JCS030445 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Specific interaction of DEN1 to Nedd8. (A) Purified GST, GST-DEN1 and GST-DEN1CA proteins (3 µg each) are shown in SDS-PAGE. (B) GST-DEN1 and GST-DEN1CA, but not GST, were able to precipitate co-incubated His-pNedd8GG, as shown in western blot using anti-His antibody. (C) Western blot by anti-His antibody shows that immobilized GST-DEN1 could precipitate co-incubated His-mNedd8, but not His-Ub or His-SUMO. Each input control included 0.5 Mg of purified proteins. GST-pull-down assays were performed with glutathione-sepharose bead-immobilized GST or GST fusion protein in the binding buffer containing 40 mM Tris-Cl (pH 7.4), 1 mM DTT, 0.1% NP40, 1mM PMSF and proteinase inhibitor cocktail.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Mapping the processing site by mass spectrometric analysis. His-pNeddGG was treated with or without DEN1 (as in Fig. 2) and then individually separated by SDS-PAGE. The bands were manually excised from the gel and subjected to in-gel digestion with sequencing grade Lys-C (Roche Applied Science, Germany) in 25 mM ammonium bicarbonate overnight at 37°C. Following digestion, the fragments were extracted twice with 50% acetonitrile (ACN) containing 5% formic acid (FA) for 15 minutes each time with moderate sonication. The extracted solutions were pooled and evaporated to dryness under vacuum. Pellets were resuspended in 10 ml of 50% ACN with 0.1% FA. 1 ml aliquot was then 1:2 mixed with matrix solution (10 mg/ml a-cyano-4-hydroxy-cinnamic acid in 50% ACN, 0.1% FA) and spotted onto the MALDI sample plate. Mass spectrometric analysis was performed with a MALDI-TOF-TOF instrument (4700 Proteomics analyzer; Applied Biosystems, MA, USA), which was pre-calibrated with digested BSA. (A) Digestion by Lys-C, which cleaved specifically at the C-terminus of lysine residues, is expected to give a peptide (L10) at m/z 2404.3, corresponding to M+H⁺ of the C-terminal peptide of pNedd-GG. Pre-treatment with DEN1, which cleaved specifically at the C terminus of Gly96, is expected to reduce the m/z of L10 to 1575.9, as indicated. Molecular ion signals corresponding to the respective L10 peptides of pNedd-GG with (C) and without (B) DEN1 processing were detected by MALDI-TOF-TOF analysis and their identities were further confirmed by manual MS/MS analysis and spectral assignment (data not shown). By contrast, pNedd-AA was shown to be resistant to DEN1 processing by similar MALDI-MS and MS/MS analyses (data not shown), therefore supporting a crucial role for Gly residues at position 95 and 96 (plus the His tag and the linker sequence) in His-pNedd8.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. DEN1 mutant alleles. (A) Diagram shows insertion sites of three EP elements, EY04455, GE11907 and GE14336, in the CG8493 locus that encodes the Drosophila DEN1, and deleted chromosomal regions of three isolated DEN1 null alleles. The EP insertion strain EY04455 was obtained from Bloomington Stock Center, and GE11907 and GE14336 were from the Genisys Collection (GenExel, Korea). The EX9 allele was isolated by imprecise excision of the EY04455 insertion, and DGE11907 and DGE14336 were isolated from GE11907 and GE14336 insertions, respectively. All three null alleles delete the coding exon 2 and are viable and fertile. The EX9 allele was used mostly and referred as DEN1^null in the study. (B) DEN1 mRNA expression levels in wild type, EY01391 and EX9 flies were examined by RT-PCR. The pair of primers produces a 500 bp fragment of DEN1 which appeared in wild type and EY01391, but not in EX9. β-Tub expression is used as an internal control.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Processing of Nedd8-CFP fusion detected by anti-GFP antibodies. Western blot using larval lysates of myc-mNedd8, myc-Nedd8GG-CFP or myc-Nedd8AA-CFP transgenes driven by tubP-GAL4 flies in wild type or DEN1^null background probed with antibodies against GFP that also recognize the CFP variant. The protein level of Myc-Nedd8GG-CFP at 40 kDa is much more reduced in wild type than in DEN1^null (lane 2, 3). However, Myc-Nedd8AA-CFP is expressed at identical levels in both wild tyep and DEN1^null (lanes 4 and 5). CFP expression in the control myc-mNedd8 (lane 1) is not detected and the bottom panel shows the control of α-Tub expression.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. Western blots to detect neddylated and unneddylated Cul1 levels in adult flies. Neddylated and unneddylated Cul1 protein levels remain almost identical in wild type and DEN1^null extracts prepared from adult flies.

• Supplemental Figure S6 (Adobe PDF) -

  Fig. S6. Specificity of anti-Nedd8 antibodies. Western blots using anti-Nedd8 antibodies (left panel) detect the expression of endogenous Nedd8 in wild-type larval extracts (lane 1), and both endogenous Nedd8 and ectopically expressed Myc-mNedd8 in tubP-GAL4;UAS-myc-mNedd8 larval extracts (lane 2). When using anti-Myc antibody (right panel), only Myc-mNedd8 signal is detected.

• Supplemental Figure S7 (Adobe PDF) -

  Fig. S7. Effects of DEN1 mutations on Cul1 and Cul3 neddylation in the absence of CSN5. The neddylation patterns were examined in larval extracts prepared from DEN1^null, CSN5^null, and DEN1^null;CSN5^null double mutants by western blot using Cul1 or Cul3 antibodies. The neddylated Cul1 and Cul3 patterns in DEN1^null;CSN5^null double mutant (lane 3) resemble those in CSN5^null mutants (lane 2).