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Localization of the PE methylation pathway and SR-BI to the canalicular membrane

2003; Elsevier BV; Volume: 44; Issue: 9 Linguagem: Inglês

10.1194/jlr.m200488-jlr200

1539-7262

Ephraim Sehayek, Rong Wang, Jennie G. Ono, Vadim S. Zinchuk, Elizabeth M. Duncan, Sarah Shefer, Dennis E. Vance, Meenakshisundaram Ananthanarayanan, Brian T. Chait, Jan L. Breslow,

Folate and B Vitamins Research

To better understand the regulation of biliary phospholipid and cholesterol excretion, canalicular membranes were isolated from the livers of C57BL/6J mice and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry. A prominent protein revealed by this analysis was betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first step in a three-enzyme pathway that promotes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Immunoblotting confirmed the presence of BHMT on the canalicular membrane, failed to reveal the presence of the second enzyme in this pathway, methionine adenosyltransferase, and localized the third enzyme of the pathway, PE N-methyltransferase (PEMT). Furthermore, immunfluorescence microscopy unambiguously confirmed the localization of PEMT to the canalicular membrane. These findings indicate that a local mechanism exists in or around hepatocyte canalicular membranes to promote phosphatidylethnolamine methylation and PC biosynthesis. Finally, immunoblotting revealed the presence and immunofluorescence microscopy unambiguously localized the scavenger receptor class B type I (SR-BI) to the canalicular membrane. Therefore, SR-BI, which is known to play a role in cholesterol uptake at the hepatocyte basolateral membrane, may also be involved in biliary cholesterol excretion.Based on these findings, a model is proposed in which local canalicular membrane PC biosynthesis in concert with the phospholipid transporter mdr2 and SR-BI, promotes the excretion of phospholipid and cholesterol into the bile. To better understand the regulation of biliary phospholipid and cholesterol excretion, canalicular membranes were isolated from the livers of C57BL/6J mice and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry. A prominent protein revealed by this analysis was betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first step in a three-enzyme pathway that promotes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Immunoblotting confirmed the presence of BHMT on the canalicular membrane, failed to reveal the presence of the second enzyme in this pathway, methionine adenosyltransferase, and localized the third enzyme of the pathway, PE N-methyltransferase (PEMT). Furthermore, immunfluorescence microscopy unambiguously confirmed the localization of PEMT to the canalicular membrane. These findings indicate that a local mechanism exists in or around hepatocyte canalicular membranes to promote phosphatidylethnolamine methylation and PC biosynthesis. Finally, immunoblotting revealed the presence and immunofluorescence microscopy unambiguously localized the scavenger receptor class B type I (SR-BI) to the canalicular membrane. Therefore, SR-BI, which is known to play a role in cholesterol uptake at the hepatocyte basolateral membrane, may also be involved in biliary cholesterol excretion. Based on these findings, a model is proposed in which local canalicular membrane PC biosynthesis in concert with the phospholipid transporter mdr2 and SR-BI, promotes the excretion of phospholipid and cholesterol into the bile. The excretion of the biliary lipids, bile acids, phospholipids, and cholesterol, is a complex process and is regulated at multiple levels. Bile acids are synthesized in hepatocytes, excreted into the bile, and enter the enterohepatic circulation, where they are taken up from the portal system by the hepatocyte basolateral membrane transporter Ntcp. The uptake of bile acids is followed by their reexcretion, along with newly synthesized bile acids, back into the bile. This recycling is considered a major driving force behind biliary excretion of phospholipids and cholesterol (1Cohen D.E. Hepatocellular transport and secretion of biliary lipids.Curr. Opin. Lipidol. 