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• W2087272783 abstract "From a total of 47 known apolipoprotein A-I (apoA-I) mutations, only 18 are linked to low plasma HDL apoA-I concentrations, and 78% of these map to apoA-I helices 6 and 7 (residues 143–186). Gene transfer and transgenic mouse studies have shown that several helix 6 apoA-I mutations have reduced hepatic HDL production. Our objective was to examine the impact of helix 6 modifications on intracellular biosynthetic processing and secretion of apoA-I. Cells were transfected with wild-type or mutant apoA-I, radiolabeled with [35S]Met/Cys, and then placed in unlabeled medium for up to 4 h. Results show that >90% of newly synthesized wild-type apoA-I was secreted by 60 min. Over the same length of time, only 20% of helix 6 deletion mutant (Δ6 apoA-I) was secreted, whereas 80% remained cell associated. Microscopic and biochemical studies revealed that cell-associated Δ6 apoA-I was located predominantly within the cytoplasm as lipid-protein inclusions, whereas wild-type apoA-I was localized in the endoplasmic reticulum/Golgi. Results using other helix deletions or helix 6 substitution mutations indicated that only complete removal of helix 6 resulted in massive cytoplasmic accumulation.These data suggest that alterations in native apoA-I conformation can lead to aberrant trafficking and accumulation of apolipoprotein-phospholipid structures. Thus, conformation-dependent alterations in intracellular trafficking and turnover may underlie the reduced plasma HDL concentrations observed in individuals harboring deletion mutations within helix 6. From a total of 47 known apolipoprotein A-I (apoA-I) mutations, only 18 are linked to low plasma HDL apoA-I concentrations, and 78% of these map to apoA-I helices 6 and 7 (residues 143–186). Gene transfer and transgenic mouse studies have shown that several helix 6 apoA-I mutations have reduced hepatic HDL production. Our objective was to examine the impact of helix 6 modifications on intracellular biosynthetic processing and secretion of apoA-I. Cells were transfected with wild-type or mutant apoA-I, radiolabeled with [35S]Met/Cys, and then placed in unlabeled medium for up to 4 h. Results show that >90% of newly synthesized wild-type apoA-I was secreted by 60 min. Over the same length of time, only 20% of helix 6 deletion mutant (Δ6 apoA-I) was secreted, whereas 80% remained cell associated. Microscopic and biochemical studies revealed that cell-associated Δ6 apoA-I was located predominantly within the cytoplasm as lipid-protein inclusions, whereas wild-type apoA-I was localized in the endoplasmic reticulum/Golgi. Results using other helix deletions or helix 6 substitution mutations indicated that only complete removal of helix 6 resulted in massive cytoplasmic accumulation. These data suggest that alterations in native apoA-I conformation can lead to aberrant trafficking and accumulation of apolipoprotein-phospholipid structures. Thus, conformation-dependent alterations in intracellular trafficking and turnover may underlie the reduced plasma HDL concentrations observed in individuals harboring deletion mutations within helix 6. Apolipoprotein A-I (apoA-I) is the major protein constituent of HDLs and is synthesized and secreted by the liver and intestine (1Higuchi K. Law S.W. Hoeg J.M. Schumacher U.K. Meglin N. Brewer Jr., H.B. Tissue-specific expression of apolipoprotein A-I (ApoA-I) is regulated by the 5′-flanking region of the human apoA-I gene.J. Biol. Chem. 1988; 263: 18530-18536Google Scholar). This unique and abundant plasma protein directs mature spherical HDL particles to the liver, where the cholesteryl ester-rich core is removed by scavenger receptor class B type I, thereby completing a reverse cholesterol cycle (2Glomset J.A. The plasma lecithin:cholesterol acyltransferase reaction.J. Lipid Res. 1968; 9: 155-167Google Scholar, 3Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Google Scholar, 4Rigotti A. Miettinen H.E. Krieger M. The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other tissues.Endocr. Rev. 2003; 24: 357-387Google Scholar). Completion of this cycle is thought to prevent the accumulation of cholesterol in the aorta and represents a mechanism explaining the high correlation between increased plasma HDL concentrations and a lower incidence of coronary heart disease in humans (5Schaefer E.J. Familial lipoprotein disorders and premature coronary artery disease.Med. Clin. North Am. 1994; 78: 21-39Google Scholar, 6Castelli W.P. Garrison R.J. Wilson P.W.F. Abbott S. Incidence of coronary heart disease and lipoprotein cholesterol levels: the Framingham Study.J. Am. Med. Assoc. 