Dual tagging as an approach to isolate endogenous chromatin remodeling complexes from Saccharomyces cerevisiae

Biochimica et Biophysica Acta 1854 (2015) 198–208

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Dual tagging as an approach to isolate endogenous chromatin remodeling complexes from Saccharomyces cerevisiae Tzong-Yuan Lin a, Andriy Voronovsky a, Monika Raabe d, Henning Urlaub d,e, Bjoern Sander b,c, Monika M. Golas a,c,⁎ a

Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, DK-8000 Aarhus C, Denmark Stereology and EM Laboratory, Department of Clinical Medicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, DK-8000 Aarhus C, Denmark Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, DK-8000 Aarhus C, Denmark d Bioanalytical Mass Spectrometry Group, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany e Bioanalytics, Institute for Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany b c

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Article history: Received 3 April 2014 Received in revised form 11 November 2014 Accepted 25 November 2014 Available online 5 December 2014 Keywords: Chromatin remodeling SWI/SNF family RSC complex Dual tagging Purification Saccharomyces cerevisiae

a b s t r a c t Affinity isolation has been an essential technique for molecular studies of cellular assemblies, such as the switch/ sucrose non-fermentable (SWI/SNF) family of ATP-dependent chromatin remodeling complexes. However, even biochemically pure isolates can contain heterogeneous mixtures of complexes and their components. In particular, purification strategies that rely on affinity tags fused to only one component of a complex may be susceptible to this phenomenon. This study demonstrates that fusing purification tags to two different proteins enables the isolation of intact complexes of remodels the structure of chromatin (RSC). A Protein A tag was fused to one of the RSC proteins and a Twin-Strep tag to another protein of the complex. By mass spectrometry, we demonstrate the enrichment of the RSC complexes. The complexes had an apparent Svedberg value of about 20S, as shown by glycerol gradient ultracentrifugation. Additionally, purified complexes were demonstrated to be functional. Electron microscopy and single-particle analyses revealed a conformational rearrangement of RSC upon interaction with acetylated histone H3 peptides. This purification method is useful to purify functionally active, structurally well-defined macromolecular assemblies. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The higher-order organization of chromatin is highly dynamic in eukaryotes. Chromatin remodeling is driven by macromolecular complexes that remodel the chromatin architecture according to the current needs of the cell [1]. In the yeast Saccharomyces cerevisiae, the switch/ sucrose non-fermentable (SWI/SNF) family of ATP-dependent chromatin remodelers [2] comprise amongst others the remodels the structure of chromatin (RSC) complex [3,4] and are conserved in humans. RSC is recruited to RNA polymerase III promoters, as well as to transcriptional activators and repressors at RNA polymerase II promoters [5]. RSC is also

Abbreviations: SWI/SNF, switch/sucrose non-fermentable; RSC, remodels the structure of chromatin; S. cerevisiae, Saccharomyces cerevisiae; PCR, polymerase chain reaction; TAP, tandem affinity purification; CBP, calmodulin-binding peptide; TEV, tobacco etch virus; IgG, immunoglobulin G; EGTA, ethylene glycol tetraacetic acid; YPD, yeast extract– peptone–dextrose; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; DTT, dithiolthreitol; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; ATP, adenosine triphosphate; TBE, Tris–borate–EDTA; PBS, phosphate buffered saline; PBST, PBS containing 0.1% Tween-20; EM, electron microscopy; CCD, charge-coupled device ⁎ Corresponding author at: Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233/1234, DK-8000 Aarhus C, Denmark. Tel.: +45 871 68239. E-mail address: [email protected] (M.M. Golas).

http://dx.doi.org/10.1016/j.bbapap.2014.11.009 1570-9639/© 2014 Elsevier B.V. All rights reserved.

involved in DNA repair [6]. Two different isoforms of RSC that are composed of up to 17 proteins exist in S. cerevisiae [7,8]: the Rsc1 complex is comprised of Rsc1p, Rsc3p, Rsc4p, Rsc6p, Rsc8p, Rsc9p, Rsc30p, Rsc58p, Sfh1p, Sth1p, Rtt102p, Npl6p, Ldb7p, Htl1p, Arp7p, Arp9p as well as actin. Rsc3p and Rsc30p are underrepresented in the Rsc2 complex, and Rsc1p is replaced by Rsc2p [9]. Together, these proteins amount to a molecular mass of approximately 0.85–1.07 MDa. In logarithmically growing yeast, the vast majority of RSC complexes represent Rsc2 complexes [10]. Structural studies of the yeast RSC complex have revealed a hole-shaped cavity that is thought to serve as a binding site for nucleosomes [11–13], but further investigations of the relationship between structure and function are necessary. In S. cerevisiae, stable fusion of an affinity tag to a protein can be accomplished by polymerase chain reaction (PCR)-based homologous recombination [14]. The tandem affinity purification (TAP) tag is the most studied and widely used tag in yeast [15–19]. The TAP tag contains a calmodulin-binding peptide (CBP) and a Protein A moiety separated by a tobacco etch virus (TEV) cleavage site [15]. An advantage of this tag is that Protein A binding to immunoglobulin G (IgG) is irreversible under physiologic conditions [20]. This strong affinity allows very low copy-number proteins to be captured from large volumes of cell extracts by using batch affinity selection and subsequent elution by

