Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae.
In addition to its role in nucleocytoplasmic transport, the nuclear pore complex (NPC) acts as a docking site for proteins whose apparent primary cellular functions are unrelated to nuclear transport, including Mad1p and Mad2p, two proteins of the spindle assembly checkpoint (SAC) machinery. To understand this relationship, we have mapped domains of yeast Saccharomyces cerevisiae Mad1p that interact with the nuclear transport machinery, including further defining its interactions with the NPC. We showed that a Kap95p/Kap60p-dependent nuclear localization signal, positioned in the C-terminal third of Mad1p, is required for its efficient targeting to the NPC. At the NPC, Mad1p interacts with Nup53p and a presumed Nup60p/Mlp1p/Mlp2p complex through two coiled coil regions within its N terminus. When the SAC is activated, a portion of Mad1p is recruited to kinetochores through an interaction that is mediated by the C-terminal region of Mad1p and requires energy. We showed using photobleaching analysis that in nocodazole-arrested cells Mad1p rapidly cycles between the Mlp proteins and kinetochores. Our further analysis also showed that only the C terminus of Mad1p is required for SAC function and that the NPC, through Nup53p, may act to regulate the duration of the SAC response.
Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.
Robert J
RJ
Scott
C Patrick
CP
Lusk
David J
DJ
Dilworth
John D
JD
Aitchison
Richard W
RW
Wozniak
D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D018386:Kinetochores / Q000378:metabolism; D022022:Nuclear Pore / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D021382:Protein Sorting Signals / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D018797:Cell Cycle Proteins; C501382:MAD1 protein, S cerevisiae; D009687:Nuclear Proteins; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins