triglyceride transfer protein (MTP), which facilitates the mobilization of triglyceride into the microsomal lumen. Recent experimental evidence has suggested that the lumenal triglyceride associated with endoplasmic reticulum (ER)/Golgi may represent an entity serving as precursors for large VLDL1. Under lipid-rich conditions, discrete triglyceride-rich lipidic bodies, termed lumenal lipid droplets, are accumulated in association with ER/Golgi microsomes. Formation of the microsome-associated lumenal lipid droplets (MALD) is dependent on the activity of MTP, and the resulting apoB-free lipidic body is associated with a variety of proteins including apolipoproteins that are components of VLDL. Formation and utilization of MALD during the assembly and secretion of VLDL1 have a profound influence on hepatic cell physiology, such as ER stress responses. This review summarizes current understanding of hepatic triglyceride homeostasis in general, and highlights the functional significance of triglyceride compartmentalization between cytosol and microsomes in particular. Understanding of MALD metabolism may shed new light on the prevention and treatment of liver diseases associated with abnormally elevated intracellular triglycerides.

Luminal accumulation of newly synthesized morphine-3-glucuronide in rat liver microsomal vesicles

Morphine is converted to morphine 3-β-D-glucuronide (M3G) by the UDP-glucuronosyltransferase Ugt2b1 in the endoplasmic reticulum (ER) of rat liver. Because of its luminal localization, UGT activity requires UDP-glucuronate import and glucuronide export across the ER membrane. The former transport is generally considered to be rate limiting and to explain the latency of UGT activities in intact microsomal vesicles. However, some observations indicate that the release of bulky glucuronides, such as M3G, might also be rate limiting for glucuronidation. This assumption was tested by characterizing the transport of M3G and its distribution between the intra- and extravesicular spaces during synthesis in rat liver microsomes. The amount of vesicle-associated M3G was measured using rapid filtration and LC-MS measurement. Our results reveal a remarkable accumulation of newly synthesized M3G in the microsomal lumen above the equilibrium. The transport showed a linear concentration-dependence in a wide range (5-200 μM). Therefore, the build-up of high (about 20 μM) luminal M3G concentration could adjust the rate of release to that of synthesis (44.85 ± 4.08 pmol/min/mg protein) during the conjugation of 100 μM morphine. These data can explain earlier findings indicative of separate intracellular pools of M3G in rat liver. Accumulation of bulky glucuronides in the ER lumen might also play an important role in their targeting and in the control of biliary excretion.

Missense Mutation in APOC3 within the C-terminal Lipid Binding Domain of Human ApoC-III Results in Impaired Assembly and Secretion of Triacylglycerol-rich Very Low Density Lipoproteins: EVIDENCE THAT ApoC-III PLAYS A MAJOR ROLE IN THE FORMATION OF LIPID PRECURSORS WITHIN THE MICROSOMAL LUMEN

Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL(1), the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys(58) with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys(58) and Lys(60) with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.

Functional analysis of the missense APOC3 mutation Ala23Thr associated with human hypotriglyceridemia

We have shown that expression of apolipoprotein (apo) C-III promotes VLDL secretion from transfected McA-RH7777 cells under lipid-rich conditions. To determine structural elements within apoC-III that confer to this function, we contrasted wild-type apoC-III with a mutant Ala23Thr originally identified in hypotriglyceridemia subjects. Although synthesis of [(3)H]glycerol-labeled TAG was comparable between cells expressing wild-type apoC-III (C3wt cells) or Ala23Thr mutant (C3AT cells), secretion of [(3)H]TAG from C3AT cells was markedly decreased. The lowered [(3)H]TAG secretion was associated with an inability of C3AT cells to assemble VLDL(1). Moreover, [(3)H]TAG within the microsomal lumen in C3AT cells was 60% higher than that in C3wt cells, yet the activity of microsomal triglyceride-transfer protein in C3AT cells was not elevated. The accumulated [(3)H]TAG in C3AT microsomal lumen was mainly associated with lumenal IDL/LDL-like lipoproteins. Phenotypically, this [(3)H]TAG fractionation profiling resembled what was observed in cells treated with brefeldin A, which at low dose specifically blocked the second-step VLDL(1) maturation. Furthermore, lumenal [(35)S]Ala23Thr protein accumulated in IDL/LDL fractions and was absent in VLDL fractions in C3AT cells. These results suggest that the presence of Ala23Thr protein in lumenal IDL/LDL particles might prevent effective fusion between lipid droplets and VLDL precursors. Thus, the current study reveals an important structural element residing within the N-terminal region of apoC-III that governs the second step VLDL(1) maturation.

