Colocalization of SCD1 and DGAT2: implying preference for endogenous monounsaturated fatty acids in triglyceride synthesis
2006; Elsevier BV; Volume: 47; Issue: 9 Linguagem: Inglês
10.1194/jlr.m600172-jlr200
ISSN1539-7262
AutoresWeng Chi Man, Makoto Miyazaki, Kiki Chu, James M. Ntambi,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoStearoyl-coenzyme A desaturase (SCD) is an endoplasmic reticulum (ER) protein that catalyzes the Δ9-cis desaturation of saturated fatty acids. Mice with targeted disruption in SCD1 (Scd1−/−) have significant reduction in the tissue content of triglycerides, suggesting that monounsaturated fatty acids endogenously synthesized by SCD1 are important for triglyceride synthesis. Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is the enzyme that catalyzes the final reaction in the synthesis of triglycerides. The lack of DGAT2, one of the two DGAT isoforms, results in almost a complete loss of tissue triglycerides. We hypothesize that SCD1 participates in triglyceride synthesis by providing a more accessible pool of monounsaturated fatty acids through substrate channeling. In this study, we test whether SCD1 is proximal to DGAT2 by colocalization study with confocal microscopy, coimmunoprecipitation, and fluorescence resonance energy transfer using HeLa cells as the model of study. All of the results suggest that SCD1 and DGAT2 are located very close to each other in the ER, which is a very important criterion for the channeling of substrate. By performing subcellular fractionation using mouse livers, we also show, for the first time, that SCD is present in the mitochondria-associated membrane. Stearoyl-coenzyme A desaturase (SCD) is an endoplasmic reticulum (ER) protein that catalyzes the Δ9-cis desaturation of saturated fatty acids. Mice with targeted disruption in SCD1 (Scd1−/−) have significant reduction in the tissue content of triglycerides, suggesting that monounsaturated fatty acids endogenously synthesized by SCD1 are important for triglyceride synthesis. Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is the enzyme that catalyzes the final reaction in the synthesis of triglycerides. The lack of DGAT2, one of the two DGAT isoforms, results in almost a complete loss of tissue triglycerides. We hypothesize that SCD1 participates in triglyceride synthesis by providing a more accessible pool of monounsaturated fatty acids through substrate channeling. In this study, we test whether SCD1 is proximal to DGAT2 by colocalization study with confocal microscopy, coimmunoprecipitation, and fluorescence resonance energy transfer using HeLa cells as the model of study. All of the results suggest that SCD1 and DGAT2 are located very close to each other in the ER, which is a very important criterion for the channeling of substrate. By performing subcellular fractionation using mouse livers, we also show, for the first time, that SCD is present in the mitochondria-associated membrane. Triglycerides are the major energy store in eukaryotic organisms. However, excessive deposition of triglycerides in the white adipose tissue results in obesity, which has evolved into a serious health problem owing to its medical complications. Therefore, understanding the molecular basis of triglyceride biosynthesis is essential.Stearoyl-coenzyme A desaturase (SCD) is an endoplasmic reticulum (ER) enzyme that is responsible for the critical committed step in the de novo synthesis of monounsaturated fatty acids (1Ntambi J.M. Miyazaki M. Regulation of stearoyl-CoA desaturases and role in metabolism..Prog. Lipid Res. 2004; 43: 91-104Crossref PubMed Scopus (537) Google Scholar, 2Miyazaki M. Ntambi J.M. Role of stearoyl-coenzyme A desaturase in lipid metabolism..Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 113-121Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 3Ntambi J.M. The regulation of stearoyl-CoA desaturase (SCD)..Prog. Lipid Res. 1995; 34: 139-150Crossref PubMed Scopus (285) Google Scholar, 4Ntambi J.M. Miyazaki M. Recent insights into stearoyl-CoA desaturase-1..Curr. Opin. Lipidol. 