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• W2034333923 abstract "In the liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is present not only in the endoplasmic reticulum but also in the peroxisomes. However, to date no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein and that is localized exclusively to peroxisomes. This cell line was obtained by growing UT2 cells (which lack the endoplasmic reticulum HMG-CoA reductase) in the absence of mevalonate. The cells exhibited a marked increase in a 90-kDa HMG-CoA reductase that was localized exclusively to peroxisomes. The wild type Chinese hamster ovary cells contain two HMG-CoA reductase proteins, the well characterized 97-kDa protein, localized in the endoplasmic reticulum, and a 90-kDa protein localized in peroxisomes. The UT2 cells grown in the absence of mevalonate containing the up-regulated peroxisomal HMG-CoA reductase are designated UT2*. A detailed characterization and analysis of this cell line is presented in this study. In the liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is present not only in the endoplasmic reticulum but also in the peroxisomes. However, to date no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein and that is localized exclusively to peroxisomes. This cell line was obtained by growing UT2 cells (which lack the endoplasmic reticulum HMG-CoA reductase) in the absence of mevalonate. The cells exhibited a marked increase in a 90-kDa HMG-CoA reductase that was localized exclusively to peroxisomes. The wild type Chinese hamster ovary cells contain two HMG-CoA reductase proteins, the well characterized 97-kDa protein, localized in the endoplasmic reticulum, and a 90-kDa protein localized in peroxisomes. The UT2 cells grown in the absence of mevalonate containing the up-regulated peroxisomal HMG-CoA reductase are designated UT2*. A detailed characterization and analysis of this cell line is presented in this study. In mammalian cells, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 1The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; kb, kilobase pair(s); ER, endoplasmic reticulum; FCS, fetal calf serum; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; LPDS, lipoprotein-deficient serum. reductase is the rate-limiting enzyme for the synthesis of mevalonic acid, the precursor of cholesterol and other non-sterol isoprenoids. We and others (1Keller G.A. Barton M.C. Shapiro D.J. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 770-774Crossref PubMed Scopus (148) Google Scholar, 2Keller G.A. Pazirandeh M. Krisans S. J. Cell Biol. 1986; 103: 875-886Crossref PubMed Scopus (112) Google Scholar, 3Volkl A. Zaar K. Stegtmeier K. Fahimi H.D. Jpn. Soc. Cell Biol. 1986; 289: 13Google Scholar, 4Appelkvist E.L. Kalen A. Eur. J. Biochem. 1989; 185: 503-509Crossref PubMed Scopus (39) Google Scholar) have demonstrated that HMG-CoA reductase is localized in two distinct intracellular compartments, endoplasmic reticulum (ER) and peroxisomes. ER HMG-CoA reductase is a 97-kDa transmembrane glycoprotein. A short non-conserved sequence links the multiple transmembrane domain to the highly conserved catalytic domain, which extends out into the cytosol. Because of its role in cholesterol biosynthesis, the regulation of HMG-CoA reductase has been intensely studied. The levels of the ER enzyme are regulated by transcription (5Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4565) Google Scholar, 6Liscum L. Luskey K.L. Chin D.J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1983; 258: 8450-8455Abstract Full Text PDF PubMed Google Scholar, 7Osborne T.F. Goldstein J.L. Brown M.S. Cell. 1985; 42: 203-212Abstract Full Text PDF PubMed Scopus (102) Google Scholar), translation (8Peffley D. Sinensky M. J. Biol. Chem. 1985; 260: 9949-9952Abstract Full Text PDF PubMed Google Scholar, 9Nakanishi M. Goldstein J.L. Brown M.S. J. Biol. Chem. 1988; 263: 8929-8937Abstract Full Text PDF PubMed Google Scholar), and enzyme degradation (10Kumagai H. Chun K.T. Simoni R.D. J. Biol. Chem. 1995; 270: 19107-19113Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 11McGee T.P. Cheng H.H. Kumagai H. Omura S. Simoni R.D. J. Biol. Chem. 1996; 271: 25630-25638Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Another critical role for this enzyme has emerged in recent years, due to the requirement of farnesyl diphosphate and geranyl-geranyl diphosphate in isoprenylation of proteins (12Maltese W.A. FASEB J. 1990; 4: 3319-3328Crossref PubMed Scopus (430) Google Scholar). Keller et al. (1Keller G.A. Barton M.C. Shapiro D.J. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 770-774Crossref PubMed Scopus (148) Google Scholar) were the first to demonstrate that in the liver HMG-CoA reductase is present not only in the ER but also within the peroxisomes. The function of the peroxisomal reductase in cholesterol/isoprenoid metabolism has yet to be defined. However, it is clear that the ER and peroxisomal HMG-CoA reductases can be regulated differently and, therefore, may play different functional roles (2Keller G.A. Pazirandeh M. Krisans S. J. Cell Biol. 1986; 103: 875-886Crossref PubMed Scopus (112) Google Scholar,13Rusnak N. Krisans S.K. Biochem. Biophys. Res. Commun. 1987; 148: 890-895Crossref PubMed Scopus (11) Google Scholar). The ER reductase has a diurnal cycle distinct from that of the peroxisomal reductase (13Rusnak N. Krisans S.K. Biochem. Biophys. Res. Commun. 1987; 148: 890-895Crossref PubMed Scopus (11) Google Scholar). However, the two reductases can also be regulated coordinately. Both reductase activities are induced by cholestyramine (a bile acid resin) (2Keller G.A. Pazirandeh M. Krisans S. J. Cell Biol. 1986; 103: 875-886Crossref PubMed Scopus (112) Google Scholar). No information is available regarding the function of the peroxisomal reductase in cholesterol/isoprenoid metabolism, nor has the structure of the peroxisomal HMG-CoA reductase been determined. Accordingly, to facilitate our studies of the function, regulation, and structure of the peroxisomal HMG-CoA reductase, we have identified a mammalian cell line that expresses only one HMG-CoA reductase protein of 90 kDa and that is localized exclusively to peroxisomes. These cells provide a model system to study the peroxisomal HMG-CoA reductase independent of the ER reductase. A detailed characterization and analysis of this cell line is presented in this study. Biochemicals were purchased from Sigma. Electrophoresis supplies, AG1-X8-200–400-mesh formate resin, Zeta Probe GT membrane (used for Northern analysis) and Trans-Blot Transfer Medium (used for Western analysis) were purchased from Bio-Rad. All cell culture media and fetal calf serum were purchased from Life Technologies, Inc. Lipoprotein-deficient media were obtained from PerImmune. 3-Hydroxy-3-methylglutaryl coenzyme A,dl-3[glutaryl-3-14C]- and (RS)-[5-3H]mevalonic acid was purchased from NEN Life Science Products. 125I-Protein A was obtained from Amersham Corp. Cholestyramine (Questran) was obtained from Bristol Laboratories, and mevinolin (Mevacor) was from Merck. Male Sprague-Dawley rats (100–180 g) were maintained on a 12-h light/dark cycle. Water was given ad libitum, and rats were treated for 7 days with a standard laboratory diet containing 5.0% cholestyramine. Rats were fasted overnight and killed by decapitation 2 h into their light cycle. UT2 cells were obtained from Dr. J. Goldstein. CHO cells were maintained in 1:1 Dulbecco's modified Eagle's media: F12, supplemented with 5% fetal calf serum (FCS), fungizone, and Pen/Strep, in a 37 °C incubator with 5% CO2. UT2 cell cultures were maintained in the same media and supplemented with 0.2 mm mevalonate. We also maintained the UT2 cells in the presence of fetal calf serum (10% FCS) but in the absence of mevalonate. After 3 days in media lacking mevalonate more than 25% UT2 cells remained. The surviving UT2 cells were single cell cloned and designated UT2* and maintained in media lacking mevalonate. Cell suspensions were pelleted and washed twice with 20 mm KPO4, pH 7.5, 150 mm NaCl, and once with homogenization buffer, 250 mm sucrose, 5 mm Tris-HCl, pH 7.5, 1 mm EDTA, 0.1% EtOH. Cells were resuspended in homogenization buffer, gently dispersed with two strokes of a glass/Teflon homogenizer, and transferred to a nitrogen cavitation bomb. After a 10-min incubation at 4 °C and 62–63 p.s.i, cells were collected dropwise from the bomb. The suspension was gently homogenized until 80% cell rupture was observed, centrifuged at 750 relative centrifugal field for 5 min, and the pellet resuspended, rehomogenized, and recentrifuged. Supernatants were combined and applied to a linear 20–50% metrizamide gradient. The gradient was centrifuged at 19,000 rpm for 80–90 min in a Sorvall TV850 rotor. Fractions were collected dropwise with a two-way needle. Assays of marker enzymes were performed as described previously (14Biardi L. Sreedhar A. Zokaei A. Vartak N.B. Bozeat R.L. Shackelford J.E. Keller G.-A. Krisans S.K. J. Biol. Chem. 1994; 269: 1197-1205Abstract Full Text PDF PubMed Google Scholar). Aliquots of each fraction were also precipitated with an equal volume of 10% trichloroacetic acid for immunoblot analysis. Cells were rinsed three times with PBS and scraped into 50 mm KPO4, pH 7.0, containing 200 mm NaCl, 30 mm EDTA, 10 mm DTT (KEND) and centrifuged at 12,000 rpm in a Sorvall microcentrifuge for 5 min. Pellets were resuspended in KEND plus 0.2% Triton, 50 μm leupeptin, 1 mm PMSF, 5 mm EGTA and homogenized by hand with 20 strokes with an Eppendorf glass pestle. Extracts were centrifuged for 5 min and supernatants used for determination of protein levels and HMG-CoA reductase activity. Only freshly isolated fractions were assayed. The samples were preincubated for 30 min at 37 °C before the addition of substrate, to ensure the inactivation of HMG-CoA lyase activity (2Keller G.A. Pazirandeh M. Krisans S. J. Cell Biol. 1986; 103: 875-886Crossref PubMed Scopus (112) Google Scholar). The preincubation mixture consisted of 150 μl of KEND buffer, pH 7.0, containing 100–200 μg of protein. After preincubation, the reaction mixture (150 μl), containing 208 μm HMG-CoA and 2 mm NADPH (final concentration) and 20,000 dpm of [3H]mevalonate, in KEND buffer, was added. The samples were incubated at 37 °C for 40 min, and the reaction was stopped by the addition of 30 μl of 10.5 n HCl. Control samples lacking either NADPH or enzyme were routinely included. After centrifugation, HMG-CoA was separated from the product (mevalonolactone) by AG 1-X8 formate resin ion exchange columns (15Edwards P.A. Lemongello D. Fogelman A.M. J. Lipid Res. 1979; 20: 40-46Abstract Full Text PDF PubMed Google Scholar). We also employed thin layer chromatagraphy to separate HMG-CoA and mevalonolactone (16Shapiro D.J. Nordstrom J.L. Mitschelen J.J. Rodwell V.W. Schmke R.T. Biochim. Biophys. Acta. 1974; 370: 369-377Crossref PubMed Scopus (331) Google Scholar). The results obtained were the same as obtained by use of the formate resin ion exchange columns. Successful separation of the 97-kDa ER reductase from the 90-kDa peroxisomal reductase is dependent upon both the length of the gel and percent of acrylamide. Standard length (12.5 cm) 10% acrylamide gels fail to adequately resolve the two proteins. 10% acrylamide gels that are 16.5 cm in length do provide the required resolution. Alternatively, 12.5-cm gels that are 7.5% acrylamide also give acceptable resolution in this molecular weight range. SDS-gel electrophoresis and immunoblotting was performed as described previously with the following modifications (17Tanaka R.D. Edwards P.A. Lan S.-F. Fogelman A.M. J. Biol. Chem. 1983; 258: 13331-13339Abstract Full Text PDF PubMed Google Scholar). Trichloroacetic acid-precipitated proteins were first resuspended in 20 μl of 125 mm Tris-HCl, pH 6.8, 1% SDS, 0.1 n NaOH, followed by addition of 130 μl of sample buffer containing 7m urea, 8% SDS, and 1.1 m2β-mercaptoethanol. Cell cultures were rinsed three times with methionine-free and cysteine-free Dulbecco's minimal Eagle's media and preincubated for 1 h in this media. Fresh media were added containing 87.5 μCi/ml [trans-35S]methionine (1227 Ci/mmol), and the cells were incubated for 3 h. Labeled proteins were extracted with buffer containing detergents and protease inhibitors as described (17Tanaka R.D. Edwards P.A. Lan S.-F. Fogelman A.M. J. Biol. Chem. 1983; 258: 13331-13339Abstract Full Text PDF PubMed Google Scholar). Aliquots of the extracts were incubated with the indicated anti-HMG-CoA reductase antisera overnight, and immunoprecipitants were isolated on Protein A-Sepharose beads. For competition experiments the anti-peptide G and anti-peptide H antisera were preincubated with the corresponding peptide for 30 min prior to addition to aliquots of the cell extracts. Beads were washed and precipitating proteins were solubilized in the sample buffer as described above. After electrophoresis, proteins were transferred to nitrocellulose, and 35S-labeled proteins were visualized with a Molecular Dynamics PhosphorImager system. The C-terminal (last 15 amino acids) polyclonal HMG-CoA reductase antibody was obtained from Dr. S. Panini (18Straka M.S. Panini S.R. Arch. Biochem. Biophys. 