1. Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle
- Author
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Alexander Dudziak, Karl Mechtler, Stefan Westermann, Miriam Böhm, Mihkel Örd, Mart Loog, Karolin Jänen, Pradeep Pant, Elsa Sanchez-Garcia, Kerstin Killinger, and Simone Hohoff
- Subjects
S. cerevisiae ,Cell Cycle Proteins ,0302 clinical medicine ,Cell division control protein 4 ,Phosphorylation ,Biology (General) ,Kinetochores ,0303 health sciences ,biology ,Protein Stability ,Chemistry ,Kinetochore ,General Neuroscience ,General Medicine ,Chromosomes and Gene Expression ,kinetochore ,Ubiquitin ligase ,Cell biology ,DNA-Binding Proteins ,Medicine ,cell cycle ,Microtubule-Associated Proteins ,Biologie ,Cell Division ,Research Article ,Saccharomyces cerevisiae Proteins ,QH301-705.5 ,Ubiquitin-Protein Ligases ,Science ,Protein subunit ,Centromere ,Kinetochore assembly ,Mutation, Missense ,Saccharomyces cerevisiae ,Spindle Apparatus ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Amino Acid Sequence ,Mitosis ,030304 developmental biology ,Cyclin-dependent kinase 1 ,Organisms, Genetically Modified ,General Immunology and Microbiology ,F-Box Proteins ,SCF ,Cell Biology ,Cytoskeletal Proteins ,biology.protein ,Degron ,030217 neurology & neurosurgery - Abstract
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.
- Published
- 2021