1986; 156: 2835-2838Google Scholar).Until recently, the origin of nascent HDL apoA-I in plasma was thought to occur by either direct secretion of “discoidal” structures from the liver, as shown by studies from perfused rat liver (7Hamilton R.L. Williams M.C. Fielding C.J. Havel R.J. Discoidal bilayer structure of nascent high density lipoproteins from perfused rat liver.J. Clin. Invest. 1976; 58: 667-680Google Scholar), or by rearrangement of intestinal remnants released from the lipolysis of triglyceride-rich lipoproteins or the association of phospholipids and apoA-I in plasma (8Eisenberg S. High density lipoprotein metabolism.J. Lipid Res. 1984; 25: 1017-1058Google Scholar). Whereas VLDL particles have been readily isolated from the Golgi (9Hamilton R.L. Moorehouse A. Havel R.J. Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions.J. Lipid Res. 1991; 32: 529-543Google Scholar, 10Rusinol A. Verkade H. Vance J.E. Assembly of rat hepatic very low density lipoproteins in the endoplasmic reticulum.J. Biol. Chem. 1993; 268: 3555-3562Google Scholar), efforts to identify HDL particles along the secretory pathway have had little success (9Hamilton R.L. Moorehouse A. Havel R.J. Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions.J. Lipid Res. 1991; 32: 529-543Google Scholar). Although these findings provided little support for the intracellular assembly of nascent HDL, a number of other studies supported the concept of an intracellular apoA-I lipidation pathway (11Banerjee D. Redman C.M. Biosynthesis of high density lipoprotein by chicken liver: nature of nascent intracellular high density lipoprotein.J. Cell Biol. 1983; 96: 651-660Google Scholar, 12LeCureux L.W. Kezdy F.J. Wattenberg B.W. The efficiency and kinetics of secretion of apolipoprotein A-I in hepatic and non-hepatic cells.Atherosclerosis. 1994; 106: 225-233Google Scholar, 13Banerjee D. Mukherjee T.K. Redman C.M. Biosynthesis of high density lipoprotein by chicken liver: intracellular transport and proteolytic processing of nascent apolipoprotein A-I.J. Cell Biol. 1985; 101: 1219-1226Google Scholar, 14Dixon J.L. Battini R. Ferrari S. Redman C.M. Banerjee D. Expression and secretion of chicken apolipoprotein AI in transfected COS cells.Biochim. Biophys. Acta. 1989; 1009: 47-53Google Scholar, 15Dixon J.L. Chattapadhyay R. Huima T.R. Redman C.M. Banerjee D. Biosynthesis of lipoprotein: location of nascent apoA-I and apoB in the rough endoplasmic reticulum of chicken hepatocytes.J. Cell Biol. 1992; 117: 1161-1169Google Scholar, 16Howell K.E. Palade G.E. Heterogeneity of lipoprotein particles in hepatic Golgi fractions.J. Cell Biol. 1982; 92: 833-845Google Scholar). With the discovery of the ABCA1 transporter, many of these concepts concerning nascent HDL assembly have been revised, and recent studies support the idea of both ABCA1-dependent and -independent mechanisms responsible for apoA-I lipidation (17Chisholm J.W. Burleson E.R. Shelness G.S. Parks J.S. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL.J. Lipid Res. 2002; 43: 36-44Google Scholar, 18Kiss R.S. McManus D.C. Franklin V. Tan W.L. McKenzie A. Chimini G. Marcel Y.L. The lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways.J. Biol. Chem. 2003; 278: 10119-10127Google Scholar).The conformation of apoA-I is believed to be an essential factor regulating the synthesis, secretion, and lipidation of nascent HDL (19Marcel Y.L. Kiss R.S. Structure-function relationships of apolipoprotein A-I: a flexible protein with dynamic lipid associations.Curr. Opin. Lipidol. 2003; 14: 151-157Google Scholar, 20Klon A.E. Segrest J.P. Harvey S.C. Comparative models for human apolipoprotein A-I bound to lipid in discoidal high-density lipoprotein particles.Biochemistry. 