Cell Lines, Transfections, and Expression Analysis
HEK293 and U2OS cells were maintained in DMEM containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin.
For transfection experiments, HEK293 and U2OS cells were transfected with TransIT-LT1 Transfection Reagent (Mirus, Madison, WI) according to the manufacturer's protocol. RNA interference (RNAi) experiments were performed with TransIT-TKO Transfection reagent according to the manufacturer's instructions (Mirus). Small interfering RNA (siRNA) duplexes targeting either a region in exon 3 of Rfp2 or a region in exon 1 of Dltet were designed and purchased from Dharmacon (Boulder, CO; http://www.dharmacon.com/sidesign
). As controls, scrambled siRNAs or siRNA duplexes against green fluorescent protein were used. siRNA sequences can be obtained from the authors upon request.
If not otherwise indicated, cells were harvested 48 h after transfection and subsequently lysed in M-RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.05% SDS, 50 mM Tris, pH 7.4). Lysis buffer was supplied with Complete protease inhibitor cocktail (Roche, Indianapolis, IN), 1 mM phenylmethylsulphonylfluoride (PMSF), 1 mM sodium orthovanadate, 5 mM sodium fluoride (Sigma, St. Louis, MO). Lysates were subjected to SDS-PAGE and transferred to PVDF membranes, and the proteins were detected by Western blot analysis using an enhanced chemiluminescence system (ECL, Amersham Biosciences, Piscataway, NJ).
For immunoprecipitation experiments, cells were lysed in M-RIPA or NP-40 (150 mM NaCl, 1% Nonidet P-40, 50 mM Tris, pH 7.4) containing 10 mM N-ethylmaleimide (Pierce, Rockford, IL). One milligram of precleared lysate was incubated with Rfp2 antibody (500 ng) at 4°C overnight, followed by incubation with Gammabind G Sepharose beads (Amersham Biosciences) for 1 h. The beads were washed five times with lysis buffer and immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis. For glutathione S-transferase (GST) pulldown, 1 mg of total protein lysate was incubated with glutathione Sepharose 4B (Amersham Biosciences) at 4°C overnight. The resin was washed five times and analyzed as described above. For GST-pulldown in denaturing conditions, cells were lysed in NP-40 buffer containing 1% SDS to disrupt noncovalent interactions. The lysate was subsequently diluted 10 times with NP-40 buffer without SDS, and pulldown was performed as described above.
For protein stability experiments, cells were treated with 30 μM MG-132 (Biomol, Plymouth Meeting, PA) for the indicated time points. For cycloheximide chase experiments, cells were treated with 100 μg/ml cycloheximide (Biomol) in the presence or absence of MG-132. Cells were harvested at the indicated time points.
Cell cycle distribution of Rfp2 expression was examined by flow cytometry analysis as described previously (Panaretakis et al., 2003
). DNA was stained by addition of propidium iodide staining solution (Nordic BioSite, Täby, Sweden) 20 min before flow analysis.
The full-length coding regions of the RFP2 and DLTET ORFs were separately PCR amplified with Advantage enzyme (BD Biosciences) and cloned into pEBG GST- and Tag2B FLAG-tagged vectors (Invitrogen, Carlsbad, CA). The sequence of the cloned cDNA was verified in its entire length by sequencing. The RFP2[C13A] mutant was constructed using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Primers used for cloning can be obtained from the authors upon request. The RFP2-ΔRING deletion mutant was generated by cutting the RFP2 construct with PstI and subsequently religating. The resulting ORF encodes 317 amino acids and completely lacks the RING domain. RFP2-ΔTM was constructed by PCR amplification using the reverse primer deltaTMR (ttttccatcgatgggcttgcaggcaaattagagg) together with a forward primer in frame with the ATG, resulting in the 3′-deletion of 131 amino acids, including the transmembrane domain.
Untagged pME18S-FL3-RFP2 expression constructs were kindly provided by Dr. Akio Matsuda (Laboratory for Biology, Institute for Life Science Research, Health Care Company, ASAH1 KASEI Corporation, Shizuoka, Japan). ECFP-VCP plasmid was kindly provided by Dr. Florian Salomons (Department of Cellular and Molecular Biology, Karolinska Institute, Solna, Sweden). CD3-δ-YFP and Ub-R-YFP expression constructs were kindly provided by Dr. Nico Dantuma (Department of Cellular and Molecular Biology, Karolinska Institute, Solna, Sweden).
The following primary antibodies were used: rabbit polyclonal against Rfp2 (see below), mouse monoclonal against GST (B-14, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal against GST (Z5, Santa Cruz Biotechnology), mouse monoclonal against FLAG (M2, Stratagene), rabbit polyclonal against hemaglutinin (HA) (Y11, Santa Cruz Biotechnology), mouse monoclonal against β-tubulin (Sigma), mouse monoclonal against β-actin (Sigma), rabbit polyclonal against GAPDH (Abcam, Cambridge, MA), mouse monoclonal against Bcl-2 (124, DAKO, Carpinteria, CA), mouse monoclonal against c-Myc (9E10, Santa Cruz Biotechnology), mouse monoclonal against green fluorescent protein (GFP; Roche), rabbit polyclonal against GFP (Sigma), and horseradish peroxidase (HRP)-conjugated goat antibody against biotin (Cell Signaling). As secondary antibodies HRP-conjugated anti-rabbit and anti-mouse antibodies (Cell Signaling, Beverly, MA) were used.
To generate a novel mono-specific antibody recognizing Rfp2, a protein fragment from the coiled-coil region of Rfp2 (amino acids 158–300) was chosen because of the low sequence similarity to all other human proteins. Subsequently, primers were designed and used in RT-PCR, resulting in a gene encoding the protein fragment (Agaton et al., 2003
). Production, purification, and characterization of the protein fragment were performed as previously described (Agaton et al., 2003
). Antibody purification was performed by a depletion chromatography step, where antibodies directed toward the His-ABP-part of the fusion protein were removed and a second purification allowed for capture of the specific antibodies (Agaton et al., 2004
). Characterization of the antibodies was done according to a previous study (Nilsson et al., 2005
Cytosolic, membranous, and nuclear extracts were prepared using the Qproteome Cell Compartment Kit according to the manufacturer's protocol (Qiagen, Chatsworth, CA). Briefly, sequential addition of different buffers to cell pellets followed by incubation and centrifugation results in the isolation of different cellular compartments. First buffer selectively disrupts the plasma membrane without solubilizing it, resulting in the isolation of cytosolic proteins. Plasma membranes and compartmentalized organelles remain intact and are pelleted by centrifugation. The pellet from the first step is resuspended in the second buffer, which solubilizes the plasma membrane as well as all organelle membranes except the nuclear membrane. This fraction contains membrane proteins (including proteins from the ER membrane) and proteins from the lumen of organelles (e.g., the ER and mitochondria). In the final step nuclei are solubilized using the third buffer in which all soluble and most membrane-bound nuclear proteins are extracted. Compartment separation was analyzed by immunodetection of stated marker proteins.
HEK293 cells were transfected with CD3-δ-YFP or Ub-R-YFP together with indicated constructs or siRNAs. After 24 h, transfectants were incubated for 30 min in methionine-/cysteine-free medium and then metabolically pulsed with 70 μCi 35S cell-labeling mix (Redivue ProMix 35S, Amersham Biosciences) at 37°C for 30 min. After the pulse, cells were washed, chased in complete medium supplemented with 10 mM methionine and 1 mM cysteine (Sigma), and harvested at the indicated time points in M-RIPA lysis buffer. CD3-δ-YFP and Ub-R-YFP were immunoprecipitated using rabbit polyclonal GFP antibody (Sigma) and the level of metabolically labeled YFP construct was subsequently analyzed by SDS-PAGE and autoradiography.
Isolation of Protein Complexes for Mass Spectrometry
To prepare protein extracts for subsequent mass spectrometric analysis, ~5 × 107 HEK293 cells were used. Because Rfp2 was shown to be a strictly membrane-associated protein, soluble cytosolic proteins were discarded in order to reduce complexity of the sample. Washed cell pellets were resuspended in hypotonic lysis buffer (10 mM NaCl, 3 mM MgCl2, 20 mM Tris, pH 7.5), incubated for 10 min with shaking, and centrifuged at 3600 rpm for 10 min. Supernatants containing soluble cytosolic proteins were discarded, and cell pellets were lysed in NP-40 buffer. Endogenous Rfp2 protein was immunoprecipitated as described above using 2 μg of Rfp2 antibody and proteins were subjected to SDS-PAGE with subsequent staining with SimplyBlue Safestain (Invitrogen). In parallel, other control proteins were similarly immunoprecipitated and analyzed. Samples were run in different gels or some distance apart to minimize cross-contamination. Entire lanes were cut into 10-kDa pieces, and proteins were identified by nanoflow liquid chromatography tandem mass spectrometry as described below. Only peptides exclusively present in Rfp2 immunoprecipitated samples were regarded as possible Rfp2 interacting proteins.
