Bacterial two-hybrid assay with hCdc34/UbcH3 as bait
We followed the manufacturer’s instructions (Stratagene, La Jolla, CA, USA). The N-terminal two-third region of Cdc34, corresponding to amino acid 1–143 and containing catalytic core, was amplified by PCR using primers with engineered restriction enzyme sites and a spacer amino-acid sequence. The primer sequences were as follows: HYYOL-58, 5′-cggcgcggccgcaggaggaggaagcggaatggctcggccgctagtg-3′; and HYYOL-10, 5′-gcggaattctcagctcacgtttgcgggcga-3′.
The PCR product was digested by NotI–EcoRI and ligated into the pBT vector. The resulting construct was used as bait (pBT-34delC). (Constructs containing full-length Cdc34 weakly activated reporter cassettes on their own and thus were not used for screening.) For prey constructs, we used a commercial expression library prepared from a human HeLa S3 cells (Stratagene). Among a few hundred carbenicillin-resistant colonies that emerged on sixty-seven 15 cm selection plates, we picked 192 colonies. Of these, 97 colonies were beta-Gal positive. We recovered 87 plasmids from the blue colonies and sequenced them. A clone containing the 24–350 amino-acid region of RING105 was identified. A cDNA containing full-length RING105 was obtained from American Type Culture Collection (ATCC) for further manipulation (ATCC number 6232371).
We prepared constructs encoding C-terminally GFP-tagged RING105 by ligating XhoI–SacII-treated PCR fragments into pEGFP-N (Clontech, Palo Alto, CA, USA). Point mutations were introduced by a site-directed mutagenesis kit (Strata-gene). We made a mammalian expression construct for RING105 without GFP by deleting the GFP coding region with SacII–NotI and religating the DNA after T4 DNA polymerase treatment.
Cell Culture and transfection
Cell culture was performed as described previously (Kallio et al., 1998
). We used FuGene 6 reagent (Roche Biochemicals, Indianapolis, IN, USA) for DNA transfection following the manufacturer’s instructions. Occasionally, we used 293T cells because of their high transfection efficiency.
RING105 antibody production and purification
The N-terminal region of RING105 cDNA, corresponding to amino acids 1–134, was amplified by PCR using the ATCC cDNA as template and ligated into XhoI site in pET28C vector (Novagen, Madison, WI, USA). The primer sequences were as follows: HYYOL-82, 5′-cggctcgagttagaccaggataacagggga-3′; and HYYOL-74: 5′-ccggctcgagcgccaccatgcaccctgcagccttcccg-3′
Bacterially expressed protein was purified by Talon Ni affinity beads (Clontech) and sent to Covance Inc. to immunize two rabbits (numbers 98 and 100). For affinity purification, each rabbit anti-RING105 sera was preincubated with CNBr beads (Pharmacia, Rockville, MD, USA) coupled with bacterial lysate containing His-tagged Uba1 to remove background. Sera were then incubated with amino-link beads bound to RING105 (1–134) and eluted following the manufacturer’s protocol (Pierce, Rockford, IL, USA). For further characterization of the antibody, see Supplementary Information 1
Cell extraction, immunoprecipitation and immunoblotting
For immunoprecipitation samples, cultured cells at 50–70% confluency cells on 10 cm plates were trypsinized, harvested and frozen at −80°C. The cells were thawed, extracted in 600 µl RIPA buffer, supplemented with 10mm
PMSF, 10 µm
MG132, 5 µg/ml protease inhibitor cocktail (Sigma, St Louis, MO, USA) and 400 nm
microcystin LR to preserve phosphorylations. The extract was cleared by centrifugation at 15 800 g
for 15 min at 4°C. The supernatant was incubated with antibody-bound protein A beads for 1.5 to 3 h at 4°C. The beads were washed with the same buffer five times. The samples were boiled for 6 min in LDS sample buffer and loaded onto SDS (4–20%)–polyacrylamide gels (Invitrogen, Carlsbad, CA, USA). Proteins were transferred to BioTrace PVDF (Pall Corporation, East Hills, NY, USA) or Immunobilon P membrane (Millipore, Billerica, MA, USA) using an electrophoretic blotting device. For TSSC5 blotting, cell extracts were mixed with SDS–Hepes sample buffer (final concentration: 50mm
Hepes, pH 8.0, 1% (w/v) SDS, 5% glycerol, 0.025% bromophenolblue, 1mm
DTT) and loaded onto SDS–PAGE gels without heating to reduce protein aggregation occasionally observed in membrane proteins extracted in conventional SDS sample buffer (Sagne et al., 1996
We used rabbit anti-GFP antibody (BabCo, Richmond, CA, USA), mouse anti-GFP antibody (Clontech), rabbit anti-HA Y-11 (Santa Cruz, Santa Cruz, CA, USA), rabbit anti-S tag (Gorbsky lab) and mouse anti-HA F7 (Santa Cruz) for immunoprecipitation (0.3 µg/sample) and/or immunoblot (2–5 µg/ml in 5% skim milk in PBS). For human tissue blotting, we used a commercial human multiple tissue blot (Oncogene Research, San Diego, CA, USA).
