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The Active Site His-460 of Human Acyl-coenzyme A:Cholesterol Acyltransferase 1 Resides in a Hitherto Undisclosed Transmembrane Domain

2005; Elsevier BV; Volume: 280; Issue: 45 Linguagem: Inglês

10.1074/jbc.m508384200

1083-351X

Zhan‐Yun Guo, Lin Song, Jennifer A. Heinen, Catherine C.Y. Chang, Ta‐Yuan Chang,

Peroxisome Proliferator-Activated Receptors

Human acyl-coenzyme A:cholesterol acyltransferase 1 (hACAT1) esterifies cholesterol at the endoplasmic reticulum (ER). We had previously reported that hACAT1 contains seven transmembrane domains (TMD) (Lin, S., Cheng, D., Liu, M. S., Chen, J., and Chang, T. Y. (1999) J. Biol. Chem. 274, 23276-23285) and nine cysteines. The Cys near the N-terminal is located at the cytoplasm; the two cysteines near the C-terminal form a disulfide bond and are located in the ER lumen. The other six free cysteines are located in buried region(s) of the enzyme (Guo, Z.-Y., Chang, C. C. Y., Lu, X., Chen, J., Li, B.-L., and Chang, T.-Y. (2005) Biochemistry 44, 6537-6548). In the current study, we show that the conserved His-460 is a key active site residue for hACAT1. We next performed Cys-scanning mutagenesis within the region of amino acids 354-493, expressed these mutants in Chinese hamster ovary cells lacking ACAT1, and prepared microsomes from transfected cells. The microsomes are either left intact or permeabilized with detergent. The accessibility of the engineered cysteines of microsomal hACAT1 to various maleimide derivatives, including mPEG5000-maleimide (large, hydrophilic, and membrane-impermeant), N-ethylmaleimide, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (small, hydrophilic, and ER membrane-permeant), and N-phenylmaleimide (small, hydrophobic, and ER membrane-permeant), were monitored by Western blot analysis. The results led us to construct a revised, nine-TMD model, with the active site His-460 located within a hitherto undisclosed transmembrane domain, between Arg-443 and Tyr-462. Human acyl-coenzyme A:cholesterol acyltransferase 1 (hACAT1) esterifies cholesterol at the endoplasmic reticulum (ER). We had previously reported that hACAT1 contains seven transmembrane domains (TMD) (Lin, S., Cheng, D., Liu, M. S., Chen, J., and Chang, T. Y. (1999) J. Biol. Chem. 274, 23276-23285) and nine cysteines. The Cys near the N-terminal is located at the cytoplasm; the two cysteines near the C-terminal form a disulfide bond and are located in the ER lumen. The other six free cysteines are located in buried region(s) of the enzyme (Guo, Z.-Y., Chang, C. C. Y., Lu, X., Chen, J., Li, B.-L., and Chang, T.-Y. (2005) Biochemistry 44, 6537-6548). In the current study, we show that the conserved His-460 is a key active site residue for hACAT1. We next performed Cys-scanning mutagenesis within the region of amino acids 354-493, expressed these mutants in Chinese hamster ovary cells lacking ACAT1, and prepared microsomes from transfected cells. The microsomes are either left intact or permeabilized with detergent. The accessibility of the engineered cysteines of microsomal hACAT1 to various maleimide derivatives, including mPEG5000-maleimide (large, hydrophilic, and membrane-impermeant), N-ethylmaleimide, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (small, hydrophilic, and ER membrane-permeant), and N-phenylmaleimide (small, hydrophobic, and ER membrane-permeant), were monitored by Western blot analysis. The results led us to construct a revised, nine-TMD model, with the active site His-460 located within a hitherto undisclosed transmembrane domain, between Arg-443 and Tyr-462. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) 2The abbreviations used are: ACATacyl-coenzyme A:cholesterol acyltransferaseAMS4-acetamido-4′maleimidylstilbene-2′disulfonic acid; CHOChinese hamster ovaryERendoplasmic reticulumHAhemagglutininIAiodoacetamideME2-mercaptoethanolNEMN-ethylmaleimideNPMN-phenylmaleimidePEG-malmPEG5000-maleimideTMDtransmembrane domainWTwild typeaaamino acid(s)PVDFpolyvinylidene difluorideHRPhorseradish peroxidase is a membrane-bound enzyme present in a variety of tissues and cells. It is mainly located at the endoplasmic reticulum, and catalyzes the biosynthesis of cholesteryl esters, using long-chain fatty acyl-coenzyme A and cholesterol as its substrates. ACAT plays important roles in cholesterol homeostasis. At the single cell level, it is a key enzyme that prevents excess free cholesterol from building up in the cell membranes. At the physiological level, it contributes cholesteryl esters as part of the neutral lipid cargo, to be packaged into the cores of very low density lipoproteins and chylomicrons. Under pathophysiological condition, in cholesterol-loaded macrophages, ACAT converts excess cholesterol into cholesteryl esters. This action reduces the amount of cholesterol available from the macrophage cell surface for efflux and converts the macrophages to foam cells, which are the hallmark of early lesions of the disease atherosclerosis (reviewed in Ref. 1Chang T.Y. Chang C.C.Y. Cheng D. Annu. Rev. Biochem. 1997; 66: 613-638Crossref PubMed Scopus (442) Google Scholar). For these reasons, ACAT has been a drug target for pharmaceutical intervention of diseases, including atherosclerosis and hyperlipidemia. In mammals, two ACAT genes exist that encode for two similar but different proteins, ACAT1 and ACAT2. Available evidence suggests that ACAT1 and ACAT2 may function in distinct and complementary manners in various tissues (reviewed in Refs. 2Chang T.Y. Chang C.C.Y. Lin S. Yu C. Li B.L. Miyazaki A. Curr. Opin. Lipidol. 2001; 12: 289-296Crossref PubMed Scopus (211) Google Scholar and 3Rudel L. Lee R. Cockman T. Curr. Opin. Lipidol. 2001; 12: 121-127Crossref PubMed Scopus (210) Google Scholar). Unlike many other enzymes/proteins involved in cellular cholesterol metabolism, neither ACAT1 nor ACAT2 is regulated at the transcription level by the cholesterol-dependent SREBP (sterol regulatory element-binding protein) cleavage-activating protein (SCAP)/sterol regulatory element-binding protein pathway. Instead, available evidence suggests that ACAT1 may contain a distinct regulatory site that specifically recognizes cholesterol as its activator (4Zhang Y. Yu C. Liu J. Spencer T.A. Chang C.C. Chang T.Y. J. Biol. Chem. 2003; 278: 11642-11647Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 5Liu J. Chang C.C.Y. Westover E.J. Covey D.F. Chang T.Y. Biochem. J. 2005; 390Google Scholar). This mechanism allows ACAT1 to be up-regulated rapidly (within minutes) by cholesterol that builds up at the ER. The enzymological and biochemical characteristics of ACAT2 significantly diverge from those of ACAT1 in several ways; however, ACAT2 may also be allosterically regulated by cholesterol (5Liu J. Chang C.C.Y. Westover E.J. Covey D.F. Chang T.Y. Biochem. J. 2005; 390Google Scholar, 6Chang C.C.Y. Sakashita N. Ornvold K. Lee O. Chang E. Dong R. Lin S. Lee C.Y.G. Strom S. Kashyap R. Fung J. Farese Jr., R.V. Patoiseau J.F. Delhon A. Chang T.Y. J. Biol. Chem. 2000; 275: 28083-28092Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). acyl-coenzyme A:cholesterol acyltransferase 4-acetamido-4′ ′ Chinese hamster ovary endoplasmic reticulum hemagglutinin iodoacetamide 2-mercaptoethanol N-ethylmaleimide N-phenylmaleimide mPEG5000-maleimide transmembrane domain wild type amino acid(s) polyvinylidene difluoride horseradish peroxidase Molecular cloning of the human ACAT1 (hACAT1) gene (7Chang C.C.Y. Huh H.Y. Cadigan K.M. Chang