2003; 14: 255-261Crossref PubMed Scopus (207) Google Scholar). The preferred substrates are palmitoyl- and stearoyl-CoAs, which are converted into palmitoleoyl- and oleoyl-CoAs (5Enoch H.G. Strittmatter P. Role of tyrosyl and arginyl residues in rat liver microsomal stearylcoenzyme A desaturase..Biochemistry. 1978; 17: 4927-4932Crossref PubMed Scopus (62) Google Scholar). These two monounsaturated fatty acids are the major fatty acids found in membrane phospholipids, triglycerides, and cholesteryl esters (6Miyazaki M. Dobrzyn A. Man W.C. Chu K. Sampath H. Kim H.J. Ntambi J.M. Stearoyl-CoA desaturase 1 gene expression is necessary for fructose-mediated induction of lipogenic gene expression by sterol regulatory element-binding protein-1c-dependent and -independent mechanisms..J. Biol. Chem. 2004; 279: 25164-25171Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Given that monounsaturated fatty acids are readily obtainable from diets, it is a surprising finding that the disruption of SCD1 in mice results in a significant reduction in the content of liver triglycerides and white adipose depots (7Ntambi J.M. Miyazaki M. Stoehr J.P. Lan H. Kendziorski C.M. Yandell B.S. Song Y. Cohen P. Friedman J.M. et al.Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity..Proc. Natl. Acad. Sci. USA. 2002; 99: 11482-11486Crossref PubMed Scopus (871) Google Scholar, 8Miyazaki M. Kim Y.C. Gray-Keller M.P. Attie A.D. Ntambi J.M. The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1..J. Biol. Chem. 2000; 275: 30132-30138Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). This suggests that SCD and its endogenous products are crucial in the regulation of lipid accumulation. Even when given a high-fat diet, mice with targeted disruption in SCD1 (Scd1−/−) are resistant to diet-induced obesity and liver steatosis (7Ntambi J.M. Miyazaki M. Stoehr J.P. Lan H. Kendziorski C.M. Yandell B.S. Song Y. Cohen P. Friedman J.M. et al.Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity..Proc. Natl. Acad. Sci. USA. 2002; 99: 11482-11486Crossref PubMed Scopus (871) Google Scholar).The final step in the synthesis of triglyceride is the addition of the third acyl chain to diacylglycerol, and this reaction is catalyzed by the membrane-bound enzyme acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) (9Weiss S.B. Kennedy E.P. Kiyasu J.Y. The enzymatic synthesis of triglycerides..J. Biol. Chem. 1960; 235: 40-44Abstract Full Text PDF PubMed Google Scholar). Two DGAT genes, DGAT1 and DGAT2, have been identified (10Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis..Proc. Natl. Acad. Sci. USA. 1998; 95: 13018-13023Crossref PubMed Scopus (859) Google Scholar, 11Cases S. Smith S.J. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Novak S. Collins C. Welch C.B. Lusis A.J. et al.Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members..J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). Although they catalyze similar reactions, they share neither nucleotide nor amino acid sequence similarity (12Stone S.J. Myers H.M. Watkins S.M. Brown B.E. Feingold K.R. Elias P.M. Farese Jr., R.V. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice..J. Biol. Chem. 2004; 279: 11767-11776Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). Studies performed using mice lacking DGAT1 (Dgat1−/−) have suggested that DGAT1 does not have a profound effect on triglyceride metabolism in general and is not essential for life (13Smith S.J. Cases S. Jensen D.R. Chen H.C. Sande E. Tow B. Sanan D.A. Raber J. Eckel R.H. Farese R.V. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat..Nat. Genet. 2000; 25: 87-90Crossref PubMed Scopus (725) Google Scholar). In contrast, mice with a disruption in the DGAT2 gene (Dgat2−/−) have severely reduced triglyceride content in their tissues and die in early postnatal periods, demonstrating that DGAT2 is essential for the fundamental synthesis of triglyceride in mammals and is crucial for survival (12Stone S.J. Myers H.M. Watkins S.M. Brown B.E. Feingold K.R. Elias P.M. Farese Jr., R.V. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice..J. Biol. Chem. 2004; 279: 11767-11776Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar).Suzuki et al. (14Suzuki R. Tobe K. Aoyama M. Sakamoto K. Ohsugi M. Kamei N. Nemoto S. Inoue A. Ito Y. Uchida S. et al.Expression of DGAT2 in white adipose tissue is regulated by central leptin action..J. Biol. Chem. 2005; 280: 3331-3337Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) predicted the subcellular localization of DGAT1 and DGAT2 based on their amino acid sequences. This prediction suggested that DGAT1 is targeted mainly to the plasma membrane and DGAT2 to the ER (14Suzuki R. Tobe K. Aoyama M. Sakamoto K. Ohsugi M. Kamei N. Nemoto S. Inoue A. Ito Y. Uchida S. et al.Expression of DGAT2 in white adipose tissue is regulated by central leptin action..J. Biol. Chem. 2005; 280: 3331-3337Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In fact, overexpression of mouse DGAT2 results in the accumulation of lipid droplets in the cytosol, whereas lipid droplets around the cell periphery are observed when DGAT1 is overexpressed (12Stone S.J. Myers H.M. Watkins S.M. Brown B.E. Feingold K.R. Elias P.M. Farese Jr., R.V. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice..J. Biol. Chem. 2004; 279: 11767-11776Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). It was proposed that the proximity of DGAT2 to the lipogenic enzymes on the ER allows it to make use of the fatty acids synthesized de novo around the ER (14Suzuki R. Tobe K. Aoyama M. Sakamoto K. Ohsugi M. Kamei N. Nemoto S. Inoue A. Ito Y. Uchida S. et al.Expression of DGAT2 in white adipose tissue is regulated by central leptin action..J. Biol. Chem. 2005; 280: 3331-3337Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar).A lipogenic high-carbohydrate diet induces SCD1 expression as well as other lipogenic genes in rodents, resulting in an increase in triglyceride synthesis (1Ntambi J.M. Miyazaki M. Regulation of stearoyl-CoA desaturases and role in metabolism..Prog. Lipid Res. 2004; 43: 91-104Crossref PubMed Scopus (537) Google Scholar). However, when the same diet is administered to mice with SCD1 deficiency, induction of triglyceride synthesis fails (6Miyazaki M. Dobrzyn A. Man W.C. Chu K. Sampath H. Kim H.J. Ntambi J.M. Stearoyl-CoA desaturase 1 gene expression is necessary for fructose-mediated induction of lipogenic gene expression by sterol regulatory element-binding protein-1c-dependent and -independent mechanisms..J. Biol. Chem. 2004; 279: 25164-25171Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Moreover, it is noteworthy that the basal mRNA expression and activity of DGAT in Scd1−/− mice is not lower than that in the wild-type counterpart, suggesting that SCD1 may serve as a key point of regulation in the synthesis of triglyceride. Despite the fact that DGAT1 and DGAT2 can use a wide range of fatty acids as substrates, oleate is still the most abundant fatty acid in tissue triglycerides, which suggests that a preference for oleate may exist in triglyceride synthesis. Therefore, we hypothesize that SCD1 is proximal to DGAT2 and that SCD1 participates in the synthesis of triglycerides by providing a more easily accessible pool of monounsaturated acyl-CoAs.In this study, we evaluated whether SCD1 is proximal to DGAT2 in vivo and in vitro. DGAT and SCD are proteins in the ER. However, it has also been shown that DGAT is enriched in a subcellular compartment known as the mitochondria-associated membrane (MAM). MAM is a special ER domain enriched with enzymes responsible for the synthesis of phospholipids and triglycerides, including DGAT, and thus has been proposed to potentially serve as a component of the secretory pathway that supplies lipids for assembly into very low density lipoproteins. By subcellular fractionation of the mouse liver, we demonstrate for the first time that SCD1 is present in MAM, which may imply that SCD1 works intimately with other enzymes in the synthesis of triglycerides and phospholipids. In vitro, we investigated whether SCD1 and DGAT2 are closely located in the ER by colocalization analysis using confocal microscopy, coimmunoprecipitation, and fluorescence resonance energy transfer (FRET) using flow cytometry. Each of these individual approaches suggested that SCD1 and DGAT2 are located in close proximity to each other in the ER.MATERIALS AND METHODSAnimals and dietsSix to 8 week old mice on a 129 SV background were used in all experiments. The mice were given ad libitum