1999; 10: 295-302Crossref PubMed Scopus (41) Google Scholar). However, the importance of the bile acid recycling in determining biliary phospholipid and cholesterol excretion has been challenged by studies in mdr2 knockouts where normal bile acid excretion is associated with a complete defect in biliary phospholipid and cholesterol excretion (2Smit J.J. Schinkel A.H. Oude Elferink R.P. Groen A.K. Wagenaar E. van Deemter L. Mol C.A. Ottenhoff R. van der Lugt N.M. van Roon M.A. et al.Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.Cell. 1993; 75: 451-462Abstract Full Text PDF PubMed Scopus (1318) Google Scholar). This animal model provides unequivocal proof that mdr2, a flippase that localizes specifically to the canalicular membrane and transfers phosphatidylcholine (PC) from the inner to the outer leaflet, has a critical role in biliary phospholipid excretion. Moreover, the failure of mdr2 knockouts to excrete cholesterol suggests that biliary phospholipid excretion plays a critical role in driving the excretion of cholesterol into the bile. The complex relationships between biliary cholesterol and phospholipid excretion has been further exemplified by studies in mice with liver and intestinal overexpression of hemitransporters implicated in the enterohepatic metabolism of plant sterols and cholesterol, ABCG5 and ABCG8 (3Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol.J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (600) Google Scholar). In this model, transgenic males display a 4- to 5-fold increase in biliary cholesterol concentrations that was associated with a modest but significant increase in phospholipid concentration, suggesting that under certain conditions, increased excretion of cholesterol may drive phospholipid excretion. Finally, Cyp27a1 knockout mice, an animal model with a severely depleted bile acid pool, maintain their biliary cholesterol levels (4Rosen H. Reshef A. Maeda N. Lippoldt A. Shpizen S. Triger L. Eggertsen G. Bjorkhem I. Leitersdorf E. Markedly reduced bile acid synthesis but maintained levels of cholesterol and vitamin D metabolites in mice with disrupted sterol 27-hydroxylase gene.J. Biol. Chem. 1998; 273: 14805-14812Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). When taken together, these studies exemplify the complexity of biliary lipid excretion and call for studies that clarify the details of these processes at the molecular level.To increase our understanding of biliary lipid excretion, canalicular membranes from the livers of C57BL/6J mice were isolated and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). One of the proteins identified was the enzyme betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first in a three-step metabolic pathway that promotes the methylation of phosphatidylethanolamine (PE) to PC. Immunoblotting confirmed the presence of BHMT and also revealed the enzyme that catalyzes the third step in this pathway, PE N-methyltransferase (PEMT). Furthermore, immunofluorescent microscopy clearly showed that PEMT is localized to the canalicular membrane. These findings suggest that canalicular localization of BHMT and PEMT contributes to local PC synthesis and secretion into the bile. In addition, immunoblotting of canalicular membranes and immunfluorescent microscopy studies clearly revealed the presence of scavenger receptor class B type I (SR-BI), which may play a role in directly transferring cholesterol from the canalicular membrane into the biliary space. These findings suggest a model in which local canalicular membrane PC biosynthesis, in concert with mdr2 and SR-BI, promotes phospholipid and cholesterol excretion into the bile.MATERIALS AND METHODSAnimalsWild-type C57BL/6J males were purchased from The Jackson Laboratories (Bar Harbor, ME) and studied at 10–13 weeks of age. Animals were housed in a temperature- and humidity-controlled room with a 12 h light-dark cycle (6 AM–6 PM light) and fed Picolab Rodent Chow 20 (5053) pellets for 3 weeks. The Rockefeller University Institutional Animal Care and Research Advisory Committee approved all experiments.Membrane