T.Y. J. Biol. Chem. 1993; 268: 20747-20755Abstract Full Text PDF PubMed Google Scholar) provided the opportunity to study its biochemical properties. The recombinant hACAT1 expressed in Chinese hamster ovary cells can be purified to homogeneity (8Chang C.C.Y. Lee C.Y.G. Chang E.T. Cruz J.C. Levesque M.C. Chang T.Y. J. Biol. Chem. 1998; 273: 35132-35141Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). However, due to the low quantities of protein derived from the purification process, current efforts in our laboratory focus on studies at the enzymological and cell biological levels, but not at the structural biology level. ACAT1 is homotetrameric in vitro and in intact cells (9Yu C. Chen J. Lin S. Liu J. Chang C.C.Y. Chang T.Y. J. Biol. Chem. 1999; 274: 36139-36145Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The region near the N-terminal contains a dimerization motif. Deleting the N-terminal region converts the enzyme into a homodimer; the dimeric enzyme is fully active catalytically, and remains to be allosterically regulated by cholesterol (10Yu C. Zhang Y. Lu X.H. Chen J. Chang C.C.Y. Chang T.Y. Biochemistry. 2002; 41: 3762-3769Crossref PubMed Scopus (34) Google Scholar). ACAT1 contains multiple transmembrane domains (TMDs). To deduce its membrane topology, we had previously inserted the nine-amino acid HA tag at various hydrophilic regions of the protein, then expressed the tagged proteins in the ACAT-deficient AC29 cells by transfection. The cells were fixed and treated with digitonin, a cholesterol binder, or with saponin, a weak detergent. Digitonin causes the plasma membranes to become permeable, allowing antibodies to gain access to the cell cytosol, including the cytoplasmic side of the ER membranes. In contrast, saponin (or other mild detergents such as Triton X-100) causes both the plasma membranes and the ER membranes to become permeable, allowing the antibodies to gain access to both the cytoplasmic and the luminal sides of the ER membranes. After selective permeabilization, immunostainings using antibodies against the HA tag were performed, and the signals were monitored under a fluorescence microscope. This method enabled us to determine the sidedness of the HA tag along the ER membranes. The results obtained, summarized below in Fig. 1A, suggested that hACAT1 contains at least seven TMDs, with the N-terminal region located in the cytoplasmic side of the ER, and the C-terminal region located in the ER lumen (11Lin S. Cheng D. Liu M.S. Chen J. Chang T.Y. J. Biol. Chem. 1999; 274: 23276-23285Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The region near the C-terminal half is very hydrophobic and may contain additional membrane embedded segment(s) (11Lin S. Cheng D. Liu M.S. Chen J. Chang T.Y. J. Biol. Chem. 1999; 274: 23276-23285Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). hACAT1 contains nine cysteines. None of the cysteines is required for catalysis (12Lu X.H. Lin S. Chang C.C.Y. Chang T.Y. J. Biol. Chem. 2002; 277: 711-718Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). mPEG5000-maleimide (PEG-mal) is a large, hydrophilic and membrane-impermeant sulfhydryl-specific reagent. The PEG attachment causes an easily detectable shift of ACAT1 band on SDS-PAGE and Western blot (13Guo Z.-Y. Chang C.C.Y. Lu X. Chen J. Li B.-L. Chang T.-Y. Biochemistry. 2005; 44: 6537-6548Crossref PubMed Scopus (26) Google Scholar). We used PEG-mal to map the disulfide linkage and to probe the environment of the free sulfhydryls of the enzyme. The results show that a disulfide bond, formed between Cys-528 and Cys-546, is located in the lumen of the ER membranes. Cys-92 is at the cytoplasmic side of the membranes. All the other remaining free cysteines (Cys-333, Cys-345, Cys-365, Cys-387, Cys-467, and Cys-516) are not accessible to PEG-mal under native condition; thus, they are either buried within the ER membranes, or are folded within various regions of the ACAT1 protein itself (13Guo Z.-Y. Chang C.C.Y. Lu X. Chen J. Li B.-L. Chang T.-Y. Biochemistry. 2005; 44: 6537-6548Crossref PubMed Scopus (26) Google Scholar). These results are consistent with a modified, 7-TMD model for ACAT1, as summarized in Fig. 1C. In contrast, Joyce and colleagues had proposed a 5-TMD model for ACAT1 (25Joyce C.W. Shelness G.S. Davis M.A. Lee R.G. Skinner K. Anderson R.A. Rudel L.L. Mol. Biol. Cell. 2000; 11: 3675-3687Crossref PubMed Scopus (102) Google Scholar). The 5-TMD model, summarized as Fig. 1B, was deduced by monitoring the sidedness of the tag at the end of the C-terminal of the protein, after successive deletions from the ACAT1 C-terminal to produce various truncated ACAT1s. Similar to the 7-TMD model (Fig. 1, A and C), the 5-TMD model also proposes the existence of the first four TMDs near the N-terminal half, and the last TMD near the C-terminal. However, it predicts that no other TMD exists, and that all of the followings cysteines: Cys-333, Cys-345, Cys-365, Cys-387, and Cys-467, are located at the cytoplasmic side the ER membranes. These predictions are incompatible with the PEG-mal modification data (13Guo Z.-Y. Chang C.C.Y. Lu X. Chen J. Li B.-L. Chang T.-Y. Biochemistry. 2005; 44: 6537-6548Crossref PubMed Scopus (26) Google Scholar). In results not shown, we had found that even modest truncation(s) from the C-terminal of ACAT1 led to total inactivation in ACAT enzyme activity. Thus, it is possible that truncation(s) from the C-terminal of ACAT1 might have caused major structural alteration(s) within the ACAT1 polypeptide. In addition to the experimentally derived 7-TMD model and the 5-TMD model, there is at least one TMD algorithm, called TMbase (28Hofmann K. Stoffel W. Biol. Chem. Hoppe-Seyler. 1993; 347: 166Google Scholar), which predicts that the hydrophobic segments (aa 354-493), depicted as a long red bar in Fig. 1C, contain regions of high helical propensity, and may form two additional TMDs; this prediction is summarized as Fig. 1D. The possible existence of these additional TMDs, depicted as TMD6 and #7 in Fig. 1D, could not be ascertained by methodologies previously employed. ACAT1 is the prototypic member of a multimembrane-spanning acyltransferase family with more than 20 members that include ACAT1, ACAT2, and diacylglycerol acyltransferase 1. Within this family, an invariant His residue (His-460 in hACAT1) exists and may constitute as part of the active site of the enzyme (14Hofmann K. Trends Biochem. Sci. 2000; 25: 111-112Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Previous work from this laboratory showed that His-434, the equivalent of His-460 in hACAT1, is essential for hACAT2 catalysis (15Lin S. Lu X. Chang C.C.Y. Chang T.Y. Mol. Biol. Cell. 2003; 14: 2447-2460Crossref PubMed Scopus (74) Google Scholar). Recently, chemical modification by using PEG-mal together with Cys-scanning mutagenesis has been used successfully to investigate the membrane topology of various membrane proteins (16Lu J. Deutsch C. Biochemistry. 2001; 40: 13288-13301Crossref PubMed Scopus (85) Google Scholar, 17Kosolapov A. Deutsch C. J. Biol. Chem. 2003; 287: 4305-4313Abstract Full Text Full Text PDF Scopus (31) Google Scholar, 18Katzen F. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10471-10476Crossref PubMed Scopus (41) Google Scholar). In the current work, we first performed site-specific mutagenesis experiments to test if the conserved His-460 is a key active site residue for hACAT1. We next re-investigated the membrane topology of hACAT1 by performing Cys-scanning mutagenesis at regions of interest, and probed the environment of the engineered cysteines by using PEG-mal and three other small, ER membrane-permeant maleimide derivatives (NEM, AMS, and NPM) with different hydrophobicity. The results led us to construct a revised, 9-TMD model in which the active site His-460 is located in a hydrophobic environment, within a newly disclosed TMD, between Arg-443 and Tyr-462. A portion of these results described herein has been published previously in abstract form (19Lu X. Lin S. Chang C.C.Y. Chang T.Y. The 3rd AHA National Conference on Atherosclerosis, Thrombosis, and Vascular Biology.Salt Lake City, UT (Abstract). 2002; Google Scholar). Materials—PEG-mal was from Watershears; N-ethylmaleimide (NEM), N-phenylmaleimide (NPM), and iodoacetamide (IA) were from Sigma; 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) was from Molecular Probes. For modification under non-denaturing condition, stock solutions (PEG-mal at 40 mm, NEM and AMS at 100 mm) were freshly prepared in Buffer A (50 mm Tris-Cl, 1 mm EDTA, pH 7.8). NPM was not soluble in water, so it was dissolved in Me2SO as a 100 mm stock solution. For modification under denatured condition, PEG-mal or IA was prepared as a fresh stock solution in Lysate Buffer (10% SDS, 50 mm Tris-Cl, 1 mm EDTA, pH 8.7). 2-Mercaptoethanol was from Sigma. FuGENE 6 Transfection Reagent was from Roche Molecular Biology. [9,10-3H]Oleic acid was from Amersham Biosciences. PVDF membrane (Immobilon P) was from Millipore. Mouse anti-BiP antibody was from BD Transduction Laboratories. The rabbit polyclonal antibodies (DM10) generated against the N-terminal fragment (1-131) of hACAT1 were described previously (20Chang C.C.Y. Chen J. Thomas M.A. Cheng D. Del Priore V.A. Newton R.S. Pape M.E. Chang T.Y. J. Biol. Chem. 1995; 270: 29532-29540Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Goat anti-rabbit IgG(L+H)-HRP conjugate and Goat anti-mouse IgG(L+H)-HRP conjugate were from Bio-Rad. The SuperSignal West Pico Chemiluminescent Substrate was from Pierce. Cell Culture—The CHO cells were cultured in F-12/Dulbecco's modified Eagle's medium (50:50) supplemented with 10% fetal bovine serum in a 5% CO2 incubator at 37 °C. The ACAT1-deficient CHO cell line AC29 (21Cadigan K.M. Heider J.G. Chang T.Y. J. Biol. Chem. 1988; 263: 274-282Abstract Full Text PDF PubMed Google Scholar) was used to express the N-terminal His6-tagged hACAT1 (His-hACAT1) or its various engineered mutants. For transfections, the AC29 cells were cultured in 6-well plates to 70-80% confluency and transfected with 2 μg of pcDNA3 vectors encoding His-hACAT1 or its mutants, using FuGENE 6 Transfection Reagent according to the manufacturer's protocols. On the second day, the cells were trypsinized, divided equally into three wells, and grown for 2 days in G-418 containing medium for ACAT enzyme activity and protein expression studies (22Yang L. Lee O. Chen J. Chen J. Chang C.C. Zhou P. Wang Z.Z. Ma H.H. Sha H.F. Feng J.X. Wang Y. Yang X.Y. Wang L. Dong R. Ornvold K. Li B.L. Chang T.Y. J. Biol. Chem. 2004; 279: 46253-46262Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Alternatively, for chemical modification studies, on the second day after transfection, the cells were trypsinized; cells from 2 to 3 wells were pooled to a single 90 mm dish, and grown for 2 days at 37 °C, with G418 (0.3 mg/ml) present in the growth medium. Cells were then harvested for chemical modification studies. Recombinant DNA Technology—All of the ACAT1 mutants were prepared using the QuikChange mutagenesis kit from Stratagene according to the manufacturer's manual. For mutants based on His-hACAT1 or [C92A]His-ACAT1 as the template, the DNA constructs pGEM-7Z(-)/His-hACAT1 or pGEM-7Z(-)/[C92A]His-hACAT1 were used as the mutagenesis template (13Guo Z.