Newly Synthesized Canalicular ABC Transporters Are Directly Targeted from the Golgi to the Hepatocyte Apical Domain in Rat Liver
2000; Elsevier BV; Volume: 275; Issue: 21 Linguagem: Inglês
10.1074/jbc.m909875199
ISSN1083-351X
Autores Tópico(s)Pancreatic function and diabetes
ResumoNewly synthesized canalicular ectoenzymes and a cell adhesion molecule (cCAM105) have been shown to traffic from the Golgi to the basolateral plasma membrane, from where they transcytose to the apical bile canalicular domain. It has been proposed that all canalicular proteins are targeted via this indirect route in hepatocytes. We studied the membrane targeting of rat canalicular proteins by in vivo [35S]methionine metabolic labeling followed by preparation of highly purified Golgi membranes and canalicular (CMVs) and sinusoidal/basolateral (SMVs) membrane vesicles and subsequent immunoprecipitation. In particular, we compared membrane targeting of newly synthesized canalicular ABC (ATP-binding cassette) transporters MDR1, MDR2, and SPGP (sister ofP-glycoprotein) with that of cCAM105. Significant differences were observed in metabolic pulse-chase labeling experiments with regard to membrane targeting of these apical proteins. After a chase time of 15 min, cCAM105 appeared exclusively in SMVs, peaked at 1 h, and progressively declined thereafter. In CMVs, cCAM105 was first detected after 1 h and subsequently increased for 3 h. This findings confirm the transcytotic targeting of cCAM105 reported in earlier studies. In contrast, at no time point investigated were MDR1, MDR2, and SPGP detected in SMVs. In CMVs, MDR1 and MDR2 appeared after 30 min, whereas SPGP appeared after 2 h of labeling. In Golgi membranes, each of the ABC transporters peaked at 30 min and was virtually absent thereafter. These data suggest rapid, direct targeting of newly synthesized MDR1 and MDR2 from the Golgi to the bile canaliculus and transient sequestering of SPGP in an intracellular pool en route from the Golgi to the apical plasma membrane. This study provides biochemical evidence for direct targeting of newly synthesized apical ABC transporters from the Golgi to the bile canaliculus in vivo. Newly synthesized canalicular ectoenzymes and a cell adhesion molecule (cCAM105) have been shown to traffic from the Golgi to the basolateral plasma membrane, from where they transcytose to the apical bile canalicular domain. It has been proposed that all canalicular proteins are targeted via this indirect route in hepatocytes. We studied the membrane targeting of rat canalicular proteins by in vivo [35S]methionine metabolic labeling followed by preparation of highly purified Golgi membranes and canalicular (CMVs) and sinusoidal/basolateral (SMVs) membrane vesicles and subsequent immunoprecipitation. In particular, we compared membrane targeting of newly synthesized canalicular ABC (ATP-binding cassette) transporters MDR1, MDR2, and SPGP (sister ofP-glycoprotein) with that of cCAM105. Significant differences were observed in metabolic pulse-chase labeling experiments with regard to membrane targeting of these apical proteins. After a chase time of 15 min, cCAM105 appeared exclusively in SMVs, peaked at 1 h, and progressively declined thereafter. In CMVs, cCAM105 was first detected after 1 h and subsequently increased for 3 h. This findings confirm the transcytotic targeting of cCAM105 reported in earlier studies. In contrast, at no time point investigated were MDR1, MDR2, and SPGP detected in SMVs. In CMVs, MDR1 and MDR2 appeared after 30 min, whereas SPGP appeared after 2 h of labeling. In Golgi membranes, each of the ABC transporters peaked at 30 min and was virtually absent thereafter. These data suggest rapid, direct