Abstract
We show that MAD3 encodes a novel 58-kD nuclear protein which is not essential for viability, but is an integral component of the spindle checkpoint in budding yeast. Sequence analysis reveals two regions of Mad3p that are 46 and 47% identical to sequences in the NH(2)-terminal region of the budding yeast Bub1 protein kinase. Bub1p is known to bind Bub3p (Roberts et al. 1994) and we use two-hybrid assays and coimmunoprecipitation experiments to show that Mad3p can also bind to Bub3p. In addition, we find that Mad3p interacts with Mad2p and the cell cycle regulator Cdc20p. We show that the two regions of homology between Mad3p and Bub1p are crucial for these interactions and identify loss of function mutations within each domain of Mad3p. We discuss roles for Mad3p and its interactions with other spindle checkpoint proteins and with Cdc20p, the target of the checkpoint.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Calcium-Binding Proteins / metabolism*
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Carrier Proteins*
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Cdc20 Proteins
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Cell Cycle Proteins / genetics*
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Cell Cycle Proteins / immunology
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Cell Cycle Proteins / metabolism*
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Cloning, Molecular
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Fungal Proteins / genetics*
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Fungal Proteins / immunology
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Fungal Proteins / metabolism*
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Genes, cdc
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Mad2 Proteins
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Nuclear Proteins
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Precipitin Tests
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Protein Kinases / genetics
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Protein Serine-Threonine Kinases
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Protein Structure, Tertiary / genetics
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Proteins / metabolism*
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Saccharomyces cerevisiae Proteins*
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Saccharomycetales / cytology
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Saccharomycetales / enzymology
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Saccharomycetales / genetics*
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Sequence Homology, Amino Acid
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Spindle Apparatus / genetics*
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Two-Hybrid System Techniques
Substances
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CDC20 protein, S cerevisiae
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Calcium-Binding Proteins
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Carrier Proteins
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Cdc20 Proteins
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Cell Cycle Proteins
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Fungal Proteins
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MAD2 protein, S cerevisiae
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Mad2 Proteins
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Nuclear Proteins
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Proteins
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Saccharomyces cerevisiae Proteins
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Protein Kinases
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Bub1 spindle checkpoint protein
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Protein Serine-Threonine Kinases