preparationMouse liver canalicular and basolateral membranes were isolated by a minor modification of the nitrogen cavitation/calcium precipitation method described by Kipp and Arias for the rat (5Kipp H. Arias I.M. Newly synthesized canalicular ABC transporters are directly targeted from the Golgi to the hepatocyte apical domain in rat liver.J. Biol. Chem. 2000; 275: 15917-15925Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Twenty mouse livers were rapidly perfused with an ice-cold SHCa buffer (0.25 M sucrose, 10 mM HEPES/Tris, pH 7.4, 0.2 mM CaCl2) to which 2 μg/ml aprotinin, 2 μg/ml pepstatin A, 2 μg/ml leupeptin, 5 μg/ml benzamidine, and 20 μg/ml phenylmethylsulfonyl fluoride were added. The livers were minced with a razor blade and groups of 10 livers homogenized in 50 ml of SHCa buffer with four strokes of a loose-fitting Dounce homogenizer. The liver suspensions were combined and filtered through a double layer of cheesecloth. The filtrate was homogenized with an additional 15 strokes, diluted with SH buffer (Ca-free SHCa buffer) to 270 ml, supplemented with EDTA (pH 7.4) to a final concentration of 1 mM, and centrifuged for 10 min at 1,880 g (Beckman Allegra 6R, 2868 rpm). After centrifugation, the tubes were allowed to come to a stop with the brake off. The pellet and fluffy layer just above the pellet were collected and used to prepare canalicular membranes, while the supernatant was used to prepare basolateral membranes.Canalicular membrane preparationPellet and fluffy layer were resuspended in 50 ml of SHCa buffer, homogenized with six strokes of a loose-fitting Dounce homogenizer, and centrifuged for 10 min at 3,000 g (Beckman Allegra 6R, 3,624 rpm). The resulting pellet was suspended in 50 ml of SHCa buffer, homogenized with six strokes of a loose-fitting Dounce homogenizer, placed in a beaker within an ice-cold high-pressure chamber (Parr Instrument Model 4635), and equilibrated for 15 min with nitrogen at 900 pounds per square inch by stirring. After 15 min the chamber's pressure was brought within 3 min to atmospheric pressure. The suspension was then homogenized with six strokes in a tight-fitting Dounce homogenizer, diluted with SHCa buffer to 120 ml, supplemented with CaCl2 to a final concentration of 10 mM, incubated for 10 min on ice, and centrifuged for 20 min at 7,600 g (Beckman L6 centrifuge, SW28 rotor, 6,500 rpm at 4°C, brake on). After centrifugation, the supernatant was filtered through fine nylon mesh and the filtrate centrifuged for 30 min at 47,000 g (Beckman SW28, 16,200 rpm at 4°C). The pellet was then homogenized in 30 ml of SHCa buffer with six strokes in a tight-fitting Dounce homogenizer and centrifuged for 10 min at 3,000 g (Beckman Allegra 6R, 3,624 rpm). The supernatant was collected and centrifuged for 30 min at 47,000 g (Beckman SW28, 16,200 rpm at 4°C) and the pellet suspended in SHCa buffer using a 1 ml syringe attached to a 28-gauge needle at a concentration of 1–2 mg/ml protein and stored at −80°C until used. In a typical preparation, the livers from 20 mice yielded 150–250 μg of canalicular membrane protein.Basolateral membrane preparationThe first supernatant was separated and centrifuged for 10 min at 5,500 g (Sorval GSA rotor, 5,800 rpm) and supernatant and fluffy layer collected and centrifuged for 30 min at 22,000 g (Sorval GSA rotor, 11,600 rpm). The pellet was resuspended in 12 ml of calcium-free SH buffer containing 1 mM EDTA (pH 7.4), homogenized with six strokes in a loose-fitting Dounce homogenizer, and layered on a discontinuous gradient consisting of 1 ml 60% sucrose, 23 ml 23% sucrose, 4% Ficoll 400, and 7 ml 20% sucrose. After 90 min centrifugation at 130,000 g (Beckman swinging bucket rotor SW 28, 27,000 rpm), the interphase between the 20% sucrose and 23% sucrose 4% Ficoll layers was aspirated, diluted 6-fold with SHCa buffer, and centrifuged for 30 min at 47,000 g (Beckman SW-28 rotor, 20,000 rpm). The pellet was resuspended in SHCa buffer and centrifugation repeated as above. The resulting pellet was resuspended in SHCa buffer with a syringe and 28-gauge needle at a concentration of ∼10 mg/ml protein and stored at −80°C