Ubiquitin-Dependent Proteolytic Pathway in Wheat Germ: Isolation of Multiple Forms of Ubiquitin-Activating Enzyme, E1

Ubiquitin is a highly conserved protein involved in several important regulatory processes through its ATP-dependent, covalent ligation to a variety of eukaryotic target proteins. We describe here the characterization of ubiquitin conjugation in wheat germ extracts and the subsequent isolation of enzymes involved in conjugation. With ¹²⁵I-ubiquitin as a substrate, wheat germ extracts form conjugates with either endogenous or added proteins. Conjugation requires ATP and has a pH optimum of ~8, and the conjugating activity is relatively stable over time. In addition, activities responsible for the ATP-dependent degradation and disassembly of ubiquitin conjugates have been detected in vitro. Ubiquitin-activating enzyme (E1) was purified from wheat germ extracts by using a modification of the covalent affinity chromatography procedure of Ciechanover et al. [(1982) J. Biol. Chem. 267, 2537-2542], E1 from wheat germ, like that from rabbit reticulocytes, formed thiol ester intermediates with ubiquitin in the presence of ATP. Purified E1 preparations contained three polypeptides of apparent molecular masses of 117, 123, and 126 kDa after NaDodS04-PAGE. Under nondenaturing conditions, these proteins have native molecular masses of ~ 115 kDa, indicating that they exist as monomers. We concluded that all three species were E1 on the basis of their coelution with E1 activity, by immunorecognition by anti-human E1 antibodies, and by the similarity of their peptide maps. Furthermore, antibodies prepared against wheat germ E1's recognized E1 from rabbit reticulocytes. All three wheat germ E1's were detected in crude extracts prepared under conditions that minimized proteolysis, suggesting that the heterogeneity of the purified E1 preparations was not the result of posthomogenization breakdown. The immunological similarity of animal and plant E1's indicates that this conjugation enzyme, like ubiquitin, has been conserved through evolution.