14‐Deoxyandrographolide desensitizes hepatocytes to tumour necrosis factor‐alpha‐induced apoptosis through calcium‐dependent tumour necrosis factor receptor superfamily member 1A release via the NO/cGMP pathway

Andrographis paniculata (AP) has been found to display hepatoprotective effect, although the mechanism of action of the active compounds of AP in this context still remains unclear. Here, we evaluated the hepatoprotective efficacy of 14-deoxyandrographolide (14-DAG), a bioactive compound of AP, particularly its role in desensitization of hepatocytes to tumour necrosis factor-alpha (TNF-alpha)-induced signalling of apoptosis. TNF-alpha-mediated ligand receptor interaction in hepatocytes in the presence of 14-DAG was studied in vitro in primary hepatocyte cultures, with the help of co-immunoprecipitation, confocal microscopy and FACS analysis. Events associated with 14-DAG-induced TNFRSF1A release from hepatocytes were determined using immunoblotting, biochemical assay and fluorimetric studies. Pulse-chase experiments with radiolabelled TNF-alpha and detection of apoptotic nuclei by terminal transferase-mediated dUTP nick-end labelling were performed under in vivo conditions. 14-DAG down-regulated the formation of death-inducing signalling complex, resulting in desensitization of hepatocytes to TNF-alpha-induced apoptosis. Pretreatment of hepatocytes with 14-DAG accentuated microsomal Ca-ATPase activity through induction of NO/cGMP pathway. This resulted in enhanced calcium influx into microsomal lumen with the formation of TNFRSF1A-ARTS-1-NUCB2 complex in cellular vesicles. It was followed by the release of full-length 55 kDa TNFRSF1A and a reduction in the number of cell surface TNFRSF1A, which eventually caused diminution of TNF-alpha signal in hepatocytes. Taken together, the results demonstrate for the first time that 14-DAG desensitizes hepatocytes to TNF-alpha-mediated apoptosis through the release of TNFRSF1A. This can be used as a strategy against cytokine-mediated hepatocyte apoptosis in liver dysfunctions.

Expression of apolipoprotein C-III in McA-RH7777 cells enhances VLDL assembly and secretion under lipid-rich conditions

Apolipoprotein (apo) C-III plays a regulatory role in VLDL lipolysis and clearance. In this study, we determined a potential intracellular role of apoC-III in hepatic VLDL assembly and secretion. Stable expression of recombinant apoC-III in McA-RH7777 cells resulted in increased secretion efficiency of VLDL-associated triacylglycerol (TAG) and apoB-100 in a gene-dosage-dependent manner. The stimulatory effect of apoC-III on TAG secretion was manifested only when cells were cultured under lipid-rich (i.e., media supplemented with exogenous oleate) but not lipid-poor conditions. The stimulated TAG secretion was accompanied by increased secretion of apoB-100 and apoB-48 as VLDL(1). Expression of apoC-III also increased mRNA and activity of microsomal triglyceride transfer protein (MTP). Pulse-chase experiments showed that apoC-III expression promoted VLDL(1) secretion even under conditions where the MTP activity was inhibited immediately after the formation of lipid-poor apoB-100 particles, suggesting an involvement of apoC-III in the second-step VLDL assembly process. Consistent with this notion, the newly synthesized apoC-III was predominantly associated with TAG within the microsomal lumen that resembled lipid precursors of VLDL. Introducing an Ala23-to-Thr mutation into apoC-III, a naturally occurring mutation originally identified in two Mayan Indian subjects with hypotriglyceridemia, abolished the ability of apoC-III to stimulate VLDL secretion from transfected cells. Thus, expression of apoC-III in McA-RH7777 cells enhances hepatic TAG-rich VLDL assembly and secretion under lipid-rich conditions.

