Mps2 (monopolar spindle protein) is a coiled-coil protein found at the spindle pole body (SPB) and at the nuclear envelope that is required for insertion of the SPB into the nuclear envelope. We identified three proteins that interact with Mps2 in a two-hybrid screen: Bbp1, Ynl107w and Spc24. All three proteins contain coiled-coil motifs that appear to be required for their interaction with Mps2. In this work, we verified the Mps2-Spc24 interaction by co-immunoprecipitation in vivo and by the in vitro interaction of recombinant proteins. Previous two-hybrid screens with Spc24 as bait had identified Spc25 and Ndc80 as putative interacting partners, and we verified these interactions in vivo by purification of TAP-tagged derivatives of Spc24 and Ndc80. Finally, we found that spc24 thermosensitive mutants had a chromosome segregation defect, but no apparent defect in SPB duplication. These results are consistent with recently published data showing that Spc24, Spc25 and Ndc80 are peripheral kinetochore com-ponents required for chromosome segregation. The Mps2-Spc24 interaction may contribute to the localization of Spc24 and other kinetochore components to the inner plaque of the SPB.
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| Gene | Phenotype | Experiment Type | Mutant Information | Strain Background | Chemical | Details |
|---|---|---|---|---|---|---|
| SPC24 | chromosome segregation: abnormal | classical genetics | conditional Allele: spc24-11 S109P, K171N | S288C | Temperature: elevated temperature, 37 °C Details: assemble and elongate spindle in absence of chromosome segregation; defect not detected by mitotic checkpoint pathways | |
| SPC24 | heat sensitivity: increased | classical genetics | conditional Allele: spc24-11 S109P, K171N | S288C | Temperature: elevated temperature, 37 °C | |
| BBP1 | protein/peptide distribution: abnormal Reporter: GFP-Mps2p | classical genetics | conditional Allele: bbp1-1 | Other | Temperature: elevated temperature, 37 °C Details: recruitment of Mps2p to the new SPB duplication plaque is defective |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Interactor | Interactor | Assay | Annotation | Action | Modification | |
|---|---|---|---|---|---|---|
| MPS2 | SPC24 | Affinity Capture-Western | manually curated | Hit-Bait | No Modification | |
| MPS2 | SPC24 | Reconstituted Complex | manually curated | Bait-Hit | No Modification | |
| MPS2 | BBP1 | Two-hybrid | manually curated | Bait-Hit | No Modification | |
| MPS2 | SPC24 | Two-hybrid | manually curated | Bait-Hit | No Modification | |
| MPS2 | YAF9 | Two-hybrid | manually curated | Bait-Hit | No Modification | |
| MPS2 | SPC24 | Co-purification | manually curated | Hit-Bait | No Modification | |
| NDC80 | SPC24 | Affinity Capture-Western | manually curated | Bait-Hit | No Modification | |
| NDC80 | SPC24 | Affinity Capture-Western | manually curated | Hit-Bait | No Modification | |
| NDC80 | SPC24 | Two-hybrid | manually curated | Hit-Bait | No Modification | |
| SPC25 | NDC80 | Affinity Capture-Western | manually curated | Hit-Bait | No Modification |