A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7
2018; Elsevier BV; Volume: 59; Issue: 3 Linguagem: Inglês
10.1194/jlr.m082354
ISSN1539-7262
AutoresSvenja Bockelmann, John G. Mina, Sergei M. Korneev, Dina Hassan, Dagmar Müller, Angelika Hilderink, Hedwich C. Vlieg, Reinout Raijmakers, Albert J. R. Heck, Per Haberkant, Joost C. M. Holthuis,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoCeramides are central intermediates of sphingolipid metabolism with dual roles as mediators of cellular stress signaling and mitochondrial apoptosis. How ceramides exert their cytotoxic effects is unclear and their poor solubility in water hampers a search for specific protein interaction partners. Here, we report the application of a photoactivatable and clickable ceramide analog, pacCer, to identify ceramide binding proteins and unravel the structural basis by which these proteins recognize ceramide. Besides capturing ceramide transfer protein (CERT) from a complex proteome, our approach yielded CERT-related steroidogenic acute regulatory protein D7 (StarD7) as novel ceramide binding protein. Previous work revealed that StarD7 is required for efficient mitochondrial import of phosphatidylcholine (PC) and serves a critical role in mitochondrial function and morphology. Combining site-directed mutagenesis and photoaffinity labeling experiments, we demonstrate that the steroidogenic acute regulatory transfer domain of StarD7 harbors a common binding site for PC and ceramide. While StarD7 lacks robust ceramide transfer activity in vitro, we find that its ability to shuttle PC between model membranes is specifically affected by ceramides. Besides demonstrating the suitability of pacCer as a tool to hunt for ceramide binding proteins, our data point at StarD7 as a candidate effector protein by which ceramides may exert part of their mitochondria-mediated cytotoxic effects. Ceramides are central intermediates of sphingolipid metabolism with dual roles as mediators of cellular stress signaling and mitochondrial apoptosis. How ceramides exert their cytotoxic effects is unclear and their poor solubility in water hampers a search for specific protein interaction partners. Here, we report the application of a photoactivatable and clickable ceramide analog, pacCer, to identify ceramide binding proteins and unravel the structural basis by which these proteins recognize ceramide. Besides capturing ceramide transfer protein (CERT) from a complex proteome, our approach yielded CERT-related steroidogenic acute regulatory protein D7 (StarD7) as novel ceramide binding protein. Previous work revealed that StarD7 is required for efficient mitochondrial import of phosphatidylcholine (PC) and serves a critical role in mitochondrial function and morphology. Combining site-directed mutagenesis and photoaffinity labeling experiments, we demonstrate that the steroidogenic acute regulatory transfer domain of StarD7 harbors a common binding site for PC and ceramide. While StarD7 lacks robust ceramide transfer activity in vitro, we find that its ability to shuttle PC between model membranes is specifically affected by ceramides. Besides demonstrating the suitability of pacCer as a tool to hunt for ceramide binding proteins, our data point at StarD7 as a candidate effector protein by which ceramides may exert part of their mitochondria-mediated cytotoxic effects. Sphingolipids are abundant components of eukaryotic membranes that participate in a wide array of cellular processes by modulating vital physical membrane properties and as signaling molecules in responses to physiological cues and stresses (1.Lingwood D. Simons K. Lipid rafts as a membrane-organizing principle.Science. 2010; 327: 46-50Crossref PubMed Scopus (3202) Google Scholar, 2.Lippincott-Schwartz J. Phair R.D. Lipids and cholesterol as regulators of traffic in the endomembrane system.Annu. Rev. Biophys. 2010; 39: 559-578Crossref PubMed Scopus (115) Google Scholar, 3.Hla T. Dannenberg A.J. 