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J. Biol. Chem., Vol. 275, Issue 22, 17130-17135, June 2, 2000

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Molecular Basis for the Transport of Cytochrome P450 2E1 to the Plasma Membrane^*

Etienne P. A. Neve and Magnus Ingelman-Sundberg

From the Division of Molecular Toxicology, National Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden

Received for publication, February 6, 2000, and in revised form, March 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Endoplasmic reticulum-resident cytochrome P450 enzymes that face the cytosol are present on the plasma membrane of hepatocytes, but the molecular origin for their transport to this compartment has until now remained unknown. The molecular basis for the transport of rat ethanol-inducible cytochrome P450 2E1 (CYP2E1) to the plasma membrane was investigated by transfection of several different mutant cDNAs into mouse H2.35 hepatoma cells. Two NH₂-terminal CYP2E1 mutants were constructed: N⁺⁺2E1, which carried two positive charges in the NH₂ terminus, and 2C-2E1, in which the transmembrane domain of CYP2E1 was replaced with that of CYP2C1, which was previously described to cause retention of CYP2C1 in the endoplasmic reticulum, as well as CYP2E1 COOH-terminally tagged with the vesicular stomatitis virus G protein (VSV-G) epitope (2E1-VSV-G). Immunofluorescent microscopy and cell surface biotinylation experiments revealed that all CYP2E1 variants were present on the extracellular side of the plasma membrane. The VSV-G epitope on CYP2E1 was detected on the outside of the plasma membrane using VSV-G-specific antibodies, indicating that the large COOH-terminal part of CYP2E1 is indeed exposed on the outside of the plasma membrane. The relative levels of CYP2E1, 2C-2E1, and 2E1-VSV-G on the cell surface were found to be about 2% of total cellular enzyme, whereas twice this amount of N⁺⁺2E1 was recovered at the cell surface. Protease protection experiments performed on microsomes isolated from cDNA transfected cells revealed that a small fraction of CYP2E1 and all variant proteins was found to be located in the lumen of the endoplasmic reticulum (type II orientation), whereas the majority of the proteins were in the expected cytosolic or type I orientation. It is concluded that the NH₂-terminal transmembrane domain of CYP2E1 plays a critical role in directing the protein to the cell surface and that topological inversion of a small fraction of CYP2E1 in the endoplasmic reticulum directs the protein to the plasma membrane.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hepatic microsomal cytochrome P450s (P450s)¹ are a large superfamily of enzymes that are known to metabolize a wide variety of both endogenous and exogenous compounds (1, 2). The majority of the P450 enzymes are membrane-bound, predominantly localized in the endoplasmic reticulum (ER) membrane by its hydrophobic NH₂ terminus (3-6), leaving the majority of the protein, including the catalytic domain, exposed on the cytoplasmic side of the ER membrane. The hydrophobic NH₂ terminus of P450 not only is responsible for targeting to and insertion in the ER but also has been shown to cause retention of the protein in the ER (7, 8). The precise mechanism by which the NH₂ terminus of P450s mediates ER retention is not clear, yet there are no known ER retention or retrieval signals present in the sequence. It has been shown that P450 can form oligomeric complexes (9, 10) and is able to form complexes with its redox partners NADPH cytochrome P450 reductase and cytochrome b₅ (11). It could be hypothesized that the formation of oligomeric protein complexes causes retention in the ER by excluding them from the export complexes, as has been demonstrated for proteins residing in the Golgi apparatus (12). However, based on the mobility of CYP2C2 in the ER membrane as determined by photobleaching/fluorescence recovery, this seems to be less probable (13).

Although xenobiotic metabolizing P450s are predominantly localized in the ER, significant levels have been shown to be distributed in mitochondria (14, 15), lysosomes (16), Golgi apparatus (17), peroxisomes (18), and the plasma membrane (PM) (19-22). Immunofluorescent microscopy has indicated that several different P450 enzymes are present on the outer surface of hepatocytes isolated from human and rat liver (19-21). In addition, it was shown that CYP2E1 and CYP2D6 are catalytically active in purified plasma membranes isolated from respectively rat liver hepatocytes and Saccharomyces cerevisiae expressing CYP2D6 cDNA (19, 23). The involvement of the constitutive secretory pathway in the transport to the PM has been suggested. Several forms of P450 have been shown to be present in Golgi apparatus isolated from rat liver (17), and Golgi transport inhibitors were shown to decrease the expression of CYP2B on the PM of rat hepatocytes (24).