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TEV cleavage. Enrichment of the protein complex of interest during the first step facilitates an effective purification in the second step. Moreover, protein contaminants that could interact with calmodulin in a calcium-dependent manner are removed during the first step, before calmodulin affinity selection and EGTA elution. In Escherichia coli and mammalian cells, Strep-based tags (i.e., the Strep-tag II and Twin-Strep tag) represent one of several alternatives for affinity isolation of proteins [21]. Proteins are bound to StrepTactin, an engineered version of streptavidin, and then eluted with desthiobiotin [22]. Both Strep-tags are small, which preserves protein function and structure, and does not reduce translation efficiency. The Strep-tag II sequence does not naturally occur in most organisms. However, biotin binds to Strep-Tactin with considerable affinity and must be blocked or removed prior to purification. Initial Strep-tag II studies in yeast reported promising results with respect to yield and purity for an overexpressed protein [23]. In contrast, isolating endogenous macromolecular complexes is challenging because they typically exist in cells at lower numbers than overexpressed proteins. Moreover, the purification of macromolecular complexes often results in a disproportionately high amount of the bait protein and partial complexes compared to the complete complex. Therefore, we hypothesized that fusing a purification tag to two different sites of a macromolecular complex would improve the isolation of the complete RSC complex and minimize the amounts of partial complexes and individual proteins. This study shows that dual affinity tagging based on a Protein A tag and a Twin-Strep tag that target different proteins of the RSC complex, enabled efficient purification of complete and functional RSC complexes from yeast extracts. 2. Materials and methods 2.1. Construction of a triple-tag and 3xFLAG-tag plasmid cassette For all PCR amplification steps, the Phusion HF master mix (Thermo, St. Leon-Rot, Germany) was used. All restriction enzymes were FastDigest enzymes (Thermo). Primer sequences are listed in the Supplementary Table S1 of Supplementary Data, and all primers were obtained from MWG Operon (Ebersberg, Germany) or Sigma (Haverhill, U.K.). All primers were HPLC-purified, except for the small sequencing primers. The Kluyveromyces lactis (K. lactis) LEU2 gene, including its terminator, was amplified from genomic DNA by using the K. lactis strain CBS2359 (CBS, Utrecht, The Netherlands) and the primers KlLEU2-for and KlLEU2-rev. The S. cerevisiae ADH1 terminator and the Ashbya gossypii TEF promoter were amplified from the plasmid pGS1613 [24] using the primers ScADH1term_for and AgTEFprom_rev. These two PCR fragments were combined by PCR using the primers ScADH1term_for and KlLEU2_rev. The resulting PCR product, termed “marker fragment”, contained the ScADH1 terminator, the AgTEF promoter, the KlLEU2 gene, and the KlLEU2 terminator. The TEV–Protein A fragment was amplified by using the plasmid LHP1481-TOPOProteinA (#32225, Addgene, Cambridge, MA; provided by Linda Hicke) and the primers ProtA_for and ProtA_rev. The 3xFLAG fragment was amplified by using the plasmid p3xFLAG-S2P (Addgene #32966; provided by Ron Prywes) as a template and the primers FLAG_for and FLAG_rev. The FLAG_for primer also comprises parts of the CBP tag. The 3xFLAG fragment and the TEV–Protein A fragment were combined by PCR using the primers FLAG_protA and ProtA_rev. The FLAG_protA primer also comprises parts of the CBP tag. The resulting PCR product was named “triple-tag fragment”. The triple-tag fragment was treated with HindIII and SalI, the marker fragment was treated with SalI and EcoRV, and the pUC57 plasmid (Thermo) was treated with HindIII and EcoRV. Fragments were inserted into the plasmid with T4 DNA ligase (Roche, Mannheim, Germany), and transformed into Top10 cells (Life Technologies, Carlsbad, CA). The 3xFLAG sequence was amplified from the CBP-3xFLAG-TEV-ProtA-KlLEU2 triple-tag plasmid using the

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primers 3xFLAG_only_for and 3xFLAG_only_rev. The 3xFLAG PCR fragment was digested with HindIII and SalI and inserted into the CBP-3xFLAG-TEV-ProtA-KlLEU2 triple-tag plasmid using HindIII and SalI sites to replace the triple-tag with a 3xFLAG-tag. Clones were confirmed by restriction enzyme digestion of the mini-prepped plasmids and sequencing (MWG Operon).

2.2. Tagging of RSC genes by PCR-based homologous recombination The Twin-Strep cassette with the TRP1 auxotrophic marker and the 5′ and 3′ homologous arms for STH1 were PCR-amplified using the primers Sth1_Twin_for and Sth1_Twin_rev and the plasmid pGS1021 as template [24]. Cassettes were purified and transformed into protease-deficient DSY-5 S. cerevisiae (MATalpha leu2 trp1 ura3-52 his3::PGAL1-GAL4 pep4 prb1-1122; Dualsystems Biotech AG, Schlieren, Switzerland) using the lithium acetate method. Yeast cells were plated on selective medium lacking tryptophan and grown at 30 °C. Clones were screened by PCR using the primers Sth1_check_for and TRP1_check_rev, and the correct sequence was confirmed by sequencing of the PCR product. To create the dual tagged strain, the triple-tag cassette with the LEU2 marker was obtained by PCR using the primers Rsc4_triple_for and Rsc4_triple_rev, and then transformed into the Twin-Strep-tagged strain as described above. The cells were plated on selective medium lacking leucine and tryptophan. Clones were screened for integration using the primers Rsc4_check_for and LEU2_check_rev, and by sequencing of the PCR product. The 3xFLAG-tag cassette including KlLEU2 marker was amplified from the 3xFLAG plasmid using the primers Rsc4_FLAG_for and Rsc4_triple_rev. This 3xFLAG cassette was transformed into both the untagged DSY-5 and the Sth1-Twin-Strep-tagged strain as described above and plated on selective medium lacking leucine and leucine/tryptophan, respectively.

2.3. Analysis of growth phenotype Yeast logarithmically growing in yeast extract–peptone–dextrose (YPD) medium were washed with imaging buffer (20 mM 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid [HEPES] pH 7.5, 1% glucose) and stained with 15 μg/mL Hoechst 33342 (Sigma) for 5 min. For fluorescence imaging, yeast were pipetted onto a drop of 1% agarose (Life Technologies) supplemented with 1% glucose and 20 mM HEPES pH 7.5 on a freshly cleaned glass slide. Specimens were imaged using a Nikon Eclipse Ti microscope equipped with a Nikon Intensilight C-HGFI unit. For the plate growth assays, yeast were grown in YPD and diluted to an OD of 3.3. Ten-fold serial dilutions of DSY-5, Sth1-TwinStrep-tagged DSY-5 and Rsc4-CBP-3xFLAG-ProteinA/Sth1-Twin-Streptagged DSY-5 were prepared in 20 mM HEPES pH 7.5, and 5 μL of each cell suspension was spotted onto different agar plates. The following media were assayed: YPD; synthetic defined medium − Trp; synthetic defined medium − Leu; synthetic defined medium + 2% sucrose (no glucose); synthetic defined medium + 2% galactose (no glucose); YPD + 6% ethanol; YPD + 5 mM CuSO4; YPD + 1.2 M NaCl; YPD + 15 mM caffeine; YPD + 2% formamide; and YPD + 150 mM hydroxyurea (dropout base with agar and complete supplement mixtures were obtained from MP Biomedicals, all other chemicals from Sigma). Agar plates were incubated at 18 °C, 30 °C, and 37 °C for 4 days and thereafter imaged. A growth curve experiment of DSY-5, Sth1-TwinStrep-tagged DSY-5 and Rsc4-CBP-3xFLAG-ProteinA/Sth1-Twin-Streptagged DSY-5 was performed by inoculating 80 mL of fresh YPD medium (BD Biosciences, Albertslund, Denmark; Sigma) with overnight yeast cultures yielding a start optical density at 600 nm [OD600] of 0.0015. Yeast were cultured at 30 °C in shaking flasks, and at the indicated time points the OD600 was measured. The analysis was performed in triplicate.

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2.4. Purification of RSC For the purification of the RSC complex composed of triple-tagged Rsc4 and Twin-Strep-tagged Sth1, transformed yeast cells were grown on a selective medium (no leucine or tryptophan). A 100-mL YPD preculture was inoculated with a single colony of cells and grown at 30 °C overnight. Fifteen to twenty liters of YPD media (BD and Sigma) were inoculated with the YPD preculture and grown at 30 °C overnight. Cells were harvested at an OD600 of 6–7 by centrifugation at 5000 rpm for 15 min in a F8-6 × 1000y rotor (Thermo) and washed with 10 mM Tris (pH 7.6). The cell pellet was resuspended in 50 mL of lysis buffer (per 15 liter of cell culture volume) containing 20 mM HEPES/KOH (pH 7.6), 700 mM potassium acetate, 0.5 mM MgCl2, 8% (v/v) glycerol, 0.25% Triton X-100, 0.5 mM dithiolthreitol (DTT), and 1 tablet of Complete Protease Inhibitor Cocktail (Roche, Mannheim, Germany). The yeast suspension was dropped into liquid nitrogen to form small beads, which were then milled in a ZM 200 ultracentifugation mill (Retsch, Haan, Germany) under cryogenic conditions. The thawed cell lysate was centrifuged in a F21-8 × 50y rotor (Thermo) at 15,000 rpm for 20 min. The supernatant was incubated with 500 μL of IgGSepharose beads (GE Healthcare, Freiburg, Germany) at 4 °C for 2 h and transferred to a gravity flow column. The IgG affinity resin was washed three times with 10 mL of IgG wash buffer (10 mM Tris [pH 8.0], 0.15 M NaCl, 0.1% of the detergent NP-40) followed by a wash with 10 mL of TEV cleavage buffer (10 mM Tris [pH 8.0], 0.15 M NaCl, 0.1% NP-40, 0.5 mM EDTA, 1 mM DTT). Subsequently, the resin was treated with TEV protease (500 U in 5 mL) at 4 °C overnight to cleave tagged Rsc4p from the Sepharose. The TEV eluate was loaded by gravity flow on a 1 mL Strep-Tactin Superflow column (IBA, Goettingen, Germany). The column was washed with three volumes of StrepTactin wash buffer (IBA) and eluted six times with 0.5 mL of StrepTactin elution buffer (IBA). The cultivation of the 3xFLAG-tagged Rsc4 yeast cells and preparation of the cell lysate were conducted as described above. The cleared cell lysate was incubated with 200 μL of anti-FLAG M2 Affinity Gel (Sigma) at 4 °C for 2 h and thereafter centrifuged at 500 ×g for 5 min. The resin was washed three times with FLAG wash buffer (1× TBS supplemented with 20% glycerol) followed by centrifugation after each wash step. The washed resin was then incubated with 2.5 column volumes of FLAG elution buffer (1× TBS, 5% glycerol, 100 μg/mL 3xFLAG peptide (Sigma)) for 30 min. The FLAG eluate was then collected by transferring the sample into a mini spin column and a subsequent centrifugation at 500 ×g for 1 min. 2.5. SDS–PAGE analysis The TEV eluate and six Strep-Tactin eluate fractions were loaded on NuPAGE Novex 4–12% Bis-Tris sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels (Life Technologies), separated by gel electrophoresis and stained with SimplyBlue SafeStain (Life Technologies). For the 3xFLAG-tagged Rsc4 complex, we used self-casted 10% SDS–PAGEs. For relative quantification of gel bands, we scanned the SDS–PAGEs with an ImageQuant 4010 instrument (GE Healthcare), and relative quantification was performed using the ImageJ software (http://imagej.nih.gov/ij/links.html). 2.6. Mass spectrometry based analysis Samples were separated by 1D SDS–PAGE (NuPAGE Novex 4–12% Bis-Tris; Life Technologies) and stained with SimplyBlue SafeStain. Each lane was cut into 23 slices, and proteins within the slices were digested with trypsin. Peptides were extracted and analyzed by LC–MSMS on an Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) under standard conditions. Data were searched against the NCBInr database (release date 09/13/2011 with 26726 yeast entries), using Mascot 2.3.02 as search engine, and were further annotated

with Scaffold 4.3.4 software (Proteome Software, Portland, OR). The following parameters were used: taxonomy yeast, oxidation and carbamidomethylation as variable modifications, max. 2 missed cleavages allowed, digestion enzyme trypsin, fragment ion mass tolerance of 0.6 Da, and parent ion tolerance of 10 ppm. 2.7. DNA-dependent ATPase assay The ATPase activity of purified RSC complexes was measured by monitoring the fluorescence changes after inorganic phosphate hydrolyzed from ATP became bound to a phosphate sensor protein (PV4406; Life Technologies). Column eluates in a buffer of 0.1 M Tris– HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, and 2.5 mM desthiobiotin were mixed with 10 μM ATP, 2.5 mM magnesium chloride and random plasmid DNA (20 ng/μL). They were incubated for 1 h at room temperature in a 384 well plate (3677; Corning, Amsterdam, The Netherlands) according to the manufacturer's instructions. ATP and DNA were not added to control samples. After 1 h of incubation, the phosphate sensor (0.5 μM) was added. Fluorescence was measured with a Flexstation 3 microplate reader (MolecularDevices, Sunnyvale CA) using an excitation wavelength of 430 nm and an emission wavelength of 450 nm. Measurements were performed in triplicate. P-values were determined by using two-sided Student's t-tests with R software (version 3.0.2) [25] . P-values b 0.05 were considered significant. Furthermore, we measured the time-dependent formation of phosphate using the above described fluorescent ATPase activity assay except for some modifications. The sodium chloride in the StrepTactin elution buffer was replaced by 50 mM potassium acetate. A measure of 5 μL of sample (18 nM) was mixed with buffer and phosphate sensor (total volume: 20 μL). The assay was performed in high KAc buffer (final concentration: 50 mM HEPES pH 7.8, 400 mM potassium acetate, 5 mM magnesium acetate) and low NaCl buffer (final concentration: 50 mM HEPES pH 7.8, 50 mM NaCl, 5 mM magnesium acetate), as increased activity of the RSC complex was observed in the presence of low chloride ion concentration buffers [26]. Random plasmid DNA was added to a concentration of 20 ng/μL. After equilibration at room temperature for 30 min, ATP was manually added to the + ATP samples to a final concentration of 0.5 mM. Fluorescence was measured every 2.5 min in a Flexstation 3 microplate reader. Measurements were performed in triplicate. 2.8. Electrophoretic mobility shift assay and nucleosome mobilization assay A DNA fragment that is comprised of a 147 bp 601 Widom DNA sequence [27] flanked by 36 bp and 93 bp of extranucleosomal DNA was generated by gene synthesis and subcloned into the pEX-K cloning vector (MWG Operon). The nucleosome cassette was amplified from the generated plasmid using the Cy5-labeled primers Nucleosome_for and Nucleosome_rev. The resulting 276 bp PCR fragment was gelpurified, and the concentration was determined by using a Nanodrop (Thermo). A measure of 2–5 μg of human histone octamers purified from HeLa cells was mixed with the Cy5-labeled nucleosome PCR fragment in high-salt buffer (10 mM Tris–HCl pH 7.5, 2 M NaCl, 1 mM EDTA, 1 mM DTT) and incubated for 30 min at room temperature. Subsequently, the sample was dialyzed in a Slide-A-Lyzer Mini dialysis unit 3.5 K MWCO (Pierce, Thermo) at room temperature in buffers with stepwise decreasing salt concentration (1 h in a 1.5 M NaCl buffer, 2 h in a 1 M NaCl buffer, 2 h in a 0.6 M NaCl buffer). The buffer for the final dialysis step was 20 mM HEPES-K pH 7.8, 50 mM potassium acetate, 4 mM magnesium acetate, and 1 mM DTT (2 h at room temperature and finally at 4 °C overnight). Precipitates were pelleted by centrifugation, and the protein concentration was determined by using a Nanodrop. Nucleosome-RSC complexes were assembled in EMSA buffer (20 mM HEPES-K pH 7.8, 50 mM potassium acetate, 5 mM magnesium acetate, 100 μg/mL BSA, 2.5% Ficoll). For EMSA, we mixed 3 nM of mononucleosomes and 3 nM, 6 nM and 12 nM of purified RSC, respectively.

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Samples were incubated at 30 °C for 30 min before loading on a prerun 5% PAGE (in 0.5× TBE buffer). For the nucleosome mobilization assay, 8 nM of mono-nucleosomes and 24 nM of purified RSC were mixed and incubated at 30 °C in the absence or presence of 0.5 mM ATP. Ten nanograms per microliter of unspecific linear dsDNA of approximately 1.5 kb was added to samples of the nucleosome mobilization assay. Samples were loaded on a prerun 5% PAGE (in 0.5× TBE buffer). The gels were run at 4 °C, and the running buffer was continuously mixed during the electrophoresis using a peristaltic pump. Upon completion of the gel electrophoresis, the gels were imaged in an ImageQuant 4010 in fluorescence mode. 2.9. Glycerol gradient ultracentrifugation Strep-Tactin and anti-FLAG elution fractions containing the purified RSC complex were loaded onto glycerol gradients of 10–30% in gradient buffer (20 mM HEPES [pH 8.0], 0.15 M NaCl, 1 mM Mg(OAc)2, 1 mM imidazole). GraFix samples for electron microscopy (EM) were ultracentrifuged on a GraFix [28] gradient in gradient buffer that also contained 0–0.1% glutaraldehyde. GraFix conditions result in a mild chemical fixation [29]. No glutaraldehyde was added to glycerol gradients subjected to biochemical analysis as well as EM of noncrosslinked samples. Gradients for biochemical analysis as well as for EM were ultracentrifuged in a TH-660 rotor (Sorvall, Thermo) at 28,500 rpm at 4 °C for 17 h. Gradients were fractionated from the bottom by using a peristaltic pump (GE Healthcare). For biochemical analyses, the gradient fractions that were not crosslinked were precipitated with trichloroacetic acid and acetone. They were then loaded on a NuPAGE Novex 4–12% Bis-Tris gel or a self-casted 10% SDS–PAGE for Coomassie staining with SimplyBlue SafeStain. For Western blotting, samples were loaded and on a 10% SDS–PAGE. 2.10. Western blotting Proteins separated on an SDS–PAGE were transferred to a nitrocellulose membrane at 4 °C overnight. The membrane was blocked with 5% nonfat milk in phosphate buffered saline (PBS) at room temperature for 1 h. Subsequently, the membrane was incubated with the antiSth1 antibody yA-13 (Santa Cruz Biotechnology, Heidelberg, Germany; dilution, 1:200) and anti-FLAG antibody M2 (Sigma; dilution, 1:2000), respectively, at room temperature for 2 h. The membrane was washed with three 5-min washes in PBS containing 0.1% Tween-20 (PBST). Then, the membrane was incubated with a secondary antibody (antigoat IgG-peroxidase and anti-mouse IgG-peroxidase; Sigma) in PBST with 5% nonfat milk for 1 h. After two 5-min washes with PBST and one wash with PBS for 5 min, the membrane was treated with SuperSignal West Pico Chemiluminescent Substrate (Thermo). The blots were imaged with an ImageQuant LAS 4010 instrument. 2.11. EM of RSC complexes GraFix gradient fractions were used directly for preparation of EM specimens. In addition, we also analyzed the peak gradient fraction of RSC in a non-crosslinked glycerol gradient. We visualized both apoRSC as well as RSC-histone H3 peptide complexes by EM. To this end, we incubated non-crosslinked peak gradient fractions of RSC with either 0.25 mM histone H3 peptide 1–20 (Anaspec, Fremont, CA) or with 0.25 mM acetylated histone H3-K9(Ac) 1–20 (Anaspec) at room temperature for 5 min before sample preparation. EM specimens of peak gradient fractions were prepared according to the sandwich negative staining approach [30]. Images were taken at room temperature under low-dose conditions on a Tecnai 12 electron microscope (Philips/FEI, Eindhoven, The Netherlands) with LaB6 filament equipped with a 794 Multiscan CCD camera (Gatan, Pleasanton, CA) at a magnification of 118,600 × at the specimen level (GraFix fractions) and at a

