Figure 3.

Modification of AtCUL1 with RUB1. (A) AtCUL1 is modified with RUB1. In vitro translated ³⁵S-H6-AtCUL1 was incubated in the reticulocyte lysate with the GST or GST-RUB1. ³⁵S-H6-AtCUL1^722M or ³⁵S-H6-AtCUL1^692M were incubated with GST-RUB1 in the reticulocyte lysate. (B) Amino acids sequences corresponding to the C-terminal region of cullin proteins from Arabidopsis (AtCUL1), humans (HsCul 4A), and S. cerevisiae (Cdc53). K1(722) and K2(692) correspond to conserved lysines in this C-terminal region. The residues conserved between AtCUL1 and HsCul-4A or Cdc53p are labeled in bold. The asterisk indicates that HsCul-4A is a partial cDNA. (C) ³⁵S-H6- AtCUL1 was purified from the reticulocyte lyaste and was added to reactions that also contained H6-AXR1 and ECR1 or H6-AXR1, ECR1, and H6-RCE1. The arrow indicates the ³⁵S-H6-AtCUL1 protein modified with GST-RUB1. (D) Western blot analysis of protein extracts from wild-type and transgenic H6-S-RUB1 seedlings. Total protein extract was probed with the antibody against AtCUL1 or with the S-peptide detection kit (Novagen). Blot containing nickel-agarose purified proteins from wild-type and transgenic H6-S-RUB1 seedlings was probed with the antibody against AtCUL1. The arrow indicates the possible AtCUL1 modified with RUB1. The asterisk indicates the position of AtCUL1 modified with H6-S-RUB1.