Homologs of the essential ubiquitin conjugating enzymes UBC1, 4, and 5 in yeast are encoded by a multigene family in Arabidopsis thaliana
The covalent attachment of the 76 amino acid protein ubiquitin is an important prerequisite for the degradation of many eukaryotic proteins. The specificity of this ligation is accomplished in part by a family of distinct ubiquitin conjugating enzymes (E2s) working in concert with specific ubiquitin‐protein ligases (E3s). Three essential E2s in yeast encoded by ScUBC1, −4, and −5 comprise a functionally overlapping E2 subfamily that appears responsible for degrading most abnormal and short‐lived proteins. A 15 kDa E2 protein homologous to this family has been identified previously in wheat germ, designated TaE215kDa (Girod and Vierstra (1993) J. Biol. Chem. 268, 955–960). This E2 is responsible for much of the ubiquitin conjugating activity observed in wheat germ extracts and works together with a unique E3 (designated E3γ) for substrate recognition. In this paper, the cloning of five genes encoding E215kDa from Arabidiopsis thaliana is described (designated AtUBC8—12). They encode 149 amino acid basic proteins 94–98% similar to each other and 88–92% similar to ScUBC4 at the amino acid sequence level. In contrast, AtUBC8—12 are only 55–65% similar to the Arabidopsis E2s encoded by AtUBC1, −4, and −7. The AtUBC8—12 proteins do not contain N‐ or C‐terminal extensions and have the active site at residue Cys‐86, based on their homology with other E2s. Analyses of genomic Southern blots are consistent with the existence of multiple members encoding this E2 subfamily. AtUBC8—12 are transcribed to yield about 800 nucleotide mRNAs that, unlike ScUBC4 and −5, are not strongly induced by heat shock. Expression of AtUBC8 in Escherichia coli results in substantial production of functional E215kDa that works together with wheat E3γ in conjugating ubiquitin to endogenous or added substrates in vitro.
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Document Type: Research Article
Affiliations: Department of Horticulture, 1575 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706, USA
Publication date: April 1, 1993