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• W2019952168 abstract "Mammalian cells synthesize significant amounts of precursor sterols, in addition to cholesterol, at the endoplasmic reticulum (ER). The newly synthesized sterols rapidly move to the plasma membrane (PM). The mechanism by which precursor sterols move back to the ER for their enzymatic processing to cholesterol is essentially unknown. Here we performed pulse-chase experiments and showed that the C29/C30 sterols rapidly move from the PM to the ER and are converted to cholesterol. The retrograde precursor sterol transport is largely independent of the Niemann-Pick type C proteins, which play important roles in late endosomal cholesterol transport. In contrast, disrupting lipid rafts significantly retards the conversion of C29/C30 and C28 sterols to cholesterol, causing the accumulation of precursor sterols at the PM. Our results reveal a previously undisclosed function of the PM lipid rafts: they bring cholesterol biosynthesis to completion by participating in the retrograde movement of precursor sterols back to the ER. Mammalian cells synthesize significant amounts of precursor sterols, in addition to cholesterol, at the endoplasmic reticulum (ER). The newly synthesized sterols rapidly move to the plasma membrane (PM). The mechanism by which precursor sterols move back to the ER for their enzymatic processing to cholesterol is essentially unknown. Here we performed pulse-chase experiments and showed that the C29/C30 sterols rapidly move from the PM to the ER and are converted to cholesterol. The retrograde precursor sterol transport is largely independent of the Niemann-Pick type C proteins, which play important roles in late endosomal cholesterol transport. In contrast, disrupting lipid rafts significantly retards the conversion of C29/C30 and C28 sterols to cholesterol, causing the accumulation of precursor sterols at the PM. Our results reveal a previously undisclosed function of the PM lipid rafts: they bring cholesterol biosynthesis to completion by participating in the retrograde movement of precursor sterols back to the ER. Cholesterol is an important lipid component of biological membranes. It also serves as an obligatory precursor for the biosyntheses of steroid hormones, bile acids, and bioactive oxysterols (1Chang T.Y. Chang C.C. Ohgami N. Yamauchi Y. Annu. Rev. Cell Dev. Biol. 2006; 22: 129-157Crossref PubMed Scopus (428) Google Scholar). In mammals, virtually every cell of the body is capable of de novo cholesterol biosynthesis. Cholesterol biosynthesis involves successive enzymatic reactions, converting the simple, 2-carbon precursor acetyl-CoA into the 30-carbon, acyclic, apolar molecule squalene. The subsequent oxidation and cyclization of squalene yields the first sterol in the biosynthetic pathway, the 30-carbon (C30) lanosterol. The conversion of the C30 lanosterol to C27 cholesterol involves at least 18 enzymatic reactions (2Gurr M.I. Harwood J.L. Gurr M.I. Harwood J.L. Lipid Biochemistry. Chapman & Hall, New York1991: 297-337Google Scholar) and can proceed through two pathways, with one involving desmosterol as the final precursor, and the other involving lathosterol and 7-dehydrocholesterol as the final precursors (Fig. 1). The biosynthetic enzymes responsible for converting squalene to cholesterol are all located in the endoplasmic reticulum (ER) 4The abbreviations used are: CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; fetal bovine serum, fetal-bovine serum; PM, plasma membrane; ER, endoplasmic reticulum; TLC, thin layer chromatography; NPC, Niemann-Pick type C; sterol regulatory element-binding protein, sterol regulatory element binding protein; sterol regulatory element-binding protein cleavage-activating protein, sterol regulatory element-binding protein cleavage activating protein; GC-MS, gas-liquid chromatography-mass spectrometry; HCD, 2-hydroxypropyl-β-cyclodextrin; MCD, methyl-β-cyclodextrin; LE, endosome; LYS, lysosome; Hf, human fibroblast. 4The abbreviations used are: CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; fetal bovine serum, fetal-bovine serum; PM, plasma membrane; ER, endoplasmic reticulum; TLC, thin layer chromatography; NPC, Niemann-Pick type C; sterol regulatory element-binding protein, sterol regulatory element binding protein; sterol regulatory element-binding protein cleavage-activating protein, sterol regulatory element-binding protein cleavage activating protein; GC-MS, gas-liquid chromatography-mass spectrometry; HCD, 2-hydroxypropyl-β-cyclodextrin; MCD, methyl-β-cyclodextrin; LE, endosome; LYS, lysosome; Hf, human fibroblast. membranes (2Gurr M.I. Harwood J.L. Gurr M.I. Harwood J.L. Lipid Biochemistry. Chapman & Hall, New York1991: 297-337Google Scholar). Previous studies showed that, in addition to synthesizing cholesterol, mammalian cells also synthesize substantial amounts of precursor sterols (3Echevarria F. Norton R.A. Nes W.D. Lange Y. J. Biol. Chem. 1990; 265: 8484-8489Abstract Full Text PDF PubMed Google Scholar, 4Lange Y. Echevarria F. Steck T.L. J. Biol. Chem. 1991; 266: 21439-21443Abstract Full Text PDF PubMed Google Scholar). Similar to cholesterol, the precursor sterols leave the ER and rapidly reach the plasma membrane (PM) within 30-min time (5Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (206) Google Scholar, 6Kaplan M.R. Simoni R.D. J. Cell Biol. 1985; 101: 446-453Crossref PubMed Scopus (168) Google Scholar). The precursor sterols at the PM are predicted to move back to the ER to be enzymatically processed to cholesterol. This retrograde movement is an essential step to complete cholesterol biosynthesis. Little is known about the mechanism(s) of retrograde movement of sterols. In yeast Saccharomyces cerevisiae, Prinz and coworkers showed that the retrograde movement of exogenously added sterols from the PM to the ER is governed by a non-vesicular mechanism that involves ATP-binding cassette transporters (7Li Y. Prinz W.A. J. Biol. Chem. 2004; 279: 45226-45234Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and oxysterol-binding protein-related proteins (8Raychaudhuri S. Im Y.J. Hurley J.H. Prinz W.A. J. Cell Biol. 2006; 173: 107-119Crossref PubMed Scopus (210) Google Scholar). In mammalian cells, Maxfield and coworkers showed that both vesicular and non-vesicular trafficking mechanisms operate to govern the transport of a naturally fluorescent cholesterol analog dehydroergosterol (9Hao M. Lin S.X. Karylowski O.J. Wustner D. McGraw T.E. Maxfield F.R. J. Biol. Chem. 2002; 277: 609-617Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 10Pipalia N.H. Hao M. Mukherjee S. Maxfield F.R. Traffic. 2007; 8: 130-141Crossref PubMed Scopus (54) Google Scholar). Earlier, based on inhibitor studies, Metherall and colleagues (11Metherall J.E. Li H. Waugh K. J. Biol. Chem. 1996; 271: 2634-2640Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and Field and colleagues (12Field F.J. Born E. Murthy S. Mathur S.N. J. Lipid Res. 1998; 39: 333-343Abstract Full Text Full Text PDF PubMed Google Scholar) suggested that multiple drug resistance proteins may be involved in the retrograde movement of precursor sterols.The Niemann-Pick type C1 (NPC1) protein is a multispan membrane protein containing a sterol-sensing domain, which plays an essential role in the ability of the protein to bind cholesterol (13Ohgami N. Ko D.C. Thomas M. Scott M.P. Chang C.C. Chang T.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12473-12478Crossref PubMed Scopus (169) Google Scholar). Studies using mutant cells that lack the NPC1 protein show that NPC1 is involved in the egress of low density lipoprotein-derived cholesterol from the late endosomes to the PM, ER, and mitochondria (14Wojtanik K.M. Liscum L. J. Biol. Chem. 2003; 278: 14850-14856Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 15Sugii S. Reid P.C. Ohgami N. Du H. Chang T.Y. J. Biol. Chem. 2003; 278: 27180-27189Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 16Frolov A. Zielinski S.E. Crowley J.R. Dudley-Rucker N. Schaffer J.E. Ory D.S. J. Biol. Chem. 2003; 278: 25517-25525Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In certain cell types, including macrophages, NPC1 is also involved in the post-PM trafficking of biosynthesized cholesterol, and possibly other C27 sterols, from the PM to the ER (17Reid P.C. Sugii S. Chang T.Y. J. Lipid Res. 2003; 44: 1010-1019Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). On the other hand, the ability of the mutant NPC cells to convert precursor sterols, especially the methylated sterols (i.e. C28, 29, and C30 sterols), to cholesterol, is unknown.In this study, we monitored the intracellular fate of biosynthesized precursor sterols by feeding cells with radioactive acetate, performed pulse-chase experiments, and used several TLC systems to analyze the composition of labeled sterols. We also employed gas chromatography-mass spectrometry (GC-MS) to identify various precursor sterols in cells grown in sterol-free media. Our results show that, upon arrival at the PM, precursor sterols rapidly move back from the PM to the ER, to be enzymatically converted to cholesterol. The retrograde movement of the precursor sterols is largely independent of NPC proteins but depends on the functionality of the PM lipid rafts.EXPERIMENTAL PROCEDURESMaterialsVarious reagents and procedures were as described previously (15Sugii S. Reid P.C. Ohgami N. Du H. Chang T.Y. J. Biol. Chem. 2003; 278: 27180-27189Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 18Sugii S. Lin S. Ohgami N. Ohashi M. Chang C.C. Chang T.Y. J. Biol. Chem. 2006; 281: 23191-23206Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar).Media, Cell Lines, and Cell CultureMedium A is Dulbecco Modified Earle's Medium (DMEM) and Ham's F12 at 1:1 (for Chinese hamster ovary (CHO) cells), or Dulbecco's modified Eagle's medium (for human fibroblasts (Hf cells)), plus 10% fetal bovine serum; Medium D is appropriate medium plus 5% delipidated fetal bovine serum, 35 μm oleic acid; Medium B is appropriate medium plus 0.2% fatty acid-free bovine serum albumin; Medium F is appropriate medium without serum. All media contain 50 units/ml penicillin and 50 μg/ml streptomycin as antibiotics. 25RA is a mutant CHO cell line that is resistant to 25-hydroxycholesterol (19Chang T.Y. Limanek J.S. J. Biol. Chem. 1980; 255: 7787-7795Abstract Full Text PDF PubMed Google Scholar) and contains a gain-of-function mutation in sterol regulatory element-binding protein cleavage-activating protein (20Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). The CT43 mutant cell line was isolated from mutagenized 25RA cells as one of the cholesterol trafficking mutants (21Cadigan K.M. Spillane D.M. Chang T.Y. J. Cell Biol. 1990; 110: 295-308Crossref PubMed Scopus (91) Google Scholar). It contains the same gain-of-function mutation in sterol regulatory element-binding protein cleavage-activating protein, and a premature translational termination mutation near the 3′-end of the npc1 coding sequence, producing a non-functional truncated NPC1 protein (22Cruz J.C. Sugii S. Yu C. Chang T.Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The A101 clone is a CHO cell mutant lacking the NPC1 mRNA and protein; it was isolated from parental CHO cells that express the murine ecotropic retrovirus receptor (JP17 cell). CHO clones A101 and JP17 were from professors Ohno and Ninomiya (Totori University School of Medicine, Japan) (23Higaki K. Ninomiya H. Sugimoto Y. Suzuki T. Taniguchi M. Niwa H. Pentchev P.G. Vanier M.T. Ohno K. J. Biochem. (Tokyo). 2001; 129: 875-880Crossref PubMed Scopus (40) Google Scholar). A normal Hf cell line (normal-1) was from Dr. Peter Pentchev (formerly National Institute of Health). A second normal Hf cell line, GM00038, was from Coriell Institute (normal-2); a mutant NPC1 human fibroblast cell line, GM03123, was also from Coriell. GM03123 contains two point mutations in the NPC1 protein: P237S and I1061T. The NPC2 human fibroblast cell line was from Dr. Yiannis Ioannou (Mount Sinai School of Medicine) (24Walter M. Davies J.P. Ioannou Y.A. J. Lipid Res. 2003; 44: 243-253Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar).Pulse-Chase Experiments with [3H]AcetateFor CHO cells, cells were seeded into 6-well plates or 100-mm dishes and grown in Medium D for 2 days as described (22Cruz J.C. Sugii S. Yu C. Chang T.Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). For Hfs, cells were plated and grown in Medium A to near confluency, washed twice with PBS, and grown for 2 days in Medium D. To label cells with [3H]acetate, cells were washed with pre-warmed (37 °C) PBS twice, then pulse-labeled with 20 μCi/ml [3H]acetate (20 μCi/well or 100 μCi/100-mm dish) for the time indicated at 37 °C. After pulse labeling, the medium was removed; the cells were washed twice with pre-warmed PBS and incubated with the chase media (pre-warmed Medium D or Medium B) for up to 24 h. In some experiments, the chase media were collected and extracted with chloroform/methanol (2:1, v/v) and analyzed for radiolabeled sterols. Cellular lipids were extracted with hexane/isopropanol (3:2, v/v), and cellular protein was solubilized in 0.1 n NaOH to determine protein content (25Cadigan K.M. Heider J.G. Chang T.Y. J. Biol. Chem. 1988; 263: 274-282Abstract Full Text PDF PubMed Google Scholar). The non-saponifiable lipids (containing the sterols) were isolated as previously described (26Limanek J.S. Chin J. Chang T.Y. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5452-5456Crossref PubMed Scopus (39) Google Scholar).Sterol Analyses by TLCThe non-saponifiable fractions were spotted onto channeled silica TLC plates and run in methylene chloride/ethyl acetate (97:3, TLC system I) to separate C29/C30, C28, and C27 sterols (27Berry D.J. Chang T.Y. Biochemistry. 1982; 21: 573-580Crossref PubMed Scopus (11) Google Scholar). Lanosterol and cholesterol were added to the samples to serve as internal standards. After chromatography, the plates were briefly stained with iodine to identify the C29/C30 sterol band and the C27 sterol band. The band located between the C29/C30 and C27 sterol bands is the C28 sterol band (27Berry D.J. Chang T.Y. Biochemistry. 1982; 21: 573-580Crossref PubMed Scopus (11) Google Scholar). The sterol bands were scraped and counted with a liquid scintillation counter. To examine the sterol composition of the C27 sterols, the C27 sterol bands separated by TLC system I were scraped and extracted with chloroform/methanol (2:1) twice. The extracts were transferred to new glass tubes, washed once with water, and dried under nitrogen. The C27 sterols were either acetylated as described (27Berry D.J. Chang T.Y. Biochemistry. 1982; 21: 573-580Crossref PubMed Scopus (11) Google Scholar) or left non-acetylated. The acetylated C27 sterols were separated by TLC system II on silver nitrate impregnated plates. The plates were prepared by rapidly dipping commercially prepared silver nitrate-impregnated TLC plates in 10% silver nitrate solution (in acetonitrile), followed by air drying. The samples were repeatedly chromatographed (three times), each time for 1 h, using the solvent hexane/benzene (80:20) (28Kammereck R. Lee W.-H. Paliokas A. Schroepfer G.J.J. J. Lipid Res. 1967; 8: 282-284Abstract Full Text PDF PubMed Google Scholar). Non-acetylated sterol samples were separated using the silver nitrate-impregnated TLC plates prepared as described above and chromatographed for 4 h in a plastic-wrap sealed glass chamber in 100% chloroform (system III). Plates were run with authentic cholesterol, desmosterol, lathosterol, and zymosterol, or their acetylated derivatives were spotted in parallel lanes as standards. After TLC, the standard-containing lanes were charred by spraying the plates with an orcinol mixture (200 mg of orcinol/100 ml of 75% sulfuric acid) and baked at 100 °C for 20 min. The appropriate radioactive bands in sample-containing lanes were scraped to determine radioactivity using a liquid scintillation counter. Table 1 shows the Rf value of each sterol in the three TLC systems.TABLE 1Rf values of various sterols on TLC systems I to III TLC systems are described under “Experimental Procedures.”System ISystem IISystem IIIC30 sterols0.53C29 sterols0.53C28 sterols0.42C27 sterols0.35Cholesterol acetate0.49Desmosterol acetate0.18Lathosterol acetate0.49Zymosterol acetate0.18Cholesterol0.42Desmosterol0.33Lathosterol0.60Zymosterol0.42 Open table in a new tab Sterol Analyses by GC-MSCellular lipids were extracted and saponified as described above, using nanograde organic solvents. The dried sterol samples were derivatized with 0.25 ml of Sigma-Sil-A (Sigma) at 60 °C for 30 min. The trimethylsilyl derivatives of sterols were injected into a Shimadzu GC-17A gas chromatograph connected to a Shimadzu QP5000 mass spectrometer equipped with an XTI-5 (30 m × 25 μm × 0.25 mm) capillary column. The injector port was set at 290 °C. The initial