targeting of newly synthesized MDR1 and MDR2 from the Golgi to the bile canaliculus and transient sequestering of SPGP in an intracellular pool en route from the Golgi to the apical plasma membrane. This study provides biochemical evidence for direct targeting of newly synthesized apical ABC transporters from the Golgi to the bile canaliculus in vivo. asialoglycoprotein receptor canalicular membrane vesicle sinusoidal membrane vesicle polyacrylamide gel electrophoresis The bile canalicular membrane of the mammalian hepatocyte contains several primary active transporters that couple ATP hydrolysis to the transport of specific substrates into the bile canaliculus (1.Nishida T. Gatmaitan Z. Che M. Arias I.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6590-6594Crossref PubMed Scopus (192) Google Scholar, 2.Nishida T. Hardenbrook C. Gatmaitan Z. Arias I.M. Am. J. Physiol. 1992; 262: G629-G635PubMed Google Scholar, 3.Stieger B. O'Neill B. Meier P.J. Biochem. J. 1992; 284: 67-74Crossref PubMed Scopus (128) Google Scholar, 4.Gatmaitan Z.C. Arias I.M. Physiol. Rev. 1995; 75: 261-275Crossref PubMed Scopus (65) Google Scholar). These transporters are members of the superfamily of ABC (ATP-binding cassette) membrane transport proteins (5.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3323) Google Scholar) and currently include P-glycoprotein or MDR1 (multidrug resistance protein; organic cations) (6.Kamimoto Y. Gatmaitan Z. Hsu J. Arias I.M. J. Biol. Chem. 1989; 264: 11693-11698Abstract Full Text PDF PubMed Google Scholar), MDR2 (phosphatidylcholine) (7.Ruetz S. Gros P. Cell. 1994; 77: 1071-1081Abstract Full Text PDF PubMed Scopus (570) Google Scholar, 8.Nies A.T. Gatmaitan Z. Arias I.M. J. Lipid Res. 1996; 37: 1125-1136Abstract Full Text PDF PubMed Google Scholar), SPGP (sister ofP-glycoprotein; bile acids) (9.Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. Hofmann A.F. Meier P.J. J. Biol. Chem. 1998; 273: 10046-10050Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar), and MRP2 (multidrug resistance-associatedprotein; non-bile acid organic anions) (10.Büchler M. König J. Brom M. Kartenbeck J. Spring H. Horie T. Keppler D. J. Biol. Chem. 1996; 271: 15091-15098Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). The amount of each ABC transporter in the canalicular membrane is regulated by the physiological demand to excrete bile acids. Intravenous administration of rats with taurocholate or dibutyryl cAMP rapidly and selectively increased the functional activity and amount of ABC transporters in the canalicular membrane. This increase was inhibited by prior administration of colchicine, which disrupts microtubules (11.Gatmaitan Z.C. Nies A.T. Arias I.M. Am. J. Physiol. 1997; 272: G1041-G1049PubMed Google Scholar), and wortmannin, which inhibits phosphatidylinositol 3-kinase (12.Misra S. Ujhazy P. Gatmaitan Z. Varticovski L. Arias I.M. J. Biol. Chem. 1998; 273: 26638-26644Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). These observations indicate that an intracellular microtubule-dependent transport mechanism that is sensitive to active phosphatidylinositol 3-kinase is required to traffic ABC transporters to the canalicular membrane. In addition, lipid products of phosphatidylinositol 3-kinase directly regulate the ATP-dependent substrate transport activity of SPGP and MRP2 in the canalicular membrane (13.Misra S. Ujhazy P. Varticovski L. Arias I.