The soybean Eu3 gene encodes an Ni‐binding protein necessary for urease activity

Abstract

SummaryMutation in Eu3 eliminates activity of both soybean ureases, the embryo-speci®c (encoded by Eu1) and the tissue-ubiquitous (encoded by Eu4). eu3-e1 is a completely recessive null allele. Eu3-e3 is a semidominant specifying 0.1% wild-type urease activity in the homozygous state and 5±10% as a heterozygote . Antibodies to plant UreG, a homologue of the bacterial urease accessory protein, revealed a 32 kDa protein (p32) in embryos of the Eu3/Eu3 precursor genotype. p32 is identical to UreG by the criteria of size, antigenicity, and its ability to bind Ni 2+ , a trait expected from the deduced histidine-rich N-terminus of UreG. UreG was absent in eu3-e1/eu3-e1, and lack of UreG co-segregated with eu3-e1. Eu3-e3 speci®ed a UreG transcript which coded valine in place of alanine at residue 142 (A142V) con®rming that Eu3 encodes UreG, which is renamed Eu3. Eu3 (A142V) retained Ni-binding ability. Eu3 is directly involved in urease activation, since anti-Eu3 (UreG) antibodies inhibited the in vitro activation of urease. Eu1 (embryo urease) and Eu3 accumulated in parallel in the developing embryo. The presence of Eu1 was not necessary for the high embryonic level of Eu3. However, the presence of Eu3 appeared to be important for accumulation of Eu1, perhaps by stabilizing it by Ni insertion. At the level of sensitivity employed Eu3 was detected in crude extracts of embryos but not non-embryonic tissues which have 1/500th the embryo urease activity. Functional Eu3, however, is necessary for activation of the ubiquitous urease in non-embryonic tissues.

Identification of Three Urease Accessory Proteins That Are Required for Urease Activation in Arabidopsis

“…We demonstrate in this study that Arabidopsis requires three accessory proteins to generate urease activity. Soybean plants with defects in urease activity were previously shown to contain mutations in the urease and ureG genes Torisky et al, 1994;Freyermuth et al, 2000). Transgenic ureG-antisense potato plants also displayed reduced urease activity (C.P.…”

“…A mutation in the corresponding gene Eu3 eliminated both urease activities of this plant (Freyermuth et al, 2000). Functional complementation of defective microbial urease activation complexes indicated a possible role of other putative plant accessory protein orthologs in urease activation.…”

“…In contrast, potato (Solanum tuberosum), tomato (Lycopersicon esculentum), several other solanaceous species, and also Arabidopsis (Arabidopsis thaliana; ecotype Columbia [Col]) possess only a single urease gene (Witte et al, 2005). Using microbial accessory protein sequences, putative plant orthologs for UreD, UreF, and UreG were identified in the databases and the corresponding cDNAs cloned (Freyermuth et al, 2000;Bacanamwo et al, 2002). Sequence identities determined from pair-wise alignments of K. aerogenes UreD and UreF with Arabidopsis UreD and UreF candidates are about 20% in both cases, providing only a weak indication that these proteins may be orthologous.…”

“…It was hypothesized that the function of the bacterial metallochaperone UreE may have been taken over in plants by the UreG protein. In comparison to bacterial UreG proteins, plant UreGs contain an extended N terminus rich in His and Asp residues and were shown to bind nickel (Freyermuth et al, 2000;.…”

Interallelic Complementation at the Ubiquitous Urease Coding Locus of Soybean

“…A mutation in either of them is pleiotropic, eliminating the activities of both ureases. Although Eu3 encodes a nickel-binding protein, the plant ortholog of bacterial UreG (Freyermuth et al, 2000), the identity and specific function of the Eu2 protein is still unknown. Eu2 and Eu3 proteins interact to activate urease; incubation of mixed crude extracts from developing embryos of eu2/eu2 and eu3/eu3 mutants slowly gain urease activity .…”