Nanoflow Liquid Chromatography Tandem Mass Spectrometry
All experiments were performed on a 7-tesla LTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany), modified with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). The high-performance liquid chromatography setup used in conjunction with the mass spectrometer (LC-MS) consisted of a solvent degasser, nanoflow pump, and thermostated microautosampler (Agilent 1100 nanoflow system, Wilmington, DE). Chromatographic separation of peptides was achieved on a 15-cm fused silica emitter (75-μm inner diameter, Proxeon Biosystems) packed in-house with a methanol slurry of reverse-phased, fully end-capped Reprosil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH, Ammerbuch-Entrigen). Briefly, the tryptic peptides were autosampled onto the packed column at a flow rate of 500 nL/min and then eluted at a flow rate of 200 nl/min using a linear gradient of 4.5–40.5% acetonitrile in 0.5% acetic acid over 90 min and ionized by an applied voltage of 1.8 kV to the emitter.
Analysis was performed using unattended data-dependent acquisition mode, in which the mass spectrometer automatically switches between a high-resolution survey scan (resolution = 100,000 at m/z 400), followed by acquisition of both an electron capture dissociation (ECD; Zubarev et al., 2000
) and collision activated dissociation (CAD) tandem mass spectra (resolution = 25,000) of the two most abundant peptides eluting at this moment from the nano-LC column. Complementary fragmentation of the same peptide yielded different fragment ions that increased the specificity of the sequence information. Using this complementary information (Horn et al., 2000
) for protein ID not only improves the confidence in protein identification performed by search engines, but for a fixed confidence level also identifies a larger number of peptides and proteins than when only one fragmentation technique is used (Nielsen et al., 2005
All data from the acquired MS/MS spectra was extracted into so-called dta-files using TurboSequest software (Thermo), and an in-house written Java program was used for extraction of complementary fragment masses before database searching, as described previously (Nielsen et al., 2005
; Savitski et al., 2005
). The new dta-files containing complementary fragment masses was merged into a single file, which was searched using the Mascot Search Engine (Matrix Science, Boston, MA; Perkins et al., 1999
) against the full IPI human database (version 3.15; downloaded February 2006) with carbamidomethyl cysteine as fixed modification and oxidized methionine and ubiquitination on lysine as variable modifications. Peptide searches were performed with an initial tolerance on mass measurement of 3 ppm in MS mode and 0.02 Da in MS/MS mode. Only proteins uniquely identified by a minimum of two significant peptides were taken into consideration for this study.
In Vitro Ubiquitination Assays
HEK293 cells were transfected with GST-tagged pEBG, DLTET, RFP2, or RFP2[C13A] expression plasmids. Forty-eight hours after transfection, cells were harvested in LSLD buffer (50 mM NaCl, 0.1% Tween-20, 10% glycerol, 50 mM HEPES) with protease inhibitor cocktail. Two milligrams of total protein was used for GST pulldown as described above. Precipitates were extensively washed (five times each in LSLD buffer and 20 mM Tris, pH 7.5), and in vitro reactions were performed directly on the beads. The in vitro reactions contained 20 ng human recombinant E1, 200 ng E2, 2 μg biotinylated ubiquitin (BioMol), 5 mM ATP, 5 mM MgCl2, 1 mM DTT, 1 U inorganic pyrophosphatase, 10 mM creatine phosphate, 2.5 U creatine phosphokinase from rabbit muscle (Sigma) in 20 mM Tris, pH 7.5, to a final volume of 50 μl. As E2s, UbcH5b and [C85A]UbcH5b were used. [C85A]UbcH5b is a catalytically inactive mutant incapable of forming thiol ester conjugates. The reactions were incubated at 37°C for 60–120 min and stopped by boiling in 1× NuPAGE LDS Sample Buffer (Invitrogen). Ubiquitinated proteins were detected by Western blot analysis using biotin antibodies.
U2OS cells and HEK293 were transfected as described. Cells on coverslips were fixed in 4% formaldehyde for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and blocked with blocking buffer (2% BSA, 0.2% Tween-20, 5% glycerol in PBS) for 30 min at room temperature. They were incubated with primary antibodies (anti-Rfp2 diluted 1:100, anti-Myc diluted 1:50) for 1 h at room temperature followed by appropriate secondary antibodies conjugated with fluorescein isothiocyanate (FITC; 1:40; DAKO), Texas Red (1:200; Vector Laboratories, Burlingame, CA) or Alexa Fluor 594 (1:500; Molecular Probes, Eugene, OR). Slides were mounted using Vectashield with DAPI (Vector Laboratories), and images were acquired on a Zeiss Axioplan 2 imaging microscope with Axiovision software (Thornwood, NY) and processed either as gray scale or dual color TIFF images in Adobe Photoshop (Adobe Systems, San Jose, CA). Mock-transfected cells were used as negative controls.