Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle
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Evaluation Summary:
This paper will be of interest to those in the fields of chromosome biology, mitotic regulation, and proteostasis. The authors put forward an interesting model of phosphodegron regulation of kinetochore assembly based on convincing genetic and biochemical data. The novel model will require some additional evidence before it can be considered well-supported, but the paper represents an advance in our knowledge of kinetochore regulation with experiments that are rigorous, well-designed and carefully conducted.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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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 Ctf19 CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1 CENP-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 Mtw1 Mis12 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.
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Reviewer #3 (Public Review):
In this manuscript, Böhm et. al. aim to understand how precise kinetochore assembly is tied to cell cycle progression in budding yeast. In this work, the authors identify CDK phosphorylation sites concentrated in the N-terminus of Ame1, a protein of the COMA complex, and set out to characterize the role these phosphorylation sites may play protein function at the kinetochore. Although phospho-null Ame1 does not affect cell viability, expressing an Ame1 mutant that lacks the phosphorylated domain results in cell death. Interestingly, overexpression of the phospho-null Ame1 mutant accumulates to a higher level than the wild type protein leading the authors to hypothesize that these phosphorylation sites function as phosphodegrons in the Ame1 protein. Through molecular modeling and genetic analysis, the authors …
Reviewer #3 (Public Review):
In this manuscript, Böhm et. al. aim to understand how precise kinetochore assembly is tied to cell cycle progression in budding yeast. In this work, the authors identify CDK phosphorylation sites concentrated in the N-terminus of Ame1, a protein of the COMA complex, and set out to characterize the role these phosphorylation sites may play protein function at the kinetochore. Although phospho-null Ame1 does not affect cell viability, expressing an Ame1 mutant that lacks the phosphorylated domain results in cell death. Interestingly, overexpression of the phospho-null Ame1 mutant accumulates to a higher level than the wild type protein leading the authors to hypothesize that these phosphorylation sites function as phosphodegrons in the Ame1 protein. Through molecular modeling and genetic analysis, the authors determine that Ame1 is a substrate of the SCF E3 ubiquitin ligase and is likely recognized by the Cdc4 F box protein. The authors go on to convincingly show that phosphorylation of what is referred to as the "CDC4 phosphodegron domain" is phosphorylated in a step-wise manner that is cell cycle dependent and that the phosphorylated Ame1 protein specifically is degraded in mitosis. In addition to Ame1 phosphorylation, the authors show that Ame1 degradation depends on whether Ame1 is bound to the Mtw1c (binding prevents degradation), which only happens at a fully assembled kinetochore. Based on these observations, the authors propose a model in which the phosphodegron motif functions to degrade any molecules of the COMA complex that are not incorporated into the kinetochore and in this way prevents kinetochore assembly at ectopic regions of the chromosome.
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Reviewer #2 (Public Review):
Böhm et al. investigated the phosphorylation of the Ctf19CCAN component Ame1CENP-U by Cdk1 which forms a phosphodegron motif recognized by the E3 ubiquitin ligase complex SCF-Cdc4. They identify phosphorylation sites on Ame1 and demonstrate that phosphorylation of Ame1 leads to its degradation by the SCF with Cdc4 in a cell-cycle dependent manner. They also demonstrate that the outer kinetochore component Mtw1c shields Ame1 from Cdk1 phosphorylation in vitro. Finally, they propose a model in which at least one component, Ame1, is present in excess at S-phase in yeast to incorporate into high levels of sub-complexes for efficient inner kinetochore formation on newly duplicated centromere DNA. Then, in mitosis, phosphodegrons serve to mediate the degradation of excess Ame1 (and presumably other CCAN …
Reviewer #2 (Public Review):
Böhm et al. investigated the phosphorylation of the Ctf19CCAN component Ame1CENP-U by Cdk1 which forms a phosphodegron motif recognized by the E3 ubiquitin ligase complex SCF-Cdc4. They identify phosphorylation sites on Ame1 and demonstrate that phosphorylation of Ame1 leads to its degradation by the SCF with Cdc4 in a cell-cycle dependent manner. They also demonstrate that the outer kinetochore component Mtw1c shields Ame1 from Cdk1 phosphorylation in vitro. Finally, they propose a model in which at least one component, Ame1, is present in excess at S-phase in yeast to incorporate into high levels of sub-complexes for efficient inner kinetochore formation on newly duplicated centromere DNA. Then, in mitosis, phosphodegrons serve to mediate the degradation of excess Ame1 (and presumably other CCAN components) and in so doing protect against the formation of ectopic outer kinetochores.
