FIGURE 3:

Ptc1 dephosphorylates Pom1 in vitro and its activity contributes to its localization. (A) In vitro phosphatase assay, using recombinant proteins. Autophosphorylated recombinant GST-Pom1 was used as substrate and incubated with recombinant GST-Ptc1 (WT or 3A mutant) or commercial PP1 as indicated. Autophosphorylated Pom1 migrates as a smear, which collapses to two fast-migrating bands upon dephosphorylation. Phosphatase-treated Pom1 was then used in a kinase assay to monitor Pom1 activity. Recombinant MBP-Cdc15C was used as Pom1 substrate, as this fragment is quantitatively phosphorylated by Pom1 leading to slower migration (Bhattacharjee et al., 2020). Note that the phosphorylation status of Pom1 does not alter its activity toward Cdc15C. (B) Cell lengths at division quantified from more than 160 cells in three independent experiments; statistical significance measured by Student’s t test against wild type or pom1∆. (C) Localization of Ptc1-GFP and Ptc1^3A-GFP (left) and quantification of cortical profiles (right). Scale bar: 5 µm.