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Pfeilschifter J. Ceramide-binding and activation defines protein kinase c-Raf as a ceramide-activated protein kinase.Proc. Natl. Acad. Sci. USA. 1996; 93: 6959-6963Crossref PubMed Scopus (184) Google Scholar), cathepsin D (19.Heinrich M. Wickel M. Schneider-Brachert W. Sandberg C. Gahr J. Schwandner R. Weber T. Saftig P. Peters C. Brunner J. Cathepsin D targeted by acid sphingomyelinase-derived ceramide.EMBO J. 1999; 18: 5252-5263Crossref PubMed Scopus (305) Google Scholar), and protein phosphatase 2A (PP2A) inhibitor, SET (20.Mukhopadhyay A. Saddoughi S.A. Song P. Sultan I. Ponnusamy S. Senkal C.E. Snook C.F. Arnold H.K. Sears R.C. Hannun Y.A. Direct interaction between the inhibitor 2 and ceramide via sphingolipid-protein binding is involved in the regulation of protein phosphatase 2A activity and signaling.FASEB J. 2009; 23: 751-763Crossref PubMed Scopus (172) Google Scholar). Identification of additional ceramide binding proteins is desirable, as this would likely lead to further mechanistic insights into ceramide-mediated signaling pathways and expand opportunities for exploiting their therapeutic potential. Several proteome-wide methods have been developed to detect specific lipid-protein interactions, which include the application of protein microarrays in a screen for novel phosphoinositide binding proteins (21.Zhu H. Bilgin M. Bangham R. Hall D. Casamayor A. Bertone P. Lan N. Jansen R. Bidlingmaier S. Houfek T. Global analysis of protein activities using proteome chips.Science. 2001; 293: 2101-2105Crossref PubMed Scopus (1935) Google Scholar). In an inverted setup, lipid strips have been used to obtain lipid-binding fingerprints for a large number of proteins with predicted lipid binding domains (22.Gallego O. Betts M.J. Gvozdenovic-Jeremic J. Maeda K. Matetzki C. Aguilar-Gurrieri C. Beltran-Alvarez P. Bonn S. Fernández-Tornero C. Jensen L.J. A systematic screen for protein-lipid interactions in Saccharomyces cerevisiae.Mol. Syst. Biol. 2010; 6: 430Crossref PubMed Scopus (128) Google Scholar). Column-based affinity purification strategies with lipids immobilized onto magnetic beads have also been utilized (23.Kota V. Szulc Z.M. Hama H. Identification of C(6)-ceramide-interacting proteins in D6P2T Schwannoma cells.Proteomics. 2012; 12: 2179-2184Crossref PubMed Scopus (15) Google Scholar, 24.Bidlingmaier S. Ha K. Lee N-K. Su Y. Liu B. Proteome-wide identification of novel ceramide-binding proteins by yeast surface cDNA display and deep sequencing.Mol. Cell. Proteomics. 2016; 15: 1232-1245Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). However, a major drawback of screens using lipids immobilized on solid supports is that such lipids are not presented in their natural state. Moreover, interactions where the lipid has to enter a deep hydrophobic binding pocket within the protein are likely to be missed. In recent years, bifunctional lipid analogs have emerged as promising new tools to circumvent some of these disadvantages, enabling global profiling of lipid-protein interactions in living cells (25.Haberkant P. Raijmakers R. Wildwater M. Sachsenheimer T. Brügger B. Maeda K. Houweling M. Gavin A-C. Schultz C. van Meer G. In vivo profiling and visualization of cellular protein-lipid interactions using bifunctional fatty acids.Angew. Chem. Int. Ed. Engl. 2013; 52: 4033-4038Crossref PubMed Scopus (89) Google Scholar, 26.Hulce J.J. Cognetta A.B. Niphakis M.J. Tully S.E. Cravatt B.F. Proteome-wide mapping of cholesterol-interacting proteins in mammalian cells.Nat. Methods. 2013; 10: 259-264Crossref PubMed Scopus (277) Google Scholar, 27.Niphakis M.J. Lum K.M. Cognetta A.B. Correia B.E. Ichu T-A. Olucha J. Brown S.J. Kundu S. Piscitelli F. Rosen H. 