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5814-5819Crossref PubMed Scopus (96) Google Scholar). These studies indicate that bile secretion requires intrahepatic trafficking and regulation of the canalicular ABC transporters. Membrane targeting of the newly synthesized canalicular ectoenzymes dipeptidyl peptidase IV, aminopeptidase N, and 5′-nucleotidase and the canalicular cell adhesion molecule cCAM105 (also known as HA4) has been studied in rat liver by in vivo metabolic pulse-chase labeling. After biosynthesis, these canalicular proteins are transferred from the Golgi to the basolateral membrane and subsequently reach the bile canaliculus only by transcytosis (14.Bartles J.R. Feracci H.M. Stieger B. Hubbard A.L. J. Cell Biol. 1987; 105: 1241-1251Crossref PubMed Scopus (258) Google Scholar, 15.Schell M.J. Maurice M. Stieger B. Hubbard A.L. J. Cell Biol. 1992; 119: 1173-1182Crossref PubMed Scopus (109) Google Scholar). Based on these results, it was proposed that all newly synthesized canalicular proteins, including canalicular ABC-type transporters, are targeted via this indirect route (16.Bartles J.R. Hubbard A.L. Trends Biochem. Sci. 1988; 13: 181-184Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 17.Roelofsen H. Soroka C.J. Keppler D. Boyer J.L. J. Cell Sci. 1998; 111: 1137-1145Crossref PubMed Google Scholar). Although the membrane targeting of newly synthesized canalicular ectoenzymes and cell adhesion molecule cCAM105 has been thoroughly studied, comparable investigations of canalicular ABC transporters have not been performed. In this study, we used metabolic pulse-chase labeling followed by subcellular fractionation of rat liver and immunoprecipitation to investigate the intracellular trafficking of newly synthesized canalicular proteins. In particular, we focused on membrane targeting of newly synthesized transporters of the MDR family, including SPGP, and compared their trafficking with that of cCAM105 and with the basolateral membrane resident asialoglycoprotein receptor (ASGP-R).1 Radiochemicals were supplied by NEN Life Science Products. All other chemicals were of the highest purity available and were purchased from Sigma. Monoclonal antibody C219 (anti-MDR1/MDR2) was from Centocor (Malvern, PA), and polyclonal anti-β-COP antibody was from Sigma. Other antibodies were kind donations: EAG15 (polyclonal, anti-MRP2), D. Keppler (10.Büchler M. König J. Brom M. Kartenbeck J. Spring H. Horie T. Keppler D. J. Biol. Chem. 1996; 271: 15091-15098Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar); Ab669 (polyclonal, anti-cCAM105), S. H. Lin, (18.Lin S.H. Culic O. Flanagan D. Hixson D.C. Biochem. J. 1991; 278: 155-161Crossref PubMed Scopus (65) Google Scholar); HA301 (monoclonal, anti-dipeptidyl peptidase IV), A. L. Hubbard (19.Bartles J.R. Braiterman L.T. Hubbard A.L. J. Biol. Chem. 1985; 260: 12792-12802Abstract Full Text PDF PubMed Google Scholar); and anti-ASGP-R, R. J. Stockert (20.Stockert R.J. Morell A.G. J. Biol. Chem. 1990; 265: 1841-1846Abstract Full Text PDF PubMed Google Scholar). A glutathione S-transferase fusion protein containing a 90-amino acid fragment of the SPGP linker region (amino acids 653–742, starting with LVT) was used as antigen to raise antibody LVT90. The corresponding coding DNA fragment was amplified from full-length SPGP cDNA (provided by P. Meier, (9.Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. Hofmann A.F. Meier P.J. J. Biol. Chem. 1998; 273: 10046-10050Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar) by polymerase chain reaction using the oligonucleotides 5′-AAT GAA TCC TGC TTG TGA CCC TGC AAA G-3′ (containing a BamHI site) and 5′-ATT GTC GAC TAC CTA ACT GGG GCA GGT TC-3′ (containing a SalI site). The polymerase chain reaction product was digested by BamHI and SalI and ligated into theBamHI/SalI sites of the pGEX-5X-3 vector (Amersham Pharmacia Biotech). In-frame cloning was confirmed by DNA sequencing. Expression of the glutathione S-transferase fusion protein in Escherichia