RESUMEN
As a surveillance mechanism, the activated spindle assembly checkpoint (SAC) potently inhibits the E3 ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) to ensure accurate chromosome segregation. Although the protein phosphatase 2A (PP2A) has been proposed to be both, directly and indirectly, involved in spindle assembly checkpoint inactivation in mammalian cells, whether it is similarly operating in the fission yeast Schizosaccharomycer pombe has never been demonstrated. Here, we investigated whether fission yeast PP2A is involved in SAC silencing by following the rate of cyclin B (Cdc13) destruction at SPBs during the recovery phase in nda3-KM311 cells released from the inhibition of APC/C by the activated spindle checkpoint. The timing of the SAC inactivation is only slightly delayed when two B56 regulatory subunits (Par1 and Par2) of fission yeast PP2A are absent. Overproduction of individual PP2A subunits either globally in the nda3-KM311 arrest-and-release system or locally in the synthetic spindle checkpoint activation system only slightly suppresses the SAC silencing defects in PP1 deletion (dis2Δ) cells. Our study thus demonstrates that the fission yeast PP2A is not a key regulator actively involved in SAC inactivation.
Asunto(s)
Schizosaccharomyces , Ciclosoma-Complejo Promotor de la Anafase/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Mamíferos/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Huso Acromático/fisiologíaRESUMEN
Using genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Single-domain camelid antibodies generated against GFP have been engineered as nanobodies or GFP-binding proteins (GBPs) that can bind GFP as well as some GFP variants with high affinity and selectivity. In this study, we have used GBP-mCherry fusion protein as a tool to perturb the natural functions of a few kinetochore proteins in the fission yeast Schizosaccharomyces pombe. We found that cells simultaneously expressing GBP-mCherry and the GFP-tagged inner kinetochore protein Cnp1 are sensitive to high temperature and microtubule drug thiabendazole (TBZ). In addition, kinetochore-targeted GBP-mCherry by a few major kinetochore proteins with GFP tags causes defects in faithful chromosome segregation. Thus, this setting compromises the functions of kinetochores and renders cells to behave like conditional mutants. Our study highlights the potential of using GBP as a general tool to perturb the function of some GFP-tagged proteins in vivo with the objective of understanding their functional relevance to certain physiological processes, not only in yeasts, but also potentially in other model systems.