Figure 3.

Requirement of 20-aa “tag” (MGSSHHHHHHSSGLVPRGSH) for PHAS-I to function as a substrate for the Tel1p kinase. (a) PHAS-I was phosphorylated by an immunoprecipitate derived from a strain with TEL1-HA (JMY48); the final two washes of the immunoprecipitate were done in the absence of PMSF. A portion of the sample was treated with thrombin (Roche Molecular Biochemicals) to remove the N-terminal 17 aa; both the untreated (lane 1) and treated (lane 2) samples were examined by gel electrophoresis. (b) Kinase reactions with immunoprecipitated Tel1p were done with two samples. One sample (lane 1) contained untreated PHAS-I. The sample in lane 2 was treated with thrombin before performing the kinase reaction. Thrombin was inactivated by addition of PMSF.