Truncation mutants of Mad1p. (A) schematic of N- and C-terminal truncations of Mad1p is shown. Amino acid residues are shown on the left. The SMART domain annotation resource (Schultz et al., 1998) predicts Mad1p contains three coiled-coil domains high-lighted in black with bordering residues indicated. The amino acid sequence of the Mad1p NLS and its position (hatched region) are shown.

In vivo localization of Mad1p truncation mutants. (A) The ORF encoding GFP was integrated after specific codons within the endogenous MAD1 gene in the strain BY4741 to produce chimeric ORFs encoding full-length Mad1-GFP (Y3028), 1-325-GFP (Y3029), and 1-250-GFP (Y3030). GFP-tagged N-terminal deletions of MAD1 encoding the truncation 250-325-GFP (pMad1-250-325-GFP), 318-749-GFP (pMad1318-749-GFP), or 475-749-GFP (pMad1-475-749-GFP) in a CEN/URA3 plasmid were introduced into mad1Δ cells (Y3021). Strains were grown to mid-log phase at 30°C and viewed directly (asynchronous [AS] cultures) or arrested in G₂/M by treatment with 12.5 μg/ml nocodazole (NOC) for ∼2 h and then examined. Arrows indicate intranuclear foci and arrowheads indicate approximate nuclear position in representative cells. Fluorescent images were captured using a confocal microscope. Note, in addition to a diffuse nuclear signal, foci of 318-749-GFP and 475-749-GFP signal colocalized with the kinetochore marker Mtw1-RFP in 13 and 61% of asynchronous cells and 62 and 83% of nocodazole arrested cells, respectively. (B) Cells producing the tagged kinetochore protein Mtw1-RFP (Y3031) and either 318-749-GFP or 475-749-GFP were arrested in G₁ phase using α-factor or in G₂/M phase by treatment with 12.5 μg/ml NOC. Images of the two fluorescent proteins were obtained as in A. Merged images are shown on the right. Bars, 5 μm.

Mad1p contains a functional NLS. (A) The nucleotides encoding amino-acid residues 499-533 of Mad1p were inserted upstream of the ORFs encoding three tandemly repeated GFP in the plasmid pMad1-NLS-GFP₃ (Mad1-NLS-GFP₃) and introduced into wild-type yeast cells (BY4741). The localization of Mad1-NLS-GFP₃ was then visualized using a confocal microscope before (untreated) and after treatment with the metabolic poisons 2-deoxy-d-glucose and sodium azide (+2DG/N₃). (B) Import of the Mad1p NLS requires Kap95p. pMad1-NLS-GFP₃ plasmid was introduced into a wild-type strain (DF5) and the temperature-sensitive karyopherin mutants kap121-34 and kap95-14. Localization of the NLS was examined at 23°C and 2.5 h after shifting cells to 37°C. (C) The NLS of Mad1p is required for the efficient localization of Mad1p to the NPC. Plasmids harboring MAD1-GFP (pMad1-GFP; +NLS) or mad1ΔNLS-GFP (pmad1ΔNLS-GFP; -NLS) were introduced into a mad1Δ strain (Y3021) and the localization of the fusion proteins was visualized with a confocal microscope. (D) Kap95p is required for the efficient localization of Mad1p to the NPC. A plasmid encoding Mad1-GFP was introduced into mad1Δ (YMB1911) and mad1Δkap94-14 (Y3032) strains. Localization of Mad1-GFP was compared in these two strains at 23°C and after cells were shifted to 37°C for 2.5 h. Bars, 5 μm.

Nup53p binds directly to Mad1p. (A) The recombinant proteins GST, GST-Mad1p, GST-1-325, or GST-318-749 were synthesized in E. coli (BL21) and purified using glutathione-Sepharose beads. The bead-bound proteins were incubated with (+) or without (-) purified, recombinant Nup53p (L). After this, the unbound fraction was collected, the beads were washed, and the bound fraction was eluted with SDS-PAGE sample buffer. Proteins present in equivalent proportions of the bound and unbound fractions were resolved using SDS-PAGE and visualized with Coomassie Blue staining. Molecular mass markers in kilodaltons are shown on the left. (B) Binding experiments were conducted using GST (our unpublished data), GST-1-325 or GST-475-749, and Nup53p as described in A). Note, GST-475-749 failed to bind Nup53p and was present in the unbound fraction. The lack of Nup53p in the bound fractions was only visible after extended periods of electrophoresis when the mobility of Nup53p is sufficiently different from that of GST-475-749 protein.