P450s localized at the cell surface have been implicated in the pathogenesis of several forms of drug-induced autoimmune hepatitis, and it is likely that PM expressed P450s play a role in the hepatotoxicity associated with this disease (25, 26). Patients suffering from drug-induced hepatitis were shown to have high levels of autoantibodies directed against certain forms of P450 in their blood (27, 28). Until now, much research has been focused on the identification of the epitopes present on P450s that are recognized by these autoantibodies, and many of these epitopes have been mapped around the catalytic site of P450 (29-31). However, the mechanism responsible for the appearance of P450 on the surface of the PM remains unknown. One puzzling aspect is the appearance of P450 on the outer surface of the PM, whereas it displays a cytoplasmic or type I (Ccyt/Nexo) orientation in the ER. In a recent investigation, topologically inverted CYP2D6 was expressed in S. cerevisiae, and it was concluded that topological inversion in the ER membrane was not responsible for directing CYP2D6 to the PM (32).

CYP2E1 plays an important role in the gluconeogenesis, especially during fasting (33, 34); is able to metabolize a wide variety of small hydrophobic compounds, including many known toxic and carcinogenic compounds (34); causes oxidative stress through the production of active oxygen species; and has been implicated in the development of alcoholic liver disease (35). Autoantibodies against both the native CYP2E1 and CYP2E1 hydroxyethyl radical adducts have been observed among alcoholic patients (31, 36).

In the present investigation, the role of the NH₂-terminal transmembrane domain of CYP2E1 for the transport from the ER to the outer surface of the PM was evaluated by monitoring the presence of the protein and NH₂-terminal and COOH-terminal mutants thereof in the ER and plasma membrane utilizing immunofluorescent microscopy, cell surface biotinylation, and protease protection experiments. It is concluded that the COOH-terminal part of CYP2E1 is localized on the outside of the PM and that the molecular mechanism underlying the transport of CYP2E1 to the PM involves the incorporation of a small fraction of CYP2E1 during translation in a lumenal or type II (Cexo/Ncyt) orientation in the ER membrane.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmid Construction-- Full-length rat CYP2E1 cDNA (wt2E1) and N⁺⁺2E1, having A2K and V3R substitutions, were cloned into the mammalian expression vector pCMV5 as described (15). The construct 2E1-VSV-G was formed by the introduction of the 11-amino acid epitope derived from the vesicular stomatitis virus G protein (VSV-G) to CYP2E1. 2E1-VSV-G was generated by polymerase chain reaction amplification using Pfu DNA polymerase (Stratagene, La Jolla, CA), the forward primer 1a, and the reverse primer 2a (Table I), containing the coding sequence for the VSV-G tag. The resulting 2E1-VSV-G cDNA was cloned between the EcoRI and XbaI sites of the pCMV5 expression vector. A chimeric construct in which the 31 NH₂-terminal amino acids of CYP2E1 were replaced by the 28 NH₂-terminal amino acids from rabbit CYP2C1 was constructed as follows. The cDNA of the NH₂ terminus of CYP2C1 was generated by polymerase chain reaction amplification using rabbit liver cDNA (kindly provided by Dr. S. Svensson, Karolinska Institutet, Stockholm, Sweden), the forward primer 1b, and the reverse primer 2b, starting 390 base pairs upstream of the initiation codon. The resulting truncated CYP2C1 cDNA was cloned in between restriction sites EcoRI and XbaI of pCMV5. The pCMV-CYP2C1 plasmid was digested with HindIII and XbaI, leaving the coding sequence for the first 28 amino acids of CYP2C1 in the plasmid. The cDNA of CYP2E1, lacking the coding region for amino acids 1-31 and containing a HindIII site at the 5'end, was generated by polymerase chain reaction amplification using the forward primer 1c and the reverse primer 2c. The resulting cDNA was ligated in between the HindIII and XbaI sites of the restricted pCMV-CYP2C1 plasmid, thereby generating the 2C-2E1 chimera. The correct DNA sequence of all inserts was confirmed by DNA sequencing using the ABI PRISM® dye terminator cycle sequencing kit from Perkin-Elmer.

Cell Culture and Western Blot Analysis-- H2.35 cells, mouse SV-40 transformed hepatocytes, were purchased from the American Type Culture Collection (Manassas, VA) and grown and transfected with the cationic lipid DMRIE-C (Life Technologies, Inc.) as described previously (15). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk and incubated with the appropriate antibodies as described previously (17). Immunoreactive bands were visualized by the enhanced chemiluminescence method (Pierce).

Immunofluorescent Microscopy-- Cells grown and transfected on glass coverslips were washed three times in phosphate-buffered saline (PBS), fixed in 2% formaldehyde in PBS for 10 min, and either permeabilized with 0.2% Triton X-100 or not permeabilized. After blocking in 10% fetal bovine serum in PBS for 2.5 h, primary antibodies were incubated in the presence of 3% bovine serum albumin (w/v) in PBS for 90 min followed by fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (1:500 dilution) in the presence of 3% bovine serum albumin in PBS for 90 min. Stained cells were carefully mounted with a drop of Vecta-Shield (Vector Laboratories, Burlingame, CA) on a glass slide. The glass slides were viewed under an Olympus BX60 microscope equipped with an Olympus PM20 camera (Olympus, Tokyo, Japan).

Chlorzoxazone Hydroxylation-- The catalytic activity of the CYP2E1 variants was determined by monitoring the hydroxylation of chlorzoxazone in the microsomal fractions isolated from transfected H2.35 cells essentially as described before (17) with some modifications. The microsomes were diluted with 50 mM phosphate buffer, pH 7.4, to a protein concentration of 0.2 mg/ml and were incubated in the presence of 0.5 mM chlorzoxazone in the presence or absence of a NADPH generating system (0.2 mM NADPH, 2.0 mM glucose-6-phosphate and 3 units/ml glucose-6-phosphate dehydrogenase). After 10 min, the reaction was terminated by the addition of orthophosphoric acid, an internal standard (0.04 µg of acetaminophen) was added, and the samples were extracted twice with 1 ml of dichloromethane. The organic phases were collected and evaporated under a nitrogen flow, and the remaining residue was dissolved into 100 µl of mobile phase. The samples were analyzed on a LKB 2150 high pressure liquid chromatography system (Amersham Pharmacia Biotech) using a LiChrospher^®100 RP-8 prepacked column (Merck, Darmstadt, Germany), and the mobile phase consisted of acetonitrile:0.5% orthophosphoric acid (0.25:0.75) at a flow rate of 1 ml/min. Both the product 6-hydroxychlorzoxazone and the internal standard were detected using a LC-4C amperometric detector (Bioanalytical Systems, West Lafayette, IN) with a potential of 0.9 V over the electrochemical cell.

Cell Surface Biotinylation-- H2.35 cells were transfected in 35-mm dishes, and 30 h posttransfection, the cells were biotinylated. Dishes containing the transfected cells were transferred to ice and washed twice with ice-cold PBS. The biotinylation reaction was carried out on ice by incubating the cells with 35 µl of the ECL protein biotinylation reagent biotinamidocaproate N-hydroxysuccinamide ester (Amersham Pharmacia Biotech) per ml of PBS under gentle shaking. After 30 min, the biotinylation reagent was removed, and cells were washed three times with 50 mM glycine in PBS to quench the remaining reagent and solubilized in 1.3 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, containing 250 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml antipain, and 10 µg/ml leupeptin). The biotinylated proteins were recovered from the precleared cell lysate by incubation with streptavidin-agarose beads (50 µl of swollen gel) in a vertical rotating platform for 2 h. After washing of the streptavidin-agarose beads, three times with lysis buffer and twice with PBS, the biotinylated proteins were eluted by boiling the beads in 100 µl of SDS-PAGE sample buffer, and the eluted proteins were analyzed by Western blotting. Protein levels were quantified by densitometric analysis on a personal densitometer (Molecular Dynamics, Sunnyvale, CA).