coli BL21 cells and purification using glutathione-Sepharose beads were performed according to protocols provided by Amersham Pharmacia Biotech. A commercial service was employed to raise antibodies in rabbits (Covance, Denver, PA) using a standard protocol for immunization and bleeding. Groups of five male Sprague-Dawley rats (300–350 g) kept on a standard diet were anesthetized with sodium pentobarbital (50 mg/kg, injected intraperitoneally) and were injected in the tail vein with 3.5 mCi of [35S]methionine/cysteine (1175 Ci/mmol; Expre35S35S protein label, NEN Life Science Products) in 1 ml of phosphate-buffered saline. 15 min later, 50 mg of unlabeled methionine and 5 mg of unlabeled cysteine in 2.5 ml of phosphate-buffered saline were injected intraperitoneally. For investigation of membrane targeting, livers were removed after 15 min, 30 min, 1 h, 2 h, and 3 h and used for subcellular fractionation. Data presented are typical results observed in at least three sets of five rats. Previously published methods were combined, modified, and optimized for a high yield of canalicular, sinusoidal/basolateral, and Golgi membranes from a single rat liver (Fig.1). After gentle homogenation of rat liver, bile canaliculi remain attached to tight junctions and sediment with the low speed nuclear pellet. Canalicular membrane vesicles were prepared from the low speed pellet by nitrogen cavitation followed by calcium precipitation (21.Inoue M. Kinne R. Tran T. Biempica L. Arias I.M. J. Biol. Chem. 1983; 258: 5183-5188Abstract Full Text PDF PubMed Google Scholar). The low speed supernatant was split and used for purification of basolateral membranes on a sucrose/Ficoll gradient (22.Inoue M. Kinne R. Tran T. Arias I.M. Hepatology. 1982; 2: 572-579Crossref PubMed Scopus (150) Google Scholar) and for preparation of Golgi membranes by floating a microsomal fraction on a discontinuous sucrose gradient (23.Subramaniam V.N. bin Mohd Yusoff A.R. Wong S.H. Lim G.B. Chew M. Hong W. J. Biol. Chem. 1992; 267: 12016-12021Abstract Full Text PDF PubMed Google Scholar). Details are as follows. Excised rat liver (10–15 g, wet weight) was rapidly perfused with ice-cold SHCa buffer (0.25 m sucrose, 10 mmHEPES/Tris, pH 7.4, and 0.2 mm CaCl2supplemented with protease inhibitors (2 μg/ml leupeptin, 2 μg/ml pepstatin A, 20 μg/ml phenylmethylsulfonyl fluoride, 5 μg/ml benzamidine, and 2 μg/ml aprotinin)) and homogenized in 50 ml of SHCa buffer with four strokes in a loose-fitting Dounce homogenizer. The suspension was filtered through a double layer of cheesecloth; homogenized again with 15 strokes; diluted with SH buffer (SHCa buffer without CaCl2) to 140 ml; supplemented with 0.1m EGTA stock solution, pH 7.4, to a final concentration of 1 mm; and centrifuged for 10 min at 1880 ×g (Beckman JA-14, 3500 rpm). The pellet and fluffy layer were collected and used for preparation of CMVs. The supernatant (∼130 ml) was split 1:1 and used for preparation of SMVs and Golgi membranes, respectively. The pellet was resuspended in 50 ml of SHCa buffer and centrifuged for 10 min at 3000 × g (Beckman JA-14, 4500 rpm). The resulting pellet was suspended in 50 ml of SHCa buffer, placed in a high pressure chamber (Parr Instrument Model 4635), and equilibrated with nitrogen at 850 p.s.i. for 15 min with shaking at 4 °C. Pressure was released within 3 min; and the contents of the chamber were homogenized with six strokes in a tight-fitting Dounce homogenizer, diluted with SHCa buffer to 120 ml, and supplemented with 1 m CaCl2 stock solution to a final concentration of 10 mm. After incubation for 10 min on ice, the suspension was centrifuged for 20 min at 7600 × g(Beckman JA-14, 7000 rpm). The supernatant was