FIGURE 4.

Cdc14 Ser(P) selectivity exists in vivo. A, schematic of Acm1 protein showing the location and sequence of the four Ser(P)-containing Cdk consensus sites and the corresponding amino acid substitutions made to create the Acm1-S4T mutant. B, extracts from asynchronous cultures of cells expressing Acm1 or Acm1-S4T from the natural ACM1 promoter before and after galactose-induced overexpression of 3HA-Cdc14 were analyzed by SDS-PAGE and immunoblotting. G6PD is a loading control. pAcm1 represents a slow mobility Cdk-phosphorylated form of Acm1. C, anti-HA antibody resin was used to isolate 3HA-Cdc14-C283S and interacting proteins from soluble extracts of asynchronous cultures expressing either wild-type Acm1 or Acm1-S4T from the natural ACM1 promoter. Immunoblotting was used to detect the indicated proteins in the initial extracts and after anti-HA IP. G6PD is a loading control.