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Acta. 2014; 1841: 1022-1030Crossref PubMed Scopus (48) Google Scholar). A recent study combined the advantages of bifunctional and coumarin caged lipids to facilitate identification of protein binding partners of the signaling lipids, sphingosine and diacylglycerol (DAG) (30.Höglinger D. Nadler A. Haberkant P. Kirkpatrick J. Schifferer M. Stein F. Hauke S. Porter F.D. Schultz C. Trifunctional lipid probes for comprehensive studies of single lipid species in living cells.Proc. Natl. Acad. Sci. USA. 2017; 114: 1566-1571Crossref PubMed Scopus (71) Google Scholar). In the present study, we report the synthesis and application of a bifunctional ceramide analog, pacCer, to search for novel ceramide binding proteins. Besides proteins involved in DNA damage response pathways, protein ubiquitination, membrane trafficking, and signal transduction, our approach yielded CERT and the CERT-related phosphatidylcholine (PC) transfer protein, steroidogenic acute regulatory protein D7 (StarD7). Using molecular modeling in combination with photoaffinity labeling and lipid transfer assays, we demonstrate that StarD7 harbors a lipid-binding pocket with dual specificity for ceramide and PC, and pinpoint structural determinants of lipid recognition. As StarD7 is required for normal respiratory activity and cristae structure of mitochondria (31.Horibata Y. Ando H. Zhang P. Vergnes L. Aoyama C. Itoh M. Reue K. Sugimoto H. StarD7 protein deficiency adversely affects the phosphatidylcholine composition, respiratory activity, and cristae structure of mitochondria.J. Biol. Chem. 2016; 291: 24880-24891Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 32.Yang L. Na C-L. Luo S. Wu D. Hogan S. Huang T. Weaver T.E. The phosphatidylcholine transfer protein Stard7 is required for mitochondrial and epithelial cell homeostasis.Sci. Rep. 2017; 7: 46416Crossref PubMed Scopus (33) Google Scholar), its ability to bind ceramides may be relevant to the mechanism by which ceramides mediate their cytotoxic effects. The 1-palmitoyl-2-oleoyl-sn-glycerol (DAG, 16:0/18:1; catalog number 800815), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), POPC, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (lactosyl-PE), C18-ceramide (d18:1/18:0; catalog number 860518), and C16:0-ceramide (d18:1/16:0; catalog number 860516) were obtained from Avanti Polar Lipids. D-erythro-sphingosine was obtained from Enzo Biochem. Alexa Fluor647-N3 and biotin-N3 were from Thermo Fischer Scientific. Other fine chemicals were from Sigma-Aldrich. The antibodies used were: rabbit polyclonal anti-StarD7 (1:1,000; catalog number 15689-1-AP, Proteintech), mouse monoclonal anti-PARP-1 (1:1,000; catalog number sc8007, Santa Cruz), mouse monoclonal anti-mitochondrial surface protein p60 (1:1,000; catalog number MAB1273, Millipore), affinity-purified rabbit polyclonal anti-SM synthase-related protein (SMSr) antibody [1:1,000; (33.Bickert A. Ginkel C. Kol M. vom Dorp K. Jastrow H. Degen J. Jacobs R.L. Vance D.E. Winterhager E. Jiang X-C. Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice.J. Lipid Res. 2015; 56: 821-835Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar)], mouse monoclonal anti-biotin antibody conjugated to horseradish peroxidase (1:1,000; catalog number 200-032-211, Jackson ImmunoResearch), goat anti-mouse and goat anti-rabbit IgG conjugated to horseradish peroxidase (1:1,000; catalog numbers 31430 and 31460, respectively, Thermo Fischer Scientific). A 15 carbon-long FA containing a photo-activatable diazerine and clickable alkyne group, pacFA, was synthesized in three steps from commercially available educts, as described in (25.Haberkant P. Raijmakers R. Wildwater M. Sachsenheimer T. Brügger B. Maeda K. Houweling M. Gavin A-C. Schultz C. van Meer G. In vivo profiling and visualization of cellular protein-lipid interactions using bifunctional fatty acids.Angew. Chem. Int. Ed. Engl. 2013; 52: 4033-4038Crossref PubMed Scopus (89) Google Scholar) (see the supplemental information for further details). Next, pacFA was coupled to D-erythro-sphingosine using a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and hydroxybenzotriazole as condensing reagents, yielding the photo-activatable and clickable C15-ceramide analog, pacCer (85% overall yield). pacPC was synthesized starting from 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (Avanti Polar Lipids) and pacFA under the action of