1995; 317: 235-243Crossref PubMed Scopus (12) Google Scholar). The polyclonal anti-peptide G (residues Arg224 through Leu242), anti-peptide H (residues Thr284 through Glu302), HMG-CoA reductase antisera, and the corresponding peptides were obtained from Dr. R. Simoni (19Roitelman J. Olender E.H. Bar-Nun S. Dunn Jr., W.A. Simoni R.D. J. Cell Biol. 1992; 117: 959-973Crossref PubMed Scopus (153) Google Scholar). Cells on coverslips were washed in PBS and fixed in 3.0%p-formaldehyde in PBS for 15 min. Cells were permeabilized with 1% Triton X-100 in PBS for 5 min and then washed with 0.1% Tween 20 in PBS (also used for subsequent washes). A mixture of mouse HMG-CoA reductase antibody (1:25) and rabbit anti-peroxisomal signal (SKL) IgG antibody (1:200) was used. The cells were washed, and a mixture of secondary reagents consisting of fluorescein conjugate of goat anti-rabbit IgG (heavy + light) antibody (at a final dilution of 1:100) and Texas Red conjugate of goat anti-mouse IgG (heavy + light) antibody (at a final dilution of 1:200) was applied to the coverslips for 60 min. The cells were washed extensively, and the coverslips were mounted on microscope slides for observation with a Nikon fluorescence microscope. UT2* cells were fixed in 3% formaldehyde and 0.5% glutaraldehyde in 0.1 m cacodylate buffer, pH 7.2, and infused with London Resin white acrylic resin (London Resin Co., London). After dehydration with ethanol, thin sections were cut on a Reichert Ultra microtome. Immunolabeled sections were poststained in 2% ethanolic uranyl acetate and observed in a Philips CM12 transmission electron microscope (20McLean I.W. Nakane P.K. J. Histochem. Cytochem. 1974; 22: 1077-1083Crossref PubMed Scopus (3200) Google Scholar, 21Keller G.-A. Tokuyase K.T. Dutton A.H. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5744-5747Crossref PubMed Scopus (84) Google Scholar). The monoclonal HMG-CoA reductase antibody (clone A-9) was obtained from Drs. Brown and Goldsteins' laboratory (6Liscum L. Luskey K.L. Chin D.J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1983; 258: 8450-8455Abstract Full Text PDF PubMed Google Scholar), and several polyclonal HMG-CoA reductase antibodies were obtained from Dr. P. Edwards (22Edwards P.A. Lan S.-F. Tanaka R.D. Fogelman A.M. J. Biol. Chem. 1983; 258: 7272-7275Abstract Full Text PDF PubMed Google Scholar). Liver homogenates were first fractionated by differential centrifugation to obtain a peroxisome-enriched fraction, (containing peroxisomes, smaller mitochondria, and microsomes), and a microsomal fraction (14Biardi L. Sreedhar A. Zokaei A. Vartak N.B. Bozeat R.L. Shackelford J.E. Keller G.-A. Krisans S.K. J. Biol. Chem. 1994; 269: 1197-1205Abstract Full Text PDF PubMed Google Scholar). The homogenization buffer contained 0.25m sucrose, 5 mm Tris-HCl, 1 mmEDTA, 0.1% EtOH, 1.28 μg/ml aprotinin, 10 μg/ml cycloheximide, 125 ng/ml pepstatin A, 250 ng/ml antipain, 125 ng/ml chymostatin, 50 μm leupeptin, 100 μm PMSF, 20 μm DTT, 2 mm methionine, 15 μg/ml calpain I, 15 μg/ml calpain II, pH 7.5. The peroxisome-enriched fraction was then further purified by equilibrium density centrifugation on a linear Nycodenz (20–45% (w/w)) gradient (14Biardi L. Sreedhar A. Zokaei A. Vartak N.B. Bozeat R.L. Shackelford J.E. Keller G.