The Effect of the MAD1-RED Interaction on Mitosis Progression.
| Autor: | Wan-Syuan Yang, 楊婉萱 |
|---|---|
| Rok vydání: | 2014 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 102 The spindle assembly checkpoint (SAC) can monitor the connection between the kinetochore and the spindle, and ensure the faithful segregation of the paired sister chromatids into two daughter cells during mitosis. The core SAC components, including MAD1, are recruited to the unattached kinetochore for activation of the SAC. The previous study shows that RED, a MAD1-interacting protein, has a functional role in the activation of SAC. Also, depletion of RED can cause a shorter mitotic timing, a failure in the localization of MAD1 in prometaphase, and a defect in the SAC. Furthermore, the two RED-binding peptides, MAD1(301-340) and MAD1(439-480), fused to EGFP, can bind to RED at the spindle poles in prometaphase, and cause a defect in the SAC. These observations raise the possibility that MAD1 binds to RED at the spindle poles is to inactive RED and hence silence the SAC in metaphase. Therefore, the aim of this study is to understand the significance of the MAD1-RED interaction at the spindle poles in mitosis. We have identified that the amino acid residues 115-155 and 435-480 in RED, designated as RED(115-155) and RED(435-480), respectively, were MAD1-interacting regions. In addition, overexpression of EGFP-RED(115-155) and EGFP-RED(435-480) can cause cell bypass the mitotic arrest induced by nocodazole. We also did sequence alignment by NCBI blast search and found that RED(115-155) and RED(435-480) could be divided into three subregions, respectively. The conserved hydrophobic amino acids in RED(115-155) and RED(435-480), including Y126, I147, F154, L155, Y445, Y449 and Y466, have been mutated to alanine, and the resultant mutant protein is designated as REDMI for analyses. Interestingly, His-HA-REDMI cannot bind to MBP-MAD1 in vitro. It indicates that the specific amino acids in RED required for interacting with MAD1 are among these hydrophobic amino acids. Taken together, we conclude that RED(115-155) and RED(435-480) are the MAD1-interacting regions in RED and are important in the SAC, and some hydrophobic amino acids in RED(115-155) and RED(435-480) contribute to bind to MAD1. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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