FIG. 5.

YPD1-dependent phosphoryl transfer from phospho-CheY to SSK1-R2. CheY (3 μM) and YPD1 (12 μM) were incubated in the presence of 0.7 mM acetyl [³²P]phosphate in a total volume of 100 μl in 50 mM Tris-HCl (pH 7.5)–5 mM MgCl₂–1 mM DTT at room temperature. Aliquots (9 μl) were removed at the indicated time points and added to 3 μl of 4× stop buffer (lanes 1 to 4). After 30 min, SSK1-R2 was added to 4 μM, incubation was continued, and aliquots (12 μl) were removed and mixed with 4 μl of 4× stop buffer at indicated time points (lanes 5 to 9). Reaction products were separated on an SDS–15% polyacrylamide gel and analyzed as described in the legend to Fig. 3. Molecular size markers are indicated on the left.