In vitro ubiquitylation assay
Bacterially expressed and affinity-purified E1 enzyme (human Uba1) and GST-tagged ubiquitin were gifts from Dr T Kamura (Kyushu University, Japan). We used a set of commercially available E2 enzymes: UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, Ubc7 and UbcH10 (Calbiochem, San Diego, CA, USA). We immunoprecipitated GFP-RING105 for 2–3 h using rabbit anti-GFP (AbCam, Cambridge, MA, USA) or mouse anti-GFP (BabCo) with extracts prepared from transfected 293T or HeLa cells that were treated with 10 µm MG132 (AbCam).
For the in vitro RING105 autoubiquitylation assay in , E1 (150 ng), E2 (400 ng) and GST-tagged ubiquitin (40 ng) were mixed in a total 10 µl of reaction buffer (50mm Tris-Cl (pH 8.0), 10mM MgCl2, 50mm NaCl, ATP 2mm, DTT 1mm). Anti-rabbit GFP immunoprecipitates (GFP-RING105) were added and incubated in 30°C for 60 min with occasional agitation. After the reaction, the anti-GFP beads were rinsed extensively with PBS to collect only GFP- or GFP-tagged RING105. Then, we added 4 × LDS buffer to samples and heated them to 95°C for 6 min. The samples were separated by SDS–PAGE and transferred to membranes. GFP-RING105 was detected with mouse anti-GFP (Clontech). GST-ubiquitin conjugates were detected with mouse anti-GST (B-14) (Santa Cruz).
For the in vitro TSSC5 ubiquitylation assay in , we mixed E1-expressing bacterial lysate (10 µl), a Ubc (850 ng), GFP-RING105 (rabbit anti-GFP immunoprecipitates), S-tagged TSSC5-C-expressing bacterial extract (25 µl), 1mm DTT, GST-tagged ubiquitin (100 ng), 5mm ATP, 20mm ZnCl2, 50 µm MG132, 50mm Tris-Cl (pH 8.0), 10mm MgCl2 and 50mm NaCl in a total of 50 µl.We incubated the samples for 2 h at 30°C, then stopped the reaction and denatured the proteins by adding 1% SDS. We then diluted the mixture 10-fold to reduce the SDS concentration to 0.1% and removed his-tagged proteins with Talon beads and GFP-tagged proteins with protein A-beads-bound anti-GFP. We took the remaining supernatant and pulled down S-tagged protein with S-protein beads (Novagen/EMD Biosciences Inc., Madison, WI, USA). Samples were separated by SDS–PAGE and transferred to membranes. We visualized ubiquitylated protein with anti-GST antibody.
FACS analysis for GFP-expressing cells
We transfected HeLa cells with plasmids encoding a variety of GFP-tagged RING105 constructs. After 24–40 h, we suspended the cells by typsinization and fixed them with ice-cold 0.5% paraformaldehyde in PBS for 20 min. Fixed cells were rinsed once with PBS and suspended in 80% ethanol (−20°C) for at least 30 min. The permeabilized cells were rehydrated for 5 min and resuspended in PBS, then treated with RNase (0.1 mg/ml) and propidium iodide (20 µg/ml) in room temperature for at least 1 h. The samples were analysed with a FACScalibur and the cell cycle profile was estimated by ModFit software with the aid of The Flow and Image Cytometry Laboratory (University of Oklahoma Health Sciences Center).
GFP-RING105-expressing cells were observed either as living cells or after fixation. For fixation, we used 1% paraformaldehyde (Sigma) in PHEM buffer (details described in Kallio et al., 2002
). The samples were analysed with a ZEISS
Axioplan IIi microscope equipped with a Hamamatsu Orca II camera and Metamorph Imaging system (Universal Imaging Corp., Downingtown, PA, USA). For live cell observation, a planapochromat × 60 (NA 1.4) objective (Nikon USA) was used with a SenSys CCD camera (Photometrics Ltd, Tucson, AZ, USA) connected to a Nikon Diaphot microscope and imaged with Metamorph software.