An alpha-tubulin mutant demonstrates distinguishable functions among the spindle assembly checkpoint genes in Saccharomyces cerevisiae

Department of Biology, Brandeis University, Волтам, Massachusetts, United States
Genetics (Impact Factor: 5.96). 08/2002; 161(3):983-94.
Source: PubMed


Cells expressing a mutant allele of alpha-tubulin, tub1-729, are cold sensitive and arrest as large-budded cells with microtubule defects. The cold sensitivity of tub1-729 is suppressed by extra copies of a subset of the mitotic checkpoint genes BUB1, BUB3, and MPS1, but not MAD1, MAD2, and MAD3. This suppression by checkpoint genes does not depend upon their role in the MAD2-dependent spindle assembly checkpoint. In addition, BUB1 requires an intact kinase domain as well as Bub3p to suppress tub1-729. The data suggest that tub1-729 cells are defective in microtubule-kinetochore attachments and that the products of specific checkpoint genes can act either directly or indirectly to affect these attachments.

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Available from: Katharine Abruzzi, Mar 06, 2014
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    • "Loss of MT-KT Attachment Activates the KTIP Studies characterizing the tub1-729 mutant reported that the mutant protein destabilized KT-MT interactions, leading to KT detachment (Abruzzi et al., 2002). Therefore, we investigated "
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    ABSTRACT: Nuclear pore complexes (NPCs) and kinetochores perform distinct tasks, yet their shared ability to bind several proteins suggests their functions are intertwined. Among these shared proteins is Mad1p, a component of the yeast spindle assembly checkpoint (SAC). Here we describe a role for Mad1p in regulating nuclear import that employs its ability to sense a disruption of kinetochore-microtubule interactions during mitosis. We show that kinetochore-microtubule detachment arrests nuclear import mediated by the transport factor Kap121p through a mechanism that requires Mad1p cycling between unattached, metaphase kinetochores and binding sites at the NPC. This signaling pathway requires the Aurora B-like kinase Ipl1p, and the resulting transport changes inhibit the nuclear import of Glc7p, a phosphatase that acts as an Ipl1p antagonist. We propose that a distinct branch of the SAC exists in which Mad1p senses unattached kinetochores and, by altering NPC transport activity, regulates the nuclear environment of the spindle.
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    • "The orthologs for kinetochore proteins found in P. brasiliensis were Spc25p (Ndc80p complex; Le Masson, 2002) and Nnf1p (MIND kinetochore complex; Westermann et al., 2003). Several transcripts involved with the mitotic spindle were also identified: Cdc31p, required for spindle pole body duplication (Paoletti et al., 2003); Cdc42p, a small GTPase of the Rho family involved in regulation of microtubule attachment to kinetochores (Yasuda et al., 2004); Cdc48p, an AAA-ATPase required for spindle disassembly (Cao et al., 2003); Cdc55p, a protein serine/threonine phosphatase 2A subunit involved in spindle checkpoint and cytokinesis (Koren et al., 2004) and Tub1p, an α-tubulin (microtubule subunit; Abruzzi et al., 2002). We also found ESTs related to the APC, namely MAD2, which encodes a protein inhibitor of APC activity (Sugimoto et al., 2004) and Cdc20p, an activator of APC (Irniger, 2002). "
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    • "Second, extra copies of BUB1 or BUB3 suppress the chromosome–MT attachment defects generated by tub1-729 mutant, independent of MAD2- dependent signaling (Abruzzi et al., 2002). Third, although the conserved kinase domain of Bub1p is not required for nocodazole arrest in yeast (Sharp-Baker and Chen, 2001; Warren et al., 2002) or the recruitment of downstream checkpoint proteins to kinetochores in Xenopus (Sharp-Baker and Chen, 2001; Warren et al., 2002), it is required for suppression of attachment defects in tub1-729 cells (Abruzzi et al., 2002) and for accurate chromosome transmission in wild-type cells (Warren et al., 2002). We find selective binding of Bub proteins, but not Mad proteins, to kinetochores in three contexts: wild-type cells early in mitosis, ipl1-321 cells, and mcd1-1 cells. "
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    ABSTRACT: Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways.
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