filtered through fine weave cloth and centrifuged for 30 min at 47,000 × g(Beckman Ti-45, 27,000 rpm). The pellet was homogenized in 30 ml of SHCa buffer with six strokes in a tight-fitting Dounce homogenizer and centrifuged for 10 min at 3000 × g (Beckman JA-17, 4500 rpm). The supernatant was collected and centrifuged for 30 min at 47,000 × g (Beckman Ti-45, 27,000 rpm). The resulting pellet was homogenized in SHCa buffer at 3–4 mg/ml protein with a syringe and 24-gauge needle and stored at −80 °C until used. 65 ml of the first supernatant was centrifuged for 10 min at 5500 × g (Beckman JA-14, 7500 rpm). The resulting supernatant and fluffy layer were collected and centrifuged for 30 min at 22,000 × g (Beckman JA-14, 12,000 rpm). The pellet was resuspended in SH buffer containing 1 mmEGTA (total volume of 12 ml) and layered on two discontinuous gradients consisting of 1 ml of 60% sucrose, 23 ml of 23% sucrose and 4% Ficoll 400, and 7 ml of 20% sucrose. After centrifugation for 90 min at 130,000 × g in a swinging bucket rotor (Beckman SW 27, 27,000 rpm), the interphase between 20% sucrose and 23% sucrose and 4% Ficoll was collected, diluted six times with SHCa buffer, and centrifuged for 30 min at 47,000 × g (Beckman Ti-45, 27,000 rpm). The resulting pellet was homogenized in SHCa buffer at ∼10 mg/ml protein with a syringe and 24-gauge needle and stored at −80 °C until used. 65 ml of the first supernatant was homogenized with 10 strokes in a tight-fitting Dounce homogenizer, supplemented with MgCl2 to a final concentration of 5 mm, and centrifuged for 10 min at 15,000 × g (Beckman JA-14, 10,000 rpm). The supernatant was saved, and the pellet was resuspended in 50 ml of SH buffer containing 5 mm MgCl2 and centrifuged again for 10 min at 15,000 × g. The two supernatants from the 15,000 × g spins were combined and centrifuged for 60 min at 140,000 × g (Beckman Ti-45, 35,000 rpm). The pellet was resuspended in 12 ml of 1.25m sucrose and overlaid with 12 ml of 1.1 msucrose and 12 ml of 0.25 m sucrose in a 36-ml tube for gradient centrifugation. After centrifugation for 90 min at 130,000 × g (Beckman SW 27, 27,000 rpm), the interphase between 0.25 m sucrose and 1.1 msucrose was collected, diluted six times with SHCa buffer, and centrifuged for 30 min at 47,000 × g (Beckman Ti-45, 27,000 rpm). The resulting pellet was homogenized in SHCa buffer at 3–4 mg/ml protein with a syringe and 24-gauge needle and stored at −80 °C until used. Typical yields were 1.0–1.3 mg of membrane protein for CMV and Golgi preparations and 5–6 mg of protein for SMV preparations. 1 mg of protein of CMV, SMV, and Golgi preparations was solubilized for 1 h at 4 °C in 1 ml of buffer containing 20 mm octyl β-d-glucopyranoside, 0.5% Triton X-100, 0.3 m NaCl, and 0.025 mNaPi, pH 7.4, containing 0.02% NaN3 and protease inhibitors (2 μg/ml leupeptin, 2 μg/ml pepstatin A, 20 μg/ml phenylmethylsulfonyl fluoride, 5 μg/ml benzamidine, and 2 μg/ml aprotinin). The homogenate (40 mg of protein) was solubilized in 10 ml of the same buffer. The mixtures were centrifuged at 150,000 × g for 60 min at 4 °C. The supernatants were precleared by adding 40 μl of protein A-Sepharose beads, shaken for 30 min at 4 °C, and centrifuged for 5 min at 16,000 ×g. The precleared supernatants were used for immunoprecipitation with monoclonal antibody C219 (10 μg of IgG) and antisera from polyclonal anti-ASGP-R (20 μl) and anti-cCAM105 (20 μl) antibodies and LVT90 (20 μl). By repeated immunoprecipitation followed by immunoblotting, it was established that these conditions are sufficient to precipitate completely the respective antigen. The lysates were employed for precipitation with each antibody in a sequential fashion. The order in which the proteins were immunoprecipitated proved to be uncritical. The detergent extracts were supplemented with bovine serum albumin to 0.5% and incubated with the respective antibody for 1 h at 4 °C with shaking. 