Temperature-sensitive ipl1-2/Aurora B mutation is suppressed by mutations in TOR complex 1 via the Glc7/PP1 phosphatase

Abstract

Ipl1/Aurora B is the catalytic subunit of a complex that is required for chromosome segregation and nuclear division. Before anaphase, Ipl1 localizes to kinetochores, where it is required to establish proper kinetochore-microtubule associations and regulate the spindle assembly checkpoint. The protein phosphatase Glc7/PP1 opposes Ipl1 for some of these activities. To more thoroughly characterize the Glc7 phosphatase that opposes Ipl1, we have identified mutations that suppress the thermosensitivity of an ipl1-2 mutant. In addition to mutations in genes previously associated with ipl1 suppression, we recovered a null mutant in TCO89, which encodes a subunit of the TOR complex 1 (TORC1), the conserved rapamycin-sensitive kinase activity that regulates cell growth in response to nutritional status. The temperature sensitivity of ipl1-2 can also be suppressed by null mutation of TOR1 or by administration of pharmacological TORC1 inhibitors, indicating that reduced TORC1 activity is responsible for the suppression. Suppression of the ipl1-2 growth defect is accompanied by increased fidelity of chromosome segregation and increased phosphorylation of the Ipl1 substrates histone H3 and Dam1. Nuclear Glc7 levels are reduced in a tco89 mutant, suggesting that TORC1 activity is required for the nuclear accumulation of Glc7. In addition, several mutant GLC7 alleles that suppress the temperature sensitivity of ipl1-2 exhibit negative synthetic genetic interactions with TORC1 mutants. Together, our results suggest that TORC1 positively regulates the Glc7 activity that opposes Ipl1 and provide a mechanism to tie nutritional status with mitotic regulation.

Fig. 1.

Reduction of TORC1 activity suppresses the temperature sensitivity of ipl1-2. (A) Cultures of WT (KT1113), YPI1-GFP tco89-71 (KT2998), ipl1-2 (KT1829), ipl1-2 YPI1-GFP (KT3007), ipl1-2 tco89-71 (KT3003), and ipl1-2 YPI1-GFP tco89-71 (KT3000) strains were serially diluted onto medium containing 1% yeast extract, 2% peptone, 2% glucose (YPD) and imaged after 40 h at the indicated temperatures. (B) Cultures of WT (KT1113), ipl1-2 (KT1829), ipl1-2 glc8Δ (KT3005), ipl1-2 tco89-71 (KT3003), ipl1-2 glc8Δ tco89-71 (KT3004), ipl1-2 YPI1-GFP tco89-71 (KT3007), ipl1-2 tor1Δ (KT3010), and ipl1-2 tor1Δ YPI1-GFP (KT3011) strains were treated as in A. (C) Cultures of WT (KT1113), ipl1-2 (KT1829), and ipl1-2 YPI1-GFP (KT3007) strains were serially diluted onto YPD medium and YPD containing 2.5 mM caffeine and treated as in A. (D) Diluted cultures of ipl1-2 (KT1829) were plated onto YPD medium, overlaid with a 7.5-mm paper disk containing 0.1 nM rapamycin or 2.5 μM caffeine, and incubated at the designated temperatures for 48 h (caffeine) or 72 h (rapamycin) before imaging. (Scale bar: 10 mm.) (Insets) Microscopic images were acquired from the designated locations on the plates after 72 h. (Scale bar: 200 μm.)

Fig. 2.

The tco89-71 mutation suppresses the chromosome loss defect due to ipl1-2. (A) WT (KT113 × KT1963), ipl1-2 (KT1963 × KT2941), and ipl1-2 tco89-71 (KT2941 × KT3002) diploid strains were grown to log phase in YPD medium at 24 °C, shifted to 30 °C for the designated times, and assayed for cell viability (Upper) and mating ability (Lower) at each time point. (B) Distribution of GFP-tagged chromosome IV in wild-type (KT2088), ipl1-2 (KT3031), and ipl1-2 tco89-71 (KT3030) cells after incubation for 2 and 4 h at 30 °C. Cells were grouped into those with a normal fluorescent pattern and those with an abnormal pattern representing loss or missegregation of chromosome IV or chromosome XV, which contains the GFP-tagged lacI gene.

