Isolation of a cDNA encoding a mammalian multiubiquitinating enzyme (E225K) 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 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).
Chen
Z J
ZJ
Department of Biochemistry, State University of New York, Buffalo 14214.
Niles
E G
EG
Pickart
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008025:Ligases / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D012689:Sequence Homology, Nucleic Acid; D044763:Ubiquitin-Conjugating Enzymes
D012313:RNA; D004247:DNA; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases