The C-Terminal Half of Saccharomyces cerevisiae Mad1p Mediates Spindle Checkpoint Function, Chromosome Transmission Fidelity and CEN Association

Abstract

The evolutionarily conserved spindle checkpoint is a key mechanism ensuring high-fidelity chromosome transmission. The checkpoint monitors attachment between kinetochores and mitotic spindles and the tension between sister kinetochores. In the absence of proper attachment or tension, the spindle checkpoint mediates cell cycle arrest prior to anaphase. Saccharomyces cerevisiae Mad1p is required for the spindle checkpoint and for chromosome transmission fidelity. Moreover, Mad1p associates with the nuclear pore complex (NPC) and is enriched at kinetochores upon checkpoint activation. Using partial mad1 deletion alleles we determined that the C-terminal half of Mad1p is necessary and sufficient for checkpoint activation in response to microtubule depolymerizing agents, high-fidelity transmission of a reporter chromosome fragment, and in vivo association with centromeres, but not for robust NPC association. Thus, spindle checkpoint activation and chromosome transmission fidelity correlate and these Mad1p functions likely involve kinetochore association but not robust NPC association. These studies are the basis for elucidating the role of protein complexes containing Mad1p in the spindle checkpoint pathway and in maintaining genome stability in S. cerevisiae and other systems. The mitotic arrest deficient (mad) and budding uninpoint monitors the interaction between the kinetohibited by benzimidazole (bub) yeast mutants, correchores, complexes of proteins bound to the centrosponding to the MAD1-MAD3 and BUB1-BUB3 genes, meres of chromosomes, and the microtubules that form were identified by genetic screens in Saccharomyces cerethe mitotic spindle (reviewed in Musacchio and Hardvisiae on the basis of the failure of the mutants to arrest wick 2002). In metaphase, sister kinetochores attach to under checkpoint-inducing conditions (Hoyt et al. 1991; microtubules emanating from opposite spindle pole bodLi and Murray 1991). MPS1, an additional checkpoint ies during alignment of the chromosomes. Tension is gene, was originally identified in a screen for mutants of generated when the pulling forces exerted by the spindle spindle pole body duplication (Weiss and Winey 1996). are opposed by cohesin proteins that hold sister chromaOrthologs of the MADs, BUBs, and MPS1 exist in other tids together. In addition to deficiencies in attachment eukaryotes, including humans (reviewed in Skibbens and of kinetochores to spindle microtubules, the spindle Hieter 1998).

Structural Integrity of Centromeric Chromatin and Faithful Chromosome Segregation Requires Pat1

“…The CF loss phenotype of pat1Δ strains is complemented by a plasmid expressing PAT1 from its native promoter ( Figure 1A). The frequency of CF loss observed in pat1Δ strain is similar to that reported for deletion of kinetochore genes (Kastenmayer et al 2005;Ma et al 2012). The chromosome-loss phenotype observed in pat1D strain ( Figure 1A) prompted us to examine whether Pat1 associates with CEN DNA.…”

Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting

“…In yeast, a C-terminal fragment of Mad1 is sufficient for kinetochore localization and checkpoint signaling (18). Binding between yeast Mad1 CTD and Bub1-Bub3 thus makes a more substantial energetic contribution to the Mad1 kinetochore localization and hence might have higher affinity, allowing its detection.…”

“…A C-terminal fragment of yeast Mad1 containing the highly conserved C-terminal domain (CTD) is necessary and sufficient for its kinetochore localization and checkpoint function (18). By contrast, an N-terminal fragment of Xenopus Mad1 lacking the CTD has been shown to localize to kinetochores (19).…”

The nucleoporin Nup153 affects spindle checkpoint activity due to an association with Mad1

“…Interestingly, this role for Nup153 in controlling the mitotic checkpoint appears evolutionarily conserved, since its yeast homologue, Nup1p, is also required for the association of Mad1p with the NPC. 27 Moreover, mutations in nup1 result in altered spindle organization with an increase in multinucleate and anucleate daughter cells as well as failure to exit from mitosis. 47,48 Given a role for Nup153 in development and cancer [17][18][19] it will be interesting to determine how this is related to Nup153's novel function in cell division.…”

“…22 The function of the SAC proteins at the NPC has remained largely elusive, although it has been suggested that the NPC may play a role in the duration of the SAC. 26,27 Here, we have examined the effect of altering Nup153 expression in HeLa cells and found that Nup153 levels affect spindle checkpoint activity due to binding of Nup153 to the SAC protein Mad1.…”