Promotion of NEDD8-CUL1 Conjugate Cleavage by COP9 Signalosome Svetlana Lyapina, Gregory Cope, Anna Shevchenko, Giovanna Serino, Tomohiko Tsuge, Chunshui Zhou, Dieter A. Wolf, Ning Wei, Andrej Shevchenko, Raymond J. Deshaies

Supplementary Material

Supplemental Figure 1. Identification of SCF-interacting proteins by Mass Spectrometry. Mouse NIH 3T3 cells were infected with retroviruses that expressed Myc₉-TEV-tagged hSKP2 or hCUL1DC. Cell extracts (500 mg) prepared from uninfected (lane 1) cells and cells infected with CUL1DC (lane 3) retroviruses were adsorbed to anti-Myc beads, eluted with TEV protease (Gibco BRL), and analyzed by SDS-PAGE as described (1). Specific bands were excised from the gel and protein identity was determined by mass spectrometry as described (2). Lane 3 is the same sample as shown in Fig. 1A of the print version, and is reproduced here for comparison. SKP2 eluates (lane 2) contained mouse HSP90b, CUL1 and SKP1. We also identified a mouse homolog of yeast SGT1, which was previously reported to bind SKP1 and have an essential, but undefined role in SCF function (3). Surprisingly, SKP2 eluates contained relatively high amounts of the cyclin-dependent kinase regulator CKS1 (4). Although CDC2 was found in SKP2 eluates, it was present in amounts insufficient to account for the level of CKS1, suggesting that CKS1 bound directly to SKP2 and is a bona fide subunit of SCF^SKP2complexes. Recent reports confirm a functional role for CKS1 in SCF^SKP2 complexes (5).

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Supplemental Figure 2. COP9 Signalosome association with hCUL1 does not depend on CUL1 neddylation. HeLa cells were transiently transfected with pCEP4 vectors that expressed full length (lane 2) or C-terminally truncated (lacking the NEDD8 modification site) forms of Myc₉-hCUL1 (lane 3). The cullins and associated proteins were retrieved on anti-Myc beads, separated by SDS-PAGE, and visualized by Western blotting with anti-CUL1, anti-SKP1, and anti-CSN8 antibodies as described previously (6).

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Supplemental Figure 3. S. Pombe cullin 1 (Pcu1) associates with COP9 Signalosome. caa1^Myc(lanes 2 and 5), and caa1⁺ cells either mock-transfected (lanes 1 and 4) or transformed with a pRep41-based plasmid that expressed HA-tagged CSN subunit Sgn2^HA (7) (lanes 3 and 6) were either evaluated as crude extracts (left panels), or were first immunoprecipitated (IP) with anti-Myc antibodies (right panels) prior to SDS-PAGE followed by Western blotting with anti-Pcu1, anti-Myc and anti-HA antibodies, as indicated. Failure to detect Pcu1 associated with Caa1^Myc (lane 5) may stem from effects of the Myc epitope tag on Caa1 function. We observed that neddylation of endogenous Pcu1, as well as Pcu1^Myc13, was increased in caa1myc13⁺ cells (lane 2 and data not shown), compared with wt cells (lane 1), suggesting that the Myc13 epitope compromises the function of Caa1 (see Fig. 2).

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Supplemental Figure 4. Western blot analysis of SCF subunits in various mutant strains. Extracts from the indicated strains were resolved by SDS-PAGE and analyzed by western blotting with a-Pcu1, a-Psh1, and a-Pip1 antibodies.

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Supplemental Figure 5. Analysis of Pcu1 localization by immunofluorescence. Pcu1 localization was visualized in pcu1myc13⁺ and D caa1 pcu1myc13⁺ strains by immunostaining with mouse anti-Myc antibodies followed by anti-mouse IgG-FITC antibodies and confocal microscopy (8). No staining was seen with untagged strains. In both strains Pcu1 was localized throughout the cell, with increased staining in the nuclear region as revealed by DAPI staining.

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Supplemental Table 1. Two-hybrid interactions of SCF components with COP9 Signalosome subunits. Proteins listed in the vertical and horizontal columns were assayed as DNA binding domain and activation domain fusions, respectively (9, 10). The - and + indicate the fold increase of b-galactosidase activity over the empty vector: ---, 1 fold; + ,2-9 fold; ++, 10-49 fold; +++, 50-99 fold; ++++, 100 fold. Note that the N-terminus of CSN1 (CSN1N) was used, because full-length CSN1 was poorly expressed in yeast.
	CSN1N	CSN2	CSN3	CSN4	CSN5	CSN6	CSN7	CSN8	SKP1
CUL1	---	+++	---	---	---	+	---	---	++++
HRT1	+	---	---	---	---	++	---	---	---
SKP1	---	---	---	---	---	---	---	---	---

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Methods

Isolation of CUL1DC-interacting proteins. CUL1-DC and SKP2 were tagged at the C-terminus with a Myc9 epitope preceded by two consecutive repeats of the Tobacco Etch Virus (TEV) protease recognition site (Glu-Asn-Leu-Tyr-Phe-Gln-Gly). To affinity purify CUL1-DC^Myc9 and SKP2^Myc9, cell extracts (from one hundred 15 cm plates) were prepared in lysis buffer (20 mM HEPES (pH 7.2), 150 mM NaCl, 0.2% triton X-100, 10 mM EDTA, 50 mM NaF, mM DTT, 1 mM PMSF, 1 mM benzamidine, 0.25 mg/ml pepstatin, and 5 mg/ml each of leupeptin, aprotinin, and chymostatin), and 500 mg of extract was adsorbed to anti-Myc beads (2 hr at 4°C). Beads were washed 4 times with lysis buffer and eluted with 66 U/ml TEV protease (Gibco BRL) (overnight at 30oC). Eluted proteins were analyzed by SDS-PAGE and silver staining as described (1).

Two-hybrid analysis of SCF-CSN interaction. Lex A fusion in the pEG202 vector of hCUL1, hHRT1, and hSKP1 were tested for interaction with the COP9 Signalosome subunits (CSN1-8) and SKP1 fused to the activation domain in the pJG4-5 vector. pEG202 constructs were transformed into the yeast strain EGY48, while the pJG4-5 constructs were transformed into the yeast strain L40. The resulting transformants were mated and the diploid strains carrying the different construct combinations indicated in the table were tested for b-galactosidase activity in both plate and liquid assay (9).

Subcellular fractionation of S. pombe spheroplast extract. Strains were grown in 50 ml YES media until OD₅₉₅ = 0.5, harvested and washed in ice cold 'stop' buffer (50 mM NaF, 10 mM TRIS 7.5, and 0.02% NaAzide) and resuspended in 600 ml Buffer S [1.4 M Sorbitol, 40 mM HEPES (pH 7.2), 0.5 mM MgCl₂]. Spheroplasts were made by incubating the cell suspension at 30°C for 40 min in the presence of 100 mg/ml zymolyase (ICN). Cells were washed twice in 1 ml of ice cold Buffer F (20 mM HEPES (pH 7.2), 0.5 mM MgCl₂) supplemented with protease inhibitor mix, (PIM; 1 mM PMSF, 1 mM benzamidine, 0.25 mg/ml pepstatin, and 5 m g/ml each of leupeptin, aprotinin, and chymostatin) and18% Ficoll 400 (w/v). Cells were gently lysed in 200 m L of the same buffer using a stainless steel pestle (VWR). To remove unlysed cells, the suspension was gently centrifuged (2000 Krpm). Intact nuclei were isolated by layering the supernatant on 100 mL Buffer F supplemented with PIM, 7% Ficoll 400 (w/v), and 20% glycerol, followed by gentle centrifugation. Nuclei were lysed in 200 mL of 20 mM HEPES (pH 7.2), 500 mM NaCl, 1% triton X-100, 1 mM EDTA, plus PIM. Cytosolic and nuclear fractions were analyzed by SDS-PAGE followed by Western blotting. Image intensity of the Pcu1 specific band was measured using NIH Image software (v. 1.62). Total protein percentages loaded and total lysate volumes were used to calculate the relative percentage of total Pcu1 protein in the cytoplasmic or nuclear lysates (error: ±5%).

Preparation of S. pombe extracts for immunoblotting and deneddylation assays. Strains were grown in YES (fission yeast) or YPD (budding yeast) media to OD595 = 0.5, harvested, and washed in ice cold 'stop' buffer (50 mM NaF, 10 mM Tris 7.5, and 0.02% Na azide). Cells were resuspended in an equal volume of Lysis Buffer (20 mM HEPES (pH 7.2), 150 mM NaCl, 0.2% triton X-100, 10 mM EDTA, 50 mM NaF, mM DTT,) supplemented with PIM. An equal volume of glass beads was added (0.5 mm, Sigma). Cells were vortexed at maximum speed 6 times for 30 seconds. Lysates were cleared by centrifugation. Protein concentrations were determined using Bio-Rad protein assay. Lysates were used in deneddylation assays as indicated. Immunoprecipitations were performed essentially as described: unless otherwise stated, 1mg of extract was used with 0.5 mL of the indicated antibodies; immunoprecipitates were collected on protein A beads, washed four times with lysis buffer and eluted by boiling in 1x SDS-PAGE buffer. Unless otherwise indicated, 30 mg of crude cell extracts were used for Western blotting.

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