Fig 1.
COP1 associates with CRY1 and CRY2 in a blue-light dependent manner in vivo.
(A, B) Co-immunoprecipitation of CRY1 (A) and CRY2 (B) by YFP-COP1. Transgenic 35S::YFP-COP1 seedlings were grown in darkness (D) for 4 days and subsequently transferred to blue light (B) of a fluence rate of 50 μmol m-2 s-1 for 1 h (A) or 5 min (B). Protein extracts were immunoprecipitated using α-GFP beads. YFP-COP1 was detected using α-GFP antibodies; CRY1 and CRY2 were detected using α-CRY1 and α-CRY2 antibodies. Asterisks likely indicate phosphorylated CRY1 and CRY2, respectively. All signals in (A) and (B) were from the same respective membrane. The YFP-COP1 signals (A, B) of the input samples were from longer exposures than those from the co-immunoprecipitates. The CRY1 signals (A) of the input samples were from shorter exposure than those from the co-immunoprecipitates.
Fig 2.
The B-induced in vivo-association of COP1 with CRY1 requires SPA proteins, while COP1 associates with CRY2 independently of SPA.
(A, B) Co-immunoprecipitation of CRY1 (A) and CRY2 (B) by YFP-COP1 in a SPA wild-type (YFP-COP1) or spa null background (YFP-COP1 spaQn). Seedlings were grown in darkness for 4 days (D) and subsequently transferred to blue light (B) of a fluence rate of 50 μmol m-2 s-1 for 1 h (A) or 5 min (B). Protein extracts were immunoprecipitated using α-GFP beads. YFP-COP1 was detected using α-GFP antibodies. YFP-COP1 expression is very low and frequently only detectable after immunoprecipitation. CRY1 and CRY2 were detected using α-CRY1 and α-CRY2 antibodies. Asterisks likely indicate phosphorylated CRY1 and CRY2, respectively. The samples shown in (B) were on the same membrane with one lane digitally removed in the center.
Fig 3.
Co-expression of SPA1 increases the interaction between COP1 and CRY1 in yeast three-hybrid experiments.
(A) Yeast two-hybrid assay analyzing the interaction between CRY1 and COP1. (B) Yeast three-hybrid assay analyzing the CRY1-COP1 interaction in the presence or absence of co-expressed SPA1. (C) Yeast three-hybrid assay analyzing the CRY1-SPA1 interaction in the presence or absence of co-expressed COP1. Co-transformed yeast cells were grown in darkness for 24 h and exposed to B (50 μmol m-2 s-1) or kept in darkness for 24 h before measuring ß-galactosidase activity. Error bars represent the SEM of three biological replicates. Asterisks indicate significant differences between the indicated comparisons (** p < 0.01, *** p < 0.001, n.s. not significant at p = 0.05).
Fig 4.
A SPA1 deletion-protein defective in COP1-interaction shows no or a strongly reduced in vivo-association with CRY1 and CRY2, respectively.
(A) ΔCC SPA1-HA lacking the coiled-coil domain fails to interact with COP1 in vivo. Seedlings were grown in darkness for 4 d. (B, C) Co-immunoprecipitation of CRY1 (B) and CRY2 (C) by SPA1-HA and ΔCC SPA1-HA. Seedlings were grown in darkness for 4 days (D) and subsequently transferred to blue light (B) of a fluence rate of 50 μmol m-2 s-1 for 1 h (B) or 5 min (C). HA-tagged proteins were immunoprecipitated using α-HA-coupled beads. SPA1-HA and ΔCC SPA1-HA were detected using α-HA antibodies. CRY1 and CRY2 were detected using α-CRY1 and α-CRY2 antibodies. Asterisks likely indicate phosphorylated CRY1 and CRY2, respectively. Images in (B) separated by a vertical bar represent the same membrane which was exposed for different periods of time.