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magnification of 74,300 × at the specimen level (non-crosslinked fractions), respectively. Images were taken in tiling mode. 2.12. Single-particle image processing Image processing was performed independently and reference-free [31] for all four data sets: we did not use class averages of one data set to align the individual images of another data set, and no prior structural information was used. In total, 15,512 individual particles were selected manually from the raw images of the GraFix gradient fractions. The particle images were subjected to a reference-free alignment using polar coordinates [32]. Images were averaged into class averages of 20 particle images per class by using multivariate statistical analysis (MSA) and classification. Alignment and classification were iteratively repeated until the result was stable. For the non-crosslinked gradient fractions, we collected 1823 apo-RSC particles, 3225 RSC-H3(1–20) particles and 3839 RSC-H3K9Ac(1–20) particles and subjected the images to iterative rounds of single-particle image processing as described for the GraFix samples. Subsequent to alignment, the images were classified in groups of 25 class members in average. In order to enable easier comparison of the different data sets, we rotated the coordinate systems of the final class averages with respect to each other. 3. Results 3.1. Dual tagging of RSC The object of this study was to purify the endogenous RSC complex from yeast cell extracts by affinity chromatography. In initial studies, we tagged the RSC complex with a Strep-based tag only. Furthermore, we also tagged RSC4 with a 3xFLAG-tag. Purified samples revealed an overrepresentation of the tagged protein in relation to the complete complex (Figure S1 of Supplementary Data). We thus decided to tag two different proteins of the RSC complex (Fig. 1). To this end, a C-terminal Twin-Strep tag was fused to the STH1 gene with PCR-based homologous recombination (Fig. 1A). Next, the RSC4 gene was tagged by a C-terminal triple-tag (Fig. 1A). This triple tag was comprised of a CBP tag, a 3xFLAG tag, a TEV cleavage site, and a Staphylococcus aureus Protein A tag, and was followed by a KlLEU2 selection marker. The triple tag cassette for C-terminal tagging was amplified by PCR, and the endogenous gene of the yeast was tagged by homologous recombination. Transformed haploid yeast were cultured on the appropriate selection medium after each step and screened by PCR. Correct integration into the genome was confirmed by sequencing. Expression of the tagged proteins was confirmed by Western blotting. 3.2. Growth phenotype of RSC-tagged yeast To assess the phenotype of the dual tagged Rsc4-CBP-3xFLAGProteinA/Sth1-Twin-Strep DSY-5 yeast in comparison to the single tagged Sth1-Twin-Strep DSY-5 and untagged DSY-5 strains, we imaged the three yeast strains, and also stained the nuclei with Hoechst 33342. In particular, we did not observe any morphological differences between the untagged and the tagged strains (Fig. 2A). Nuclear staining revealed a normal nuclear morphology, and in particular no fragmentation of nuclei has been observed (Fig. 2B). We furthermore monitored the growth of the three strains at 30 °C. To measure the growth in liquid YPD medium, we determined a growth curve for the three yeast strains. Both, the single tagged and the dual tagged strains apparently grew faster than the untagged DSY-5 yeast strain (Fig. 2C), which was in line with the growth behavior observed on agar plates (compare Fig. 2D). The apparently slower growth of the untagged strain was caused by a longer lag phase rather than a slower doubling time. In particular, we estimated the doubling time during the logarithmic growth in liquid YPD medium at 30 °C to 83.8 ± 4.3 min, 86.6 ± 2.6 min and 89.4 ± 0.3 min for the untagged, the single

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containing medium as compared to the untagged strain. None of the strains grew on salt stress medium (1.2 M NaCl) or caffeine containing medium (Fig. 2D and data not shown), and we did not observe any growth of the three strains at 37 °C for some of the media. Together, we concluded that the tagging of the RSC subunits had no major adverse effect on the phenotype of the cells as studied by the described assays. 3.3. Purification of RSC Endogenous RSC complexes were purified from yeast under mild conditions, aimed at preserving the function and structure of complexes. All steps were performed at 4 °C, and all buffers were near physiological pH. Yeast cells were lysed under cryogenic conditions, and the cell lysate was then centrifuged. The clarified extract was incubated with IgG-Sepharose (Fig. 1B). After washing the resin, the complexes were released from the affinity resin by TEV cleavage. The volume of the starting material was reduced by a factor of approximately 20, which enabled an efficient purification by Strep-Tactin affinity column chromatography and elution with desthiobiotin (Fig. 1B). The yield of the dual step purification of RSC was up to approximately 100 nM of the RSC complex in a volume of 1000 μL using 15 L cell culture volume as starting material. 3.4. Compositional characterization of RSC

Fig. 1. C-terminal tagging and purification strategy. A. A triple tag, consisting of CBP, 3xFLAG, and the S. aureus Protein A, was fused to RSC4. A Twin-Strep tag containing a tandem copy of the Strep-tag II was fused to STH1. B. The RSC complex is purified from yeast cell extract by IgG affinity chromatography, TEV cleavage, and Strep-Tactin affinity chromatography followed by competitive elution using desthiobiotin. The first affinity chromatography step results in the isolation of a mixture of particles, ranging from the individual Rsc4 protein and its subcomplexes, to the complete RSC complexes. Subsequent to TEV cleavage, Strep-Tactin affinity chromatography then isolates RSC complexes that comprised both the Rsc4 and Sth1 proteins as well as additional proteins of the RSC complex.

tagged and the dual tagged yeast, respectively. This analysis also indicated that the dual tagged Rsc4-CBP-3xFLAG-ProteinA/Sth1-TwinStrep DSY-5 yeast strain grows logarithmically up to an OD600 of about 12, which corresponds to a growth period of approximately 21 h. Moreover, we performed a plate growth assay on different media tested at 18 °C, 30 °C and 37 °C (Fig. 2D). All three strains grew on YPD at 18 °C, 30 °C and 37 °C, although the untagged strains grew somewhat slower. As expected, only the Sth1-Twin-Strep and the Rsc4-CBP3xFLAG-ProteinA/Sth1-Twin-Strep DSY-5 yeast strain grew on medium lacking tryptophan, while only the Rsc4-CBP-3xFLAG-ProteinA/Sth1Twin-Strep DSY-5 yeast strain grew on medium lacking leucine. Except for a slightly slower growth on sucrose and galactose medium at 37 °C, the single tagged and dual tagged strains generally grew equally fast or faster on ethanol medium, CuSO4 medium, formamide and hydroxyurea

