Figure 5.

Phosphorelay experiments. (a) Schematic diagram representing the purified components of the SLN1 pathway. For phosphorelay experiments, one can start with ³²P-labeled GST-Sln1-HK or ³²P-labeled Sln1-Rec as the initial phosphodonor. Experiments are conducted with all phosphorelay components in the reaction mix in order to determne the total amount of radiolabel transmitted to the terminal phosphoreceiver (either Ssk1 or Skn7). (b) An example of a phosphorelay assay to examine phosphorylation of GST-Skn7 comparing WT Sln1-Rec to the Sln1* receiver mutant (P1148S). Equimolar concentrations of GST-Skn7, GST-Sln1-HK, Ypd1 and ³²P-labeled Sln1-Rec (WT) or Sln1*-Rec were incubated together for 15 minutes (lanes 2 and 4, respectively). Lanes 1 and 3 are the ³²P-labeled Sln1-Rec (WT) and Sln1*-Rec, respectively, incubated alone in reaction buffer. This in vitro experiment supported the in vivo genetic results that indicated that the P1148S mutation in the Sln1 receiver domain results in a hyperactive phenotype, i.e. an increase in phosphorelay activity leading to a higher level of Skn7 phosphorylation. No reverse phosphotransfer from Sln1-Rec to GST-Sln1-HK was observed (lanes 2 and 4) (reproduced from Ault et al., 2000). (c) An example phosphorelay experiment examining Ypd1-mediated phosphotransfer from Sln1-Rec to Ssk1-Rec. Equimolar concentrations of WT Ypd1 (lane 2) or a H64Q mutant Ypd1 (lane 3) was incubated with ³²P-labeled Sln1-Rec and Ssk1-Rec for 5 minutes Lane 1 contains radiolabeled Sln1-Rec incubated alone. Using WT Ypd1, virtually all the radiolabel is transferred to Ssk1-Rec within 5 minutes (lane 2). In contrast, when the H64 residue within Ypd1 is mutated, little or no phosphotransfer is observed (reproduced from Janiak-Spens et al., 2000).