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  • Title: Isolation of a cDNA encoding a mammalian multiubiquitinating enzyme (E225K) and overexpression of the functional enzyme in Escherichia coli.
    Author: Chen ZJ, Niles EG, Pickart CM.
    Journal: J Biol Chem; 1991 Aug 25; 266(24):15698-704. PubMed ID: 1714895.
    Abstract:
    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 epsilon-amino group of Lys-48 of Ubn, and the COOH-terminal Gly residue of Ubn+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).
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