access to food and water and were housed in the animal facility of the Biochemistry Department at the University of Wisconsin-Madison. The breeding and care of the animals were according to the protocols approved by the Animal Care Research Committee of the University of Wisconsin-Madison.MaterialsHeLa cells were a generous gift from Dr. Ann Palmenberg at the University of Wisconsin-Madison. Tissue culture medium and reagents were purchased from Life Technologies-Invitrogen. Monoclonal antibodies against the epitopes were obtained as follows: anti-myc and Alexa-Fluor 488 goat anti-rabbit IgG was a gift from Dr. Anant Menon at Cornell University; anti-KDEL was from Stressgen (Victoria, British Columbia, Canada); anti-FLAG was from Sigma (St. Louis, MO); and Alexa-Fluor 594 goat anti-mouse IgG was from Molecular Probes (Eugene, OR). Protein A/G agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). [14C]stearoyl-CoA and [14C]oleoyl-CoA were purchased from American Radiolabeled Chemicals (St. Louis, MO). TransIT-LT1 was purchase from Mirus (Madison, WI). All other reagents were from Fisher or Sigma.PlasmidsThe pcDNA3 plasmid expressing SCD1 with the myc epitope tagged to the C terminus (SCD1-myc-C) was provided by Dr. Travis Knight (Iowa State University). The FLAG-tagged DGAT2 cloned into a pcDNA plasmid was a gift from Dr. Robert Farese (Gladstone Institute of Cardiovascular Disease, University of California-San Francisco). pECFP-N1 and pEYFP-C1 were generous gifts from Dr. Tom Martin (University of Wisconsin-Madison). The constructs cyan fluorescent protein (CFP)-SCD1 and yellow fluorescent protein (YFP)-DGAT2 were synthesized by cloning a PCR fragment encoding the open reading frame of SCD1 or DGAT2 into pECFP-N1 and pEYFP-C1, respectively. The primers used to synthesize the SCD1 PCR fragment were as follows: forward primer, 5′-ATT CTC GAG ATG CCG GCC CAC ATG CTC-3′; reverse primer, 5′-AAT AAG CTT GCT ACT CTT GTG ACT CCC-3′. For DGAT2, the primers used were as follows: forward primer, 5′-ATT CTC GAG CTA TGA AGA CCC TCA TCG CC-3′; reverse primer, 5′-TAA TCT AGA TCA GTT CAC CTC CAG CAC-3′. The SCD1 PCR fragment was cloned into pECFP-N1 using restriction sites XhoI and HindIII, whereas the DGAT2 PCR fragment was inserted into pEYFP-C1 at the restriction sites XhoI and XbaI.Isolation and characterization of MAM, mitochondria, and ER from mouse liversThe procedures for preparing the subcellular fractions were based on those described by Vidugiriene et al. (15Vidugiriene J. Sharma D.K. Smith T.K. Boumann N.A. Menon A.K. Segregation of glycosylphosphatidylinositol biosynthetic reactions in a subcompartment of the endoplasmic reticulum..J. Biol. Chem. 1999; 274: 15203-15212Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and Vance (16Vance J.E. Phospholipid synthesis in a membrane fraction associated with mitochondria..J. Biol. Chem. 1990; 265: 7248-7256Abstract Full Text PDF PubMed Google Scholar). The mice were euthanized by carbon dioxide asphyxiation, and the livers were quickly removed and immersed in ice-cold isolation buffer (250 mM mannitol, 5 mM HEPES, pH 7.4, 0.5 mM EGTA, and 0.1% BSA). Livers were minced and then homogenized using a Potter-Elvehjem motor-driven homogenizer. The homogenate was then centrifuged at 600 g twice to remove the large debris. The supernatant was then centrifuged at 10,000 g to obtain the crude mitochondria, and the resultant supernatant was layered on a discontinuous sucrose gradient (2 ml of 30% sucrose and 4 ml of 38% sucrose prepared in 10 mM HEPES, pH 7.4) and subjected to centrifugation at 100,000 g for 2 h to obtain the ER, which precipitated as a red pellet. For the isolation of MAM and mitochondria, the crude mitochondrial pellet was washed and resuspended in isolation medium. A small amount (0.5 ml) of the suspension was layered on top of each of the 30% Percoll gradients in polycarbonate ultracentrifuge tubes. The tubes were then centrifuged at 95,000 gmax for 33 min. The band corresponding to mitochondria was approximately two-thirds down the ultracentrifuge tube, with the MAM band lying immediately above it. The two bands were removed by transfer pipette, diluted with isolation buffer, and subjected