-A. Krisans S.K. J. Biol. Chem. 1994; 269: 1197-1205Abstract Full Text PDF PubMed Google Scholar). The gradient contained all of the above protease inhibitors except PMSF, DTT, and methionine. Isolated peroxisomes were at least 94% pure as determined by marker enzyme distribution and contained <1% mitochondrial contamination and 3–6% microsomal protein. Total RNA was extracted from 80% confluent cultured cells as described (23Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63228) Google Scholar). mRNA was isolated using Collaborative Biochemical type 3 oligo(dT)-cellulose, and Northern blots were performed using standard molecular biology protocols. Gels were run at 65 V for 18 h. Blots were hybridized at 42 °C and washed at 55 °C with 0.5 × SSC, 0.1% SDS. Probes were labeled according to Boehringer Mannheim Nick Translation kit with [α-32P]dCTP (Amersham Corp.) and purified using Stratagene Nuctrap protocol. UT2 cells are a mutant clone of CHO cells that require cholesterol and low levels of mevalonate for growth due to a deficiency of the 97-kDa ER HMG-CoA reductase (24Mosley S.T. Brown M.S. Anderson R.G.W. Goldstein J.L. J. Biol. Chem. 1983; 258: 13875-13881Abstract Full Text PDF PubMed Google Scholar). This cell line has been stable for over 12 years, and the calculated spontaneous reversion rate is less than 1.5 × 10−7 (24Mosley S.T. Brown M.S. Anderson R.G.W. Goldstein J.L. J. Biol. Chem. 1983; 258: 13875-13881Abstract Full Text PDF PubMed Google Scholar). However, the UT2 cells have some HMG-CoA reductase activity, which is “bona fide” because the activity is totally inhibited when a competitive HMG-CoA reductase inhibitor (compactin) is added to the assay mixture (24Mosley S.T. Brown M.S. Anderson R.G.W. Goldstein J.L. J. Biol. Chem. 1983; 258: 13875-13881Abstract Full Text PDF PubMed Google Scholar). The enzymes of the cholesterol synthesis pathway preceding and following HMG-CoA reductase are normal, thus the UT2 cells are only deficient in HMG-CoA reductase (24Mosley S.T. Brown M.S. Anderson R.G.W. Goldstein J.L. J. Biol. Chem. 1983; 258: 13875-13881Abstract Full Text PDF PubMed Google Scholar). We have identified the mutation in the ER reductase gene responsible for this defect. 2W. H. Engfelt, K. Masuda, V. G. Paton, and S. K. Krisans, manuscript in preparation. The UT2 cells contain a mutation in the 5′ splice junction between exons 11 and 12. This results in exon 11 skipping and insertion of stop codons. Thus, this mutation prevents the production of the 97-kDa ER HMG-CoA reductase. We maintained the UT2 cells (3.5 × 106) in the presence of fetal calf serum (10% FCS) but in the absence of mevalonate. After 3 days in media lacking mevalonate, more than 25% of the UT2 cells remained. The surviving UT2 cells were single cell cloned and exhibited a marked increase in HMG-CoA reductase activity compared with that measured in UT2 cells cultured in the presence of mevalonate. The UT2 cells grown in the absence of mevalonate are designated UT2*. Fig. 1 compares the reductase activities of UT2, UT2*, and CHO cells maintained for 24 h in the presence of 5% FCS, 5% lipoprotein-deficient serum (LPDS) or in the presence of LPDS plus 0.5 μm lovastatin (I), a competitive inhibitor of HMG-CoA reductase activity, which induces synthesis of HMG-CoA reductase in vivo. The HMG-CoA reductase activity of UT2 cells was 2.0 pmol/min/mg of whole cell extract, compared with 80 pmol/min/mg for UT2* cells, in the presence of LPDS, and 200 pmol/min/mg for UT2* cells in the presence of LPDS plus 0.5 μm lovastatin, reflecting a 40–100-fold increase over the levels observed in UT2 cells. As expected, the HMG-CoA reductase activity in CHO cells is down-regulated by the addition of FCS and up-regulated by the addition of LPDS, and further increased by the addition of LPDS and lovastatin (I). Very similar regulation is observed in the UT2* cells. When whole cell extracts of CHO cells (prepared in the presence of 50 μm leupeptin, 1 mm PMSF, 5 mm EGTA) were immunoblotted for HMG-CoA reductase, two proteins reacted with the reductase antibody, one of identical mobility to the 97-kDa ER reductase and a second protein at 90-kDa (Fig.2). However, in whole cell extracts from the UT2* cells (treated identically), we only observed the 90-kDa protein band, which was found at elevated levels compared with the 90-kDa band in CHO cells. UT2 whole cell extracts contained no visible protein band reacting with the reductase antibodies (data not shown), consistent with previous reports (24Mosley S.T. Brown M.S. Anderson R.G.W. Goldstein J.L. J. Biol. Chem. 1983; 258: 13875-13881Abstract Full Text PDF PubMed Google Scholar). As illustrated in Fig. 3, the amount of the 97-kDa HMG-CoA reductase protein in CHO cells and the 90-kDa protein in UT2* cells was reduced by the addition of FCS and up-regulated by the