temp of the oven was 110 °C and was increased at 15 °C/min to 290 °C and held for 10 min. Helium flow was set at 1.3 ml/min. The sterol masses were quantified by using epicoprostanol as an internal standard.Lipid Analyses by Enzymatic AssaysThe cellular free cholesterol and choline-containing phospholipids were determined as described before (29Yamauchi Y. Chang C.C. Hayashi M. Abe-Dohmae S. Reid P.C. Chang T.Y. Yokoyama S. J. Lipid Res. 2004; 45: 1943-1951Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).Cell Fractionation after Cellular Labeling with [3H]Acetic AcidTwo different methods were used to prepare the PM and the internal membrane (IM) fractions.Method I—The PM/IM fractions were prepared by using the 30% Percoll gradient centrifugation method as previously described (15Sugii S. Reid P.C. Ohgami N. Du H. Chang T.Y. J. Biol. Chem. 2003; 278: 27180-27189Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 30Smart E.J. Ying Y. Donzell W.C. Anderson R.G.W. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). Cell homogenates from two 100-mm dishes of cells were prepared and spun at 1,000 × g for 5 min twice, and the resulting 1 ml of post nuclear supernatant was placed onto a 9-ml 30% Percoll solution and centrifuged at 84,000 × g for 30 min at 4 °C using a Beckman Type 70.1 Ti rotor. Afterward, ten 1-ml fractions were collected from the top. Lipids from each Percoll fraction were extracted with chloroform/methanol (2:1), saponified, and analyzed by TLC system I. To analyze the purity of the PM/IM fractions, cell surface proteins were biotinylated for 30 min on ice by using the EZ-Link Sulfo-NHS-Biotin kit (Pierce); the cell homogenates were then prepared and fractionated by using 30% Percoll gradient centrifugation. Afterward, the detergent Nonidet P-40 was added to each fraction at 1% final concentration; the fractions were centrifuged at 200,000 × g for 30 min twice to remove the Percoll particles. Aliquots of each supernatant were subjected to SDS-PAGE followed by immunoblotting with the monoclonal anti-HMG-CoA reductase IgG-A9 (obtained from ATCC). Biotinylated proteins were detected with the Vectastain ATP-binding cassette kit (Vector Laboratories). Densitometric analyses of protein bands were performed by using NIH Image version 1.61.Method II—The PM, IM, and cytosol fractions were prepared by using the procedure previously described (31Metherall J.E. Waugh K. Li H. J. Biol. Chem. 1996; 271: 2627-2633Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) with minor modification. Briefly, post nuclear supernatant was prepared from one 100-mm dish of cells as described above, and centrifuged at 16,000 × g for 20 min to collect the PM-rich fraction as a pellet. The supernatant was further spun at 200,000 × g for 45 min to yield the cytosol fraction as supernatant and the IM-rich fraction as a pellet. The purities of the PM/IM fractions and the labeled lipids present in these fractions were analyzed in a similar manner as described above. The amounts of free cholesterol and choline-containing phospholipids in these fractions were determined by methods described previously (29Yamauchi Y. Chang C.C. Hayashi M. Abe-Dohmae S. Reid P.C. Chang T.Y. Yokoyama S. J. Lipid Res. 2004; 45: 1943-1951Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We also employed the 11% Percoll gradient centrifugation method (22Cruz J.C. Sugii S. Yu C. Chang T.Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) to isolate various crude subcellular fractions. This method efficiently separated late endosomes/lysosomes from other membranes. Ten 1-ml fractions were collected from the top of the tube. The endosomes/lysosomes (LE/LYS) were concentrated in fractions 8, 9, and 10. Lipids from each fraction were extracted, saponified, and analyzed with the TLC as described above.CalculationsData were presented as means ± S.D. unless specified in the figure legends. Statistical analyses of results were performed using a two-tailed, unpaired Student’s t test. The difference between two sets of values was considered significant when the p value was <0.05.RESULTSER-to PM Anterograde Transport of Biosynthesized Precursor Sterols—To monitor the distribution of newly synthesized precursor sterols inside the cells, WT CHO cells grown in Medium D were incubated with [3H]acetate for 1 h. The labeled cell homogenates were subjected to subcellular fractionation analysis. Initially, two methods were employed (described under “Experimental Procedures”) to prepare the PM, IM, and cytosol fractions. To evaluate the purity of the PM fraction and IM fraction, we biotinylated the cell surface proteins as the PM marker and used the HMG-CoA reductase protein, which resides in the ER where sterols were synthesized, as the IM marker. In Method I, the distribution analyses of these markers (Fig. 2A) show that fractions 1–3 contain ∼75% of the total PM signal. Fractions 4 and 5 contain ∼90% of the total HMG-CoA reductase signal. The labeled sterols present in fractions 1–10 were extracted and analyzed. The results show that the majority (>70%) of newly synthesized C29/C30, C28, and C27 sterols were present in fractions 1–3; <25% were present in fractions 4 and 5 (Fig. 2A). Cholesterol mass analysis showed that its distribution was similar to that of newly synthesized sterols; roughly 70% of cholesterol was present in fractions 1–3, whereas <25% was present fractions 4 and 5 (data not shown). In Method II, the analysis showed that the PM-rich fraction contained 60% of the total cell surface biotinylated protein signals, the IM-rich fraction contained 80% of the total HMG-CoA reductase signal (data not shown). The results of the labeled sterol distribution analysis (Fig. 2B) show that >60% of the newly synthesized C29/C30, C28, and C27 sterols were present in the PM fraction, whereas ∼25% were present in the IM fraction. Small but significant amounts of 3H-labeled biosynthetic precursor sterols were recovered in the cytosol fraction. Cholesterol mass analysis showed that it was mainly recovered in the PM fraction as expected (Fig. 2B, white bar). These results show that the second method tended to underestimate the proportion of labeled sterols present in the PM fraction by ∼10–12% compared with the first method. We sought to test the findings described in Fig. 2 (A and B) by using another approach. Cyclodextrin is a water-soluble molecule that has high affinity for sterols. We and others had previously shown that when 2-hydroxypropyl-β-cyclodextrin (HCD) is added to the medium of intact CHO cells for 10 min or less, it efficiently removes cell surface cholesterol; in contrast, under the same condition, cholesterol sequestered in the internal membrane compartment(s) is very resistant to extraction by HCD (15Sugii S. Reid P.C. Ohgami N. Du H. Chang T.Y. J. Biol. Chem. 2003; 278: 27180-27189Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). We pulse-labeled the parental 25RA CHO cells and the NPC1-deficient mutant CT43 cells with [3H]acetate for 1 h, then exposed these cells to HCD for 10 min. Labeled lipids in cells and in the media were extracted and analyzed by TLC. The results show that, in both cell types, >35% of the total newly synthesized C27 sterols were accessible to HCD (Fig. 2C). These results, together with Fig. 2 (A and B), suggest that after 1 h of synthesis, most of the biosynthesized C27 sterols is located at the PM. The results in Fig. 2C show that newly synthesized C28 and C29/C30 sterols were also extractable by HCD, although less so than C27 sterols, suggesting that HCD may bind with less affinity toward C28 and C29/C30 sterols than C27 sterols. It is possible that the additional methyl groups present in steroid ring A (the 4,4-methyl moieties) and/or present in the junction between rings C/D (the 14-α-methyl moiety) may hinder the binding between HCD and the sterol molecule. However, we cannot rule out the possibility that the C28 and C29/C30 sterols may reside in a microdomain of the PM different from where the C27 sterols reside. The results presented in Fig. 2C do imply that after 1 h of synthesis, a substantial amount of the biosynthesized sterols is located at or near the PM to be extractable to HCD. These results also show that the availability of various newly synthesized precursor sterols to HCD was almost identical between the 25RA and the CT43 cells, indicating that the movement of C28 sterols and C29/C30 sterols to the cell surface was independent of