The localization of Mad1-GFP is altered in mutants of nup60Δ, mlp1Δ, and mlp2Δ. (A) The GFP ORF was integrated after the last amino acid codon of the chromosomal copy of MAD1 in haploid strains containing null mutations in NUP53 (nup53Δ), POM34 (pom34Δ), NUP2 (nup2Δ), NUP60 (nup60Δ), MLP1 (mlp1Δ), MLP2 (mlp2Δ), and the MLP1 MLP2 double mutant (mlp1Δmlp2Δ) to produce the strains Y3067, Y3037, Y3036, Y3040, Y3065, Y3066, and Y3061, respectively. Logarithmically growing cultures of these strains and an isogenic wild-type counterpart (Y3028) were examined by fluorescence microscopy, and images were acquired using a confocal microscope. (B) The localization of Mad1-GFP was examined in the nup60Δ strain. Logarithmically growing asynchronous (AS) nup60Δ cells expressing genomically integrated MAD1-GFP and MTW1-RFP (Y3057) were directly visualized. Note, RFP and GFP foci did not colocalize. Y3057 cells also were synchronized in G₁ with α-factor and then released into media containing 12.5 μg/ml nocodazole at 25°C. Cells were examined 60 min after addition of nocodazole. Arrows in the merged panel point to foci where Mtw1-RFP colocalizes with Mad1p-GFP. In cells containing two GFP and two RFP foci, colocalization of two foci is observed in 94% of cells. (C) In asynchronous cell cultures of the nup60Δ mutant, Mad1p colocalizes with Mlp2p. Logarithmically growing nup60Δ cells expressing genomically integrated MAD1-GFP and MLP2-RFP (Y3062) were visualized using fluorescence microscopy. Arrows indicate colocalization of Mad1-GFP with Mlp2-RFP in the merged panel. (D) Localization of Mad1-GFP in a mlp1Δmlp2Δ mutant. The plasmid pMad1-GFP was introduced into an mlp1Δmlp2Δ double mutant containing a genomically integrated MTW1-RFP (Y3064). Mad1-GFP and Mtw1-RFP were visualized in this background in both AS and in cells treated with 12.5 μg/ml nocodazole for 2 h. Bars, 5 μm.

Mad1p dynamically associates with nocodazole treated kinetochores and requires energy for this association. (A) The strain Y3057 was arrested in G₁ phase with α-factor and released into media containing 12.5 μg/ml nocodazole at 25°C. After 60 min, cells were harvested and placed on agarose slides. The kinetochore-associated Mad1-GFP was identified based on colocalization with Mtw1-RFP (prebleach). Kinetochore Mad1-GFP was bleached by applying 50 iterations of full intensity 488-nm light. GFP images only were acquired every 12 s. At the end of the experiment, Mtw1-RFP was once again visualized (8′). Arrows point to the bleached focus. Note, bleached Mtw1-RFP did not recover during the time course of the experiments. Bar, 2 μm. (B) Plots of the integrated fluorescent intensities at both kinetochore (circles) and Mlp protein foci (squares) are shown on the left, and the natural log plot of kinetochore recovery against time are shown on the right. The normalized fluorescent recovery of Mad1-GFP at kinetochores fit a single exponential function. The natural log plot was used to calculate the first order rate constant, k, and the t_1/2 of Mad1-GFP recovery. (C) Y3057 cells were arrested in nocodazole containing medium (12.5 μg/ml) for 60 min. The culture was then split and harvested cells were suspended in media containing nocodazole and glucose (Noc + glucose) or with nocodazole, 2-deoxy-d-glucose and sodium azide (Noc + 2DG/N₃) for 10 min. Images were collected using a confocal microscope. Bar, 5 μm.

The C terminus of Mad1p is required for SAC function. Plasmids encoding Mad1p or the indicated truncations, either alone (A) or as fusions with GFP (B), were introduced into the mad1Δ strain Y3021. After overnight growth in liquid cultures, equal amounts of cells were serially diluted 10-fold onto YPD plates containing vehicle alone (dimethyl sulfoxide) or 12.5 μg/ml benomyl and grown at 25°C for 2-3 d. The growth characteristics of these strains were compared with wild-type (WT) cells (BY4741) and the mad1Δ strain containing the empty vector pYEX-Bx (A) or pRS316 (B). The ΔNLS constructs lack the coding region for amino acid residues 506-527 of Mad1p.

Termination of the SAC is delayed in nup53Δ cells. (A) WT (BY4741) cells and the mutant strains nup53Δ (CPL32), nup59Δ (Y3068), nup60Δ (3058), nup53Δ + pNUP53 (CPL32 containing pNUP53), and nup53Δ + pNUP53ΔKBD (CPL32 containing pNUP53ΔKBD) were synchronized in G₁ phase using α-factor and released into media containing 12.5 μg/ml nocodazole. At the indicated time points, cells were fixed with 70% ethanol and stained with DAPI to visualize nuclei and monitor progression from metaphase to telophase. The number of large budded (G₂/M) cells containing two DAPI-staining bodies was scored and is presented graphically as a percentage of total cells verses time (minutes) after release into nocodazole-containing media. (B) WT (CPL61), nup53Δ (CPL62), nup59Δ (CPL63), and mad1Δ (CPL64) strains containing an extra copy of the MPS1 ORF chromosomally integrated behind a galactose-inducible promoter were grown in media containing 2% raffinose. The cells were synchronized in G₁ phase with α-factor at 30°C and released into media containing 2% raffinose and 3% galactose in the presence of α-factor to induce the synthesis of excess Mps1p. After 2 h, cells were released from G₁ and G₂/M-phase arrest was monitored by scoring the number of large budded cells at the indicated time points (minutes). These numbers are shown graphically as a percentage of total cells versus time.