RUB1-AMP is an intermediate product of RUB1 activation. (A) Thiolester reactions containing radiolabeled RUB1 and 5 mM ATP or different AMP-PNP concentrations were performed. These reactions contained protein extract prepared from bacteria expressing recombinant H6-AXR1 and H6-ECR1 (see Experimental Procedures). The reaction products were separated by SDS/PAGE in the absence of DTT. (B) Thiolester assay of ECR1 or AXR1 subunits. Radiolabeled RUB1 was used in thiolester reactions that contained 10 mM ATP and bacterial protein extract containing recombinant H6-AXR1, H6-ECR1, or H6-ECR1^215A proteins. The reactions were stopped with 4× SDS/DTT loading buffer. The DTT-resistant band at 25–35 kDa was formed when the reaction was performed with ECR1 or ECR1^215A. Asterisks indicate residual GST-³²P-RUB1 remaining after thrombin digestion. (C) Purified H6-AXR1 or ECR1 were incubated with [α-³²P]ATP or [γ-³²P]ATP and cold RUB1 protein. A DTT-resistant product at ≈25 kDa (arrow) was generated only when ECR1 was incubated with [α-³²P]ATP. The label at the bottom of the gel is unincorporated ATP. (D) AMP-³²P-RUB1 (arrow) was generated by incubation of ECR1 with ³²P-RUB1 and ATP. After removing the ATP by ammonium sulfate precipitation, either buffer (lane 1) or H6-AXR1 (lane 2) was added to the reactions.

Identification of an Arabidopsis RUB E2 enzyme. (A) Alignment of UBC12 proteins and RCE1. Amino acid sequences corresponding to the RCE1 protein in Arabidopsis (accession no. AF202771), human (Hs-UBC12), S. pombe (Sp-Ubc12p), and S. cerevisiae (Sc-Ubc12p) were aligned by using the pileup program of GCG. RCE1 contains the conserved cysteine (position 112) implicated in thiolester bond formation within the highly conserved UBC domain. The residues conserved between RCE1 and these proteins are labeled in bold. (B) Thiolester formation between RUB1 and the Arabidopsis E2, RCE1. Thiolester reactions containing purified H6-AXR1, ECR1, and radiolabeled RUB1 and with or without H6-RCE1 were performed. Half of the reactions were stopped with 4× SDS loading buffer for 10 min, and the other half were stopped with 4× SDS/DTT loading buffer and were boiled for 10 min. (C) RCE1 forms a thiolester with RUB1 but not ubiquitin. Thiolester reactions containing ³²P-ubiquitin, wheat E1 UBA1, and the Arabidopsis E2 UBC1 or H6-RCE1 were performed. Half of the reactions were stopped with 4× SDS loading buffer, and the other half were stopped with 4× SDS/DTT loading buffer and were boiled for 10 min. The asterisks indicate GST-³²P-RUB1 or GST-³²P-UBQ, which remained after thrombin digestion.

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.

The site of RUB-conjugation is highly conserved among cullins from diverse organisms. Residues conserved with AtCUL1 are in bold. Boxes indicate residues that are identical in all proteins shown. AtBAC-ChrI (A. thaliana, GenBank accession no. AC002330), CeCUL-4 (Caenorhabditis elegans, GenBank accession no. U58086), MmCUL-1 (Mus musculus, GenBank accession no. AF083216), At-BAC-ChrV (A. thaliana, GenBank accession no. AB025620), HsCUL-1 (Homo sapiens, GenBank accession no. AF062536), HsCUL-4A (H. sapiens, GenBank accession no. U58090), LeCUL (Lycopersicon esculentum, European Molecular Biology Laboratory accession no. Y16124), DmChr2 (Drosophila melanogaster, accession no. AC005473), HsCul-4B (H. sapiens, accession no. U58091), CeCUL-1 and -2 (C. Elegans, accession nos. U58083 and U58085), HsCUL3 and -2 (H. Sapiens, accession nos. U58089 and gbU83410), and CeCUL-2 (C. Elegans, accession no. U58084).