to further centrifugation at 6,300 g. The mitochondria pelleted after centrifugation as a yellowish brown pellet. The supernatant from the mitochondrial fraction was combined with the supernatant from the MAM fraction and further centrifuged at 100,000 g to obtain MAM.Characterization of the subcellular fractionsTo characterize the subcellular fractions, the following marker enzyme assays were performed. Cytochrome C oxidase and NADPH-cytochrome C reductase were chosen as the marker enzymes for mitochondria and ER, respectively. For the cytochrome C oxidase assay, 100 μl of 0.1 M phosphate buffer, pH 7.0, 30 μl of 0.5% Triton X-100, 800 μl of water, and subcellular fraction proteins (25 μg or less) were combined and incubated at 37°C for 5 min. Seventy microliters of reduced ferrocytochrome C at a concentration of 20 mg/ml was then added to the mixture to start the reaction. A decrease in optical density at 550 nm was measured for 3 min. For the NADPH-cytochrome C reductase assay, the subcellular fraction proteins were added to 100 μl of 0.5 M phosphate buffer and 45 μl of 1% cytochrome C and incubated at room temperature for 3 min before 100 μl of 10 mM NADPH was added to start the reaction. An increase in optical density at 550 nm was measured for 3 min. In both assays described above, the rate of change in optical density was used to calculate the specific activity of the marker enzyme.Cell culture and transient transfection using electroporationHeLa cells were maintained in high-glucose DMEM supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin. The cells were incubated at 37°C in a humidified atmosphere with 5% CO2. The transfection of the different constructs into the HeLa cells was performed as described (17Pottekat A. Menon A.K. Subcellular localization and targeting of N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the second enzyme in the glycosylphosphatidylinositol biosynthetic pathway..J. Biol. Chem. 2004; 279: 15743-15751Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Briefly, HeLa cells were cultured to 80–100% confluence. The cells were harvested and pelleted. The cell pellet was then washed by resuspending in 50 ml of cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 25 mM HEPES/KOH, pH 7.6, 25 mM EGTA, and 25 mM MgCl2). The cells were then pelleted, and 500 μl of cytomix was added to resuspend the cells. The cell suspension was then transferred to an electroporation cuvette. Twenty-five micrograms of DNA plasmid was added to the cell suspension, and the mixture was exposed to a single electric pulse of 300 V with a capacitance of 1,000 μF using the Bio-Rad pulse system. The cell mixture was transferred back to a 100 mm culture plate, and the cells were incubated in culture medium as described above for 24 or 48 h as specified before analysis was performed. For confocal microscopy study, cells were plated onto polylysine-coated cover slips.Analysis of triglyceride synthesisHeLa cells were transfected with pcDNA, SCD1-myc only, DGAT2-FLAG only, or SCD1-myc and DGAT2 using TransIT-LT1. The cells were incubated with serum-free medium containing [14C]stearate coupled to BSA. Forty-eight hours after transfection, the cells were lysed with lysis buffer and total lipids were extracted and separated by thin-layer chromatography, with petroleum hexane-diethyl ether-acetic acid (80:30:1) as the developing solvent. The counts in the triglyceride fractions (cpm) were determined by autoradiography using Packard Instant Imager. To determine the ratio of oleate (18:1) to stearate (18:0), the triglyceride fraction was scraped from the TLC plate. The lipids were extracted according to the method of Bligh and Dyer (18Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification..Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42174) Google Scholar). Fatty acids were released from the triglyceride and separated on a silver nitrate-impregnated silica gel as described below for SCD activity assay.Oil Red O stainingThe cells were fixed with 10% buffered neutral formalin and then stained with the Oil Red O solution containing Oil Red O (1.5 mg/100 ml solution), 50% (v/v) alcohol and 50% (v/v) acetone.Confocal microscopyApproximately 24 h after transfection, the cover slips were taken from the culture plate and washed with PBS three times. The cells were then fixed with 4% paraformaldehyde at room temperature for 10 min, followed by an additional three washes with PBS. For visualization of the distribution of SCD1-myc and DGAT2-FLAG, the cells expressing these recombinant proteins were permeabilized with 0.3% Triton X-100 in PBS for 25 min at 4°C. After permeabilization with detergents, the cells were washed with PBS three times and blocked with 10% (v/v) fetal bovine serum in PBS for 1 h at room temperature. The permeabilized cells were then incubated with rabbit anti-myc antibody at 1:500 dilution and anti-FLAG monoclonal antibody at 2 μg/ml for 1 h at room temperature. After incubation with primary antibodies, the cells were washed three times with PBS and incubated with Alexa-Fluor 594 goat anti-mouse IgG or Alexa-Fluor 488 goat anti-rabbit IgG at a dilution of 1:250 for 1 h at room temperature. After three more washes with PBS, the cover slips were mounted on microscopy slides with a drop of Vectorshield (Vector Laboratories) added between the slides and the cover slips. Confocal microscopy was performed to visualize the staining pattern. For cells expressing pECFP-SCD1 and pEDGAT2-YFP, after washing with PBS and fixation with 4% paraformaldehyde, the cover slips were mounted onto the slides and analyzed. Images were obtained using a confocal microscope (Nikon Eclipse TE 2000-U).CoimmunoprecipitationHeLa cells were transfected with SCD1-myc and DGAT2-FLAG by electroporation. Forty-eight hours after transfection, the cells were harvested and lysed with lysis buffer [40 mM HEPES-KOH, pH 7.4, 150 mM NaCl, and 0.5% (w/v) Nonidet P-40] containing protease inhibitors at the following concentrations: 10 μg/ml leupeptin, 5 μg/ml pepstatin A, 2 μg/ml aprotinin, and 2 mM PMSF. The lysis was allowed to proceed for 40 min on ice, after which the lysate was spun at 16,000 g for 20 min. The supernatant was transferred to a new microcentrifuge tube. Protein A/G bead slurry was added and incubated at 4°C for 1 h with gentle rotation. After centrifugation at 2,000 g for 1 min, monoclonal myc antibody or monoclonal FLAG antibody was added to the supernatant and incubated at 4°C for 1 h with gentle rotation. Protein A/G bead slurry was then added and incubated at 4°C overnight with rotation. The next day, the protein A/G beads were washed with lysis buffer three times and subjected to boiling for 5 min to release the proteins, which were then separated by SDS-PAGE.Preparation of microsomesThe cells expressing the desired recombinant constructs were harvested 48 h after transfection and washed with 0.1 M potassium phosphate buffer, pH 7.2. The cells were then disrupted with a variable-speed tissue disruptor and centrifuged at 10,000 g for 15 min at 4°C. The supernatant was then subjected to further centrifugation at 100,000 g for 1 h at 4°C. The pellet obtained was the microsomes.DGAT activity assayThe DGAT activity assay was performed as described (19Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members..J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar) by incubating the subcellular fractions or microsomes with 10 μl of 40 mM DTT, 5 μl of 8 mM diacylglycerol in acetone, 5 μl of 1.5 mM oleoyl-CoA, 10 μl of 2.5 mg/ml BSA, 2 μl of [14C]oleoyl-CoA, and reaction buffer to give a total reaction volume of 200 μl for 5 min at 37°C. The reaction buffer was composed of 250 mM sucrose, 1 mM EDTA, and 100 mM Tris-HCl, pH 7.5. Chloroform-methanol (0.75 ml; 1:2, v/v) was added to the mixture and vortexed. Chloroform (0.25 ml) and 0.25 ml of water were added, vortexed, and centrifuged at 13,000 g for 2 min. A total of 250 μl of the chloroform (bottom) layer was taken out and dried by nitrogen. The lipid was redissolved in 50 μl of chloroform-methanol (2:1, v/v). The lipid was then separated by thin-layer chromatography (hexane-ether-acetic acid, 90:20:1 or heptane-isopropyl ether-acetic acid, 60:40:3). The TLC plate was then subjected to autoradiography. The ratio of the cpm corresponding to the triacylglycerol band to the total cpm was used to determine the DGAT activity of the