a functional NPC1 protein.FIGURE 2Presence of biosynthetic precursor sterols at the PM. A, on day 0, WT CHO cells were plated in triplicate at 1 × 106 cells per 100-mm dish and grown in Medium A (8 ml/dish). On day 1, the medium was switched to Medium D, and the cells were grown for another 2 days. On day 3, the cells were incubated with [3H]acetic acid (20 μCi/ml, 5 ml/dish) for 1 h in Medium F at 37 °C. Cell homogenate was subjected to the 30% Percoll gradient centrifugation analysis (Method I) as described under “Experimental Procedures.” Afterward, lipids were extracted from each fraction, and the non-saponifiable lipids were analyzed by the TLC system I to identify radiolabeled biosynthetic precursor sterols. The distribution of biotinylated proteins and HMG-CoA reductase in each fraction were analyzed as described under “Experimental Procedures.” Data were reported as % of total in each sterol species as indicated. The results shown are averages of two experiments; the error bars indicate the sizes of difference between the average values. B, WT CHO cells were set up and radiolabeled as described above. Cell homogenates were subjected to subcellular fractionation Method II to prepare the plasma membrane (PM), intracellular membranes (IM), and cytosol (CS) fractions (described under “Experimental Procedures”). Afterward, lipids were extracted from each fraction, the non-saponifiable lipids were analyzed by the TLC system I to determine [3H]sterols. Mass of free cholesterol (FC) was determined by the colorimetric assay as described under “Experimental Procedures.” Data are reported as % total in each lipid species as indicated. The results shown are averages ± S.D. and are from one of two separate experiments with similar results. C, on day 0, the 25RA and CT43 CHO cells were seeded in triplicate into 6-well trays at a density of 1 × 105 (for 25RA) or 2 × 105 (for CT43) cells per well and grown in 1.5 ml/well in Medium A. On day 1, the medium was changed to Medium D, and the cells were grown for another 2 days. On day 3, the cells were radiolabeled with [3H]acetic acid (20 μCi/ml; 1 ml/well) Medium F for 1 h, sterol efflux was then induced by adding 4% HCD to Medium F for 10 min. Afterward, lipids were extracted from the cells and from the medium, and the non-saponifiable lipids were analyzed by the TLC system I. Data are reported as % total (cell plus medium) in each sterol species present in the medium as indicated. The results shown are averages ± S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The results presented in Fig. 2, together with previous studies (5Heino S. Lusa S. Somerharju P. Ehnholm C. Olkkonen V.M. Ikonen E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8375-8380Crossref PubMed Scopus (206) Google Scholar), show that immediately after biosynthesis, the majority of precursor sterols are transported from the ER to the PM. Thus, one or more PM-to-ER retrograde sterol movement systems are needed to bring precursor sterols arriving at the PM back to the ER, where all the post-squalene cholesterol biosynthetic enzymes reside, for their eventual conversion to cholesterol.Role of NPC Proteins in the PM-to-ER Retrograde Transport of Biosynthesized Precursor Sterols—Because the NPC proteins play an important role in endosomal cholesterol transport, we examined whether the NPC-dependent sterol transport system is involved in the retrograde transport of newly synthesized precursor sterols. It is known that all the enzymes responsible for converting C29/C30 or C28 sterols to C27 sterols are located at the ER. This allowed us to use the conversions of C29/C30 or C28 sterols to C27 sterols as a biological assay to examine the retr" @default.
• W2019952168 created "2016-06-24" @default.
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• W2019952168 creator A5005218140 @default.
• W2019952168 creator A5013353605 @default.
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• W2019952168 date "2007-11-01" @default.
• W2019952168 modified "2023-10-17" @default.
• W2019952168 title "Plasma Membrane Rafts Complete Cholesterol Synthesis by Participating in Retrograde Movement of Precursor Sterols" @default.
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