-Y. Chang C.C.Y. Lu X. Chen J. Li B.-L. Chang T.-Y. Biochemistry. 2005; 44: 6537-6548Crossref PubMed Scopus (26) Google Scholar). All of the expected mutations were confirmed by DNA sequencing. The mutated ACAT1 gene was transferred from pGEM-7Z(-) vector to pcDNA3 vector as follows: The 1.7-kb DNA fragment encoding ACAT1 mutants was released from the pGEM-7Z(-) vector by HindIII cleavage, filled in by T4 DNA polymerase, and then cleaved with EcoRI. Subsequently, the 1.7-kb DNA fragment was ligated into the pcDNA3 vector pretreated with EcoRI and EcoRV. For the mutants based on [C2 (528/546)]His-hACAT1 as the template (12Lu X.H. Lin S. Chang C.C.Y. Chang T.Y. J. Biol. Chem. 2002; 277: 711-718Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 13Guo Z.-Y. Chang C.C.Y. Lu X. Chen J. Li B.-L. Chang T.-Y. Biochemistry. 2005; 44: 6537-6548Crossref PubMed Scopus (26) Google Scholar), the construct pcDNA3/[C2 (528/546)]His-hACAT1 was used as the mutagenesis template, and the expected mutations were confirmed by DNA sequencing. For the mutants based on hACAT1-HA6, containing a HA tag (with an amino acid sequence of YPYDVPDYA) between Tyr-404 and Lys-405, the construct pcDNA3/ACAT1-HA6 (11Lin S. Cheng D. Liu M.S. Chen J. Chang T.Y. J. Biol. Chem. 1999; 274: 23276-23285Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) was used as the mutagenesis template. The Cys-92 residue in ACAT1-HA6 was mutated to Ala-92 by replacing the DNA fragment (cut by KpnI and Eco47III) with the same fragment released from pcDNA3/[C92A]His-ACAT1. ACAT Activity Assay in Intact Cells—This method measures the rate of [3H]cholesteryl oleate synthesis in intact cells (23Chang C.C.Y. Doolittle G.M. Chang T.Y. Biochemistry. 1986; 25: 1693-1699Crossref PubMed Scopus (56) Google Scholar). The transiently transfected AC29 cells were cultured in 6-well plates at 37 °C as described above. The cells were given a fresh media change (1 ml/well) 2 h before the assay. Then, 20 μl of 10 mm [3H]oleate in 10% bovine serum albumin was added to the media at 37 °C for 30 min. ACAT1 Protein Content Analysis after Transfection—The cells in 6-well plates were washed with 2 ml of phosphate-buffered saline and lysed by 240 μl of Lysate Buffer containing 10 mm IA. The cell lysates were transferred to Eppendorf tubes, 60 μl/tube of SDS-PAGE Loading Buffer (10% SDS, 20% glycerol, 0.05% bromphenol blue, 50 mm Tris-Cl, pH 6.8) was added, and the samples were mixed well by vigorous vortexing. Then, 60 μl of the sample mixture were loaded onto a 9% SDS-gel. After electrophoresis, the proteins were transferred to a PVDF membrane, and the ACAT1 protein bands were visualized by Western blot using DM10 as the primary antibodies. The relative amount of the hACAT1 mutants versus that of the WT hACAT1 was analyzed by densitometry. Permeability Analysis of Microsomal Vesicles—The AC29 cells that stably express hACAT1 were cultured in 90-mm dishes and lysed by hypotonic shock followed by scraping according to the previously published procedure (24Chang T.Y. Limanek J.S. Chang C.C.Y. Anal. Biochem. 1981; 116: 298-302Crossref PubMed Scopus (33) Google Scholar). After centrifugation (800 × g, 5 min) to remove the unbroken cells and nuclei, the whole cell homogenates underwent ultracentrifugation (100,000 × g, 30 min) at 4 °C. After ultracentrifugation the pellets that contain microsomal vesicles were gently resuspended in the cold buffer A. Then, the microsomal vesicles were treated with or without saponin, and/or with or without PEG-mal as indicated at 4 °C for 1 h, and then 2-mercaptoethanol (from a freshly prepared 100 mm stock solution in Buffer A) was added to the final concentration of 10 mm (to react with the excess amount of PEG-mal). The samples underwent ultracentrifugation (100,000 × g, 1 h) at 4 °C to collect the microsomal vesicles. After ultracentrifugation, the supernatants were carefully removed, and the pellets were resuspended in buffer A containing 10 mm 2-mercaptoethanol at the same volume as the supernatants. Then, Lysate buffers containing 40 mm IA were added to the supernatants and the pellets at a volume equal to that of the supernatants and the pellets. The reactions were incubated at room temp for 30 min. Next, SDS-PAGE loading buffer at 1/4 volume of the sample mixtures were added. The final supernatant and the pellet modification mixtures were loaded in equal amounts onto a SDS-gel (9%) for SDS-PAGE. After electrophoresis, the proteins were transferred to a PVDF membrane, and the BiP and ACAT1 protein bands were visualized by Western blot using anti-BiP and anti-ACAT1 antibodies, respectively. PEG-mal Modification under Non-denaturing Condition—The AC29 cells transiently expressing His-hACAT1 mutants were cultured in 90-mm dishes. The whole cell homogenates were prepared by the same method as described earlier. Then, the 40 mm stock PEG-mal solution dissolved in Buffer A (50 mm Tris-Cl, 1 mm EDTA, pH 7.8) was added to the cell lysates, in the absence or presence of saponin (which permeabilizes the microsomal membranes) (15Lin S. Lu X. Chang C.C.Y. Chang T.Y. Mol. Biol. Cell. 2003; 14: 2447-2460Crossref PubMed Scopus (74) Google Scholar, 13Guo Z.-Y. Chang C.C.Y. Lu X. Chen J. Li B.-L. Chang T.-Y. Biochemistry. 2005; 44: 6537-6548Crossref PubMed Scopus (26) Google Scholar). The final concentration of PEG-mal was 4 mm, and the final concentration of saponin was 5 mg/ml. The modification reactions were carried out at 4 °C for 1.5 h. After incubation, 2-mercaptoethanol was added to the final concentration of 10 mm (to react with the excess amount of PEG-mal). The reactions were carried out at 4 °C for an additional 30 min. Subsequently, 40 mm IA in Lysate Buffer at volumes equal to the sample volume was added (to modify the remaining unreacted thiol groups of hACAT1 under denatured condition). Finally, one-half to one-fourth of the total modification mixtures were used for analysis by SDS-PAGE; the proteins were transferred to a PVDF membrane after electrophoresis. The ACAT1 bands were visualized by Western blot using DM10 as the primary antibodies. NEM, AMS, and NPM Modifications under Nondenaturing Condition—The whole cell homogenates of AC29 cells that transiently express ACAT1 mutants based on [C2 (528/546)]His-ACAT1 were prepared by the same method as described earlier. The 800 × g supernatants that contain the microsomes were treated with NEM, or AMS, or NPM as indicated at 4 °C for 1.5 h. The final concentration of NEM, AMS, or NPM was 4 mm. For parallel NEM/AMS modifications, the reactions were carried out in Buffer A; for parallel NPM/NEM modifications, because NPM is relatively insoluble in buffer A, the modifications with NEM or with NPM were carried out in buffer A containing 4% Me2SO. After incubation, the modification mixture was diluted 40-fold and incubated at 4 °C for 30 min (to quench the reactions between free cysteines in ACAT1 and the maleimides). For parallel NEM/AMS modification mixtures, Buffer A was used for dilution; for parallel NPM/NEM modification mixtures, Buffer A containing 4% Me2SO was used for dilution. Afterward, the microsomes in the modification mixtures were separated from the maleimide used by ultracentrifuge (100,000 × g, 30 min) at 4 °C. The pellets were resuspended in 50 μl of Buffer A. Then, 50 μl of Lysate Buffer containing 10 mm PEG-mal was added per sample at room temperature for 30 min (to modify the residual free cysteines that did not react with NEM or with AMS). Finally, the modification