Fig. 3.

The tco89-71 mutation partially restores phosphorylation of Ipl1 substrates. (A) Immunoblot analysis of WT (KT1113), glc7-127 (KT1969), glc7-127 ipl1-2 (KT1968), ipl1-2 (KT1829), tco89-71 ipl1-2 (KT3003), and tco89-71 (KT2959) extracts with phospho-specific antibody to histone H3 serine 10 and with antibody to total histone H3. (B) Dam1 phosphorylation on Ser257 of WT (KT1113), glc7-127 (KT1969), glc7-127 ipl1-2 (KT1968), ipl1-2 (KT1829), tco89-71 ipl1-2 (KT3003), and tco89-71 (KT2959) extracts was examined by immunoblotting with anti-S257 P and the overall level of Dam1 was examined using anti-Dam1. The ratio of the signal from the anti-S257 P antibody to the anti-Dam1 antibody is presented below each lane in A and B. (C) The fold increase in phosphorylation of H3 Ser10 and Dam1 Ser257 in extracts from ipl1-2 tco89-71 and ipl1-2 glc7-127 strains over extracts from ipl1-2 strains. The results represent the average of three separate experiments for histone H3 and four separate experiments for Dam1. Error bars, SD.

Fig. 4.

Correlation between suppression of ipl-2 and sensitivity to TORC1 depletion. (A) Cultures of WT (KT1112), ipl1-2 (KT1963), ipl1-2 tco89-71 (KT3002), ipl1-2 glc7-129, (KT3076), ipl1-2 glc7-127 (KT1965), and ipl1-2 glc7-109 (KT1964) strains were serially diluted onto YPD medium and imaged after 40 h at the indicated temperatures. (B) Cultures of WT (KT1112), tco89-71 (KT2958), glc7-129 (KT1774), glc7-127 (KT1967), and glc7-109 (KT1966) strains were serially diluted onto YPD medium containing 5 nM rapamycin or 5 mM caffeine and imaged as in A.

Fig. 5.

Tco89 is required for the normal nuclear localization of Glc7. (A) Subcellular distribution of Glc7-mCitrine in WT (KT3252), tco89-71 (KT3245), YPI1-GFP (KT3247), and tco89-71 YPI1-GFP (KT3250) strains at log phase in YPD medium grown at 30 °C. WT and mutant cells were imaged in the same field and distinguished by Pom34-mCherry fluorescence in the mutant cells. (Top panels) Fluorescence images of Pom34-mCherry overlaid on differential interference contrast (DIC) images. (Bottom and Middle panels) Fluorescence images of Glc7-mCitrine. The images in the lowest panel are displayed using a six-shade look-up table to better distinguish fluorescence levels between WT and mutant cells. (Scale bar: 5 μm.) (B) Quantitative analysis of nuclear and cytoplasmic Glc7-mCitrine fluorescence of cells (n ≥ 67 cells) imaged in A. The ratio of nuclear-to-cytoplasmic fluorescence is presented for each panel in A. Error bars, SD. Significantly different levels from WT, *P < 10⁻²⁰, according to the unpaired Student's t test. (C) Immunoblot analysis with anti-GFP antibody of extracts from strains imaged in A. Glc7-mCitrine levels are relative to the loading control (Pgk1) and normalized to the WT. (D) Quantitative analysis of nuclear and cytoplasmic Glc7-mCitrine fluorescence in WT cells (KT3242) after treatment with 10 mM caffeine or 10 nM rapamycin. n ≥ 50 cells. Error bars, SD. Significantly different levels from YPD-grown control, *P < 10⁻¹⁹, according to the unpaired Student's t test.