Specific Features of the Prion Protein Transmembrane Domain Regulate Nascent Chain Orientation

The sequence of a transmembrane (TM) domain and the adjacent regions are important for recognition, orientation, and integration at the translocon during membrane protein biosynthesis. However, the sequences of individual TM domains vary considerably. Although some general effects of electrostatic and hydrophobic interactions have been observed, it is still not clear what features of diverse sequences influence TM domain orientation. Here we utilized the ability of the prion protein (PrP) to be synthesized in multiple topological forms to assay the effects of substitutions and mutations on TM domain orientation. Several of the TM domains we tested appear to contain no inherent information regulating orientation. In contrast, we found that the middle region of the PrP TM domain significantly reduces the ability of the chain to invert its orientation in the translocon. We also observed that the C-terminal region of the PrP TM domain influences orientation, and we characterized the orientation differences between two forms of a physiologically relevant polymorphism in this region. Specifically, we found that the identity of a single amino acid, that at position 129, can significantly alter PrP TM domain orientation. Because position 129 is the location of the disease-associated Met/Val polymorphism, we discuss both how this small change may affect TMD orientation and the larger biological implications of these results.

Human GM 3 synthase: a new mRNA variant encodes an NH 2-terminal extended form of the protein

All human GM 3 synthase mRNA variants until now identified predict a protein of 362 amino acids having substrate activity highly restricted to lactosylceramide. In this report we describe the identification of a new GM 3 synthase transcript containing an additional translation start codon, located upstream and in-frame with that up to now considered unique translation initiation site in the human GM 3 synthase gene. In vitro expression studies showed that the new transcript produces a longer form of human GM 3 synthase, that is efficiently translocated into the microsomal lumen and glycosylated. Moreover, stable cDNA transfection into mammalian cells gives rise to a threefold increase of GM 3 synthase activity, associated to a broader substrate specificity. Although this transcript has been initially identified in the human placenta, RT-PCR analyses verified the expression of an identical mRNA also in undifferentiated HL60 cells, but not in the monocytic lineage. Altogether, these results are the first demonstration of the existence of a new isoform of human GM 3 synthase, which could play an important role during HL60 cell differentiation. The functional relevance of the existence of two isoforms of GM 3 synthase is also discussed.

Intestinal assembly and secretion of highly dense/lipid-poor apolipoprotein B48–containing lipoprotein particles in the fasting state: Evidence for induction by insulin resistance and exogenous fatty acids

Emerging evidence suggests that overproduction of intestinally derived apolipoprotein (apo) B48–containing lipoprotein particles may be an important contributor to both fasting and postprandial dyslipidemia in insulin-resistant states. Mechanisms regulating the assembly and secretion of apoB48-containing lipoproteins are not fully understood particularly in the diabetic/insulin-resistant intestine. In the present study, we have investigated the density profile of apoB48 lipoproteins assembled in primary hamster enterocytes. Both intracellular and secreted apoB48 particles were examined in intestinal enterocytes isolated from normal or insulin-resistant fructose-fed hamsters, as well as in enterocytes treated with exogenous oleic acid. Microsomal luminal contents and culture media were analyzed by discontinuous and sequential ultracentrifugation on sucrose and KBr gradients, respectively. ApoB48 was mostly secreted on VLDL-, LDL-, and denser HDL-sized particles in the fasting state. In pulse-chase labeling experiments, nascent apoB48-containing particles initially accumulated in the microsomal lumen as HDL-sized particles, with subsequent formation of apoB48-VLDL particles, with only a minute amount of chylomicrons observed. Treatment with 720 μmol/L of oleic acid, increased microsomal apoB48 HDL synthesis, and induced a marked shift toward lighter more buoyant particles. A marked enhancement in assembly of apoB48-containing lipoproteins was also observed in the microsomal lumen of fructose-fed hamster enterocytes, suggesting facilitated assembly and secretion of dense intestinal lipoprotein particles in insulin-resistant states. Overall, these observations suggest that a major proportion of apoB48-containing lipoprotein particles is assembled and secreted as highly dense, HDL-sized particles. The production of these small, dense, and potentially atherogenic apoB48 particles can be stimulated by increased free fatty acid flux as well as in insulin-resistant diabetes.

Inhibitory activity of diacylglycerol acyltransferase (DGAT) and microsomal triglyceride transfer protein (MTP) by the flavonoid, taxifolin, in HepG2 cells: potential role in the regulation of apolipoprotein B secretion

The purpose of the present study was to examine the role of taxifolin, a plant flavonoid, on several aspects involving apolipoprotein B (apoB) secretion and triglyceride (TG) availability in HepG2 cells. Taxifolin was shown by ELISA to markedly reduce apoB secretion under basal and lipid-rich conditions up to 63% at 200 μmol/L. As to the mechanism underlying this effect, we examined whether taxifolin exerted its effect by limiting TG availability in the microsomal lumen essential for lipoprotein assembly. Taxifolin was shown to inhibit microsomal TG synthesis by 37% and its subsequent transfer into the lumen (−26%). The reduction in synthesis was due to a decrease in diacylglycerol acyltransferase (DGAT) activity (−35%). The effect on DGAT activity was found to be non-competitive and non-transcriptional in nature. Both DGAT-1 and DGAT-2 mRNA expression remained essentially unchanged suggesting the point of regulation may be at the post-transcriptional level. Evidence is accumulating that microsomal triglyceride transfer protein (MTP) is also involved in determining the amount of lumenal TG available for lipoprotein assembly and secretion. Taxifolin was shown to inhibit this enzyme by 41%. Whether the reduction in TG accumulation in the microsomal lumen is predominantly due to DGAT and/or MTP activity remains to be addressed. In summary, taxifolin reduced apoB secretion by limiting TG availability via DGAT and MTP activity.

The Chalcone Xanthohumol Inhibits Triglyceride and Apolipoprotein B Secretion in HepG2 Cells

The present study examined the role of xanthohumol (XN), a plant chalcone, on apolipoprotein B (apoB) and triglyceride (TG) synthesis and secretion, using HepG2 cells as the model system. The data indicated that XN decreased apoB secretion in a dose-dependent manner under both basal and lipid-rich conditions (as much as 43% at 15 micromol/L). This decrease was associated with increased cellular apoB degradation. To determine the mechanism underlying this effect, we examined triglyceride availability, a major factor in the regulation of apoB secretion. XN inhibited the synthesis of TG in the microsomal membrane and the transfer of this newly synthesized TG to the microsomal lumen (decreases of 26 and 64%, respectively, under lipid-rich conditions), indicating that TG availability is a determining factor in the regulation of apoB secretion under the experimental conditions. The inhibition of TG synthesis was caused by a reduction in diacylglycerol acyltransferase (DGAT) activity, which corresponded to a decrease in DGAT-1 mRNA expression, but not DGAT-2 expression. Microsomal triglyceride transfer protein (MTP) may also control the rate of TG transfer from the microsomal membrane to the active lumenal pool. XN decreased MTP activity in a dose-dependent manner (as much as 30%). Whether the reduction in TG accumulation in the microsomal lumen is predominantly due to DGAT and/or MTP activity remains unknown. In summary, the data suggest that xanthohumol is a potent inhibitor of apoB secretion.

Overexpression of the endoplasmic reticulum 60 protein ER-60 downregulates apoB100 secretion by inducing its intracellular degradation via a nonproteasomal pathway: evidence for an ER-60-mediated and pCMB-sensitive intracellular degradative pathway.

