Ubiquitin-dependent proteolytic pathway in wheat germ: isolation of multiple forms of ubiquitin-activating enzyme, E1

Abstract

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 (El) 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], El from wheat germ, like that from rabbit reticulocytes, formed thiol ester intermediates with ubiquitin in the presence of ATP. Purified El 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 El on the basis of their coelution with El activity, by immunorecognition by anti-human El antibodies, and by the similarity of their peptide maps. Furthermore, antibodies prepared against wheat germ El's recognized El from rabbit reticulocytes. All three wheat germ El's were detected in crude extracts prepared under conditions that minimized proteolysis, suggesting that the heterogeneity of the purified El preparations was not the result of posthomogenization breakdown. The immunological similarity of animal and plant El's indicates that this conjugation enzyme, like ubiquitin, has been conserved through evolution. e highly conserved, 76 amino acid protein ubiquitin is present in eukaryotic cells in a free form and covalently bound to various intracellular proteins (Finley & Varshavsky, 1985;Hershko & Ciechanover, 1986). As a consequence of ligation, ubiquitin is involved in several regulatory processes. Best characterized of these is its role in selective protein degradation, where conjugation serves to commit proteins to degradation (Hershko & Ciechanover, 1986; Hough & Rechsteiner, 1986). Less characterized functions include the possible regulation of chromatin structure (Goldknopf & Busch, 1977), DNA repair (Jentsch et al., 1987), and cell surface recognition (Siegelman et al., 1986).Ligation of ubiquitin to cellular proteins requires ATP and is catalyzed by a multienzyme system initially observed in rabbit reticulocyte lysates (Hershko et al., 1983). In this series of reactions, an unusual peptide bond is formed between the C-terminal glycine carboxyl group of ubiquitin and primary amino groups on the target protein. In the initial step of ligation, ubiquitin-activating enzyme (El) adenylates the carb...

Proteolytic activity during senescence of plants

“…Vierstra's group has isolated the Ub-activating enzyme. El (Hatfield & Vierstra, 1989) and the Ub carrier or conjugating protein, E2 (Sullivan & Vierstra, 1989) from wheat germ. Determination of the Ub DNA sequences from oat and Arabidopsis (Vierstra, Langan & Schaller, 1986;Burke, Callis & Vierstra, 1989), and sunflower (Binet, Steinmetz & Tessier, 1989) show that it is probably as highly conserved as that from mammals.…”

Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway

“…Ubiquitin thiolester formation to the different E2 isozymes occurs by transfer of this moiety from a ternary complex of ubiquitin-activating enzyme (E1) containing two forms of activated ubiquitin polypeptide: a tightly bound ubiquitin adenylate intermediate and a covalent E1-ubiquitin thiolester (19,20). Plants inexplicably contain multiple E1 isozymes (21), whereas other eukaryotes possess a single gene that is transcribed into a 3.5-kb message subject to translation at alternate start sites to yield cytoplasmic and nuclear isoforms of 110 and 117 kDa, respectively (22). The additional amino-terminal 40 residues of the 117-kDa E1 contain a nuclear localization signal and multiple phosphorylation sites that promote sequestering of this isozyme within the nucleus (22).…”

Conjugation of Ubiquitin to Proteins from Green Plant Tissues

“…A pH of 7.6 is commonly used for the ubiquitin binding assay in reticulocytes, and an optimum for extracts from wheat germ was approximately pH 8 (10). When in the pH range of 5.5 to 9.5, optimum binding occurred between pH 8.0 and 8.5 (Fig.…”