Supplementary Methods
Molecular biology techniques
Details of plasmid construction and primer sequences are available upon request. C. elegans dcn-1 cDNA was amplified by PCR from a first strand worm cDNA template. Amplified full-length cDNA was sub-cloned into pGEM-T (Promega). To obtain N-terminal GST::DCN-1, the cDNA was cloned into pGEX6P-1 (Amersham Pharmacia Biotech).
GST::DCN-1 was expressed in BL21 CodonPlus RP E.coli (Stratagene) and purified under native condition using glutathione agarose beads (Sigma). GST::DCN-1 1-295aa or control GST::Y39G10AR.12 (1-123aa) were dialysed into binding buffer (50mM Hepes pH 7.4, 100mM KCl, 10% glycerol, 0.05% NP-40), 60mg of dialysed protein was bound to 45 ml glutathione agarose beads in 1 ml binding buffer. After binding for 1 hour at 4°C, the resin was washed twice with binding buffer and twice with lysis buffer (1 mM EGTA, 1 mM MgCl2, 50 mM HEPES pH 7.4, 100 mM KCl, 10% glycerol, 0.05% NP-40, 1 mM DTT). Worm extract was prepared by sonicating 1g frozen worm pellets in equal volume of lysis buffer containing protease inhibitors. Worm extract was incubated with the resin for 1 hour at 40C, washed 4 times with lysis buffer and bound proteins were eluted in 45ml 2xSDS loading buffer. 10ml from each sample was loaded on 10% SDS gel and analysed by western blotting with affinity purified CUL-3 antibody.
S. cerevisiae DCN1 contains one intron from nucleotides 4 to 97 of the genomic sequence. To obtain the DCN1 cDNA the ORF was amplified from haploid genomic DNA using primers that correspond to the second exon with a three-nucleotide 5' extension containing the ATG start codon. The full length cDNA was subsequently cloned into pBluescript (Stratagene).
To obtain N-terminal HA-tagged Cdc53p, the CDC53 DNA was cloned into the pRD54 plasmid.
Microscopy, immunocytochemistry and GFP imaging
Microscopy and immunocytochemistry were performed as previously described1. The following antibodies were used: monoclonal mouse anti-tubulin (Sigma; clone DM1a), affinity-purified rabbit anti-DCN-1. DNA was labelled with TOTO3 (Molecular Probes) or 1 µg/ml Hoechst. Fluorescent images were taken with a BioRad Radiance confocal microscope or a DeltaVision deconvolution microscope (Applied Precision) using a CoolSnap CCD camera (Roper Scientific). Three-dimensional wide-field datasets were computationally deconvolved and projected.
The TH29 strain expressing GFP::DCN-1 was constructed as described previously2. Full-length genomic locus of H38K22.2a was amplified by PCR. The amplified product was digested and cloned into the PAZ-132 plasmid to generate an N-terminal GFP::DCN-1 fusion gene under control of the pie-1 promoter. The plasmid, containing unc-119(+) as selection marker was bombarded into unc-119(ed3) worms. TH29 likely contains an extrachromosomal array of the rescuing plasmid pAZ-132 [GFP::DCN-1] due to the observed non-Mendelian segregation of GFP expression and unc-119(+) markers to F1 progeny.
The GFP::DCN-1 expressing strain TH29 and the GFP::MEI-1 expressing strain EU1065, were examined by spinning disc confocal microscopy. Images were acquired with an ORCA (Hamamatsu) or a MicroMax-EBF 512 CCD camera using MetaMorph Imaging Software.
Two-hybrid assays
Two-hybrid assays were performed by transforming pEG203-based plasmids expressing LexA DNA-binding domain fusions, and pJG4-6 based plasmids containing transcriptional activation domain fusions into the yeast strain EGY48. LacZ reporter activity was measured as described previously3.
1. Kurz, T. et al. Cytoskeletal regulation by the Nedd8 ubiquitin-like protein modification pathway. Science 295, 1181-1412 (2002).
2. Praitis, V., Casey, E., Collar, D. & Austin, J. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 1217-26 (2001).
3. Breeden, L. & Nasmyth, K. Regulation of the yeast HO gene. Cold Spring Harb Symp Quant Biol 50, 643-50 (1985).