Co- and posttranslational regulation of apolipoprotein B (apoB) has been postulated to involve degradation by both proteasomal and nonproteasomal pathways; however, nonproteasomal mechanisms of apoB degradation are currently unknown. We have previously demonstrated an intracellular association of newly synthesized apoB with endoplasmic reticulum (ER)-60, an ER-localized protein, possessing both proteolytic and chaperone activities. In the present paper, adenoviral expression vectors containing rat ER-60 cDNA were used to achieve dose- and time-dependent overexpression of ER-60 to investigate its role in apoB100 turnover. Overexpressed ER-60 accumulated in the microsomal lumen of HepG2 cells and was associated with apoB100 in dense lipoprotein particles. Overexpression of ER-60 in HepG2 cells significantly reduced both intracellular and secreted apoB100, with no effect on the secretion of a control protein, albumin. Similar results were obtained in McA-RH7777 rat hepatoma cells. ER-60-stimulated apoB100 degradation and inhibition of apoB100 secretion were sensitive to the protease inhibitor, p-chloromercuribenzoate (pCMB), in a dose-dependent manner but were unaffected by the proteasomal or lysosomal protease inhibitors, N-acetyl-leucinyl-leucinyl-nor-leucinal, E64, and leupeptin. Interestingly, enhanced expression of ER-60 induced apoB100 fragmentation in permeabilized HepG2 cells and resulted in detection of a unique 50 kDa degradation intermediate, a process that could be inhibited by pCMB. Intracellular stability and secretion of apoB100 in primary hamster hepatocytes were also found to be sensitive to pCMB. When taken together, the data suggest an important role for ER-60 in promoting apoB100 degradation via a pCMB-sensitive process in the ER. ER-60 may act directly as a protease or may be involved indirectly as a chaperone/protein factor targeting apoB100 to this nonproteasomal and pCMB-sensitive degradative pathway.

Transmembrane Lipid Transfer Is Crucial for Providing Neutral Lipids during Very Low Density Lipoprotein Assembly in Endoplasmic Reticulum

Very low density lipoprotein (VLDL), a large particle containing apolipoprotein B (apoB) and large amounts of neutral lipids, is formed in the luminal space within the endoplasmic reticulum (ER) of hepatic cells. The assembly mechanism of VLDL particles is a tightly regulated process where apoB, associated with an insufficient amount of lipids, is selectively degraded intracellularly. In this study we found that treatment of HuH-7 human hepatoma cells with verapamil inhibited secretion of apoB-containing lipoprotein particles through increasing degradation of apoB. Addition of N-acetylleucyl-leucyl-norleucinal, an inhibitor of proteasome and other cysteinyl proteases that are responsible for apoB degradation, restored apoB recovery from verapamil-treated cells. De novo synthesis of lipids from [14C]acetate was increased in the presence of verapamil, suggesting that verapamil decreases lipid availability for apoB thus leading to the secretion of apoB-containing lipoprotein. We prepared cytosolic fractions from cells preincubated with [14C]acetate and used as a donor of radioactive lipids. When this cytosolic fraction was incubated with microsomes isolated separately, radioactive triglyceride (TG) accumulated in the luminal space of the microsomes. The transfer of radioactive TG from the cytosolic fraction to the microsomal lumen was inhibited in the presence of verapamil, suggesting that there is a verapamil-sensitive mechanism for TG transfer across ER membranes that is involved in formation of apoB-containing lipoprotein particles in ER. Verapamil showed no inhibitory effect on microsomal TG transfer protein, a well known lipid transfer protein in ER. We propose from these results that there is novel machinery for transmembrane movement of neutral lipids, which is involved in providing TG for apoB during VLDL assembly in ER.