N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine (DMAP) with satisfactory yield (39%). pacDAG was synthesized in three steps starting from 1-oleoyl-sn-glycerol (Santa Cruz Biotechnology). First, the primary HO-group was protected with the triphenylmethyl protecting group (trityl-chloride/pyridine; 92% overall yield). The glycerol obtained was coupled with the pacFA using EDCI/DMAP activation (58% overall yield). The final deprotection step was achieved using trifluoroacetic acid to generate pacDAG (28% overall yield). pacPE was synthesized in three steps starting from 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids). First, the amino-group was protected with the tert-butoxycarbonyl protecting group (di-tert-butyl dicarbonate/triethylamine; 98% overall yield). The ethanolamine obtained was coupled with pacFA using EDCI/DMAP activation in a good yield (52%). The final deprotection step was achieved with trifluoroacetic acid to generate pacPE (35% overall yield). pacSM was synthesized starting from sphingosylphosphorylcholine (lyso-SM d18:1; Avanti Polar Lipids) and pacFA under the action of EDCI/hydroxybenzotriazole (78% overall yield). pacGlcCer was synthesized from 1-β-D-glucosylsphingosine (Matreya) and pacFA in the presence of triphenylphosphine and dithiopyridine, essentially as described by (34.Kishimoto Y. Costello C. Rearrangement of 3-ketoceramide.Chem. Phys. Lipids. 1975; 15: 27-32Crossref PubMed Scopus (5) Google Scholar) (62% overall yield). The synthesis of C1-deoxy-pacCer, C3-deoxy-pacCer, and C3-deoxy-N-methyl-pacCer is described in the supplemental information. All synthetic compounds were purified by thin-layer chromatography to a high degree (purity >98%) and their structures were confirmed by 1H and 13C NMR and ESI MS. Bacterial maltose binding protein (MBP) expression construct pMAL-c5X was obtained from New England Biolabs. A DNA insert encoding the steroidogenic acute regulatory transfer (START) domain of human CERT was amplified from cDNA (kindly provided by K. Hanada) and cloned into the BamHI and NotI restriction sites of bacterial expression vector pET24a(+). DNA inserts encoding full-length human StarD7 (StarD7 isoform-I) and StarD7 lacking the N-terminal mitochondrial targeting sequence [StarD7 isoform-II; (35.Horibata Y. Sugimoto H. StarD7 mediates the intracellular trafficking of phosphatidylcholine to mitochondria.J. Biol. Chem. 2010; 285: 7358-7365Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar)] were PCR amplified from IMAGE clone 3842611 and cloned into the SalI and XhoI restriction sites of pET24a(+). DNA inserts encoding full-length human StarD2 and StarD10 were PCR amplified from IMAGE clones 4575824 and 4301295, respectively, and cloned into the EcoRI and XhoI restriction sites of pET24a(+). Single amino acid substitutions were introduced by site-directed mutagenesis according to the QuickChangeII™ manual (Agilent Technologies) with modifications. All expression constructs were verified by DNA sequencing. Escherichia coli BL21(DH3) pLysS cells transformed with the expression construct were grown in LB medium supplemented with 0.1 mM isopropyl-D-thiogalactoside for 2 h at 30°C. MBP was purified from cell lysates in-batch using amylose resin (New England Biolabs) according to the manufacturer's instructions. Poly-His-tagged proteins were purified by Ni2+-NTA affinity (Qiagen) using an in-batch protocol, eluted in 50 mM Tris/HCl (pH 7,4), 300 mM NaCl, 300 mM imidazole, 2.5 mM β-mercaptoethanol, and protease inhibitor cocktail (150 nM aprotinin, 1 μM leupeptin, 1.5 μM pepstatin, 7.5 μM antipain, and 1 mM benzamidine), supplemented with 10% glycerol (volume), aliquoted, and stored at −80°C until further use. Protein concentrations were determined by SDS-PAGE and Coomassie staining using BSA as reference protein. StarD7 isoform-I was expressed and purified as described in (25.Haberkant P. Raijmakers R. Wildwater M. Sachsenheimer T. Brügger B. Maeda K. Houweling M. Gavin A-C. Schultz C. van Meer G. In vivo profiling and visualization of cellular protein-lipid interactions using