Protease Protection Assay-- The membrane topology of the CYP2E1 variants in the ER was determined by a protease protection assay as described (15). Briefly, cells transfected with the CYP2E1 variants were harvested and homogenized in microsome isolation buffer (50 mM Hepes, pH 7.4, containing 0.25 M sucrose, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride) by Dounce homogenization. After removal of the nuclear and mitochondrial fractions, the microsomal fraction was isolated by ultracentrifugation (60 min at 100,000 × g). The microsomal fraction was diluted with 50 mM Tris-HCl buffer, pH 8.0, containing 10 mM CaCl₂ and 150 mM NaCl to a protein concentration of 1 mg/ml. The samples were incubated with proteinase K (83 µg/ml) (Roche Molecular Biochemicals) in the presence or absence of 0.5% Triton X-100 at 37 °C. After 30 min, the reaction was stopped by the addition of an equal volume of ice-cold 50% trichloroacetic acid, and proteins were allowed to precipitate on ice for 30 min. The precipitated proteins were centrifuged down, washed with ice-cold acetone, dissolved in SDS-PAGE sample buffer, and analyzed by Western blotting.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transient Expression of the CYP2E1 Variants in H2.35 Cells-- The mammalian expression vector pCMV-5 containing mutant cDNAs encoding the CYP2E1 variants was transiently expressed in a mouse hepatoma cell line, H2.35 cells. The constructs made were as follows (Fig. 1): (i) N⁺⁺2E1, which contains two positively charged amino acid residues in the NH₂ terminus, a modification that was demonstrated in CYP2D6 and CYP2C11 to result in lumenal or type II topology in the ER membrane in COS cells (32, 37); (ii) 2C-2E1, in which the NH₂-terminal transmembrane domain of CYP2E1 had been replaced with that of CYP2C1, which previously has been shown to be sufficient for retention of CYP2C1 in the ER membrane in COS cells (7); and (iii) 2E1-VSV-G, CYP2E1 COOH-terminally tagged with the epitope from VSV-G in order to determine the localization of the COOH terminus using antibodies directed toward the VSV-G tag. The intracellular distribution of the various CYP2E1 proteins expressed was determined by immunofluorescent microscopy performed on fixed permeabilized cells stained with CYP2E1-specific antibodies (Fig. 2). wt2E1 (Fig. 2B) and N⁺⁺2E1 (Fig. 2D) were predominantly localized in the ER, whereas cells transfected with empty plasmid (Fig. 2A) contained no significant levels of CYP2E1 (15). The other two CYP2E1 variants, 2C-2E1 (Fig. 2C) and 2E1-VSV-G (Fig. 2E), exhibited the same intracellular distribution as wt2E1, indicative of their ER localization. Analysis by Western blotting of microsomes isolated from cells transfected with these CYP2E1 variants revealed that all of them were expressed at their correct size (data not shown). When permeabilized cells expressing 2E1-VSV-G were stained with antibodies specifically recognizing the VSV-G tag, a staining pattern identical to that seen in Fig. 2E was observed (not shown).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   The primary structure of the CYP2E1 variants used. Only the regions in CYP2E1 that were modified are shown. In N++2E1, the amino acid substitutions at positions 2 and 3, A2K and V3R, are depicted in a black box. The NH2 terminus of CYP2C1 is shown in a gray box, and the COOH-terminal VSV-G tag is shown in a white box.

View larger version (59K): [in this window] [in a new window]   Fig. 2.   The intracellular distribution of wt2E1, 2C-2E1, N++2E1, and 2E1-VSV-G as determined by immunofluorescent microscopy. H2.35 cells were transfected with empty plasmid (A), wt2E1 (B), 2C-2E1 (C), N++2E1 (D), and 2E1-VSV-G (E), fixed, permeabilized, and incubated with CYP2E1-specific antibodies (1:5000 dilution), followed by anti-rabbit fluorescein isothiocyanate-conjugated antibodies (1:500 dilution).

The catalytic activity of CYP2E1 and its variants was determined by measuring the NADPH-supported formation of 6-hydroxychlorzoxazone in microsomes isolated from the transfected cells. Fig. 3 shows that CYP2E1 and all the CYP2E1 variants displayed similar catalytic activities. The fact that all CYP2E1 variants were catalytically active indicated that they were correctly folded and incorporated into the ER membrane and were able to interact with NADPH cytochrome P450 reductase. Cells transfected with empty plasmid displayed no significant formation of 6-hydroxychlorzoxazone (not shown), indicating no endogenous CYP2E1 in these cells.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Catalytic activity of wt2E1, 2C-2E1, N++2E1, and 2E1-VSV-G in microsomes isolated from transfected H2.35 cells. The NADPH-supported formation of 6-hydroxychlorzoxazone (6-OH-CZN) was determined as described under "Experimental Procedures," and the results represent the mean ± S.E. of three independent determinations.

Cell Surface Expression of the CYP2E1 Variants-- The distribution of wt2E1 and the CYP2E1 variants on the outside of the PM was also studied by immunofluorescent microscopy performed on fixed nonpermeabilized cells. Fig. 4 shows transfected H2.35 cells stained with CYP2E1-specific antibodies. wt2E1 (Fig. 4B), 2C-2E1 (Fig. 4C), N⁺⁺2E1 (Fig. 4D), and 2E1-VSV-G (Fig. 4E) all displayed a weak but significant cell surface staining, which was absent in cells transfected with empty plasmid (Fig. 4A). Typically, the staining pattern observed was not uniformly distributed over the entire surface of the cell but appeared as discrete patches on the cell surface. The extracellular localization of CYP2E1 was further confirmed by using the COOH-terminally VSV-G-tagged form of CYP2E1. When nonpermeabilized cells transfected with 2E1-VSV-G were stained with antibodies specifically recognizing the VSV-G epitope, a staining pattern identical to that seen with CYP2E1-specific antibodies was observed (cf. Fig. 5B and Fig. 4E). Again, no staining was observed in cells transfected with the empty plasmid (Fig. 5A). This clearly demonstrated that the COOH-terminal part of the CYP2E1 protein is located on the outer surface of the PM and that the COOH terminus of CYP2E1 is exposed at the surface of the protein.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Cell surface localization of wt2E1, 2C-2E1, N++2E1, and 2E1-VSV-G as determined by immunofluorescent microscopy performed on nonpermeabilized H2.35 cells. Cells were transfected with empty plasmid (A), wt2E1 (B), 2C-2E1 (C), N++2E1 (D), and 2E1-VSV-G (E), fixed, and incubated with CYP2E1-specific antibodies followed by anti-rabbit fluorescein isothiocyanate-conjugated antibodies.

View larger version (62K): [in this window] [in a new window]   Fig. 5.   The COOH terminus of CYP2E1 is exposed on the outside of the plasma membrane. H2.35 cells were transfected with empty plasmid (A) and 2E1-VSV-G (B), fixed, and incubated with antibodies specifically recognizing the VSV-G epitope (1:1000 dilution), followed by anti-rabbit fluorescein isothiocyanate-conjugated antibodies (1:500 dilution).

To study the expression of CYP2E1 on the outer surface of the PM in a more quantitative manner, a protein biotinylation method was developed. After transfection, cells were either permeabilized, to permit biotinylation of total cellular protein, or not permeabilized, to enable it to biotinylate cell surface proteins only. Fig. 6A shows biotinylated proteins isolated from permeabilized (lanes 1) and nonpermeabilized (lanes 2) H2.35 cells expressing wt2E1. The isolated biotinylated proteins were subjected to Western blot analysis for CYP2E1 and the ER resident proteins reductase and calnexin. Only CYP2E1 was detected on the cell surface (lanes 2), whereas reductase and calnexin were absent. These results revealed not only that reductase and calnexin were absent from the outside of the PM but also that there was no significant contamination from intracellular proteins. CYP2E1 present at the cell surface was shown to have an electrophoretic mobility on SDS-PAGE similar to that of CYP2E1 detected in the microsomal fraction isolated from transfected cells, indicating that there was no posttranslational modification (Fig. 6A). The relative protein levels of the transfected CYP2E1 and its variants present on the PM of the cells as revealed by Western blot analysis were expressed as a percentage of the total cellular amount of the CYP2E1 variant proteins (Fig. 6B). There were no significant differences observed between the levels of wt2E1, 2C-2E1, and 2E1-VSV-G present on the PM, all of them constituting around 2-3% of the total cellular amount. The amount of N⁺⁺2E1 present on the PM, however, was found to be more than twice the amount of wt2E1 present on the PM; 4.7 ± 0.9% (mean ± S.E.) of N⁺⁺2E1 was present on the PM, whereas 1.8 ± 0.2% of wt2E1 was found (p < 0.005).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Cell surface expression of CYP2E1. A, cell surface biotinylation of H2.35 cells expressing wt2E1. H2.35 cells were transfected with wt2E1, either permeabilized (lane 1) or not permeabilized (lane 2) and biotinylated as described under "Experimental Procedures." The biotinylated proteins, which were recovered with streptavidin-agarose, were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with CYP2E1-specific antibodies (left panel), NADPH cytochrome P450 reductase-specific antibodies (middle panel), or calnexin-specific antibodies (right panel). Microsomes (mic) isolated from wt2E1 transfected cells are included as a reference. Calnexin is indicated by an asterisk. B, relative amounts of wt2E1, 2C-2E1, N++2E1, and 2E1-VSV-G expressed at the cell surface of H2.35 cells as determined by biotinylation. The levels of the transfected proteins were determined by Western blotting using CYP2E1-specific antibodies and quantified by densitometric analysis. The amount of transfected protein present on the outside of the cell surface (nonpermeabilized cells) was expressed as a percentage of the total cellular amount of transfected protein (permeabilized cells). Values are mean ± S.E. of at least three independent transfections. The amount of N++2E1 was significantly different when compared with wt2E1 (p < 0.005).

Protease Protection Assay-- The topology of wt2E1, N⁺⁺2E1, 2C-2E1, and 2E1-VSV-G in the ER membrane was studied by a protease protection assay. Microsomes isolated from transfected cells were incubated with or without proteinase K in the presence or absence of detergent (Fig. 7). As expected, the majority of the CYP2E1 protein was digested in the presence of proteinase K only, and the same was observed for all the CYP2E1 variants, indicating that the majority of these proteins are exposed at the cytosolic side of the ER membrane. However, consistently a small fraction of the expressed proteins was resistant toward digestion in the presence of proteinase K alone, whereas NADPH cytochrome P450 reductase, a protein that faces the cytosol, was completely digested (Fig. 7, bottom panel). Complete digestion of CYP2E1 and its variants could only be achieved in the presence of both proteinase K and detergent, conditions that were required to completely digest ERp29 (Fig. 7, middle panel), a protein residing in the lumen of the ER similar to protein-disulfide isomerase (38, 39). These results indicate that CYP2E1 and its variants are incorporated in a dual topology in the ER membrane. Most of the protein is incorporated in a cytosolic or type I (Ccyt/Nexo) orientation, whereas a small fraction of the protein is in a lumenal or type II (Cexo/Ncyt) orientation. Interestingly, the proteinase K resistant CYP2E1 and its variants appeared to display a slightly higher molecular weight, as judged by its mobility on SDS-PAGE. Treatment of these protease resistant fractions with the enzyme N-glycosidase F (overnight incubation of 0.1 mg of the proteinase-treated microsomes with 80 units of N-glycosidase F/ml at 37 °C, as recommended by the manufacturer), which is able to cleave all types of Asp-bound N-glycans, did not result in a decrease in apparent molecular weight (data not shown), indicating that N-glycosylation was not responsible for this decrease in mobility observed on SDS-PAGE. At present, we do not know the molecular basis for this difference in mobility, but we cannot exclude other types of posttranslational modifications, such as phosphorylation or alkylation.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Protease protection assay performed on microsomes isolated from H2.35 cells transfected with wt2E1, 2C-2E1, 2E1-VSV-G, and N++2E1. Microsomal membranes were incubated with or without proteinase K (Prot K) in the presence or absence of 0.5% Triton X-100, as indicated in the figure. The transfected proteins were analyzed by Western blotting for the presence of CYP2E1 reactive protein (top panel). In addition, the presence of the ER resident lumenal protein ERp29 (middle panel) and the ER resident protein NADPH cytochrome P450 reductase (RED) (bottom panel) were determined by Western blotting.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The presence of CYP2E1 and the three CYP2E1 variants 2C-2E1, N⁺⁺2E1, and 2E1-VSV-G on the extracellular side of the PM was clearly demonstrated by the results obtained with both immunofluorescent microscopy and cell surface biotinylation experiments. In addition, the VSV-G tag present at the COOH terminus of CYP2E1 could be detected on the PM of nonpermeabilized 2E1-VSV-G transfected cells, indicating that the large COOH-terminal domain, including the catalytical part of CYP2E1, is indeed exposed at the cell surface.

