Abstract
Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta, we demonstrate that a urease–UreD–UreF–UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation in vitro was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated in vitro by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential HXH metal-binding sites. Complementing the ureG-1 mutant of Arabidopsis with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first HXH motif is highly beneficial and up to the second HXH motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo. Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process.
Highlights
The urease accessory proteins (UAPs) interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site
Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta, we demonstrate that a urease–UreD– UreF–UreG complex exists in plants and show its stepwise assembly
We show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo
Summary
To obtain a complete interaction map of a plant urease and its accessory proteins (UAPs), the respective Arabidopsis cDNAs were cloned into binary vectors for transient expression in Nicotiana benthamiana. Co-purification of urease with tagged UreF was only possible in the presence of UreD (lane 10) and with tagged UreG only in the presence of UreD and UreF (lane 4) These findings in dicotyledonous Arabidopsis prompted us to investigate the urease activation complex in rice as an important representative of the monocots. From leaves of N. benthamiana transiently expressing rice urease and the three rice UAPs (with UreD tagged), a urease–UreD–UreF–UreG complex could be purified (supplemental Fig. S4). An assembly model derived from the co-purification data (Fig. 2) suggests that there are two main routes toward the urease–UreD–UreF–UreG complex: (i) binding of UreD to urease followed by UreF and UreG or (ii) the formation of a UreD–UreF–UreG complex by binding of UreG to preformed UreD–UreF, which can interact with urease. Urease could be recovered after incubation with a matrix loaded with UreD–UreF– UreG (Fig. 3, lane 5) but not in a control experiment with empty matrix (Fig. 3, lane 6) demonstrating that urease can bind to a preformed UreD–UreF–UreG complex
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