2002; 41: 10895-10905Google Scholar, 21Brouillette C.G. Anantharamaiah G.M. Engler J.A. Borhani D.W. Structural models of human apolipoprotein A-I: a critical analysis and review.Biochim. Biophys. Acta. 2001; 1531: 4-46Google Scholar, 22Klon A.E. Jones M.K. Segrest J.P. Harvey S.C. Molecular belt models for the apolipoprotein A-I Paris and Milano mutations.Biophys. J. 2000; 79: 1679-1685Google Scholar). Cataloging naturally occurring human apoA-I mutations associated with low HDL concentrations suggests that disruption of apoA-I’s native structure dramatically alters HDL’s intravascular metabolism (23Sorci-Thomas M.G. Thomas M.J. The effects of altered apolipoprotein A-I structure on plasma HDL concentration.Trends Cardiovasc. Med. 2002; 12: 121-128Google Scholar). Of 18 documented apoA-I mutants associated with low HDL, 78% are amino acid substitutions/deletions within helices 6 and 7 (23Sorci-Thomas M.G. Thomas M.J. The effects of altered apolipoprotein A-I structure on plasma HDL concentration.Trends Cardiovasc. Med. 2002; 12: 121-128Google Scholar). The association between mutant forms of helix 6 and 7 and aberrant HDL metabolism has been studied in mice by several different approaches. In one study, transgenic mice were created expressing Δ6 apoA-I, a mutant form of apoA-I lacking the entire proline-punctuated 22 amino acid helix 6 (residues 143–164) (24Sorci-Thomas M.G. Thomas M. Curtiss L. Landrum M. Single repeat deletion in apoA-I blocks cholesterol esterification and results in rapid catabolism of D6 and wild-type apoA-I in transgenic mice.J. Biol. Chem. 2000; 275: 12156-12163Google Scholar, 25Baralle M. Baralle F.E. Genetics and molecular biology.Curr. Opin. Lipidol. 2000; 11: 653-656Google Scholar). In other studies, an adenoviral construct expressing the dominant-negative helix 6 point mutation, L159R apoA-I, was expressed in apoA-I knockout mice (26McManus D.C. Scott B.R. Franklin V. Sparks D.L. Marcel Y.L. Proteolytic degradation and impaired secretion of an apolipoprotein A-I mutant associated with dominantly inherited hypoalphalipoproteinemia.J. Biol. Chem. 2001; 276: 21292-21302Google Scholar), and in yet another study, a targeted replacement for the dominant-negative mutant apoA-I Milano (R173C apoA-I), a helix 7 point mutation, was examined (27Parolini C. Chiesa G. Zhu Y. Forte T. Caligari S. Gianazza E. Sacco M.G. Sirtori C.R. Rubin E.M. Targeted replacement of mouse apolipoprotein A-I with human ApoA-I or the mutant ApoA-IMilano. Evidence of APOA-IM impaired hepatic secretion.J. Biol. Chem. 2003; 278: 4740-4746Google Scholar). In each of these studies, the data suggest that mutations within apoA-I helices 6 and/or 7 cause decreased hepatic production and contribute to the dominant-negative phenotype observed in individuals heterozygous for these mutations (23Sorci-Thomas M.G. Thomas M.J. The effects of altered apolipoprotein A-I structure on plasma HDL concentration.Trends Cardiovasc. Med. 2002; 12: 121-128Google Scholar, 25Baralle M. Baralle F.E. Genetics and molecular biology.Curr. Opin. Lipidol. 2000; 11: 653-656Google Scholar).Thus, the current studies were conducted to probe how apoA-I conformation affects intracellular trafficking and secretion by measuring the in vitro secretion efficiency of mutant forms of apoA-I compared with wild-type apoA-I. Combining biochemical and microscopy studies, we found that although wild-type apoA-I was localized mainly in the endoplasmic reticulum (ER)/Golgi compartment and was secreted efficiently, the helix 6 deletion mutation, Δ6 apoA-I, was poorly secreted and accumulated within the cytosol as novel lipid-apolipoprotein structures. These studies show that deletion of a structurally essential helix results in the disruption of protein conformation and its accumulation and retention within the cell cytosol.MATERIALS AND METHODSDMEM, DMEM/Coon’s F12 (50:50), and G418 sulfate were obtained from Mediatech, Inc. Trypsin was obtained from JRH Biosciences, Inc. FuGene® was obtained from Roche Diagnostics Corp. Saponin was purchased from Sigma Chemicals. The polyclonal antibody to human apoA-I raised in goat was obtained from Chemicon, Inc. Rhodamine-conjugated antibody to goat IgG, mouse anti-cathepsin