Figure 2.

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.