until used. A typical preparation yielded 2.5–5 mg of basolateral membrane protein.Preparation of liver cytosolLiver tissue (0.5 g) was homogenized on ice in 7 ml of ice-cold homogenization buffer (100 mM K2HPO4, 1 mM EDTA, 5 mM DTT, 50 mM KCl, 5% glycerol, pH 7.4) by seven strokes of a tight-fitting Dounce homogenizer. The homogenate was spun for 10 min at 2,000 g at 4°C (Beckman Allegra 6R, 2,960 rpm), and the resultant supernatant spun for 20 min at 10,000 g (Beckman SW55 rotor, 10,300 rpm at 4°C). The supernatant of the second spin was subjected to ultracentrifugation at 100,000 g for 90 min (Beckman SW55 rotor, 32,500 rpm at 4°C), and cytosol (supernatant) was collected and stored at −80°C.Alkaline phosphatase activityAlkaline phosphatase activity was measured in whole-liver homogenate and canalicular or basolateral membrane fractions. Aliquots containing 10–20 μg of protein were assayed colorimetrically using a commercially available kit (Sigma kit Cat # 245-10). Activity was expressed as units/mg protein/min.Biliary protein extractionThe gallbladder bile of C57BL/6J males was aspirated as previously described (6Sehayek E. Shefer S. Nguyen L.B. Ono J.G. Merkel M. Breslow J.L. Apolipoprotein E regulates dietary cholesterol absorption and biliary cholesterol excretion: Studies in C57BL/6 apolipoprotein E knockout mice.Proc. Natl. Acad. Sci. USA. 2000; 97: 3433-3437Crossref PubMed Scopus (63) Google Scholar) and biliary proteins were isolated following bile delipidation. Briefly, 50 μl of bile were mixed in an Ependorf tube with 1 ml of ice-cold ethyl-ether/ethanol (1:3; v/v), vortexed, incubated for 1 h at −20°C, spun for 30 min at room temperature, the supernatant discarded, and the pellet resuspended in 1 ml of ice-cold ethyl-ether/ethanol (2:3; v/v). Resuspended pellet was incubated for 1 h at −20°C, spun for 30 min at 4°C, the supernatant discarded, and the pellet resuspended in 1 ml of ice-cold ethyl-ether, vortexed, incubated for 1 h at −20°C, spun for 30 min at 4°C, and the last ethyl-ether extraction repeated one more time as above. The final pellet was allowed to evaporate at room temperature, dissolved in ddH2O, and protein concentration measured using the bicinchoninic protein assay (Pierce Biotechnology, Rockford, IL).SDS-PAGE, Coomassie staining, zinc staining, and Western blottingTen to fifty micrograms of biliary proteins or canalicular or basolateral membrane fractions were resuspended in an equal volume of DTT containing Novex Tris-Glycine Sample Buffer, boiled for 5 min, and loaded on a Novex polyacrylamide Tris-Glycine gel and subjected to electrophoresis at 100 V for 2–3 h. For Coomassie staining, gels were soaked in a Coomassie blue solution for 1 h and destained with 10% PBS-methanol. For zinc staining, a Bio-Rad zinc staining and destaining kit was used according to the manufacturer's Instruction Manual (Bio Rad Laboratories, Hercules, CA). For Western blots, proteins were subjected to overnight transfer at 110 mA onto a nitrocellulose membrane, prehybridized in casein blocker, and hybridized with either anti-mdr2 (monoclonal C219, Signet Laboratories, Dedham, MA), anti-Ntcp, anti-SR-BI (polyclonal NB 400-104, Novus Biologicals, Inc., Littleton, CO), anti-apolipoprotein A-I (apoA-I) (polyclonal K23500R, BIODESIGN International, Saco, ME), anti-BHMT (a generous gift of Dr. Timothy A. Garrow, University of Illinois, Urbana, IL), anti-methionine adenosyltransferase (MAT) (a generous gift of Dr. Jose M. Mato, Universidad de Navarra, Pamplona, Spain), or anti-PEMT2 antibodies, washed in PBS Tween buffer, incubated with the appropriate secondary antibody, and detected by chemiluminescence using NEN Luminol and Oxidizing reagents.Mass spectrometryThe protein bands separated by SDS-PAGE were visualized by zinc staining (Bio Rad Laboratories). The bands of interest were excised, subjected to in-gel digestion with trypsin, and the resulting peptide mixtures extracted as described (7Qin J. Fenyo D. Zhao Y. Hall W.W. Chao D.M. Wilson C.J. Young R.A. Chait B.T. A strategy for rapid, high-confidence protein identification.Anal. Chem. 1997; 69: 3995-4001Crossref PubMed Scopus (98) Google Scholar). Peptide mixtures were analyzed with MALDI-TOF MS using a delayed ion extraction and ion mirror reflector mass spectrometer (Voyager-DE STR; Perseptive Biosystems). The measured masses of the tryptic peptides (tryptic peptide map) were used to search for protein candidates in the nonredundant protein sequence database with the program ProFound (ProteoMetrics, New York, NY) (8Fenyo D. Qin J. Chait B.T. Protein identification using mass spectrometric information.Electrophoresis. 1998; 19: 998-1005Crossref PubMed Scopus (174) Google Scholar). To confirm the protein identification results obtained from tryptic peptide mapping, peptide mixtures were analyzed by tandem MS fragmentation analyses using HPLC-ion trap mass spectrometry (LCQ, Finnigan MAT, San Jose, CA) equipped with a capillary HPLC (Magic 2002, Michrom BioResources, Auburn, CA) (9Qin J. Herring C.J. Zhang X. De novo peptide sequencing in an ion trap mass spectrometer with 18O labeling.Rapid Commun. Mass Spectrom. 1998; 12: 209-216Crossref PubMed Scopus (65) Google Scholar) and the protein search algorithm PepFrag (ProteoMetrics) (8Fenyo D. Qin J. Chait B.T. Protein identification using mass spectrometric information.Electrophoresis. 1998; 19: 998-1005Crossref PubMed Scopus (174) Google Scholar).Immunofluorescent microscopy and confocal imagingLivers were cut into pieces less than 1 mm thick, embedded in OCT compound (Miles, Elkhart, IN), and frozen in liquid nitrogen. Cryostat sections (8–10 μm in thickness) were picked up on poly-l-lysine-coated glass slides, air-dried, and fixed in acetone for 15 min at −20°C. Ten percent goat serum in Tris-buffered saline was applied to block nonspecific binding. Samples were then incubated with individual or mixtures of anti-Mrp2 (Alexis Biochemicals, San Diego, CA), anti-PEMT2, and/or immunopurified anti-SR-BI antibodies (Novus Biologicals) diluted 1:100, 1:200, and 1:400, respectively, for 30–40 min (PEMT studies) and 1 h (SR-BI studies) at room temperature. After rinsing with Tris-buffered saline, the samples were incubated with the mixture of corresponding secondary antibodies (conjugated with Alexa 594 and Alexa 488, respectively) (Molecular Probes, Eugene, OR), and diluted 1:400 for 1 h at room temperature. In controls, specimens were either incubated with nonimmune IgG or the primary antibodies were omitted from the labeling process. After a final washing step, the sections were mounted with a ProLong antifade reagent (Molecular Probes, Leiden, The Netherlands), covered with glass, and examined using a confocal laser scanning microscope LSM 410 (Carl Zeiss, Jena, Germany). Obtained images were saved to MO disk and processed on a Macintosh Dual G4 Power PC (Apple Computer, Cupertino, CA). Adobe Photoshop 7 and Adobe Illustrator 10 software (Adobe Systems, San Jose, CA) was used to adjust brightness and contrast of the images and to compose them in plates. Plates were printed directly using a Fuji Pictrography 3000 printer (Fuji Photo Film, Tokyo, Japan).RESULTSA number of tests were performed to assess the quality and purity of the isolated liver canalicular membrane fraction. Kipp and Arias reported that liver canalicular membranes are enriched in alkaline phosphatase (5Kipp H. Arias I.M. Newly synthesized canalicular ABC transporters are directly targeted from the Golgi to the hepatocyte apical domain in rat liver.J. Biol. Chem. 2000; 275: 15917-15925Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Therefore, this enzymatic activity was assessed in whole-liver homogenate and canalicular or basolateral membrane fractions and found to be 0.06 ± 0.05, 2.66 ± 1.36, and 0.11 ± 0.03 U/mg protein/min, respectively (mean ± SD of 10 different preparations). Thus the nitrogen cavitation/calcium precipitation method yielded canalicular membranes that were 44- and 24-fold enriched in alkaline phosphatase compared with whole-liver homogenates and basolateral membrane fractions, respectively. To assess cross-contamination between membrane fractions, immunoblotting was carried out for the canalicular membrane marker, mdr2, and the basolateral membrane marker, Ntcp, the