The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly

Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1–GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions

Comment on "A centrosome-independent role for gamma-TuRC proteins in the spindle assembly checkpoint"

Müller et al. (Reports, 27 October 2006, p. 654) showed that inhibition of the γ-tubulin ring complex (γ-TuRC) activates the spindle assembly checkpoint (SAC), which led them to suggest that γ-TuRC proteins play molecular roles in SAC activation. Because γ-TuRC inhibition leads to pleiotropic spindle defects, which are well known to activate kinetochore-derived checkpoint signaling, we believe that this conclusion is premature

Cdc28/Cdk1 Regulates Spindle Pole Body Duplication through Phosphorylation of Spc42 and Mps1

AbstractDuplication of the Saccharomyces cerevisiae spindle pole body (SPB) once per cell cycle is essential for bipolar spindle formation and accurate chromosome segregation during mitosis. We have investigated the role that the major yeast cyclin-dependent kinase Cdc28/Cdk1 plays in assembly of a core SPB component, Spc42, to better understand how SPB duplication is coordinated with cell cycle progression. Cdc28 is required for SPB duplication and Spc42 assembly, and we found that Cdc28 directly phosphorylates Spc42 to promote its assembly into the SPB. The Mps1 kinase, previously shown to regulate Spc42 phosphorylation and assembly, is also a Cdc28 substrate, and Cdc28 phosphorylation of Mps1 is needed to maintain wild-type levels of Mps1 in cells. Analysis of nonphosphorylatable mutants in SPC42 and MPS1 indicates that direct Spc42 phosphorylation and indirect regulation of Spc42 through Mps1 are two overlapping pathways by which Cdc28 regulates Spc42 assembly and SPB duplication during the cell cycle

Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I

Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore

Ybp2 Associates with the Central Kinetochore of Saccharomyces cerevisiae and Mediates Proper Mitotic Progression

The spindle checkpoint ensures the accurate segregation of chromosomes by monitoring the status of kinetochore attachment to microtubules. Simultaneous mutations in one of several kinetochore and cohesion genes and a spindle checkpoint gene cause a synthetic-lethal or synthetic-sick phenotype. A synthetic genetic array (SGA) analysis using a mad2Δ query mutant strain of yeast identified YBP2, a gene whose product shares sequence similarity with the product of YBP1, which is required for H2O2-induced oxidation of the transcription factor Yap1. ybp2Δ was sensitive to benomyl and accumulated at the mitotic stage of the cell cycle. Ybp2 physically associates with proteins of the COMA complex (Ctf19, Okp1, Mcm21, and Ame1) and 3 components of the Ndc80 complex (Ndc80, Nuf2, and Spc25 but not Spc24) in the central kinetochore and with Cse4 (the centromeric histone and CENP-A homolog). Chromatin-immunoprecipitation analyses revealed that Ybp2 associates specifically with CEN DNA. Furthermore, ybp2Δ showed synthetic-sick interactions with mutants of the genes that encode the COMA complex components. Ybp2 seems to be part of a macromolecular kinetochore complex and appears to contribute to the proper associations among the central kinetochore subcomplexes and the kinetochore-specific nucleosome

Two Pathways Recruit Telomerase to Saccharomyces cerevisiae Telomeres

The catalytic subunit of yeast telomerase, Est2p, is a telomere associated throughout most of the cell cycle, while the Est1p subunit binds only in late S/G2 phase, the time of telomerase action. Est2p binding in G1/early S phase requires a specific interaction between telomerase RNA (TLC1) and Ku80p. Here, we show that in four telomerase-deficient strains (cdc13-2, est1Ä, tlc1-SD, and tlc1-BD), Est2p telomere binding was normal in G1/early S phase but reduced to about 40–50% of wild type levels in late S/G2 phase. Est1p telomere association was low in all four strains. Wild type levels of Est2p telomere binding in late S/G2 phase was Est1p-dependent and required that Est1p be both telomere-bound and associated with a stem-bulge region in TLC1 RNA. In three telomerase-deficient strains in which Est1p is not Est2p-associated (tlc1-SD, tlc1-BD, and est2Ä), Est1p was present at normal levels but its telomere binding was very low. When the G1/early S phase and the late S/G2 phase telomerase recruitment pathways were both disrupted, neither Est2p nor Est1p was telomere-associated. We conclude that reduced levels of Est2p and low Est1p telomere binding in late S/G2 phase correlated with an est phenotype, while a WT level of Est2p binding in G1 was not sufficient to maintain telomeres. In addition, even though Cdc13p and Est1p interact by two hybrid, biochemical and genetic criteria, this interaction did not occur unless Est1p was Est2p-associated, suggesting that Est1p comes to the telomere only as part of the holoenzyme. Finally, the G1 and late S/G2 phase pathways for telomerase recruitment are distinct and are likely the only ones that bring telomerase to telomeres in wild-type cells

A Genome-Wide Screen for Regulators of TORC1 in Response to Amino Acid Starvation Reveals a Conserved Npr2/3 Complex

TORC1 is a central regulator of cell growth in response to amino acid availability, yet little is known about how it is regulated. Here, we performed a reverse genetic screen in yeast for genes necessary to inactivate TORC1. The screen consisted of monitoring the expression of a TORC1 sensitive GFP-based transcriptional reporter in all yeast deletion strains using flow cytometry. We find that in response to amino acid starvation, but not to carbon starvation or rapamycin treatment, cells lacking NPR2 and NPR3 fail to fully (1) activate transcription factors Gln3/Gat1, (2) dephosphorylate TORC1 effector Npr1, and (3) repress ribosomal protein gene expression. Both mutants show proliferation defects only in media containing a low quality nitrogen source, such as proline or ammonia, whereas no defects are evident when cells are grown in the presence of glutamine or peptone mixture. Proliferation defects in npr2Δ and npr3Δ cells can be completely rescued by artificially inhibiting TORC1 by rapamycin, demonstrating that overactive TORC1 in both strains prevents their ability to adapt to an environment containing a low quality nitrogen source. A biochemical purification of each demonstrates that Npr2 and Npr3 form a heterodimer, and this interaction is evolutionarily conserved since the human homologs of NPR2 and NPR3 (NPRL2 and NPRL3, respectively) also co-immunoprecipitate. We conclude that, in yeast, the Npr2/3 complex mediates an amino acid starvation signal to TORC1

Bub1-Mediated Adaptation of the Spindle Checkpoint

During cell division, the spindle checkpoint ensures accurate chromosome segregation by monitoring the kinetochore–microtubule interaction and delaying the onset of anaphase until each pair of sister chromosomes is properly attached to microtubules. The spindle checkpoint is deactivated as chromosomes start moving toward the spindles in anaphase, but the mechanisms by which this deactivation and adaptation to prolonged mitotic arrest occur remain obscure. Our results strongly suggest that Cdc28-mediated phosphorylation of Bub1 at T566 plays an important role for the degradation of Bub1 in anaphase, and the phosphorylation is required for adaptation of the spindle checkpoint to prolonged mitotic arrest