Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle

• DOI: 10.7554/elife.67390
• Publication Date: 2021-07-26T12:00:32Z
• Authors: Miriam Böhm; Kerstin Killinger; Alexander Dudziak; Pradeep Pant; Karolin Jänen; Simone Hohoff; Karl Mechtler; Mihkel Örd; Mart Loog; Elsa Sanchez-Garcia; Stefan Westermann
• Keywords: Protein Degradation; Protein Stability

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. It is still poorly understood how efficient, centromere-dependent kinetochore assembly accomplished from hundreds individual building blocks in a cell cycle-dependent manner. Here, by combining comprehensive phosphorylation analysis native Ctf19CCAN subunits with biochemical functional assays model system budding yeast, we demonstrate Cdk1 activates phospho-degrons on essential subunit Ame1CENP-U, which recognized E3 ubiquitin ligase complex SCF-Cdc4. Gradual degron motifs culminates M-phase targets for degradation. Binding Mtw1Mis12 shields proximal phospho-degron, protecting kinetochore-bound Ame1 degradation machinery. Artificially increasing strength partially suppresses temperature sensitivity cdc4 mutant, while overexpression Ame1-Okp1 toxic SCF mutants, demonstrating physiological importance this mechanism. We propose phospho-regulated clearance excess CCAN facilitates efficient assembly. Our results suggest novel strategy can be used regulate complexes.