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• W2023308622 abstract "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. 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. cytochrome P450 plasma membrane endoplasmic reticulum NADPH cytochrome P450 reductase phosphate-buffered saline vesicular stomatitis virus G protein polyacrylamide gel electrophoresis wild-type 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.Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. Pharmacogenetics. 1996; 6: 1-42Crossref PubMed Scopus (2620) Google Scholar, 2.Omura T. Biochem. Biophys. Res. Commun. 1999; 266: 690-698Crossref PubMed Scopus (190) Google Scholar). The majority of the P450 enzymes are membrane-bound, predominantly localized in the endoplasmic reticulum (ER) membrane by its hydrophobic NH2terminus (3.Bar-Nun S. Kreibich G. Adesnik M. Alterman L. Negishi M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 965-969Crossref PubMed Scopus (129) Google Scholar, 4.Monier S. Van Luc P. Kreibich G. Sabatini D.D. Adesnik M. J. Cell Biol. 1988; 107: 457-470Crossref PubMed Scopus (118) Google Scholar, 5.Sakaguchi M. Mihara K. Sato R. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3361-3364Crossref PubMed Scopus (100) Google Scholar, 6.Sakaguchi M. Mihara K. Sato R. EMBO J. 1987; 6: 2425-2431Crossref PubMed Scopus (181) Google Scholar), 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.Ahn K. Szczesna-Skorupa E. Kemper B. J. Biol. Chem. 1993; 268: 18726-18733Abstract Full Text PDF PubMed Google Scholar, 8.Murakami K. Mihara K. Omura T. J. Biochem. 1994; 116: 164-175Crossref PubMed Scopus (68) Google Scholar). 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.Myasoedova K.N. Berndt P. FEBS Lett. 1990; 275: 235-238Crossref PubMed Scopus (25) Google Scholar, 10.Schwarz D. Pirrwitz J. Meyer H.W. Coon M.J. Ruckpaul K. Biochem. Biophys. Res. Commun. 1990; 171: 175-181Crossref PubMed Scopus (35) Google Scholar) and is able to form complexes with its redox partners NADPH cytochrome P450 reductase and cytochromeb 5 (11.Ingelman-Sundberg M. Ortiz de Montellano P.R. Cytochrome P450 Structure, Mechanisms and Biochemistry. Plenum Press, New York1986: 119-160Google Scholar). 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.Munro S. Trends Cell Biol. 1998; 8: 11-15Abstract Full Text PDF PubMed Scopus (218) Google Scholar). However, based on the mobility of CYP2C2 in the ER membrane as determined by photobleaching/fluorescence recovery, this seems to be less probable (13.Szczesna-Skorupa E. Chen C.D. Rogers S. Kemper B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14793-14798Crossref PubMed Scopus (55) Google Scholar). Although xenobiotic metabolizing P450s are predominantly localized in the ER, significant levels have been shown to be distributed in mitochondria (14.Addya S. Anandatheerthavarada H.K. Biswas G. Bhagwat S.V. Mullick J. Avadhani N.G. J. Cell Biol. 1997; 139: 589-599Crossref PubMed Scopus (133) Google Scholar, 15.Neve E.P.A. Ingelman-Sundberg M. FEBS Lett. 1999; 460: 309-314Crossref PubMed Scopus (58) Google Scholar), lysosomes (16.Ronis M.J. Johansson I. Hultenby K. Lagercrantz J. Glaumann H. Ingelman-Sundberg M. Eur. J. Biochem. 1991; 198: 383-389Crossref PubMed Scopus (76) Google Scholar), Golgi apparatus (17.Neve E.P.A. Eliasson E. Pronzato M.A. Albano E. Marinari U. Ingelman-Sundberg M. Arch. Biochem. Biophys. 1996; 333: 459-465Crossref PubMed Scopus (47) Google Scholar), peroxisomes (18.Pahan K. Smith B.T. Singh A.K. Singh I. Free Radic. Biol. Med. 1997; 23: 963-971Crossref PubMed Scopus (43) Google Scholar), and the plasma membrane (PM) (19.Wu D. Cederbaum A.I. Hepatology. 1992; 15: 515-524Crossref PubMed Scopus (61) Google Scholar, 20.Loeper J. Descatoire V. Maurice M. Beaune P. Feldmann G. Larrey D. Pessayre D. Hepatology. 1990; 11: 850-858Crossref PubMed Scopus (79) Google Scholar, 21.Loeper J. Descatoire V. Maurice M. Beaune P. Belghiti J. Houssin D. Ballet F. Feldmann G. Guengerich F.P. Pessayre D. Gastroenterology. 1993; 104: 203-216Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 22.Eliasson E. Kenna J.G. Mol. Pharmacol. 1996; 50: 573-582PubMed Google Scholar). Immunofluorescent microscopy has indicated that several different P450 enzymes are present on the outer surface of hepatocytes isolated from human and rat liver (19.Wu D. Cederbaum A.I. Hepatology. 