Abstract
We show here that the fission yeast gene products Cut9 and Nuc2 are the subunits of the 20S complex, the putative APC (anaphase promoting complex)/cyclosome which contains ubiquitin ligase activity required for cyclin and Cut2 destruction. The assembly of Cut9 into the 20S complex requires functional Nuc2, and vice versa. The size of fission yeast APC/cyclosome is similar to that of higher eukaryotes, but differs greatly from that (36S) of budding yeast. The 20S complex is present in cells arrested at different stages of the cell cycle, and becomes slightly heavier in mitosis than interphase. Cut9 in the 20S complex is hyperphosphorylated specifically at the time of metaphase. The truncated forms of Cut9 block entry into mitosis, however. The 20S assembly impaired in the cut9 mutant can be restored by elevating the level of a novel gene product Hcnl, similar to budding yeast Cdc26. Furthermore, deletion of protein kinase PKA (Pkal) suppresses the phenotype of the cut9 mutation and reduces phosphorylation of Cut9. In contrast, PP1 (Dis2) phosphatase mutation shows the reverse effect on the phenotype of cut9. The Cut9 subunit is likely to be a target for regulating APC/ cyclosome function through protein-protein interactions and phosphorylation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Anaphase*
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Anaphase-Promoting Complex-Cyclosome
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Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome
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Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome
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Base Sequence
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Centrifugation, Density Gradient
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Cyclic AMP-Dependent Protein Kinases / metabolism
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Fungal Proteins / chemistry
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Fungal Proteins / genetics
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Fungal Proteins / metabolism*
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G1 Phase
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Gene Expression Regulation
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Genes, Suppressor
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Ligases / chemistry
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Ligases / metabolism*
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Mitosis
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Molecular Sequence Data
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Mutation
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Phosphoprotein Phosphatases / metabolism
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Phosphorylation
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Repressor Proteins / chemistry
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Repressor Proteins / genetics
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Repressor Proteins / metabolism
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Schizosaccharomyces / cytology
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Schizosaccharomyces / genetics
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Schizosaccharomyces / metabolism*
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Schizosaccharomyces pombe Proteins*
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Ubiquitin-Protein Ligase Complexes*
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Ubiquitin-Protein Ligases
Substances
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Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome
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Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome
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CUT9 protein, S pombe
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Fungal Proteins
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NUC2 protein, S pombe
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Nuclear Proteins
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Repressor Proteins
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Schizosaccharomyces pombe Proteins
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hcn1 protein, S pombe
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Ubiquitin-Protein Ligase Complexes
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Anaphase-Promoting Complex-Cyclosome
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Ubiquitin-Protein Ligases
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Cyclic AMP-Dependent Protein Kinases
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Phosphoprotein Phosphatases
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Ligases