40 μl of protein A-Sepharose beads was added, and incubation with shaking at 4 °C was continued for 1 h. The beads were sedimented for 1 min at 16,000 × g, washed four times by repeated suspension in 1 ml of the lysis buffer (without octyl glucoside and protease inhibitors), and centrifuged for 1 min at 16,000 ×g. 30 μl of gel loading buffer (10 mmTris-HCl, pH 6.5, 3% SDS, 10% glycerol, 5% β-mercaptoethanol, 8m urea, and 0.025% bromphenol blue) was added to the washed beads and heated to 95 °C for 5 min. To remove the Sepharose beads, the suspension was applied to a microcentrifuge column (Bio-Rad) and centrifuged for 2 min at 16,000 × g. The flow-through fraction was subjected to SDS-PAGE on an 8% gel (24.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). The gel was fixed with 1-propanol/water/acetic acid (25:65:10), stained with 0.25% Coomassie Blue in methanol/water/acetic acid (45:45:10), destained with methanol/water/acetic acid (20:75:5), and dried on filter paper (80 °C, 2 h, vacuum). The dried gel was exposed in a PhosphorImager cassette and read after 1 week with a Molecular Dynamics PhosphorImager. For Western blotting, polypeptides were electrotransferred (25.Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44644) Google Scholar) onto nitrocellulose membranes (Schleicher & Schüll). Antibodies were detected by incubation with horseradish peroxidase-conjugated secondary antibody, followed by detection with an enhanced chemiluminescence system (NEN Life Science Products). The method of Lowry et al. (26.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) was used for protein measurements with bovine serum albumin as the standard. The activities of marker enzymes were determined according to the following protocols: γ-glutamyl transpeptidase (27.Orlowski M. Meister A. Biochim. Biophys. Acta. 1963; 73: 679-681Crossref PubMed Google Scholar), alkaline phosphatase (28.Walter K. Schütt C. Bergmeyer H.U. Methods of Enzymatic Analysis. II. Academic Press, New York1974: 856-864Google Scholar), Na,K-ATPase (29.Heidrich H.G. Kinne R. Kinne-Saffran E. Hannig K. J. Cell Biol. 1972; 54: 232-245Crossref PubMed Scopus (160) Google Scholar), and galactosyltransferase (30.Sztul E. Kaplin A. Saucan L. Palade G. Cell. 1991; 64: 81-89Abstract Full Text PDF PubMed Scopus (71) Google Scholar). The quality of the membrane preparations was determined by measuring the enrichment of marker enzymes (Table I). Marker enzymes for the canalicular membrane γ-glutamyl transpeptidase and alkaline phosphatase were highly enriched only in CMVs (∼45-fold) as compared with the homogenate. A marker for the basolateral membrane Na,K-ATPase was slightly increased in Golgi fractions, below the detection limit in CMVs, and enriched 16-fold in SMVs. The enrichment of marker enzymes was comparable to that reported previously (21.Inoue M. Kinne R. Tran T. Biempica L. Arias I.M. J. Biol. Chem. 1983; 258: 5183-5188Abstract Full Text PDF PubMed Google Scholar, 22.Inoue M. Kinne R. Tran T. Arias I.M. Hepatology. 1982; 2: 572-579Crossref PubMed Scopus (150) Google Scholar). Galactosyltransferase, a marker for Golgi membranes, was slightly enriched in SMVs, below the detection limit in CMVs, and 24-fold enriched in Golgi preparations. The yields of specific marker enzyme activities in CMVs and SMVs are consistent with data reported earlier (21.Inoue M. Kinne R. Tran T. Biempica L. Arias I.M. J. Biol. Chem. 1983; 258: 5183-5188Abstract Full Text PDF PubMed Google Scholar, 22.Inoue M. Kinne R. Tran T. Arias I.M. Hepatology. 1982; 2: 572-579Crossref PubMed Scopus (150) Google Scholar).Table ISpecific activities of marker enzymesMarker enzymeHOMCMVSMVGolgiγ-Glutamyl transpeptidase (μmol/h/mg)0.3 ± 0.114.1 ± 1.90.7 ± 0.40.6 ± 0.2(1) [100%](47) [5.7%](2) [1.1%](2) [0.2%]Alkaline phosphatase (μmol/h/mg)0.4 ± 0.117.6 ± 3.11.0 ± 0.31.2 ± 0.4(1) [100%](44) [5.4%](3) [1.1%](3) [0.4%]Na,K-ATPase (μmol/h/mg)0.3 ± 0.2ND4.7 ± 1.40.7 ± 0.4(1) [100%](16) [7.2%](2) [0.3%]Galactosyltransferase (nmol/h/mg)0.8 ± 0.3ND1.3 ± 0.319.3 ± 1.0(1) [100%](2) [0.7%](24) [2.9%]Values represent means ± S.E. from four to seven determinations. The fold enrichment of marker enzymes compared with the homogenate (HOM) given in parentheses, and the yield of a specific activity is given in brackets. ND, not detected. Open table in a new tab Values represent means ± S.E. from four to seven determinations. The fold enrichment of marker enzymes compared with the homogenate (HOM) given in parentheses, and the yield of a specific activity is given in brackets. ND, not detected. The quality of membrane preparations was further established by immunochemical methods. Monoclonal anti-β-COP antibody recognizes an epitope shared by the Golgi β-COP protein (110 kDa). Immunoblots probed with anti-β-COP antibody showed significant enrichment in Golgi membranes as compared with the homogenate, whereas β-COP was absent from canalicular and basolateral membranes (Fig.2). CMV and SMV preparations were further characterized by probing with antibodies against apical and basolateral membrane resident proteins in immunoblots. As shown in Fig. 4, basolateral ASGP-R was detected only in SMVs and was absent from CMVs, whereas antibodies against the canalicular transporters MDR1 and MDR2 and SPGP and MRP2 reacted exclusively with antigens in CMVs.Figure 4CMV/SMV distribution of hepatic membrane proteins. Rat liver CMVs (C) and SMVs (S), 10 mg each, were separated by SDS-PAGE; blotted onto nitrocellulose membrane; and probed with antibodies against the basolateral membrane resident ASGP-R and the bile canaliculus resident proteins MDR1 and MDR2 (detected with the C219 antibody (anti-MDR1/MDR2)), SPGP, the organic anion transporter MRP2, the ectoenzyme dipeptidyl peptidase IV (DPP IV), and the canalicular cell adhesion molecule cCAM105. Arrowheads indicate the positions of antigens.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We were interested in antibodies directed against canalicular ABC-type transporters that were suitable for immunoprecipitation. Among available antibodies, only monoclonal antibody C219 (anti-MDR1/MDR2) met this property. Therefore, we generated a polyclonal antibody against the recently cloned canalicular bile acid transporter SPGP. Alignments of amino acid sequences revealed high similarity/homology among rat MDR1 (31.Silverman J.A. Raunio H. Gant T.W. Thorreirsson S.S. Gene (Amst.). 1991; 106: 229-236Crossref PubMed Scopus (103) Google Scholar), MDR2 (32.Brown P.C. Thorgeirsson S.S. Snorri S. Silverman J.A. Nucleic Acids Res. 1993; 21: 3885-3891Crossref PubMed Scopus (81) Google Scholar), and SPGP (9.Gerloff T. Stieger B. Hagenbuch B. Madon J. Landmann L. Roth J. Hofmann A.F. Meier P.J. J. Biol. Chem. 1998; 273: 10046-10050Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar): MDR1 versusSPGP, 48/68%; MDR1 versus MDR2, 69/82%; and MDR1versus SPGP, 46/66%. A 90-amino acid peptide from the SPGP "linker" region that showed the lowest similarity/homology to the other MDR transporters was chosen as immunogen for the generation of anti-SPGP antibody LVT90. Monoclonal antibody C219 was raised against MDR1 constitutively overexpressed in Chinese hamster ovary cells and