1992; 15: 515-524Crossref PubMed Scopus (61) Google Scholar, 20.Loeper J. Descatoire V. Maurice M. Beaune P. Feldmann G. Larrey D. Pessayre D. Hepatology. 1990; 11: 850-858Crossref PubMed Scopus (79) Google Scholar, 21.Loeper J. Descatoire V. Maurice M. Beaune P. Belghiti J. Houssin D. Ballet F. Feldmann G. Guengerich F.P. Pessayre D. Gastroenterology. 1993; 104: 203-216Abstract Full Text PDF PubMed Scopus (122) Google Scholar). 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.Wu D. Cederbaum A.I. Hepatology. 1992; 15: 515-524Crossref PubMed Scopus (61) Google Scholar, 23.Loeper J. Louerat-Oriou B. Duport C. Pompon D. Mol. Pharmacol. 1998; 54: 8-13Crossref PubMed Scopus (33) Google Scholar). 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.Neve E.P.A. Eliasson E. Pronzato M.A. Albano E. Marinari U. Ingelman-Sundberg M. Arch. Biochem. Biophys. 1996; 333: 459-465Crossref PubMed Scopus (47) Google Scholar), and Golgi transport inhibitors were shown to decrease the expression of CYP2B on the PM of rat hepatocytes (24.Robin M.A. Maratrat M. Loeper J. Durand-Schneider A.M. Tinel M. Ballet F. Beaune P. Feldmann G. Pessayre D. Gastroenterology. 1995; 108: 1110-1123Abstract Full Text PDF PubMed Scopus (54) Google Scholar). 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.Pirmohamed M. Park B.K. Ioannides C. Cytochromes P450 Metabolic and Toxicological Aspects. CRC Press, New York1996: 329-354Google Scholar, 26.Beaune P. Pessayre D. Dansette P. Mansuy D. Manns M. Adv. Pharmacol. 1994; 30: 199-245Crossref PubMed Scopus (86) Google Scholar). Patients suffering from drug-induced hepatitis were shown to have high levels of autoantibodies directed against certain forms of P450 in their blood (27.Bourdi M. Larrey D. Nataf J. Bernuau J. Pessayre D. Iwasaki M. Guengerich F.P. Beaune P.H. J. Clin. Invest. 1990; 85: 1967-1973Crossref PubMed Scopus (191) Google Scholar, 28.Beaune P. Dansette P.M. Mansuy D. Kiffel L. Finck M. Amar C. Leroux J.P. Homberg J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 551-555Crossref PubMed Scopus (300) Google Scholar). 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.Belloc C. Gauffre A. Andre C. Beaune P.H. Pharmacogenetics. 1997; 7: 181-186Crossref PubMed Scopus (38) Google Scholar, 30.Lecoeur S. Andre C. Beaune P.H. Mol. Pharmacol. 1996; 50: 326-333PubMed Google Scholar, 31.Lytton S.D. Helander A. Zhang-Gouillon Z.Q. Stokkeland K. Bordone R. Arico S. Albano E. French S.W. Ingelman-Sundberg M. Mol. Pharmacol. 1999; 55: 223-233Crossref PubMed Scopus (63) Google Scholar). 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 inS. cerevisiae, and it was concluded that topological inversion in the ER membrane was not responsible for directing CYP2D6 to the PM (32.Loeper J. Le Berre A. Pompon D. Mol. Pharmacol. 1998; 53: 408-414Crossref PubMed Scopus (20) Google Scholar). CYP2E1 plays an important role in the gluconeogenesis, especially during fasting (33.Bondoc F.Y. Bao Z. Hu W.Y. Gonzalez F.J. Wang Y. Yang C.S. Hong J.Y. Biochem. Pharmacol. 1999; 58: 461-463Crossref PubMed Scopus (74) Google Scholar, 34.Ronis M.J.J. Lindros K.O. Ingelman-Sundberg M. Ioannides C. Cytochromes P450 Metabolic and Toxicological Aspects. CRC Press, Inc., New York1996: 211-239Google Scholar); is able to metabolize a wide variety of small hydrophobic compounds, including many known toxic and carcinogenic compounds (34.Ronis M.J.J. Lindros K.O. Ingelman-Sundberg M. Ioannides C. Cytochromes P450 Metabolic and Toxicological Aspects. CRC Press, Inc., New York1996: 211-239Google Scholar); causes oxidative stress through the production of active oxygen species; and has been implicated in the development of alcoholic liver disease (35.Ingelman-Sundberg M. Johansson I. Yin H. Terelius Y. Eliasson E. Clot P. Albano E. Alcohol. 1993; 10: 447-452Crossref PubMed Scopus (211) Google Scholar). Autoantibodies against both the native CYP2E1 and CYP2E1 hydroxyethyl radical adducts have been observed among alcoholic patients (31.Lytton S.D. Helander A. Zhang-Gouillon Z.Q. Stokkeland K. Bordone R. Arico S. Albano E. French S.W. Ingelman-Sundberg M. Mol. Pharmacol. 1999; 55: 223-233Crossref PubMed Scopus (63) Google Scholar, 36.Clot P. Albano E. Eliasson E. Tabone M. Arico S. Israel Y. Moncada C. Ingelman-Sundberg M. Gastroenterology. 1996; 111: 206-216Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). 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. 