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Interactions of the COP9 Signalosome with the E3 Ubiquitin Ligase SCFTIR1 in Mediating Auxin Response
Claus Schwechheimer, Giovanna Serino, Judy Callis, William L. Crosby, Svetlana Lyapina, Raymond J. Deshaies, William M. Gray, Mark Estelle, and Xing-Wang Deng
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Supplementary Material
Probes used for northern blot in Fig. 2E:
The probes used for hybridization were EST clones (E6D11T7, IAA1; E7A12T7, IAA2; 36C1T7, IAA3; 119J5T7, IAA7; 117C3T7, IAA8; 113C1T7, IAA9; and 86G4T7, IAA16) and a cDNA clone encoding IAA5.
Experimental conditions for PSIAA6LUC degradation experiment in Fig. 2F:
Luciferase activity was measured from plants expressing the PSIAA6LUC reporter construct or the LUC reporter construct in a wild-type and CSN5 transgenic (antisense line) background, respectively (29). Protein extracts were prepared from young floral tissue as described in (3) using a buffer containing 50 mM Tris-HCl pH 7.4, 200 mM NaCl, 5 MgCl2, 1 mM ATP, 10% glycerol. The extracts were incubated at room temperature and luciferase activity was measured in duplicate at specific time points using Promega Luciferase Assay reagent. Luciferase activity at time point 0 was set as 100%. The experiments were repeated three times using protein extracts from two different plant lines. The result of one representative experiment is shown.
Experimental conditions for Fig. 3:
(A) Equal amounts of CSN5 affinity-purified COP9 signalosome from cauliflower were separated on a 12% SDS-PAGE according to (28) and used for silver staining and immunoblots, respectively. (B) Experimental conditions for the coimmunoprecipitation were as described by (27) except that the extraction/washing buffer was: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 1 mM PMSF, 1× Complete Inhibitor (Roche), 1 mM Na-vanadate, 60 mM b-glycerophosphate, 50 mM NaF, 0.1% NP-40. Antibodies as in (15, 28). (C) Expression of TIR1 is inducible by dexamethasone and induction conditions were as previously described (15). Extracts prepared from uninduced seedlings were used as negative control. Immunoprecipitation conditions were as described in (C) using a MYC-affinity resin from BabCo, Berkeley. The TBP antibody served as a negative control. (D) For the two-hybrid assays, cDNAs encoding the eight Arabidopsis COP9 signalosome subunits were inserted into the yeast two-hybrid vector pJG4-5 and tested for interaction with the SCFTIR1 subunits AtCUL1, AtRBX1 and ASK1 in pEG202. Empty vectors served as negative control (28, 32). (E) The COP9 signalosome is absent from cop9-1 mutants and reduced in the csn5 tg seedlings as exemplified by the immunoblot with the antibody against CSN8. CSN5 levels are strongly reduced in csn5 tg but still detectable in cop9-1 as a monomeric form of CSN5 is still present in the COP9 signalosome mutants that affect subunits of the COP9 signalosome other than CSN5 (6). cop/det/fus mutants where the COP9 signalosome is not affected, e.g. cop1-6, have a distribution of RUB1-modified and unmodified AtCUL1 that is identical to the wild-type. The RPT5 antibody served as a loading control (32).
Experimental conditions for Fig. 4:
A CSN5 co-suppressing line (X1#7, csn5 tg) with a weak phenotype was crossed to an axr1-3 mutant to generate axr1-3/csn5 tg lines. Root length measurements were performed as described in (11, 29).
References and Notes
3. M. T. Osterlund, C. S. Hardtke, N. Wei, X.-W. Deng, Nature 405, 462 (2000).
6. S. F. Kwok et al., Plant Cell 10, 1779 (1998).
11. C. Lincoln, J. H. Britton, M. Estelle, Plant Cell 2, 1071 (1990).
15. W. M. Gray et al., Genes Dev. 13, 1678 (1999).
27. J. M. Staub, N. Wei, X.-W. Deng, Plant Cell 8, 2047 (1996).
28. G. Serino et al., Plant Cell 11, 1967 (1999).
29. For root length measurements, seedlings were scanned and root length was measured with NIH Image software using the digital images. The average and standard error of 10 seedlings is shown for each experimental condition.
32. S. F. Kwok, J. M. Staub, X.-W. Deng, J. Mol. Biol. 285, 85 (1999).
