Isolation of a cDNA encoding a mammalian multiubiquitinating enzyme (E2_25K) and overexpression of the functional enzyme in Escherichia coli

The ubiquitin (Ub)-conjugating enzyme E2_25K catalyzes the synthesis of multi-Ub chains in which successive Ub units are linked by an isopeptide bond involving the ε-amino group of Lys-48 of Ub_n, and the COOH-terminal Gly residue of Ub_n+1 (Chen, Z., and Pickart, C. M. (1990) J. Biol. Chem., 265, 21835-21842). We now describe the polymerase chain reaction (PCR)-based cloning of an E2_25K-encoding cDNA from a bovine thymus library, using degenerate oligonucleotide primers based on the sequences of two E2_25K peptides. The cDNA encodes a 200-residue protein whose sequence bears similarities of 66 and 59%, respectively, to the sequences of the Ub-conjugating enzymes encoded by the UBC1 and UBC4/UBC5 genes of the yeast Saccharomyces cerevisiae. These three yeast E2s play key roles in Ub-dependent proteolysis (Seufert, W., McGrath, J. P., and Jentsch, S. (1990) EMBO J. 9, 4535-4541). Comparison of the amino acid sequence of E2_25K with other known E2 sequences strongly suggests that Cys-92, one of two E2_25K Cys residues, forms the Ub thiol ester adduct that is an intermediate in E2-catalyzed multiubiquitination. The E2_25K-encoding cDNA was overexpressed in Escherichia coli, and the recombinant E2_25K protein was purified to electrophoretic homogeneity; enzymatic assays showed that its multiubiquitinating activity was quantitatively identical with that of the native protein. The availability of a cloned cDNA will allow us to assess the physiological role of E2_25K.