The protein composition of the TEV eluates collected in the first purification step and the Strep-Tactin eluates collected in the second purification step was analyzed by Coomassie staining of gels after SDS–PAGE. While a large number of protein bands were seen in the TEV eluate, the typical protein band pattern of yeast RSC was observed in the Strep-Tactin elution fractions (Fig. 3). RSC complexes were mostly eluted in fractions 2–4 from the Strep-Tactin resin. We subjected the protein gel to mass spectrometry and data base search to identify the purified proteins (compare Table 1 and Supplementary Table S2). RSC proteins were identified in all analyzed samples (i.e. TEV eluate as well as Strep-Tactin elution fractions 2 and 3). RSC proteins were identified with an average sequence coverage of 47.6– 58.6% in the three samples. The overall total spectrum count of RSC proteins was 2903 for the TEV eluate, while the overall total spectrum count of RSC proteins was 5810 and 7655 for Strep-Tactin elution fractions 2 and 3. In line with the protein band pattern observed in the SDS–PAGE (Fig. 3), we identified a large number of non-RSC proteins in the TEV eluate, while considerably fewer non-RSC proteins were found in the Strep-Tactin eluates (Table 1 and Supplementary Table S2). The overall total spectrum count of non-RSC proteins was 17,127 for the TEV eluate, 2858 for Strep-Tactin elution fraction 2 and 2698 for Strep-Tactin elution fraction 3. We conclude that the nonRSC proteins are effectively depleted during the second purification step, while core RSC proteins are enriched in the Strep-Tactin eluate samples. In total, we identified 17 RSC proteins in all of the analyzed samples. Of note, we did not find actin peptides in our analysis, and repeated analyses indicated that actin was only irreproducibly identified (0–2 exclusive unique peptides). We identified both Rsc1p and Rsc2p peptides in our samples, although we observed a clear predominance of Rsc2p peptides in the samples (compare e.g. the total spectrum count of Rsc1p and Rsc2p in the Strep-Tactin eluate fractions). To analyze the presence of Rsc1p and Rsc2p in the Strep-Tactin elution fractions, we quantified the protein bands by densitometric analysis of the Coomassie stained protein bands. We estimated that 82.5 ± 6.8% of assemblies are Rsc2 complexes, which is in agreement with previous reports [10]. Furthermore, we found a clear underrepresentation of Rsc3p and Rsc30p: we estimated a relative abundance of 33.5 ± 7.8% for the two proteins. Note that Rsc3p and Rsc30p are not separated as individual bands in the SDS-PAGEs; thus, the precise contribution of the proteins cannot be

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Fig. 2. Growth phenotypes of untagged DSY-5, single tagged Sth1-Twin-Strep-tagged DSY-5, and dual tagged Rsc4-CBP-3xFLAG-ProteinA/Sth1-Twin-Strep-tagged DSY-5 S. cerevisiae. A. Cell morphology of the three yeast strains. B. Nuclear staining of the three yeast strains using Hoechst 33342. C. Growth curve of the three yeast strains (n = 3, each). D. Plate growth assay on YPD, synthetic defined medium −Trp, synthetic defined medium −Leu, synthetic defined medium + 2% sucrose, synthetic defined medium + 2% galactose, YPD + 6% ethanol, YPD + 5 mM CuSO4, YPD + 1.2 M NaCl, YPD + 2% formamide, and YPD + 150 mM hydroxyurea (HU). Ten-fold serial dilutions of the three yeast strains were spotted on the respective agar plates (top row, untagged DSY-5; middle row, single tagged yeast; bottom row, dual tagged yeast). Images were taken after 4 days of incubation at 18 °C, 30 °C and 37 °C, respectively.

determined from the gel. Together, these analyses indicate that the vast majority of the complexes represent Rsc2 complexes. 3.5. Functional characterization of RSC Next, the ATPase activity of purified RSC complexes was evaluated using a phosphate sensor assay that measures the production of inorganic phosphate with a fluorescent sensor protein. Fractions containing almost no RSC complexes by SDS–PAGE had no significant ATPase activity (Fig. 4A). In contrast, a fluorescence signal was observed when the peak fractions were assayed. This analysis was performed in the presence of 150 mM NaCl. Moreover, we measured the timedependent ATP hydrolysis in a low chloride buffer (50 mM NaCl) and a high potassium acetate buffer (400 mM) (Fig. 4B). In line with previous results [26], purified RSC complexes were more active in the presence of low chloride and high potassium acetate. A strong

time-dependent increase in the fluorescence was observed for both samples as compared to –ATP samples. Furthermore, we tested whether the RSC complexes can bind and remodel nucleosomes using a gel shift assay. We found a concentrationdependent shift upon incubation of Cy-5-labeled mono-nucleosomes with purified RSC (Fig. 4C), and nucleosomes were mobilized by purified RSC upon addition of ATP (Fig. 4D). Thus, functionally active RSC complexes were isolated by two-step affinity selection. 3.6. Glycerol gradient fractionation of RSC The peak eluate fractions were loaded on glycerol gradients and ultracentrifuged to separate complexes according to size and shape. We fractioned the gradients and analyzed the gradient fractions by Coomassie-stained SDS–PAGE. This analysis revealed that RSC had an apparent Svedberg value of approximately 20S (Fig. 5A for the Strep-

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Fig. 3. Purification of Rsc4-CBP-3xFLAG-ProteinA/Sth1-Twin-Strep-tagged RSC from S. cerevisiae. Coomassie-stained gel of the eluates after SDS–PAGE. Lane TEV shows the TEV eluate (first purification step), and lanes 1–6 represent Strep-Tactin elution fractions 1–6 (second purification step), respectively; “M” indicates the marker.

Table 1 Mass spectrometric protein identification of the purified complexes. Protein

GI number

Mol. mass [kDa]

Sth1p Rsc1p Rsc2p Rsc3p Rsc30p Rsc4p Rsc6p Rsc8p Rsc9 Rsc58p Arp7p Arp9p Actin Npl6p Sfh1p Ldb7p Rtt102p Htl1p Total (RSC) Total (other)

6322065 6321493 6323389 398366459 41629685 398364889 6319900 14318562 6323508 6323061 6325291 6323676 14318479 6323738 6323354 41629675 37362657 6319868