subcellular fraction.SCD activity assayThe SCD activity in subcellular fractions/microsomes was estimated by measuring the conversion of stearoyl-CoA (18:0 CoA) to oleoyl-CoA (18:1 CoA) (20Miyazaki M. Jacobson M.J. Man W.C. Cohen P. Asilmaz E. Friedman J.M. Ntambi J.M. Identification and characterization of murine SCD4, a novel heart-specific stearoyl-CoA desaturase isoform regulated by leptin and dietary factors..J. Biol. Chem. 2003; 278: 33904-33911Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The assay was performed by combining the subcellular fractions/microsomes with 6 nmol of stearoyl-CoA, 0.03 μCi of [14C]stearoyl-CoA, 2 mM of NADH, and 0.1 M potassium phosphate buffer, pH 7.2, to give a total reaction volume of 200 μl. For assaying SCD activity of microsomes expressing recombinant SCD1 construct, cold stearoyl-CoA was omitted from the reaction. The reaction was carried out at room temperature for 15 min and was stopped by the addition of 200 μl of a mixture of 2.5 ml of 10 M KOH, 500 μl of 5 mg/ml butyl-hydroxytoluene, and 7.0 ml of ethanol. The reaction mixture was then incubated at 80°C for 45 min. Formic acid (280 μl) was then added to protonate free fatty acids. Hexane (700 μl) was added to extract fatty acids, followed by vigorous vortexing. Two hundred to 500 μl of the hexane (upper) layer was taken out and dried under nitrogen gas. The lipid was resuspended in 50 μl of hexane and spotted on a 10% silver nitrate-impregnated TLC plate. The lipid was separated using chloroform-methanol-acetic acid-water (90:8:1:0.8). The plate was then subjected to autoradiography using Packard Instant Imager. The ratio of the cpm in the band corresponding to the oleic acid to the total cpm was used to calculate the SCD activity.Flow cytometryFlow cytometry was performed based on the protocol described by Dye et al. (21Dye B.T. Schell K. Miller D.J. Ahlquist P. Detecting protein-protein interaction in live yeast by flow cytometry..Cytometry. 2005; 63: 77-86Crossref PubMed Scopus (21) Google Scholar). Briefly, HeLa cells were transfected with the constructs using electroporation. Twelve hours after transfection, the cells were harvested for flow cytometry analysis. The data were collected with an LSRII flow cytometer (Becton Dickinson, San Jose, CA). Propidium iodide was added to the cell suspension to differentiate live cells from dead cells, as live cells would not pick up propidium iodide. Laser line 488 nm was used to excite propidium iodide, the fluorescence of which was detected using a 630/30 band-pass filter. Two lasers tuned to 488 and 403 nm were used for flow cytometry analysis. The 403 laser line was used for CFP, and the cyan fluorescence was detected using a 470/40 band-pass filter. The yellow fluorescence resulting from direct excitation by the 488 laser line was measured using a 560/10 band-pass filter, to differentiate from the yellow fluorescence resulting from FRET, which was detected using a 530/30 band-pass filter. Data analysis was performed using FlowJo (Tree Star, Inc., Ashland, OR).Western blot analysisFor immunoblot analysis, protein samples were mixed with SDS loading buffer, loaded, and separated on a 10% SDS-PAGE gel, after which the proteins were transferred to Immobilon-P transfer membranes at 4°C. The membrane was blocked in 10% nonfat dry milk in TBS-T overnight at 4°C. It was then washed with TBS-T and incubated with the primary antibody. The membrane was again washed with TBS-T and incubated with the secondary antibody. Visualization of the SCD1 protein was performed using the Super Signal West Pico kit (Pierce Biotechnology, Rockford, IL).RESULTSSCD1 is present in the MAMMAM is the region of the ER that is in close proximity to the outer membrane of the mitochondria (22Rusinol A.E. Cui Z. Chen M.H. Vance J.E. A unique mitochondria-associated membrane fraction from rat liver has a high capacity for lipid synthesis and contains pre-Golgi secretory proteins including nascent lipoproteins..J. Biol. Chem. 1994; 269: 27494-27502Abstract Full Text PDF PubMed Google Scholar). MAM has been shown to be enriched with many enzymes responsible for the synthesis of pho
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