Assembly and Secretion of Very Low Density Lipoproteins Containing Apolipoprotein B48 in Transfected McA-RH7777 Cells: LACK OF EVIDENCE THAT PALMITOYLATION OF APOLIPOPROTEIN B48 IS REQUIRED FOR LIPOPROTEIN SECRETION

We examined the role of S-linked palmitoylation of human apolipoprotein (apo) B in the assembly and secretion of very low density lipoproteins using recombinant human apoB48. There are four free cysteine residues (Cys(1085), Cys(1396), Cys(1478), and Cys(1635)) within apoB48 that potentially can be palmitoylated. All four cysteine residues were substituted with serine by site-specific mutagenesis. The mutant protein was expressed in transfected rat hepatoma McA-RH7777 cells. Metabolic labeling of the stably transfected cells with iodopalmitic acid analog showed that the mutant apoB48 lacked palmitoylation. The lack of palmitoylation had little impact on the ability of apoB48 to assemble and secrete very low density lipoproteins or high density lipoproteins. Immunocytochemistry experiments using confocal microscopy failed to reveal any major alterations in the intracellular distribution of the mutant apoB48 at steady state. Pulse-chase analysis combined with subcellular fractionation showed no apparent deficiency in the movement of the mutant apoB48 protein from the endoplasmic reticulum to cis/medial Golgi. However, the mutant apoB48 lacking palmitoylation showed retarded movement toward the distal Golgi and increased association (>2-fold) with the membranes of the secretory compartments. A marginal decrease (by 15-20%) in secretion efficiency as compared with that of wild type apoB48 was also observed. These results suggest that lack of palmitoylation may influence the partitioning of apoB48 between microsomal membranes and microsomal lumen, but it does not compromise the ability of apoB48 to assemble lipoproteins.

Protein Unfolding Is Not a Prerequisite for Endoplasmic Reticulum-to-Cytosol Dislocation

We examined the effects of protein folding on endoplasmic reticulum (ER)-to-cytosol transport (dislocation) by exploiting the well-characterized dihydrofolate reductase (DHFR) domain. DHFR retains the capacity to bind folate analogues in the lumen of microsomes and in the ER of intact cells, upon which it acquires a conformation resistant to proteinase K digestion. Here we show that a Class I major histocompatibility complex heavy chain fused to DHFR is still recognized by the human cytomegalovirus-encoded glycoproteins US2 and US11, resulting in dislocation of the fusion protein from the ER in vitro and in vivo. A folded state of the DHFR domain does not impair dislocation of Class I MHC heavy chains in vitro or in living cells. In fact, a slight acceleration of the dislocation of DHFR heavy chain fusion was observed in vitro in the presence of a folate analogue. These results suggest that one or more of the channels used for dislocation can accommodate polypeptides that contain a tightly folded domain of considerable size. Our data raise the possibility that the Sec61 channel can be modified to accommodate a folded DHFR domain for dislocation, but not for translocation into the ER, or that a channel altogether distinct from Sec61 is used for dislocation.

Microsomal Triacylglycerol Transfer Protein Is Required for Lumenal Accretion of Triacylglycerol Not Associated with ApoB, as Well as for ApoB Lipidation

The assembly of very low density lipoproteins in hepatocytes requires the microsomal triacylglycerol transfer protein (MTP). This microsomal lumenal protein transfers lipids, particularly triacylglycerols (TG), between membranes in vitro and has been proposed to transfer TG to nascent apolipoprotein (apo) B in vivo. We examined the role of MTP in the assembly of apoB-containing lipoproteins in cultured murine primary hepatocytes using an inhibitor of MTP. The MTP inhibitor reduced TG secretion from hepatocytes by 85% and decreased the amount of apoB100 in the microsomal lumen, as well as that secreted into the medium, by 70 and 90%, respectively, whereas the secretion of apoB48 was only slightly decreased and the amount of lumenal apoB48 was unaffected. However, apoB48-containing particles formed in the presence of inhibitor were lipid-poor compared with those produced in the absence of inhibitor. We also isolated a pool of apoB-free TG from the microsomal lumen and showed that inhibition of MTP decreased the amount of TG in this pool by approximately 45%. The pool of TG associated with apoB was similarly reduced. However, inhibition of MTP did not directly block TG transfer from the apoB-independent TG pool to partially lipidated apoB in the microsomal lumen. We conclude that MTP is required for TG accumulation in the microsomal lumen and as a source of TG for assembly with apoB, but normal levels of MTP are not required for transferring the bulk of TG to apoB during VLDL assembly in murine hepatocytes.