bifunctional fatty acids.Angew. Chem. Int. Ed. Engl. 2013; 52: 4033-4038Crossref PubMed Scopus (89) Google Scholar), and used in the experiments shown in Fig. 3. All other experiments were performed with StarD7 isoform-II. Human cervical carcinoma HeLa (ATCC-CCL2), Chinese hamster ovary (CHO)-K1 (ATCC-CCL-61), and mouse melanoma GM95 cells (kindly provided by Hein Sprong, University of Utrecht, The Netherlands) were grown in Dulbecco's Modified Eagle's medium supplemented with 4.5 g/l glucose, 10% FCS, and GlutaMAX™ (Invitrogen) at 37°C with 5% CO2. To knock out StarD7 in HeLa cells, we obtained a mix of three different CRISPR/Cas9 plasmids and the corresponding HDR plasmids from Santa Cruz (sc-405820). The StarD7-specific gRNA sequences were: A/sense, 5′-ATCCAACTAACACAGTAGCG-3′ B/sense, 5′-GCTCACCTCGGTACTGGTAA-3′ and C/sense, 5′-ACCCACCTTTACCAGTACCG-3′. HeLa cells were cotransfected with both plasmid mixes using Effectene (Qiagen) and grown for 48 h without selection. Next, the cells were grown for 2 weeks under selection pressure with 2 μg/ml puromycin. Individual drug-resistant clones were picked and analyzed for StarD7 expression by immunoblot analysis using anti-StarD7 antibody. Two independent StarD7−/− cell lines, StarD7-KO#1 and StarD7-KO#2, were used for subsequent RNAi experiments. To this end, cells were transfected with siRNA (Qiagen) using Oligofectamine reagent (Invitrogen) as described previously (36.Tafesse F.G. Vacaru A.M. Bosma E.F. Hermansson M. Jain A. Hilderink A. Somerharju P. Holthuis J.C.M. Sphingomyelin synthase-related protein SMSr is a suppressor of ceramide-induced mitochondrial apoptosis.J. Cell Sci. 2014; 127: 445-454Crossref PubMed Scopus (56) Google Scholar). The siRNA target sequences used were: nonsilencing RNA (siNS) (nonsense), 5′-AAUUCUCCGAACGUGUCACGU-3′, and siSMSr, 5′-CAAGAAGCUGGAAUUUCUUGC-3′. Both adherent and nonadherent cells were harvested 72 h posttransfection, washed twice in ice-cold 0.25 M sucrose, and homogenized in ice-cold IM buffer [5 mM HEPES-KOH (pH 7.0), 250 mM mannitol, and 0.5 mM EGTA] supplemented with 0.1 mM phenylmethanesulfonyl fluoride and protease inhibitor cocktail (150 nM aprotinin, 1 μM leupeptin, 1.5 μM pepstatin, 7.5 μM antipain, and 1 mM benzamidine). To this end, cells were flushed through a Balch homogenizer 20–30 times using a 2 ml syringe. Cell homogenates were centrifuged twice at 600 g maximum for 5 min at 4°C to remove nuclei. The protein concentration of postnuclear supernatants was determined by Bradford assay (Bio-Rad). Postnuclear supernatants were normalized for total protein content prior to immunoblot analysis. Liposomes used in the photoaffinity experiments with cytosolic fractions were prepared in PBS (1.4 M NaCl, 27 mM KCl, 18 mM KH2PO4, and 126 mM Na2HPO4) from a mixture of egg-PC and pacLipid (95/5 mol%). Liposomes used in the photoaffinity experiments with purified recombinant proteins were prepared from a defined lipid mixture (DOPC/DOPE/pacLipid, 80/20/1 mol%) in CHCl3/methanol (9/1, v/v). For competition assays, 0.5 or 0.25 mol% pacCer was used and C16:0 ceramide was added in 10- to 40-fold molar excess at the expense of DOPC and DOPE, keeping the DOPC/DOPE ratio constant. In brief, 10 μmol of total lipid were dried in a Rotavap and the resulting lipid film was resuspended in 1 ml buffer L [50 mM Tris-HCl (pH 7.4) and 50 mM NaCl] by vigorous vortexing and sonication, yielding a 10 mM lipid suspension. Liposomes with an average diameter of ∼100 nm were obtained by sequential extrusion of the lipid suspension through 0.4, 0.2, and 0.1 micron track-etched polycarbonate membranes (Whatman-Nuclepore) using a mini-extruder (Avanti Polar Lipids). Acceptor liposomes used in lipid transfer assays were prepared in buffer L using a mixture of DOPG and DOPE (80/20 mol%). Donor liposomes were prepared using a mixture of DOPG, DOPE, lactosyl-PE, and either C16:0-ceramide or DOPC (65/16/10/10 mol%). Donor liposomes used in competition assays were prepared using a mixture of DOPG, DOPE, lactosyl-PE, DOPC, and C16:0-ceramide or DAG (69/17/10/5/10 mol%). All liposomes were stored under N2 at 4°C and used within 2–3 days after preparation. Five 15 cm dishes each of GM95 and HeLa cells and two 15 cm dishes of CHO cells were resuspended in 2 ml of ice-cold lysis buffer [50 mM Tris (pH 6.8), 1 mM EDTA, 0.3 M sucrose, 1 mM PMSF, and 1× protease inhibitor cocktail). Cells were homogenized by passing them through a 26Gx1″ (0.45 × 25 mm) needle using a 1 ml syringe. The suspension was centrifuged at 100,000 g for 1 h at 4°C to remove cell debris, nuclei, and membranes. The protein concentration in the obtained cytosol was determined by Bradford assay and adjusted to 1.2 mg/ml with PBS. Fifty-five microliters of cytosol were mixed with 55 μl of liposome suspension containing 5 mol% of pacLipid and incubated for 30 min at room temperature with gentle shaking. Subsequently, the samples were placed on ice and irradiated for 60 s using a 1,000 W mercury lamp equipped with a dichroic mirror and a 345 nm bandpass filter (Newport) at 30 cm distance. Protein was recovered by chloroform-methanol precipitation and the air-dried protein pellet was dissolved in 20 μl of 1% SDS in PBS with vigorous shaking for 10 min at 70°C. Click reactions were performed by adding 8 μl of a freshly prepared "click" mix {40 μl of 25 mM Tris(2-carboxyethyl)phosphine hydrochloride, 40 μl of 2.5 mM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine, 40 μl of 25 mM CuSO4, and 40 μl of 25 mM biotin-N3} per sample followed by incubation at room temperature for 2 h while shaking. After addition of 0.25 vol of 5× sample buffer [0.3 M Tris/HCl (pH 6.8), 10% SDS, 50% glycerol, 0.025% bromphenol blue, and 10% β-mercaptoethanol], samples were boiled for 5 min at 95°C and subjected to SDS-PAGE and immunoblotting or Coomassie staining. For the identification of ceramide binding proteins, a cytosolic fraction was prepared from twenty 15 cm dishes of GM95 cells and diluted to a protein concentration of 1.4 mg/ml in PBS. Four hundred and fifty microliters of cytosol were mixed with 450 μl of a liposome suspension containing 5 mol% of pacCer or pacGlcCer and incubated for 30 min at room temperature with gentle shaking. Samples were split into four portions of 200 μl, UV irradiated, and subjected to chloroform-methanol precipitation as above. Protein pellets were combined, resuspended in 400 μl 1% SDS in PBS, and solubilized for 10 min at 70°C. Click reactions were performed by adding 80 μl of freshly prepared click mix containing biotin-N3, as above. Samples were split in two portions of 240 μl and subjected to chloroform-methanol precipitation twice. Protein pellets were resuspended in 200 μl of 1% SDS in PBS, solubilized by vigorous shaking for 10 min at 70°C, and then diluted 5-fold in PBS. After centrifugation at 20,000 g for 1 min at room temperature, supernatants were collected and combined. Supernatant (1,600 μl) was mixed with 50 μl of a 50% slurry of NeutrAvidin beads (NeutrAvidin™ agarose resin; Thermo Scientific) equilibrated in 0.2% SDS in PBS and incubated at room temperature with rotation. The beads were collected by centrifugation (100 g, 1 min, room temperature) and washed three times with 1 ml 0.1% SDS in PBS and three times with 1 ml of PBS. To elute bound proteins, the beads were incubated in SDS-PAGE sample buffer for 5 min at 95°C. The eluates were analyzed by SDS-PAGE and Coomassie staining. Lanes of interest were cut into 10 equal sections, cut from the gel, and protein in each section was subjected to trypsin digestion and peptides were analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer (Thermo Scientific) connected to an Agilent 1200 series nano LC system, as described in (25.Haberkant P. Raijmakers R. Wildwater M. Sachsenheimer T. Brügger B. Maeda K. Houweling M. Gavin A-C. Schultz C. van Meer G. In vivo profiling and visualization of cellular protein-lipid interactions using bifunctional fatty acids.Angew. Chem. Int. Ed. Engl. 2013; 52: 4033-4038Crossref PubMed Scopus (89) Google Scholar). The following cr
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