FIG. 3.

Characterization of Pho85p-Pcl10p kinase reconstituted from polypeptides produced in E. coli. (A) Phosphorylation of Gsy2p by Pho85p requires Pcl10p. Purified Pho85p (80 nM) was incubated together with Gsy2p (3 μM) and the indicated concentrations of Pcl10p. After 10 min at 30°C, the phosphorylation reaction was initiated by the addition of [γ-³²P]ATP and MgCl₂. After a further 10-min incubation, the reaction was terminated and the incorporation of ³²P into Gsy2p was determined by liquid scintillation counting. The data were analyzed as described in Materials and Methods. The concentration of Pcl10p required to give half the maximal rate was calculated to be ∼0.2 μM. (B) Effect of Gsy2p concentration on the rate of phosphorylation. Pho85p (50 nM) and either 50 nM (□) or 2 μM (●) Pcl10p were incubated with the indicated concentrations of Gsy2p, and the initial rate of Gsy2p phosphorylation was determined.