This manuscript puts forth well-designed and thorough experiments characterizing the phosphorylation of Ame1 and its regulation by the SCF-Cdc4 complex. The writing is clear and the figures are generally easy to understand. The authors succeed in asking pertinent questions, designing experiments to answer them, and considering potential alternative explanations or confounding factors. As a whole this creates a generally convincing study regarding the phospho-regulation of Ame1. However, I also have some important concerns:
The authors begin the manuscript by mapping phosphorylation sites across Ctf19CCAN components but then largely narrow their experimental focus to Ame1 and to a lesser extent its binding partner Okp1. Without mutation of other components, the Ame1 mutant phenotypes are either absent or very mild. This would seem to implicate that, if this is an important process, that other targets for this quality control mechanism must exist. As it stands now, the focused investigation does not make the most compelling case for the broad conclusions that are claimed. More extensive investigation of phosphoregulation of CCAN subunits beyond Ame1 would certainly help justify the claim that phosphoregulation is used to clear excess CCAN subunits and protect against ectopic kinetochore assembly. Is there another lead from their initial mass spec work that could provide some molecular evidence that this is a general process? Failing that, the discussion could at least provide some hint at how the model could be tested in future studies.
The conclusion that the binding of the Mtw1 complex shields Ame1 phosphodegrons is arguably one of the most significant and interesting claims made in this paper. However, the evidence presented to support this claim seems to rely exclusively on in vitro data. Thus, this part is out of balance with other parts of the paper where some in vivo correlations are attempted/made.
The central model mentioned at the outset strongly predicts that the mitotic degradation of Ame1 doesn't impact its abundance at centromeres. That is not the only possibility, though, and some measurement (fluorescence of a tagged Ame1 or a ChIP on centromere DNA) of Ame1 at centromeres before and through mitosis would help instill confidence in the proposal.
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Reviewer #1 (Public Review):
Kinetochores are huge protein assemblies on chromosomes which are used as attachment point for microtubules and allow microtubules to pull chromosomes into daughter cells during cell division. The proteins that form the kinetochore are well known, but the temporal regulation of the assembly of all these proteins into functional kinetochores is less understood.
In this paper the authors have identified phosphorylation sites in the 'CCAN' of budding yeast, the 'inner', i.e. chromatin-proximal, part of the kinetochore. They characterize in detail the function of phosphorylation of Ame1 (CENP-U in humans), which is part of CCAN. The data support the idea that a cluster of phosphorylation sites in Ame1 is phosphorylated by mitotic CDK1 and serves as phospho-degron for the E3 ligase SCF/Cdc4.
The authors show …
Reviewer #1 (Public Review):
Kinetochores are huge protein assemblies on chromosomes which are used as attachment point for microtubules and allow microtubules to pull chromosomes into daughter cells during cell division. The proteins that form the kinetochore are well known, but the temporal regulation of the assembly of all these proteins into functional kinetochores is less understood.
In this paper the authors have identified phosphorylation sites in the 'CCAN' of budding yeast, the 'inner', i.e. chromatin-proximal, part of the kinetochore. They characterize in detail the function of phosphorylation of Ame1 (CENP-U in humans), which is part of CCAN. The data support the idea that a cluster of phosphorylation sites in Ame1 is phosphorylated by mitotic CDK1 and serves as phospho-degron for the E3 ligase SCF/Cdc4.
The authors show phosphorylation of these CDK1 consensus sites in vivo and their phosphorylation by CDK1/Clb2 in vitro. Genetic experiments and molecular dynamics simulations support the idea that phosphorylation sites on Ame1 can serve as phospho-degron for SCF/Cdc4. Even the non-phosphorylatable mutant of Ame1 is stabilized in an SCF mutant background, though, suggesting that this phospho-degron is not the only way in which SCF influences kinetochore protein levels.
Mutants in the characterized phosphorylation sites do not impair budding yeast growth. This suggests that the degron characterized in this paper may be important for fine-tuning, but is not essential for the proper execution of mitosis. The observations overall add to prior evidence that kinetochore assembly can be regulated by phosphorylation and/or ubiquitination.
Interestingly, the authors find that phosphorylation of Ame1 by CDK1 in vitro is impaired when Ame1 binds Mtw1, another kinetochore protein. The fact that Mtw1 seems to shield these sites from phosphorylation leads the authors to put forward an interesting model: they propose that cell cycle-dependent phosphorylation and SCF-dependent degradation of kinetochore subunits allows for excess subunits during kinetochore assembly in S-phase (which will speed up assembly) while depleting any excess subunits after assembly, when the kinetochore needs to be functional.
This is an interesting model. The in vivo evidence is still limited, though. For now, it remains unknown whether the phosphorylation status of kinetochore-bound and free Ame1 is indeed different, whether more soluble Ame1 exists in S-phase, whether too early degradation of Ame1 (or possibly other kinetochore proteins) indeed impairs kinetochore assembly, or whether a failure to remove the soluble pool after assembly leads to mitotic defects. It is an attractive proposal, though, that can now be further explored experimentally.
In addition to the specific characterization of Ame1 sites, the paper also includes comprehensive data on CCAN phosphorylation sites obtained by mass spectrometry which can serve as basis for future studies.
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Evaluation Summary:
This paper will be of interest to those in the fields of chromosome biology, mitotic regulation, and proteostasis. The authors put forward an interesting model of phosphodegron regulation of kinetochore assembly based on convincing genetic and biochemical data. The novel model will require some additional evidence before it can be considered well-supported, but the paper represents an advance in our knowledge of kinetochore regulation with experiments that are rigorous, well-designed and carefully conducted.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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