sodium-dependent bile acid transporter. As shown in Fig. 1, mdr2 localized exclusively to the canalicular membrane, and Ntcp is mainly in basolateral membrane (276-fold enrichment by scan densitometry). These results clearly indicate that the mouse liver canalicular membrane fraction isolated using the protocol described in this paper is highly concentrated with no evidence of major cross-contamination with basolateral membrane fractions.Proteins residing in the canalicular membrane fraction were next identified. Two different preparations of canalicular and basolateral membrane fractions were subjected to SDS-PAGE and the results shown in Fig. 2. As shown, many differences exist in the protein-banding pattern between the two membrane fractions. Three discrete canalicular protein bands labeled in this figure as 1 (108 kDa), 2 (54 kDa), and 3 (50 kDa) were analyzed with MALDI-TOF MS. As shown in Table 1, these bands corresponded to radixin, BHMT, and γ-actin, respectively. The identities of the BHMT and γ-actin bands were confirmed by trypsinization and tandem-MS fragmentation analysis. Radixin is an actin barbed-end capping protein present at the undercoat of the cell-to-cell adherence junction and has been previously shown to occur at high concentrations in canalicular membrane preparations (10Fouassier L. Duan C.Y. Feranchak A.P. Yun C.H. Sutherland E. Simon F. Fitz J.G. Doctor R.B. Ezrin-radixin-moesin-binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia.Hepatology. 2001; 33: 166-176Crossref PubMed Scopus (93) Google Scholar). γ-Actin is a cytoskeletal protein. BHMT is an enzyme that catalyzes the transfer of a methyl group from betaine to homocysteine. Immunoblotting was used to further characterize the subcellular localization of BHMT and the results shown in Fig. 3. BHMT is present in canalicular membranes, to a lesser extent in basolateral membranes, and, as expected, to a greater extent in cyotsol (11Delgado-Reyes C.V. Wallig M.A. Garrow T.A. Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney.Arch. Biochem. Biophys. 2001; 393: 184-186Crossref PubMed Scopus (84) Google Scholar).Fig. 2SDS-PAGE protein profiles of BLM and CM preparations. Samples (10 μg protein/lane) of two different BLM preparations and two different canalicular membrane preparations were loaded on a 10–20% tris-glycine gel and Coomassie stained as described in Materials and Methods. CM bands 1 (Radixin), 2 [betaine homocysteine methyltransferase, (BHMT)], and 3 (γ-actin) were characterized using mass spectrometry analysis as described in Materials and Methods and detailed in Table 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Mass spectrometry analysis of canalicular membrane proteins (see also Fig. 2)Protein Identification by MS (GenBank Acc. #)Molecular Weight (kDaaMolecular weight based on SWISS-PROT database.)Protein Identification Confirmed by MS/MSBandPeptide Molecular WeightSequence1 Radixin (gi|131820|) 68.4 (108bMolecular weight based on SDS-PAGE (Fig. 2).)——2 BHMT (gi|2645804|) 45.0 (54bMolecular weight based on SDS-PAGE (Fig. 2).)1,097.6AIAEELAPER1,136.6KEYWQNLR1,860.9QGFIDLPEFPFGLEPR1,934.0LNAGEVVIGDGGFVFALEK3 γ-Actin (gi|809561|) 41.8 (50bMolecular weight based on SDS-PAGE (Fig. 2).)1,131.5GYSFTTTAER1,160.6EITALAPSTMK1,170.6HQGVMVGMGQK1,197.5DSYVGDEAQSK1,789.9SYELPDGQVITIGNER1,959.9YPIEHGIITNWDDMEKBHMT, betaine homocysteine methyl transferase; MS/MS, tandem mass spectrometry.a Molecular weight based on SWISS-PROT database.b Molecular weight based on SDS-PAGE (Fig. 2). Open table in a new tab Fig. 3Cellular localization of (A) BHMT, (B) methionine adenosyltransferase (MAT), and (C) phosphatidylethanolamine N-methyltransferase (PEMT). Samples (10 μg protein/lane in BHMT and MAT and 50 μg protein/lane in PEMT blots) of CM, BLM, and cytosol (CYT) used for Western blot using specific antibodies as described in Materials and Methods.View Large Image Figure ViewerDownload Hi-res image Download (PPT)BHMT catalyzes the first step in a pathway that promotes the methylation of PE to PC. Immunoblotting was also used to localize the other two enzymes of this pathway. As shown in Fig. 3, the second