mixtures were analyzed by SDS-PAGE, and the proteins were transferred to a PVDF membrane after electrophoresis. The ACAT1 bands were visualized by Western blot using DM10 as the primary antibodies. Determining the Importance of the Conserved His-460 and the Conserved Ser-269 in hACAT1—Bioinformatic analysis suggested that for the membrane-bound O-acyl transferase superfamily, the absolutely conserved His residue (His-460 in hACAT1), located within a long stretch of hydrophobic amino acid residues, might constitute the active site (14Hofmann K. Trends Biochem. Sci. 2000; 25: 111-112Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). In contrast, Joyce and colleagues (25Joyce C.W. Shelness G.S. Davis M.A. Lee R.G. Skinner K. Anderson R.A. Rudel L.L. Mol. Biol. Cell. 2000; 11: 3675-3687Crossref PubMed Scopus (102) Google Scholar) proposed that a Ser residue (Ser-269 in hACAT1), conserved within ACAT1, ACAT2, and diacylglycerol acyltransferase 1, might be the active site. The proposal by Joyce and colleagues was based on the fact that when the mutant ACAT1 with Leu replacing Ser-269 was expressed in AC29 cells, it provided no measurable ACAT enzyme activity. These investigators did not report the protein expression level of the S269L ACAT1 mutant. In principle, it is possible that either Ser-269 or His-460, or both, may serve as the active site of ACAT1. For example, carnitine acyltransferase utilizes a His residue as the catalytic site (26Gogl G. Hsiao Y.-S. Tong L. Ann. N. Y. Acad. Sci. 2004; 1033: 17-29Crossref PubMed Scopus (101) Google Scholar), whereas lecithin:cholesterol acyltransferase utilizes a Ser residue as part of the Ser/His/Asp catalytic triad (27Jonas A. Biochim. Biophys. Acta. 2000; 1529: 245-256Crossref PubMed Scopus (310) Google Scholar). To determine the importance of Ser-269 and His-460, we replaced His-460 and Ser-269 individually with different amino acids by site-specific mutagenesis, expressed the mutants in AC29 cells by transient transfections, and analyzed the activities and the protein expression levels of the individual mutant enzymes. As shown in Fig. 2 (lanes 6 and 7), when His-460 was replaced by Ala or by Asn, the mutant enzymes retained nearly normal protein expression levels, but they completely lost enzymatic activity. However, when Ser-269 was replaced by Ala or by Thr (Fig. 2, lanes 3 and 5), the mutant retained nearly normal enzyme activity as well as nearly normal protein expression levels. Interestingly, when Ser-269 was replaced by Leu (Fig. 2, lane 4), the mutant had no detectable enzymatic activity, nor did it exhibit any detectable protein expression. Therefore, the loss of activity of the S269L mutant was likely caused by the loss of ACAT1 protein expression in CHO cells. In results not shown, we found that the S269L mutant hACAT1 could be expressed in insect cells, and it still retained ∼70% of the activity expressed by the wild-type hACAT1. Therefore, we conclude that His-460 is the active site of hACAT1. Ser-269 is not the active site, but it may play important role(s) in maintaining the structural integrity/protein stability of ACAT1 in mammalian cells. These results are consistent with our earlier findings in hACAT2: mutation in the conserved His-434 led to complete loss in enzyme activity without significant loss in protein expression, whereas mutating the conserved Ser-245 to Leu led to significant loss in protein expression in CHO cells (15Lin S. Lu X. Chang C.C.Y. Chang T.Y. Mol. Biol. Cell. 2003; 14: 2447-2460Crossref PubMed Scopus (74) Google Scholar). Rationale for Developing a New Strategy to Study the ACAT1 Membrane Topology—We aimed at developing a new strategy to resolve the ACAT1 topology that covers the uncertainty region aa 354-493. We