D, affinity-purified species-specific rabbit antiglobins conjugated with rhodamine or fluorescein, and BSA (IgG-free, protease-free) were purchased from Jackson ImmunoResearch Labs, Inc. Brefeldin A was obtained from Epicentre Technologies. Protein G-Sepharose 4 Fast Flow was purchased from Amersham Biosciences.Preparation of CMV5 human apoA-I cDNA-containing plasmidsWild-type and all mutant forms of human apoA-I cDNA, as well as the human serum albumin cDNA, were cloned into the pCMV5 vector using previously established methods (28Sorci-Thomas M.G. Parks J.S. Kearns M.W. Pate G.N. Zhang C. Thomas M.J. High level secretion of wild-type and mutant forms of human proapoA-I using baculovirus-mediated Sf-9 cell expression.J. Lipid Res. 1996; 37: 673-683Google Scholar, 29Sorci-Thomas M. Kearns M.W. Lee J.P. Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships.J. Biol. Chem. 1993; 268: 21403-21409Google Scholar, 30Li H.H. Thomas M.J. Pan W. Alexander E. Samuel M. Sorci-Thomas M.G. Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL.J. Lipid Res. 2001; 42: 2084-2091Google Scholar). PCR primers (CMV 5′, 5′-GCCTGCAGTCCCCCACGGCCCTT-3′; CMV 3′, 5′-GCGGATCCCACTTTGGAAACG-3′) containing embedded PstI and BamHI restriction sites, respectively, were used in the preparation of all clones. Introduction of apoA-I mutations was carried out as described previously (29Sorci-Thomas M. Kearns M.W. Lee J.P. Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships.J. Biol. Chem. 1993; 268: 21403-21409Google Scholar, 30Li H.H. Thomas M.J. Pan W. Alexander E. Samuel M. Sorci-Thomas M.G. Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL.J. Lipid Res. 2001; 42: 2084-2091Google Scholar, 31Sorci-Thomas M.G. Curtiss L. Parks J.S. Thomas M.J. Kearns M.W. Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase reactivity.J. Biol. Chem. 1997; 272: 7278-7284Google Scholar, 32Sorci-Thomas M.G. Curtiss L. Parks J.S. Thomas M.J. Kearns M.W. Landrum M. The hydrophobic face orientation of apolipoprotein A-I amphipathic helix domain 143–164 regulates lecithin:cholesterol acyltransferase activation.J. Biol. Chem. 1998; 273: 11776-11782Google Scholar). Constructs lacking the signal peptide sequence were made from human and mutant forms of the human apoA-I cDNA using the 5′ primer 5′-GCCTGCAGCGGCCCTTCAGGCGGCATTTCTGGCAG-3′ and the same CMV 3′ primer listed above. This 5′ primer removes the entire 18 amino acid pre-apoA-I sequence, leaving the 6 amino acid “pro sequence” intact. All plasmids were grown and purified using the Maxi-prep kit (Promega) as previously described (28Sorci-Thomas M.G. Parks J.S. Kearns M.W. Pate G.N. Zhang C. Thomas M.J. High level secretion of wild-type and mutant forms of human proapoA-I using baculovirus-mediated Sf-9 cell expression.J. Lipid Res. 1996; 37: 673-683Google Scholar, 30Li H.H. Thomas M.J. Pan W. Alexander E. Samuel M. Sorci-Thomas M.G. Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL.J. Lipid Res. 2001; 42: 2084-2091Google Scholar).Cell culture maintenance and transfectionHepG2, COS-1, CHO-K1, and McArdle RH-7777 cells were obtained from the American Type Culture Collection. COS, HepG2, and McArdle RH-7777 cells were maintained in DMEM, whereas CHO cells were maintained in DMEM/Coon’s F12 (50:50, v/v). All culture media contained essential vitamins and a final concentration of 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, and 10% FBS. Cells were maintained at 37°C in an atmosphere of 5% CO2.Cells were routinely grown to near confluence (>95%) in T-75 flasks and then split every third day. The day before COS-1 cells were transfected, nearly confluent flasks were treated with trypsin (0.25% porcine trypsin, 0.02% EDTA) and seeded onto 35 mm dishes using a 1:60 dilution. Five to 6 h after seeding, cell monolayers at 25–30% confluence were transfected at a FuGene®-to-cDNA volume-to-mass ratio of 6:1 (μl/μg).HepG2 cells were seeded onto 35 mm plates using a 1:40 dilution. Before plating, cells were passed through a 25 g syringe to prevent clumping. Approximately 24 h after plating, HepG2 cell monolayers reached 85% confluence and were transfected at a FuGene®-to-cDNA ratio of 2:1.25 (μl/μg). To reduce plate-to-plate transfection variability, duplicate plates of HepG2 cells per cDNA clone transfected were combined and replated 24 h after transfection.CHO cells were stably transfected with the human apoA-I Δ6 gene as previously described (29Sorci-Thomas M. Kearns M.W. Lee J.P. Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships.J. Biol. Chem. 1993; 268: 21403-21409Google Scholar). These cells were routinely maintained in DMEM/Coon’s F12 (50:50, v/v) containing a final concentration of 10% FBS, essential vitamins, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, and 0.94 mg/ml G418.Experiments involving brefeldin A were conducted on CHO Δ6 apoA-I stably expressing cells that had been seeded onto 35 mm dishes at a 1:100 dilution from confluent T-75 flasks. Five to 6 h after plating, the cells were approximately 25–30% confluent. Cells were then treated with a final concentration of 2.5 μM brefeldin A and then placed back into the incubator for 0, 24, and 48 h.Pulse-chase labeling and apoA-I immunoprecipitationForty-eight hours after transfection, metabolic labeling studies were performed. Cell monolayers were washed once with PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.46 mM KH2PO4, pH 7.4). Labeling was carried out by adding 0.5 ml of methionine-free DMEM (COS cells and HepG2 cells) or DMEM/Coon’s F12 (50:50) (CHO cells) containing 10% fetal calf serum and 200 μCi/ml (18 μl/35 mm dish) trans 35S label or [35S]Met/Cys for 10 min as previously reported (33Ingram M.F. Shelness G.S. Folding of the amino-terminal domain of apolipoprotein B initiates microsomal triglyceride transfer protein-dependent lipid transfer to nascent very low density lipoprotein.J. Biol. Chem. 1997; 272: 10279-10286Google Scholar, 34Shelness G.S. Morris-Rogers K.C. Ingram M.F. Apolipoprotein B48-membrane interactions. Absence of transmembrane localization to nonhepatic cells.J. Biol. Chem. 1994; 269: 9310-9318Google Scholar). In some cases, metabolic labeling was continuous for up to 4 h or the indicated time. The chase was begun by aspirating the labeling medium and adding 0.5 ml of fresh methionine-free DMEM (COS cells and HepG2 cells) or DMEM/Coon’s F12 (50:50) (CHO cells) containing 10% fetal calf serum only.At the indicated times, the culture medium was removed and centrifuged to pellet cell debris. The medium was transferred to a new tube until immunoprecipitation. Cells monolayers were washed twice with PBS, scraped in 15 ml of PBS, and collected by centrifugation at 400 g for 5 min at 4°C. Unless otherwise indicated, all subsequent centrifugation steps were performed at 4°C. Cell pellets were suspended in 750 μl of ice-cold lysis buffer (25 mM Tris-HC1, pH 7.4, 300 mM NaCl, 1% Triton X-100, 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml pepstatin). After a 10 min incubation on ice, samples were centrifuged at 14,000 rpm for 2 min. The supernatants were transferred to a fresh microcentrifuge tube and adjusted to a final concentration of 2.5 mg/ml BSA. Cell pellets were immunoprecipitated by adding 30 μl (2–3 mg/ml) of goat anti-human apoA-I antibody (Chemicon International, Inc.) and 35 μl of Protein G-Sepharose beads suspended (50:50, v/v) in TBS-C (25 mM Tris-HC1, 140 mM NaCl, and 1 mM CaCl2). After incubating for 10–28 h at 4°C on a rotating wheel, immune complexes were recovered by centrifugation at 14,000 rpm for 30 s. Immune-complexed beads were washed twice with 1 ml of lysis buffer and once with 1 ml of TBS-C. Immunoprecipitation of conditioned culture medium was carried out exactly as with cell pellets except that 0.5 ml of medium was adjusted to 1 mg/ml BSA, then 30 μl of goat anti-human apoA-I antibody and 35 μl of Protein G-Sepharose were added. These pellets were washed a total of three times with TBS-C and then prepared for SDS-PAGE.SDS polyacrylamide gel electrophoresis, fluorography, and data quantificationApoA-I eluted from the Protein G-Sepharose beads was separated by 15% SDS-PAGE as previously described (34Shelness G.S. Morris-Rogers K.C. Ingram M.F. Apolipoprotein B48-membrane interactions. Absence of transmembrane localization to nonhepatic cells.J. Biol. Chem. 1994; 269: 9310-9318Google Scholar). Gels containing 35S-labeled proteins were dried and exposed to film at −80°C using Kodak Biomax MS film. Images were imported into Scion6 Image, and the signal areas were obtained from the gray scale image after calibrating the scanner as previously described (17Chisholm J.W. Burleson E.R. Shelness G.S. Parks J.S. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL.J. Lipid Res. 2002; 43: 36-44Google Scholar).Preparation of postnuclear cell membranesTransfected cells from 100 mm dishes were labeled as described above, their conditioned media were collected, and cells were then scraped from the dish using 1 ml of ice-cold hypotonic buffer (10 mM HEPES, pH 7.4, 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml pepstatin). The cells were recovered by centrifugation for 45 s at 14,000 rpm. After centrifugation, the supernatant was aspirated, and cell pellets were suspended in 1 ml of fresh hypotonic buffer and then incubated on ice for 15 min (33Ingram M.F. Shelness G.S. Folding of the amino-terminal domain of apolipoprotein B initiates microsomal triglyceride transfer protein-dependent lipid transfer to nascent very low density lipoprotein.J. Biol. Chem. 1997; 272: 10279-10286Google Scholar). The cell suspension was transferred to a 2 ml Dounce homogenizer and subjected to 20 strokes using a tight-fitting pestle. The homogenate was immediately adjusted to 250 mM (final concentration) sucrose and centrifuged at 3,000 rpm (700 g) for 10 min. The postnuclear supernatant was then transferred to a fresh microcentrifuge tube and subjected to an additional round of centrifugation as described above to ensure the complete removal of nuclei and unbroken cells. Pellets containing nuclei and unbroken cells were combined and saved for immunoprecipitation, whereas the supernatants from both spins were centrifuged at 200,000 g for 15 min using a TLA100.3 fixed-angle rotor to isolate the microsomal fraction. After this step, an aliquot of the supernatant or cell cytosolic fraction was saved for immunoprecipitation. The microsomal fraction or pellet from this spin was suspended using Dounce homogenization (tight pestle, 10 strokes) in 0.5 ml of 10 mM HEPES, pH 7.4, 250 mM sucrose, 10 mM NaCl, 10 mM KCl, 2.5 mM MgCl2, and 2.5 mM CaCl2, with the following final concentrations of protease inhibitors: 20 μg/ml soybean trypsin inhibitor, 200 μg/ml aprotinin, 2.5 mM PMSF, 25 μg/ml leupeptin, and 25 μg/ml pepstatin.Carbonate extraction of postnuclear cell membranesThe microsomal fraction was suspended in a final volume of 2 ml of ice-cold 0.1 M sodium carbonate, pH 11.5, using Dounce homogenization. The homogenized membranes were incubated on ice for 1 h and then centrifuged for 30 min at 200,000 g in a TL-100 tabletop ultracentrifuge. The microsomal pellet containing noncarbonate-extractable proteins was saved for immunoprecipitation. The supernatant containing the carbonate-extractable microsomal proteins was also saved for immunoprecipitation. All fractions to be immunoprecipitated were adjusted to the following conditions: 50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 70 mM Na2CO3, 1 mM CaCl2, and 1% Triton X-100. The final pH was adjusted to 7.4 by adding dilute HCl. Both supernatant and pellet fractions were adjusted to a final concentration of 1 mg/ml BSA, 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml pepstatin. The supernatant and pellet fractions were suspended in 800 μl and 750 μl of lysis buffer, respectively. Both suspensions were immunoprecipitated using 30 μl of goat anti-human apoA-I polyclonal antibody and 70 μl of Protein G-Sepharose according to the incubation conditions described above.Isolation of phospholipid:apoA-I complexes from postnuclear supernatant and slot-blot/Western analysisTo characterize cytoplasmic inclusion from the postnuclear supernatant, aliquots were adjusted to a density of 1.30 g/ml, overlaid with KBr solutions at 1.166, 1.066, and 1.006 g/ml, and then centrifuged using a SW-41 rotor for 44 h at 40,000 rpm at 15°C. The density of each fraction was obtained by weighing known volumes of the fractions recovered from blank gradients.Phospholipid:apoA-I inclusions were isolated from HepG2 cell cytoplasmic extracts in hypotonic buffer after elution from an anti-human apoA-I affinity column as previously described (35Colvin P. Moriguchi E. Barrett H. Parks J. Rudel L. Production rate determines plasma concentration of large high density lipoprotein in non-human primates.J. Lipid Res. 1998; 39: 2076-2085Google Scholar). Both bound and unbound fractions were characterized for phospholipid and apoA-I content (36Koritnik D.L. Rudel L.L. Measurement of apolipoprotein A-I concentration in nonhuman primate serum by enzyme-linked immunosorbent assay (ELISA).J. Lipid Res. 1983; 24: 1639-1645Google Scholar).Slot-blot/Western analyses were carried out as previously described (24Sorci-Thomas M.G. Thomas M. Curtiss L. Landrum M. Single repeat deletion in apoA-I blocks cholesterol esterification and results in rapid catabolism of D6 and wild-type apoA-I in transgenic mice.J. Biol. Chem. 2000; 275: 12156-12163Google Scholar). Briefly, aliquots (100 μl) from each fraction from the density gradient was applied to nitrocellulose (0.2 μm) and then incubated with a polyclonal antibody to human apoA-I (1:2,000; Chemicon) and then to anti-goat IgG conjugated to alkaline phosphatase (1:4,000) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Promega). The phospholipid content was determined on a pooled density fraction using established procedures (37Fiske C.A. SubbaRow Y. The colorimetric determination of phosphorus.J. Biol. Chem. 1925; 66: 375-400Google Scholar).Data analysisImages from X-ray film or nitrocellulose blots were scanned on a Linotype-Hell Ultra Scanner. Images were imported into Scion Image, and the densities were obtained from the gray scale image after calibrating the scanner as previously described (17Chisholm J.W. Burleson E.R. Shelness G.S. Parks J.S. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL.J. Lipid Res. 2002; 43: 36-44Google Scholar).Immunofluorescence microscopy and morphometryCells were prepared for immunofluorescence microscopy as previously described (38Willingham M.C. Pastan I. Immunoflourescence techniques.in: Pastan I. Willingham Mark C. An Atlas of Immunofluorescence in Cultured Cells. Academic Press, Orlando, FL1985: 1-13Google Scholar, 39Willingham M.C. Pastan I. Morphologic methods in the study of endocytosis in cultured cells.in: Pastan I. Willingham M.C. Endocytosis. Plenum Publishing, New York1985: 281-321Google Scholar, 40Willingham M.C. Rutherford A.V. The use of the osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells.J. Histochem. Cytochem. 1984; 32: 455-460Google Scholar). First, the monolayer was washed twice with PBS, and the cells were then treated with 1 ml of 3.7% formaldehyde in PBS for 10 min at room temperature. After fixing, the cells were washed three times with a solution of 1% BSA and 0.1% saponin prepared in PBS (BSA-saponin). The dishes were incubated for 10 min at room temperature to permeabilize the cell membrane and then were treated with a 1:2,500 dilution of anti-human apoA-I diluted in BSA-saponin for 30 min at room temperature. Cells were washed three times with PBS and incubated with a final concentration of 25 μg/ml affinity-purified anti-goat IgG conjugated with rhodamine in BSA-saponin for 30 min. The cells were washed three times with PBS to remove unbound antibody and then fixed again with 1 ml of 3.7% formaldehyde for 10 min at room temperature.For double-label experiments, cells were sequentially incubated in goat anti-apoA-I followed by mouse anti-cathepsin D (lysosomal marker). This step was followed by treatment with an affinity-purified species-specific rabbit antiglobin labeled with rhodamine. Lipid droplets were visualized by including the fluorescent lipid dye Nile Red or osmium-thiocarbohydrazide-osmium that selectively labeled classic neutral lipid droplets (40Willingh" @default.
• W2087272783 created "2016-06-24" @default.
• W2087272783 creator A5005935544 @default.
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• W2087272783 creator A5057354124 @default.
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• W2087272783 date "2004-07-01" @default.
• W2087272783 modified "2023-10-18" @default.
• W2087272783 title "Quality control in the apoA-I secretory pathway" @default.
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