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Interaction of the pim1/spi1 mitotic checkpoint with a protein phosphatase
1Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, New York 11724.
Abstract:
Loss of p58pim1, a homolog of human RCC1, results in uncoupling of mitosis from the completion of DNA replication in fission yeast. An extragenic suppressor of a mutant allele of pim1, esp1, has been isolated and characterized. esp1 encodes a predicted product of 305 amino acid residues, which shares 71% identity with budding yeast SIT4, a type2A related protein phosphatase. p58pim1 binds p25spi1, a 25-kd ras-related GTPase previously isolated as a high dosage suppressor of pim1. The complex dissociates in the presence of guanine nucleotides and Mg2+. The mutant p58pim1 is defective in its ability to bind p25spi1, suggesting that the physical interaction is essential for the maintenance of the interdependency of cell cycle event. In the esp1 pim1 double mutant, the mutant p58pim1 protein is still defective in its ability to bind to p25spi1. However, pmi1 induced premature mitosis is completely suppressed, suggesting that esp1 may act downstream of the p58pim1/p25spi1 physical interaction but upstream of the activation of the M-phase specific histone H1 kinase.
Insights
Loss of p58pim1 causes cell cycle errors. The esp1 gene suppresses these errors by acting downstream of the p58pim1/p25spi1 interaction, restoring normal mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- p58pim1, a homolog of human RCC1, is crucial for coordinating DNA replication and mitosis in fission yeast.
- Loss of p58pim1 function leads to cell cycle dysregulation, specifically uncoupling mitosis from DNA replication completion.
Purpose of the Study:
- To investigate the function of esp1, an extragenic suppressor of pim1 mutants.
- To elucidate the molecular mechanism by which esp1 suppresses the cell cycle defects caused by pim1 mutations.
Main Methods:
- Isolation and characterization of the esp1 gene and its product.
- Analysis of protein-protein interactions between p58pim1 and p25spi1.
- Genetic analysis of esp1 pim1 double mutants.
Main Results:
- esp1 encodes a protein phosphatase with homology to budding yeast SIT4.
- p58pim1 forms a complex with p25spi1, a ras-related GTPase, which is essential for cell cycle coordination.
- Mutant p58pim1 shows defective binding to p25spi1.
- esp1 suppresses premature mitosis in pim1 mutants, even when p58pim1 binding to p25spi1 remains defective.
Conclusions:
- The physical interaction between p58pim1 and p25spi1 is critical for maintaining the interdependence of cell cycle events.
- esp1 likely acts downstream of the p58pim1/p25spi1 complex formation but upstream of M-phase specific histone H1 kinase activation to suppress cell cycle defects.