enzyme, MAT, is cytosolic and not present on either the canalicular or basolateral membranes, whereas the third enzyme, PEMT, is present in canalicular membranes, to a lesser extent in basolateral membranes, and not present in cytosol (12McKeever M.P. Weir D.G. Molloy A. Scott J.M. Betaine-homocysteine methyltransferase: organ distribution in man, pig and rat and subcellular distribution in the rat.Clin. Sci. 1991; 81: 551-556Crossref PubMed Google Scholar).For further characterization of PEMT localization, we applied immunofluorescent microscopy and compared the hepatocytic localization of PEMT to that of MRP2 (multidrug resistance-associated protein 2), an ABC transporter that is confined to the canalicular membrane. As shown in Fig. 4A, anti-MRP2 antibodies specifically stained the canalicular membranes (arrows). It is of note that the staining with anti-PEMT antibodies revealed a pattern that resembled the staining with anti-MRP2, with the most intense staining localized to the canalicular membranes (Fig. 4B, arrows). Moreover, as shown in Fig. 4C, the colocalization of PEMT and MRP2 is most evident when the staining of these proteins is overlapped. These results unequivocally indicate that PEMT localizes to the canalicular membrane. It is important to note, however, that the staining with anti-PEMT antibodies is not confined to the canalicular membrane and staining was clearly detected in areas that may correspond to a specific endoplasmic reticulum (ER) membrane referred to as mitochondria-associated membrane (MAM) (13Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. Cloning and expression of a novel phosphatidylethanolamine N-methyltransferase. A specific biochemical and cytological marker for a unique membrane fraction in rat liver.J. Biol. Chem. 1993; 268: 16655-16663Abstract Full Text PDF PubMed Google Scholar). Finally, to exclude the possibility that the immunofluorescent microscopy studies detected PEMT in the biliary canalicular space, we extracted the bile proteins and tested for the presence of PEMT. Whereas immunoblotting studies clearly revealed the presence of the biliary resident protein apoA-I, anti-PEMT antibodies failed to find PEMT in the bile (data not shown).Fig. 4Colocalization of PEMT and MRP2 to liver canalicular membranes. Liver sections where prepared and processed for immunofluorescent microscopy as described in Materials and Methods. Confocal laser scanning micrographs show fluorescence for MRP2 (A, red signals) and PEMT (B, green signals). The protein specifically visualized is indicated by colored text. C: Overlapped staining for both MRP2 and PEMT (yellow staining). Thin and thick arrows point to hepatocytes canaliculi, whereas arrowheads point to intracellular localization of PEMT, presumably to mitochondrial-associated membranes. Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Several studies provide evidence that SR-BI is involved in biliary cholesterol excretion. Induced mutant mice overexpressing SR-BI in liver have increased biliary cholesterol concentrations (14Kozarsky K.F. Donahee M.H. Rigotti A. Iqbal S.N. Edelman E.R. Krieger M. Overexpression of the HDL receptor SR-BI alters plasma HDL and bile cholesterol levels.Nature. 1997; 387: 414-417Crossref PubMed Scopus (626) Google Scholar, 15Sehayek E. Ono J.G. Shefer S. Nguyen L.B. Wang N. Batta A.K. Salen G. Smith J.D. Tall A.R. Breslow J.L. Biliary cholesterol excretion: a novel mechanism that regulates dietary cholesterol absorption.Proc. Natl. Acad. Sci. USA. 1998; 95: 10194-10199Crossref PubMed Scopus (151) Google Scholar), and SR-BI-mediated HDL particle uptake by liver results in selective sorting of HDL cholesterol from protein and polarized cholesterol excretion (16Silver D.L. Wang N. Xiao X. Tall A.R. High density lipoprotein (HDL) particle uptake mediated by scavenger receptor class B type 1 results in selective sorting of HDL cholesterol from protein and polarized cholesterol secretion.J. Biol. Chem. 2001; 276: 25287-25293Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). As shown in Fig. 5, immunoblotting indicates that SR-BI is present at roughly equal concentrations in