Demonstration of ATP-Dependent, Ubiquitin-Conjugating Activities in Higher Plants.

Ubiquitin is a highly conserved, 76-amino acid polypeptide with several important regulatory functions in both plants and animals that all arise from its covalent ligation to other cellular proteins. Here, we demonstrate that higher plants have the capacity to conjugate ubiquitin to other plant proteins in vitro. Using (125)I-labeled human ubiquitin as a substrate, conjugating activities were observed in crude etiolated tissue extracts from all species tested, including oats, rye, barley, corn, zucchini squash, pea, soybean, and sunflower. The reaction has a soluble distribution, is specific for ATP, and requires the protease inhibitor, leupeptin, to protect ubiquitin from inactivation during the assay. Conjugation is inhibited by N-ethylmaleimide and high concentrations of 2-mercaptoethanol suggesting that the mechanism of ubiquitin ligation in plants involves a similar thiolester intermediate to that found in the mammalian pathway. The conjugating activity in etiolated oat extracts is extremely labile with a half-life of about 20 minutes at 30 degrees C. Detectable but low ATP-stimulated, conjugating activities were also observed in extracts from dry seeds and green leaves of oats. In addition to this conjugating activity, crude plant extracts have the capacity to degrade ubiquitin-protein conjugates formed in vitro. These results demonstrate that higher plants contain several of the enzymic activities necessary for ubiquitin's functions and provide a method for assaying ubiquitin conjugation in vitro.