Unlike other cell surface proteins, such as the transferrin receptor, CYP2E1 and its variants were not uniformly distributed over the PM but instead located at discrete patches on the cell surface. It is well documented that certain proteins, such as influenza virus hemagglutinin, are found at discrete patches on the PM, also called rafts, which are structures consisting of sphingolipids and cholesterol with associated proteins (40). The reason for the clustered PM distribution of CYP2E1 is not known, but a similar pattern has been observed for PM-localized CYP2E1 in constitutive FGC4 cells and in V79 cells stably transfected with CYP2E1 cDNA (22).

By using cell surface biotinylation and subsequent treatment of the membranes with streptavidin beads followed by Western blot analysis, the relative levels of wt2E1, 2C-2E1, and 2E1-VSV-G present on the cell surface were found to be very similar (2-3% of the total cellular content), whereas the amount of N⁺⁺2E1 present on the cell surface was found to be significantly higher (4.7% of the total cellular N⁺⁺2E1 content). This might indicate a higher tendency for the double NH₂-terminally charged CYP2E1 variant to be incorporated in the lumenal type II orientation during translation. Also using cell surface biotinylation, Amarneh and Simpson (41) reported similar levels of CYP19 to be present at the PM when CYP19 cDNA was transfected into COS cells. Other reports described that the relative level of CYP2E1 present in purified plasma membranes isolated from rat hepatocytes was approximately 16% of the level present in the microsomal fraction, as determined by Western blot analysis (19).

The molecular mechanism responsible for the transport of CYP2E1 from the ER to the outer surface of the PM was shown to involve topological inversion of a small fraction of CYP2E1 in the ER membrane. The protease protection data clearly demonstrate that a small part of the CYP2E1 protein and its variants are incorporated in an opposite topology in the ER membrane. The CYP2E1 having a type II topology is apparently not effectively retained in the ER and subsequently transported to the outer surface of the PM through the constitutive secretory pathway. By analogy, NADPH-cytochrome P450 reductase was found to be completely digested in the ER by proteinase K alone and not to be present on the outside of the PM as revealed by the biotinylation experiments. Recently, a similar conclusion was reached concerning the PM transport for microsomal epoxide hydrolase (42). A N-glycosylation site was engineered into the protein and based on the appearance of glycosylated epoxide hydrolase at the PM, a dual topology for the ER integration was suggested. The incorporation of membrane proteins in a dual ER topology has also been observed for ductin (43) and the prion protein (44). Ductin in its lumenal orientation was shown to be a component of the vacuolar H⁺-ATPase, and in its cytosolic orientation, it serves as part of a connexon channel of gap junctions. The introduction of positive charges in the NH₂ terminus of CYP2D6 (32) and CYP2C11 (37) resulted in a lumenal (type II) orientation in the ER membrane when the mutant cDNAs were expressed in COS cells. The introduction of two positive charges in the NH₂ terminus of CYP2E1 (N⁺⁺2E1), however, did not result in complete inversion in the ER membrane in the hepatoma cells used; the proteolytic data demonstrated that the majority of the protein still is incorporated in the ER in a cytosolic orientation. The cell surface biotinylation experiments, on the other hand, indicated a higher extent of lumenal orientation of the N⁺⁺2E1 variant, as revealed by its higher expression level at the PM. The low levels of the lumenally oriented CYP2E1 variants made it difficult to accurately quantify the relative amounts of the lumenally oriented protein. It appeared that the N⁺⁺2E1 variant displayed slightly more resistance toward proteolytic digestion in the absence of detergent than the other CYP2E1 variants, further indicating a higher extent of lumenal orientation (see Fig. 7). Despite the presence of two potential glycosylation signals (Asp-238 and Asp-288) in CYP2E1 and its lumenal orientation, no glycosylation could be observed because the CYP2E1 present on the PM displayed the same electrophoretic mobility as the CYP2E1 present in the ER. In addition, CYP2E1 present in the Golgi apparatus was previously shown to have mobility in SDS-PAGE identical to that of ER-localized CYP2E1 (17).