addition of LPDS to the media. The protein levels were increased further by the addition of LPDS + lovastatin (I). The levels of the 90-kDa protein in CHO cells were also regulated. The levels of the 97-kDa protein in CHO and the 90-kDa protein in UT2* cells correlated with the reductase activity within each cell line, as illustrated in Fig. 1. We utilized density gradient centrifugation to determine whether the 90-kDa HMG-CoA reductase protein is localized to the ER or peroxisomes. UT2* cell organelles were isolated from a post-nuclear fraction on a metrizamide linear gradient. Fig.4 illustrates one of three typical gradients. The separation of endoplasmic reticulum (as determined by cytochrome c reductase), peroxisomes (as determined by catalase), and the distribution of HMG-CoA reductase activity is shown. As can be seen from the catalase distribution, the majority of the intact peroxisomes are found at the dense end (right) of the gradient, well separated from the peak ER fractions. A portion of the catalase activity is solubilized, as a result of rupture of the peroxisomes during the isolation procedure and migrates at the light end of the gradient. The distribution of HMG-CoA reductase activity parallels the distribution of the peroxisomal marker, catalase. The cytosolic fraction is located at the light end of the gradient, and the mitochondrial fraction is also well separated from the peroxisomal fractions (data not shown). In contrast to UT2* cells, CHO cell HMG-CoA reductase activity was localized to both the ER and peroxisomes. Fig.5 illustrates the separation of the organelles in CHO cells. One of three typical gradients is represented. There is a slight contamination of the peroxisomal fractions by the ER, as indicated by the distribution of cytochrome c reductase (panel A). HMG-CoA reductase activity is localized both in the peak ER fractions as well as the peak peroxisomal fractions (panel C). Fig. 6 shows the results of a typical study in which the density gradient fractions from UT2* and CHO cells were analyzed for both HMG-CoA reductase activity and protein (immunoblot). The results clearly demonstrate that UT2* cells (panel A) express an HMG-CoA reductase that is both localized to the peroxisomes and is smaller in size than that observed in the ER (fractions 5–8) of normal CHO cells (panel B). Immunoblots of the fractions from CHO cells (panel B) demonstrate that a 97-kDa HMG-CoA reductase is predominantly localized to ER fractions with some contamination in peroxisomal fractions. In contrast, the 90-kDa HMG-CoA reductase is localized exclusively to the peroxisomal fractions (fractions 13–15). To demonstrate localization of the organelles on the gradient, equal volumes of the fractions (instead of equal protein) were loaded on the gel. This resulted in a slight downward shift of the 97-kDa protein in the ER fractions 5–8, in panel B, due to the high levels of protein in these fractions. In addition, there is an excellent correlation with reductase activity and immunoblot density levels in both cell lines. The known 97-kDa ER HMG-CoA reductase protein was not observed in any fraction in the UT2* cells (panel A). To verify further the subcellular localization of HMG-CoA reductase in UT2* cells, we examined the immunofluorescence pattern obtained with an HMG-CoA reductase antibody. CHO cells and UT2* cells were simultaneously labeled for HMG-CoA reductase and for peroxisomal proteins. A rabbit polyclonal antibody made against the peroxisomal targeting signal (SKL, at the C terminus) was used to label peroxisomal proteins. The SKL antibody has been shown to be specific for peroxisomal proteins (26Gould S.J. Krisans S. Keller G.A. Subramani S. J. Cell Biol. 1990; 110: 27-34Crossref PubMed Scopus (92) Google Scholar). The immunofluorescence pattern obtained for HMG-CoA reductase in CHO cells (Fig.7, panel A) was consistent with ER labeling; however, the pattern obtained for HMG-CoA reductase in UT2* cells was consistent with peroxisomal labeling (Fig. 7,panel B). The majority of the immunofluorescence pattern of HMG-CoA reductase in UT2* cells was directly superimposable over that for the peroxisomal targeting signal antibody (arrowheads inpanels B and D). We also observe some co-localization of HMG-CoA