Figure 2.
ULP1a Targets BZR1 for DeSUMOylation
(A) BZR1-GFP interacts with HA-ULP1a. N. benthamiana leaves transiently expressing BZR1-GFP or BZR12K/R-GFP with HA-ULP1a were collected for immunoprecipitation. Subsequently, total protein was subjected to immunoprecipitation with anti-GFP immunoaffinity beads (IP: αGFP) followed by immunoblot analysis with anti-HA (IB: αHA) antibodies to detect HA-ULP1a and αGFP (IB: αGFP) antibodies to detect BZR1-GFP. Total protein of all samples was probed with anti-HA antibody to determine ULP1a protein levels (HA-ULP1a input). GFP was used as a negative control.
(B) ULP1a is a bona fide SUMO protease that deSUMOylates BZR1 in vitro. High-molecular-weight conjugates of His-SUMO1-modified, GST-tagged BZR1 were formed by incubating purified SUMO E1 (SAE1 and 2) and E2 (SCE1) with BZR1-GST in the presence or absence of His-ULP1a and subsequently immunoblotted with anti-SUMO1 (αSUMO1) (upper panel) to detect SUMO chains, anti-His (αHis) (middle panel) to detect His-ULP1a, and anti-GST (αGST) to detect BZR1-GST (lower panel).
(C) N. benthamiana leaves transiently expressing BZR1-GFP and SUMO-HA with or without HA-ULP1a were collected for immunoprecipitation. Subsequently, total protein was subjected to immunoprecipitation with αGFP immunoaffinity beads (IP: αGFP) followed by immunoblot analysis with αSUMO1 (IB: αSUMO1) antibodies to detect HA-SUMO and αGFP (IB: αGFP) antibodies to detect BZR1-GFP. Equal amounts of HA-ULP1A protein were ascertained by probing with anti-HA antibodies. GFP was used as a negative control.
(D) ULP1a colocalizes with BZR1 in the cytoplasm. N. benthamiana leaves co-infiltrated with ULP1a-mCherry and BZR1-GFP were analyzed for fluorescence after 3 days. Before imaging, the leaves were infiltrated with 1 μM BL or 2 μM BRZ and incubated for 1 h to see the effect of the treatments. Images were obtained using confocal laser scanning microscope Carl Zeiss Airyscan 880. Scale bar, 20 μm.
(E) The accumulation of ULP1a protein is affected by brassinosteroid availability. 10-day-old 35S::ULP1a-HA transgenic Arabidopsis seedlings were treated with a combination of 200 μM cycloheximide + 1 μM BL or 2 μM BRZ. Total proteins extracted at indicated time points were immunoblotted with anti-HA (IB: αHA) antibody.
See also Figure S2.