is directed against two hexapeptides located close to the ATP-binding sites in CHO-MDR1, VQAALD and VQEALD (33.Georges E. Bradley G. Gariepy J. Ling V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 152-156Crossref PubMed Scopus (375) Google Scholar). Both recognition motifs are present in rat MDR1 and MDR2 at the same position. In rat SPGP, the corresponding sites are altered to VQEALN and VQTALD. In the first recognition sequence, aspartic acid, which is critical for antigen recognition (33.Georges E. Bradley G. Gariepy J. Ling V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 152-156Crossref PubMed Scopus (375) Google Scholar), was replaced by asparagine. In the second recognition sequence, glutamic acid was replaced by threonine. In immunoblots, the polyclonal anti-SPGP antibody LVT90 showed strong reaction with a 170-kDa protein in CMVs that was absent from SMVs (see Fig. 4). Preimmune serum did not show reactions with CMVs or SMVs. Reaction of LVT90 with CMVs was competed by addition of the glutathioneS-transferase fusion protein (data not shown). To test for possible cross-reactivity between C219 and LVT90, we performed immunoprecipitations followed by immunoblotting (Fig.3). A lysate of rat liver CMVs (500 μg) was immunoprecipitated first with anti-SPGP antibody LVT90 (lanes A) followed by anti-MDR1/MDR2 antibody C219 (lanes B). In a similar experiment, CMV lysate was first immunoprecipitated with C219 (lanes C) followed by LVT90 (lanes D). Immunoprecipitates were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and probed with either LVT90 or C219. Reaction of LVT90 in immunoblots was observed only when LVT90 was used for immunoprecipitation. Whether LVT90 immunoprecipitation was before or after precipitation with C219 was uncritical. Furthermore, C219 immunoprecipitates showed no reaction with LVT90 in immunoblots. The same applied for C219 immunoblots. Positive reactions were observed only when C219 was used for the initial precipitation, and no reaction was observed with LVT90 precipitates. The sequence in which antibodies were employed for precipitation was not critical. These immunoprecipitation/blotting experiments indicate that there is no cross-reactivity between the C219 and LVT90 antibodies with regard to immunoprecipitation and immunoblotting. Thus, C219 is specific for rat MDR1 and MDR2, and LVT90 is specific for rat SPGP. Western blots of purified CMVs and SMVs that were probed with antibodies against canalicular proteins are shown in Fig.4. Anti-dipeptidyl peptidase IV and anti-cCAM105 antibodies predominantly reacted with CMVs, and a small amount was regularly observed in SMVs. We explain the presence of these "canalicular" proteins in SMVs by the fact that these membrane proteins are initially transferred to the basolateral membrane after biosynthesis and subsequently reach the apical pole by transcytosis (14.Bartles J.R. Feracci H.M. Stieger B. Hubbard A.L. J. Cell Biol. 1987; 105: 1241-1251Crossref PubMed Scopus (258) Google Scholar). This scenario is in good accordance with detectable steady-state levels in SMVs. Under the same conditions, antibodies against the canalicular ABC-type transporters, C219 (anti-MDR1/MDR2), LVT90 (anti-SPGP), and EAG15 (anti-MRP2), exclusively recognized antigens in CMVs, which suggests that newly synthesized canalicular ABC transporters may not be initially trafficked to the basolateral plasma membrane. To test the hypothesis of direct apical targeting of canalicular ABC transporters in rat hepatocytes, we performed metabolic pulse-chase labeling experiments. Rats were injected with [35S]methionine, and labeling of newly synthesized prote
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