reductase labeling with that of the peroxisomal marker in the CHO cells (arrowheadsin Fig. 7, panels A and C). These results indicate that, in CHO cells, HMG-CoA reductase is localized to both the ER and peroxisomes. In contrast, in UT2* cells, HMG-CoA reductase appears to be exclusively localized to peroxisomes. To further confirm the localization of HMG-CoA reductase to peroxisomes in UT2* cells, these cells were processed for immunoelectron microscopy. As expected, indirect gold immunolabeling for catalase showed specific immunolabeling of peroxisomes (Fig. 8,panel A). Panel B, demonstrates the localization of HMG-CoA reductase to a similar organelle, utilizing a polyclonal HMG-CoA reductase antibody. The immunolabeling is restricted to the matrix of organelles that morphologically resemble peroxisomes. To unambiguously determine that HMG-CoA reductase is contained in the peroxisomes, we also performed double labeling experiments using both rabbit anti-catalase and a monoclonal antibody against HMG-CoA reductase. Panel C shows that 5-nm gold particles representing antigenic sites for HMG-CoA reductase (arrowheads) are present in catalase-positive organelles (10-nm gold particles), demonstrating the co-localization of catalase and HMG-CoA reductase to the same organelle, and confirming the localization of HMG-CoA reductase to peroxisomes. The small peroxisome in panel C shows immunolabeling for catalase but not for HMG-CoA reductase. Taken together, all of the above data indicate that UT2* cells contain an HMG-CoA reductase localized only to the peroxisomes. When peroxisomal fractions are isolated from UT2 cells (suppressed conditions) and immunoblotted for HMG-CoA reductase, a 90-kDa protein band and HMG-CoA reductase activity can also be detected in the peak peroxisomal fractions (Fig. 9,fractions 13–15). However, since in the UT2 cells the reductase is not up-regulated, the levels of the reductase protein are very low. We tested the abilities of a number of HMG-CoA reductase antibodies to immunoprecipitate the 90-kDa protein from UT2* cells, as well as the 97- and 90-kDa proteins from CHO cells (Fig. 10 , panel A). The polyclonal, anti-C-terminal, anti-G peptide, and the anti-H peptide antibodies all immunoprecipitated the 97- and 90-kDa proteins from 35S-labeled CHO cell lysates and the 90-kDa protein from 35S-labeled UT2* cell lysates. These proteins were specifically precipitated as they were competed by an excess of the corresponding free peptides (Fig. 10, panels B andC). Clearly, the 90-kDa protein is antigenically similar to the ER HMG-CoA reductase and must contain multiple conserved antigenic sites. To determine if a precursor-product relationship existed between the 97- and 90-kDa bands in CHO cells, a pulse-chase experiment was performed in CHO cells (Fig. 11). The results indicate that there is no precursor-product relationship between the 97- and 90-kDa reductase proteins. The data are expressed as the percentage of HMG-CoA reductase remaining at each time point. The estimated half-life from the slope of the 97-kDa band agreed well with published reports (18Straka M.S. Panini S.R. Arch. Biochem. Biophys. 1995; 317: 235-243Crossref PubMed Scopus (12) Google Scholar), and the estimated half-life of the 90-kDa band in CHO cells appears to be similar. Successful and reproducible separation of the 97- and 90-kDa reductase proteins requires specific conditions that include the presence of 7 m urea, 8% SDS, and 1.1m 2-mercaptoethanol in the sample buffer, and either 10% acrylamide gels that are 16.5 cm in length or 7.5% acrylamide gels that are 12.5 cm in length. In unpublished studies we ha" @default.
• W2034333923 created "2016-06-24" @default.
• W2034333923 creator A5003300621 @default.
• W2034333923 creator A5012835227 @default.
• W2034333923 creator A5018051897 @default.
• W2034333923 creator A5022524996 @default.
• W2034333923 creator A5036215026 @default.
• W2034333923 creator A5049471502 @default.
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• W2034333923 creator A5084805468 @default.
• W2034333923 date "1997-09-01" @default.
• W2034333923 modified "2023-09-27" @default.
• W2034333923 title "Characterization of UT2 Cells" @default.
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