157 107 102 102 101 72 54 63 65 58 54 53 42 50 49 20 18 9

TEV eluate

Elution fraction 2

Elution fraction 3

Peak glycerol gradient fraction

TSC

EUP

SC%

TSC

EUP

SC%

TSC

EUP

SC%

TSC

EUP

SC%

584 126 235 169 79 558 74 283 107 172 53 140 0 216 47 14 31 15 2903 17,127

80 31 44 54 22 42 20 40 22 32 17 21 0 27 13 5 9 5 484 9319

48 40 55 52 27 59 43 59 33 60 34 38 0 62 33 33 69 64 47.6 16.7

1117 172 500 268 124 492 242 657 347 466 211 375 0 497 153 37 109 43 5810 2858

87 34 60 46 21 46 24 45 28 40 25 26 0 29 16 4 10 5 546 1341

50 46 62 49 23 59 53 64 38 75 41 36 0 64 40 28 75 64 51.0 16.7

1536 274 612 293 128 632 334 906 457 608 293 501 0 630 199 67 127 58 7655 2698

92 40 66 50 21 43 31 49 38 43 32 29 0 29 20 5 13 6 607 1431

51 53 71 50 26 64 57 74 56 75 57 49 0 70 62 33 84 65 58.6 16.5

1025 286 483 153 63 654 245 949 385 586 244 399 0 557 158 69 114 44 6414 408

80 39 60 36 14 38 26 44 28 39 28 27 0 30 15 5 10 5 524 250

48 51 62 43 18 52 55 63 42 73 52 42 0 65 43 33 71 64 51.6 13.7

Listed are the protein name along with its GI number and theoretical molecular mass (mol. mass). For the four samples (TEV eluate, Strep-Tactin elution faction 2 and elution fraction 3, peak glycerol gradient fraction), the total spectrum count (TSC), the exclusive unique peptide count (EUP) and the sequence coverage in % (SC%) is given. Furthermore, the overall total spectrum count, the total exclusive unique peptide count as well as the average sequence coverage in % are given for the RSC core complex (without actin) and other proteins identified (see also Supplementary Table S2).

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Fig. 4. Activity assays of the purified RSC complexes. A. ATPase assay of the first fraction and the peak fraction in a chloride buffer (150 NaCl). Dark grey columns represent control samples without ATP and DNA, while blue columns indicate experimental samples that included ATP and DNA. ***p b 0.0005 (n = 3, each). B. Time-dependent formation of phosphate upon addition of ATP and DNA in a high potassium acetate buffer (blue points in the left graph) and low sodium chloride buffer (purple points in the right graph). Samples without addition of ATP (grey points in both graphs) were used as control. n = 3, each. C. Gel shift assay using dual tag purified RSC. In total, 3 nM of Cy5-labeled mono-nucleosomes was incubated with 3 nM, 6 nM, and 12 nM of RSC, respectively (lanes 2–4). Lane 1 shows the mono-nucleosome substrate. Positions of the RSC-nucleosome complex (black arrowhead) and of the mono-nucleosome (white arrowhead) are indicated. D. Nucleosome mobilization assay using dual tag purified RSC. A measure of 24 nM of purified RSC was incubated with 8 nM Cy5labeled mono-nucleosomes in the absence (lane 1) or presence of 0.5 mM ATP (lane 2). To stop the reaction, RSC was competed off the nucleosomes by the addition of an excess of unspecific DNA. The band positions of the non-mobilized and the mobilized nucleosomes are indicated by white and black arrowheads, respectively.

Tactin eluate, and Figure S2 of Supplementary Data for the TEV eluate), and that intact RSC complexes were purified from yeast cell extracts. In contrast to the glycerol gradient of the TEV eluate, where only a minor portion of the proteins represent complete RSC (Figure S2 of Supplementary Data), the vast majority of proteins of the Strep-Tactin eluate comigrated as 20S RSC complex (Fig. 5A). A small fraction of dual step purified particles appears to migrate up to the bottom of the gradient, which suggests that some higher-order complexes of RSC with other factors may exist. Due to their underrepresentation, however, these complexes are challenging to analyze at present. Western blotting of the glycerol gradient fractions was used to test for the presence of RSC proteins. Specifically, the Sth1 protein was found in the 20S region of the gradient (Fig. 5B, upper row). As the 3xFLAG tag is retained on the Rsc4 protein after purification, Western blotting for FLAG was used to confirm the presence of the Rsc4 protein in the 20S region of the glycerol gradient (Fig. 5B, lower row). The Sth1 and Rsc4 proteins were not observed in the top gradient fractions, as it would be expected for individual proteins (compare also Figure S1 B for RSC purified by anti-FLAG affinity selection). This finding demonstrates that the proteins were purified predominantly in RSC complexes and not as isolated bait protein. The RSC complex was thus isolated as a high molecular weight complex of approximately 20S. Furthermore, we also analyzed the protein composition of the peak gradient fraction by mass spectrometry and database search. Again, all core RSC proteins were identified (Table 1 and Supplementary Table S2). The overall total spectrum count was 6414 and 408 for the RSC core proteins and the non-RSC proteins, respectively. This indicates that the RSC complexes are further enriched in the glycerol gradient. Densitometric quantification of Rsc1p, Rsc2p, and Rsc3p/Rsc30p indicated that 93 ± 3.6% of the complexes represented Rsc2 complexes. The relative contribution of the Rsc3p/Rsc30p proteins was estimated to 8.7 ± 2%. Assuming that Rsc3p and Rsc30p exist as heterodimer in RSC, 4.4% of the RSC complexes might contain the Rsc3p/Rsc30p heterodimer. We concluded that the RSC complexes, in particular Rsc2 complexes, were highly enriched in the peak glycerol gradient fractions, while the non-RSC proteins were efficiently depleted. 3.7. EM characterization of RSC The peak fractions of the GraFix gradient were imaged by molecular EM to visualize the RSC complex. EM revealed a monodisperse population of particles with a maximum diameter of about 20 nm (Fig. 6A). The

fine structural details of the particles were well defined. Therefore, an EM data set of the RSC complex was collected for single-particle image processing. Upon iterative alignment and classification, the main views of the RSC complex were obtained. The class averages showed a particle with multiple well-defined domains (Fig. 6B). In particular, characteristic views of the complex repeatedly appeared in the data set. These main views represented those that were commonly observed during manual particle selection. The typical views of the yeast RSC complex revealed three to four interconnected domains that surrounded a central area of lower density (Fig. 6B). In particular, we found that the particles could adopt different conformations: a closed conformation (Fig. 6B, upper row) and an open conformation (Fig. 6B, lower row). Both conformations were approximately equally abundant. In addition, we also studied non-crosslinked RSC gradient fractions in the absence (apo-RSC) or presence of H3 peptides (unacetylated H3(1–20) and acetylated H3K9Ac(1–20), respectively). Overall, we observed the same projection views in the non-crosslinked gradient as in our GraFix samples. In agreement with the GraFix data set, the apoRSC data set showed an approximately equal abundance of the closed and open conformation (Fig. 6C). In the presence of unacetylated H3 peptide, the ratio of closed to open conformation shifted somewhat towards the closed conformation (Fig. 6D). In the presence of acetylated H3 peptide, almost all particles had a closed conformation (Fig. 6E; a schematic representation of the closed and open conformation is given in Fig. 6F). Therefore, intact RSC complexes were purified by the double affinity approach. We furthermore conclude that acetylated H3 peptides induce a closed conformation of the RSC complex. 4. Discussion This study demonstrates the isolation of endogenous RSC complexes from yeast by a tagging approach, in which two complementary purification strategies are used. Initial purification attempts using a single tag fused to one protein of the complex resulted in an overrepresentation of that protein in the isolates (Figure S1). However, fusing a second tag to another protein in the RSC complex improved the yield of the complete RSC complex and decreased the unwanted isolation of an individual protein and partial complexes (Fig. 1B). In particular, the Strep-tag II based Twin-Strep tag was used. Promising preliminary results were obtained with a yeast overexpression system, suggesting that Strep-tag II based tags might be instrumental for this task [23]. This tag was fused to STH1, which is the catalytic ATPase subunit of the RSC complex [33].

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Fig. 5. Glycerol gradient ultracentrifugation of RSC. A. Coomassie-stained gel after SDS–PAGE of glycerol gradient fractions. Lanes 1–11 represent glycerol gradient fractions 1–11, respectively; “M” indicates the marker. “Top” and “bottom” indicate the top and bottom fraction of the glycerol gradient, respectively. B. Western blotting of the glycerol gradient fractions using antibodies against Sth1 (upper row) and FLAG (lower row) that is fused to the Rsc4 protein.

An additional tag was fused to the Rsc4 protein of the RSC complex that has been shown to specifically crosslink to nucleosomes [34]. The tag, which we call the triple tag, contained a CBP tag, a 3xFLAG tag, a TEV cleavage site, and a Protein A tag in case of C-terminal tagging. For purification purposes, we utilized only the Protein A tag and the TEV cleavage site, but both the 3xFLAG tag and the CBP can be used for detection by Western blotting [16]. Although the CBP and 3xFLAG tags could also be used for purification [14,35], we observed that the yield of the complete complexes was reduced with CBP affinity chromatography (see Figure S1 and data not shown). In contrast, our dual tagging approach that involves Strep-Tactin affinity selection improved yields of complete RSC complexes. By mass spectrometry, we confirmed that the clear enrichment of the RSC complex was accompanied by a depletion of proteins that do not belong to the core proteins of the RSC complex. This approach was designed to purify protein complexes that are endogenously expressed by their native promoters. In contrast to overexpressed proteins that exist at a high number per cell, these types of macromolecules are uniquely challenging to isolate. The number of endogenous complexes is usually low. It is often in the range of hundreds to few thousands of complexes per cell [36]. Consequently,

large volumes of cell extract are required to obtain sufficient quantities of protein for experiments. This problem is compounded by the relatively high dissociation constant of many affinity chromatography resins and/or bleeding phenomena that typically limit the volume of the cell extract that can be purified to about 10 column volumes [22]. Therefore, we attempted a purification approach that could efficiently capture tagged assemblies from large volumes and be performed as batch purification. The strong IgG–Protein A interaction made the system an excellent choice as a first step [20]. Accordingly, TAP affinity purification and related techniques are widely used in the literature [15,37]. As the tagged macromolecular assemblies cannot efficiently be released from the resin under mild, non-denaturing conditions, the complexes are eluted by TEV protease cleavage [15]. Importantly, TEV cleavage can be performed with a small sample volume. Reducing the volume is necessary for the second purification step to be efficient [38]. In contrast to other two-step purification approaches [39], we fused the second purification tag (Twin-Strep tag) to another protein of the RSC complex. This strategy favors the isolation of intact macromolecular complexes and reduces the isolation of individual proteins and smaller complexes that lack one or more of the components

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protein to Strep-Tactin, while the Twin-Strep tag can still be eluted under mild conditions by using desthiobiotin [38]. Previous structural studies of yeast RSC complexes isolated by chromatography or TAP selection have shown differences in the structural details of the assemblies [11–13]. These structural differences might reflect differences in the protein composition that occur using the different purification methods. All previous studies performed EM directly on eluate fractions without using a dual tagging approach and without making use of glycerol gradient centrifugation. Our dual tagging purification protocol was investigated with glycerol gradient centrifugation. This centrifugation further separates the complexes and thus represents a third purification step. Accordingly, we observed a highly pure RSC complex in peak glycerol gradient fraction as studied by mass spectrometry. In line with previous results [10], the isolates purified using our dual tagging approach predominantly represent Rsc2 complexes, and a further enrichment for the Rsc2 complex has been achieved by glycerol gradient centrifugation. In contrast to the Rsc2 complex, the Rsc1 complex is enriched in the Rsc3p/Rsc30p heterodimer [9], which increases the molecular mass by about 25%. We suggest that this difference in the molecular mass of the Rsc1 and Rsc2 complexes favors the separation of both RSC complexes during the sedimentation in the glycerol gradient. Moreover, we stabilized the RSC complexes by crosslinking. Importantly, sample instability in the absence of a crosslinker has been observed in EM studies before [12], and we have likewise seen this phenomenon in non-GraFix fractions. By using a GraFix gradient [28,30], we could effectively stabilize our complexes by mild crosslinking that allowed visualization of intact particles. The very low concentration of glutaraldehyde applied at 4 °C results in a mild crosslinking of the assemblies that still leaves many lysine residues unmodified [29]. Accordingly, the RSC complexes were observed to be structurally welldefined by EM and single-particle image processing. Similar views of the RSC complexes were also observed in our non-GraFix gradient. In line with a previous report [12], our comparison of apo-RSC and histone H3 peptide bound RSC demonstrated a structural rearrangement of RSC towards a closed conformation upon binding of acetylated H3 peptide. Together, these finding suggests that our dual tagging approach could be useful to isolate other endogenous assemblies from yeast for biochemical, functional and structural studies. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2014.11.009. Acknowledgements

Fig. 6. EM of the yeast RSC complex. Scale bars represent 50 nm. A. Representative EM image of the peak GraFix gradient fraction. B. Class averages of the GraFix-treated RSC complex after single-particle image processing. Class averages were grouped into closed conformations (upper row) and open conformations (lower row). Open and closed conformations of RSC are approximately equally abundant. C. Class averages of noncrosslinked apo-RSC in the absence of histone peptides. D. Class averages of noncrosslinked RSC in the presence of histone H3(1–20) peptide. E. Class averages of noncrosslinked RSC in the presence of acetylated histone H3K9Ac(1–20) peptide. F. Schematic drawing of RSC in its closed (left) and open (right) conformation. The arrowheads point towards the conformational rearrangement.

(Fig. 1B). We selected the Twin-Strep tag as the second purification tag because it has favorable binding characteristics for the Strep-Tactin affinity resin compared to the Strep-tag II [38]. The presence of two copies of Strep-tag II within the Twin-Strep tag favors binding of the tagged

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