The NH₂-terminal transmembrane domain of CYP2C1 was demonstrated to be sufficient for retention in the ER membrane in COS cells. Thus, CYP2C1 was found not to be present in the Golgi apparatus or on the PM when transiently expressed (7). However, when the NH₂-terminal transmembrane domain of CYP2E1 was replaced with the transmembrane anchor of CYP2C1, no effect on the PM expression was observed. Similar levels of wt2E1 and 2C-2E1 were detected on the cell surface. The inability of the transmembrane domain of CYP2C1 to retain the 2C-2E1 chimera in the ER membrane may suggest additional signals that could be present in the large COOH-terminal part of the CYP2E1 protein and that direct the 2C-2E1 protein to the PM. This is despite the efficient ER retention signal present in the transmembrane domain of CYP2C1.

Several forms of P450, such as CYP2E1, CYP1A2, and CYP2D6, have been demonstrated to exist in a catalytically active form when present at the PM (19, 20, 23). This implies that the reductase is also present at the PM in order to supply the P450 with reducing equivalents. Our cell surface biotinylation experiments revealed that reductase is not present on the outer surface of the PM in H2.35 cells (Fig. 6A) and accordingly was also completely digested in the protease protection experiments in the absence of detergent. In S. cerevisiae, the reductase was found to be present on the PM facing the cytoplasm, whereas CYP2D6 faced the extracellular space (23). Similar results regarding the relative orientation of CYP2E1 and reductase in the PM of the H2.35 cells were also obtained. In order for the reductase to transfer its reducing equivalents to the P450 present on the extracellular side of the PM, the presence of an electron carrier to transfer the electrons from reductase to CYP2D6 was postulated by Loeper et al. (32). The presence of a catalytically active CYP2E1 at the cell surface could be of major importance from a toxicological point of view because it is well known that CYP2E1 is involved in the bioactivation of a great number of carcinogenic and toxic compounds (34), and adducts formed by CYP2E1 have been implicated to be of importance for the formation of autoantibodies (36).

In conclusion, the data presented here demonstrate a mechanism underlying the transport of CYP2E1 to the outside of the PM. Protease protection studies showed that CYP2E1 is incorporated in the ER membrane in two different topologies. The topologically inverted fraction of CYP2E1 appears to be transported via the constitutive secretory pathway, involving the Golgi apparatus, to the outer surface of the PM, where its appearance might have important toxicological consequences.

	ACKNOWLEDGEMENTS

We thank Dr. Souren Mkrtchian for valuable discussions, Dr. Mikael Oscarson for help with cloning, Dr. Stefan Svensson for supplying the rabbit cDNA, and Prof. Sandra Cecatelli for use of the immunofluorescent microscope.

	FOOTNOTES

^* This study was supported by grants from the Swedish Medical Research Council and AstraZeneca.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 46-8-7287762; Fax: 46-8-337327; E-mail etienne.neve@imm.ki.se.

Published, JBC Papers in Press, March 19, 2000, DOI 10.1074/jbc.M000957200

	ABBREVIATIONS

The abbreviations used are: P450, cytochrome P450; PM, plasma membrane; ER, endoplasmic reticulum; reductase, NADPH cytochrome P450 reductase; PBS, phosphate-buffered saline; VSV-G, vesicular stomatitis virus G protein; PAGE, polyacrylamide gel electrophoresis; wt, wild-type.

	REFERENCES

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