Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex

Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase. Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14. Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m') lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow normally and exhibit only minor defects in releasing Cdc14 during anaphase. However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene).We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets. Our study suggests that Cdc5/Polo is unusually promiscuous and highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments.In the budding yeast Saccharomyces cerevisiae, the mitotic exit network (MEN) plays a pivotal role in driving cells from mitosis to G1. Identified components of the MEN include Tem1 (a GTP-binding protein), Lte1 (a putative guanine nucleotide releasing factor), Nud1 (a spindle-pole-body protein), Cdc15 (a protein kinase), Dbf2/Mob1 (a protein kinase complex), Cdc5 (a Polo-like protein kinase), and Cdc14 (a protein phosphatase). When cells harboring conditional-lethal temperature-sensitive (ts) mutations in any of these proteins are shifted to the restrictive temperature, they uniformly arrest in late anaphase as large-budded cells with segregated chromosomes, fully elongated microtubule spindles, and in all tested cases, elevated Cdc28/Clb2 protein kinase activity (Cdc28 is the major cyclin-dependent