Fig. 6.
The SUMO proteases OTS1 and OTS2 mediate de-SUMOylation of phyB. (A) The ots1/ots2 mutant is hyposensitive to RL. Fluence rate-dependent relative inhibition of hypocotyl elongation of Col-0 (black square), phyB-9 (star symbol), ots1/ots2 (gray square), and ots1/ots2/phyB-9 (white triangle) seedlings grown in RL is shown. For additional details, see the Fig. 2 legend. (B) The ots1/ots2 mutant exhibits normal photomophogenesis in FRL or BL. Hypocotyl elongation inhibition in FRL-grown (Col-0, black triangle; ots1/ots2, white triangle) and BL-grown (Col-0, black square; ots1/ots2, white square) seedlings is shown. For additional details, see the Fig. 2 legend. (C) Accumulation of the SUMOylated form of phyB is enhanced in transgenic seedlings lacking functional OTS1/OTS2 SUMO proteases. Transgenic phyB-9 (lane 1) and ots1/ots2/phyB-9 (lane 2) seedlings expressing LIP1:PHYB-YFP were grown under 12 h L/12 h D cycles for 5 d, and samples were harvested on the sixth day at MOD. Accumulation of the SUMOylated phyB-YFP was detected as described in Fig. 1. (D) Recombinant OTS1 deSUMOylates phyB-GFP in vitro. SUMOylated phyB-GFP protein was affinity purified from total protein extracts with anti-GFP antibody and incubated with an identical amount of recombinant OTS1 (His:OTS1) (lane 1) or catalytically inactive OTS1 (His:OTS1CS) (lane 2) proteins. After incubation (typically 4 h at room temperature), the beads were reapplied to the column [the flow-through was used to determine 6xHis-OTS1 levels with anti-Histidine tag antibodies (IB:αHis)] and washed, and bound proteins were eluted with SDS/PAGE loading buffer and separated by electrophoresis, blotted, and probed with anti-GFP (IB:αGFP) and anti-SUMO1 (IB:αSUMO1) antibodies.