RESUMO
Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.
Assuntos
Proteínas de Ciclo Celular , Cinetocoros , Microtúbulos , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Cinetocoros/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Microtúbulos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas NuclearesRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Divisão Celular , Centrômero/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/química , Proteínas F-Box/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação de Sentido Incorreto , Organismos Geneticamente Modificados , Fosforilação , Estabilidade Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Fuso Acromático/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Kinetochores are chromatin-bound multi-protein complexes that allow high-fidelity chromosome segregation during mitosis and meiosis. Kinetochore assembly is exclusively initiated at chromatin containing Cse4/CENP-A nucleosomes. The molecular mechanisms ensuring that subcomplexes assemble efficiently into kinetochores only at centromeres, but not anywhere else, are incompletely understood. Here, we combine biochemical and genetic experiments to demonstrate that auto-inhibition of the conserved kinetochore subunit Mif2/CENP-C contributes to preventing unscheduled kinetochore assembly in budding yeast cells. We show that wild-type Mif2 is attenuated in its ability to bind a key downstream component in the assembly pathway, the Mtw1 complex, and that addition of Cse4 nucleosomes overcomes this inhibition. By exchanging the N-terminus of Mif2 with its functional counterpart from Ame1/CENP-U, we have created a Mif2 mutant which bypasses the Cse4 requirement for Mtw1 binding in vitro, thereby shortcutting kinetochore assembly. Expression of this Mif2 mutant in cells leads to mis-localization of the Mtw1 complex and causes pronounced chromosome segregation defects. We propose that auto-inhibition of Mif2/CENP-C constitutes a key concept underlying the molecular logic of kinetochore assembly.