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.Neve E.P.A. Ingelman-Sundberg M. FEBS Lett. 1999; 460: 309-314Crossref PubMed Scopus (58) Google Scholar). 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 (TableI), containing the coding sequence for the VSV-G tag. The resulting 2E1-VSV-G cDNA was cloned between theEcoRI 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 andXbaI of pCMV5. The pCMV-CYP2C1 plasmid was digested withHindIII 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 theHindIII 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.Table ISequences of the oligonucleotides used as polymerase chain reaction primersPrimerSequence (5′ to 3′)Primer 1aGAC GAA TTC ATG GCG GTT CTT GGC ATCPrimer 1bGAC GAA TTC ATG GAT CCT GTG GTG GTG CTG GGGPrimer 1cCTC AAG CTT CCC CCA GGA CCT TTC CCTPrimer 2aGAT TCT AGA TCA CTT GCG CAG CCT ATT CAT CTC TAT ATC GGT GTA AAT TGA ACG GGG AAT GAC ACA GAGPrimer 2bGAC TCT AGA GGT CAT GAG CGA GAA GCG CCG GGTPrimer 2cGAC TCT AGA TCA TGA ACG GGG AAT GACThe coding sequence for the VSV-G epitope is underlined, and the restriction sites are depicted in italics. Open table in a new tab The coding sequence for the VSV-G epitope is underlined, and the restriction sites are depicted in italics. 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.Neve E.P.A. Ingelman-Sundberg M. FEBS Lett. 1999; 460: 309-314Crossref PubMed Scopus (58) Google Scholar). 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.Neve E.P.A. Eliasson E. Pronzato M.A. Albano E. Marinari U. Ingelman-Sundberg M. Arch. Biochem. Biophys. 1996; 333: 459-465Crossref PubMed Scopus (47) Google Scholar). Immunoreactive bands were visualized by the enhanced chemiluminescence method (Pierce). 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). 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.Neve E.P.A. Eliasson E. Pronzato M.A. Albano E. Marinari U. Ingelman-Sundberg M. Arch. Biochem. Biophys. 1996; 333: 459-465Crossref PubMed Scopus (47) Google Scholar) with some modifications. The microsomes were diluted with 50 mmphosphate 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. 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 biotinamidocaproateN-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 mmglycine 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). The membrane topology of the CYP2E1 variants in the ER was determined by a protease protection assay as described (15.Neve E.P.A. Ingelman-Sundberg M. FEBS Lett. 1999; 460: 309-314Crossref PubMed Scopus (58) Google Scholar). 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. 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.Loeper J. Le Berre A. Pompon D. Mol. Pharmacol. 1998; 53: 408-414Crossref PubMed Scopus (20) Google Scholar, 37.Sato T. Sakaguchi M. Mihara K. Omura T. EMBO J. 1990; 9: 2391-2397Crossref PubMed Scopus (80) Google Scholar); (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.Ahn K. Szczesna-Skorupa E. Kemper B. J. Biol. Chem. 1993; 268: 18726-18733Abstract Full Text PDF PubMed Google Scholar); 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. 2 B) and N++2E1 (Fig. 2 D) were predominantly localized in the ER, whereas cells transfected with empty plasmid (Fig.2 A) contained no significant levels of CYP2E1 (15.Neve E.P.A. Ingelman-Sundberg M. FEBS Lett. 1999; 460: 309-314Crossref PubMed Scopus (58) Google Scholar). The other two CYP2E1 variants, 2C-2E1 (Fig. 2 C) and 2E1-VSV-G (Fig. 2 E), 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.2 E was observed (not shown).Figure 2The 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 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. 4 B), 2C-2E1 (Fig. 4 C), N++2E1 (Fig. 4 D), and 2E1-VSV-G (Fig. 4 E) all displayed a weak but significant cell surface staining, which was absent in cells transfected with empty plasmid (Fig. 4 A). 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.5 B and Fig. 4 E). Again, no staining was observed in cells transfected with the empty plasmid (Fig. 5 A). 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.Figure 5The 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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.6 A 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 transfecte" @default.
• W2023308622 created "2016-06-24" @default.
• W2023308622 creator A5012663065 @default.
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• W2023308622 date "2000-06-01" @default.
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