Conjugation of multiple ubiquitins serves as a committed step in the degradation of a variety of intracellular eukaryotic proteins by the 26S proteasome. Conjugates are formed via a three-enzyme cascade; the initial step requires ubiquitin-activating enzyme (E1), which couples ubiquitin activation to ATP hydrolysis. Previously, we showed that many higher plants contain multiple E1 proteins and described several E1 genes from wheat. To facilitate understanding of the roles of the different plant E1s, we characterized the E1 gene and protein family from Arabidopsis thaliana. Arabidopsis E1s are encoded by two genes (AtUBA1 and AtUBA2) that synthesize approximately 123-kDa proteins with 81% amino acid sequence identity to each other and 44-75% sequence identity with confirmed E1s from other organisms. Like other E1 proteins, AtUBA1 and 2 contain a cysteine residue in the putative active site for forming the ubiquitin thiol-ester intermediate. Enzymatic analysis of the corresponding proteins expressed in Escherichia coli demonstrated that both proteins activate ubiquitin in an ATP-dependent reaction and transfer the activated ubiquitin to a variety of Arabidopsis E2s with near equal specificity. Expression studies by quantitative RT-PCR and histochemistry with transgenic plants containing AtUBA promoter-beta-glucuronidase-coding region fusions showed that the AtUBA1 and 2 genes are co-expressed in most, if not all, Arabidopsis tissues and cells. Collectively, the data indicate that E1 proteins, and presumably the rest of the ubiquitin pathway, are present throughout Arabidopsis. They also show that the AtUBA1 and 2 genes are not differentially expressed nor do they encode E1s with dramatically distinct enzymatic properties.

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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...

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Phytochromes are a family of related chromoproteins that regulate photomorphogenesis in plants. Ectopic overexpression of the phytochrome A in severa1 plant species has pleiotropic effects, including substantial dwarfing, increased pigmentation, and delayed leaf senescence. We show here that the dwarf response is related to a reduction in active gibberellins (CAs) in tobacco (Nicofiana fabacum) overexpressing oat phytochrome A under the control of the cauliflower mosaic virus (CaMV) 35s promoter and can be suppressed by foliar applications of gibberellic acid. In transgenic seedlings, high concentrations of oat phytochrome A were detected in stem and petiole vascular tissue (consistent with the activity of the CaMV 35s promoter), implicating vascular tissue as a potential site of phytochrome A action. To examine the efficacy of this cellular site, oat phytochrome A was also expressed using Arabidopsis chlorophyll a/b-binding protein (CAB) and the Arabidopsis ubiquitin (UBQ7) promoters. Neither promoter was as effective as CaMV 35s in expressing phytochrome in vascular tissue or in inducing the dwarf phenotype. Collectively, these data indicate that the spatial distribution of ectopic phytochrome i s important in eliciting the dwarf response and suggest that the phenotype is invoked by elevated levels of the far-red-absorbing form of phytochrome within vascular tissue repressing CA biosynthesis.Phytochromes are a family of regulatory photoreceptors in plants that measure both the quantity and quality of available light (for reviews, see Quail, 1991;Vierstra, 1993;Kendrick and Kronenberg, 1994). They are dimeric, cytoplasmic proteins, with each subunit consisting of a linear tetrapyrrole chromophore covalently linked to an approx-