An Arabidopsis SUMO E3 Ligase, SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity
Fig 3
SIZ1 physically interacts with COP1, and mediates SUMO modification of COP1 at K193.
(A) BiFC assay indicating that SIZ1-YFPC interacts with COP1-YFPN (left panel) in the nucleus of N. benthamiana leaf cells in the light (1 h white-light). N. benthamiana cells co-expressing SIZ1-YFPC and YFPN (middle panel) and YFPC and COP1-YFPN (right panel) were used as negative controls. Bar = 10 μm. (B) Co-immunoprecipitation analysis showing that SIZ1-GFP is associated with Myc-COP1. SIZ1-GFP and Myc-COP1 were transiently co-expressed in Col-0 protoplasts. Co-immunoprecipitated SIZ1-GFP was detected with anti-GFP antibody. Empty Myc vector (Vector) was used as a negative control. (C) In vitro sumoylation of COP1. Sumoylated COP1 was detected with anti-FLAG and anti-SUMO1 antibodies. Arrowheads indicate possible sumoylated COP1. (D) In vivo sumoylation of COP1. Myc-COP1 and FLAG-SUMO1 were transiently co-expressed in Col-0 protoplasts. Myc-COP1 was immunoprecipitated with anti-Myc antibody and sumoylated COP1 (SUMO1-COP1) was detected with anti-FLAG antibody. FLAG-SUMO1AA was co-transformed with Myc-COP1 as a negative control. Input Myc-COP1 was detected with anti-Myc antibody. Input FLAG-SUMO1 and FLAG-SUMO1 AA were detected with anti-FLAG antibody in a separate blot, shown in S5A Fig. (E) Sumoylation of COP1 in planta. Total proteins were extracted from five-day-old dark-grown 35S-Myc-COP1 and Col-0 (control) seedlings, and anti-Myc antibody was used to immunoprecipitate Myc-COP1. Anti-SUMO1 antibody was used to determine sumoylated COP1. Input and immunoprecipitated Myc-COP1 were detected with anti-Myc antibody. Arrowhead indicates non-sumoylated COP1 band. Asterisks indicate sumoylated COP1 bands. (F) Light exposure reduces sumoylation levels of COP1. Myc-COP1 and FLAG-SUMO1 co-expressing N. benthamiana leaves were incubated under darkness for 12 h, and then exposed to white light (150 μmol m-2 s-1) for 12 h. The nuclear proteins were isolated at the end-of-dark (0 h) and the end-of-light (12 h) period, and the sumoylation level of COP1 was analyzed as described in (D). Input FLAG-SUMO1 and FLAG-SUMO1 AA were detected with anti-FLAG antibody in a separate blot, shown in S5B Fig. (G) The level of COP1 sumoylation was substantially lower in siz1-2 than in Col-0. Myc-COP1 and FLAG-SUMO1 were transiently co-expressed in Col-0 or siz1-2 protoplasts, and the sumoylation level of COP1 was analyzed as described in (D). (H) K193 is a primary sumoylation site in COP1. FLAG-SUMO1 was transiently co-expressed with Myc-COP1, Myc-COP1K14R, Myc-COP1K193R, or Myc-COP1K653R in Col-0 protoplasts, and immunoprecipitation was performed as described in (D).