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

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 is accomplished from hundreds of individual protein building blocks in a cell cycle-dependent manner. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits ...
with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U, which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-phase and targets the protein for degradation. Binding of the Mtw1Mis12 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic in SCF mutants, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits facilitates efficient centromere-dependent kinetochore assembly. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.
Mesh Terms:
Amino Acid Sequence, Cell Cycle Proteins, Cell Division, Centromere, Cytoskeletal Proteins, DNA-Binding Proteins, F-Box Proteins, Kinetochores, Microtubule-Associated Proteins, Mutation, Missense, Organisms, Genetically Modified, Phosphorylation, Protein Stability, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spindle Apparatus, Ubiquitin-Protein Ligases
Elife
Date: Dec. 26, 2020
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