Figure 3.

Phosphorylation of receiver domains. (a) Schematic showing strategy for co-incubation of bead-bound GST-Sln1-HK and receiver domains in the presence of [γ-³²P]ATP for a period of time (~30 minutes) that allows for autophosphorylation of GST-Sln1-HK and rapid phosphotransfer to the receiver domain. The phosphorylated receiver domain can then be recovered in the supernatant by pelleting the bead-bound GST-Sln1-HK. The isolated P~Rec can then be used for various in vitro biochemical studies. (b) An example phosphotransfer experiment using Sln1-Rec~P as a phosphodonor to Ypd1 (reproduced from Janiak-Spens et al., 2000). Isolated ³²P-labeled Sln1-Rec (1.6 μM) was incubated with 2 μM Ypd1 and aliquots were removed at the indicated time points. This time course experiment showed that with respect to distribution of radiolabel for WT proteins, equilibrium is reached at the earliest time point taken (8 sec.). For mutant studies, it is useful to establish a time frame for optimal phosphotransfer.