Xpo1p-dependent targeting of Mad1p to kinetochores. (A) MAD1-GFP and MTW1-RFP in nup60Δ strains containing an otherwise WT genotype (Y3057) or the mutant alleles kap95-14 (Y3161), msn5Δ (Y3116), xpo1-T539C (Y3156), or prp20-7 (Y3091) were examined under the indicated conditions of temperature or LMB addition and after nocodazole addition. The localization of Mad1-GFP and the kinetochore protein Mtw1-RFP was examined by confocal microscopy. Note that as described previously (Gillett et al., 2004), some nocodazole-treated cells contain two kinetochore foci, with one lacking attached microtubules and being capable of binding Mad1p. Arrows point to overlapping Mad1-GFP and Mtw1-RFP foci. Arrowheads point to kinetochores devoid of Mad1-GFP. (B) Quantification of Mad1-GFP and Mtw1-RFP colocalization from the experiments presented in A. The results of three experiments were combined, and SD (error bars) is shown in each case. Bars, 2 μm.

Xpo1p associates with spindle pole bodies in logarithmically growing cells and redistributes to kinetochores during SAC arrest. (A–D) Cells synthesizing Xpo1-GFP and Mtw1-RFP (Y3109) or Xpo1-GFP and Spc42-RFP (Y3101) were analyzed by epifluorescence microscopy after either logarithmic growth (A and B) or nocodazole-induced arrest (C and D). Note that the insets in B show a G1 cell. (E and F) A cdc26Δ mutant was introduced into Y3109 to produce Y3131. Cultures of these cells were shifted to 37°C, and the localization of Mtw1-RFP and Xpo1-GFP was assessed in the absence (E) and presence (F) of nocodazole. The arrows in C and F highlight colocalization of Xpo1-GFP and Mtw1-RFP, and the arrowheads in A and E point to kinetochores lacking Xpo1-GFP signal. The arrowheads in D indicate spindle poles devoid of Xpo1-GFP signal. (G) Quantification of Xpo1-GFP colocalization with either Spc42-RFP or Mtw1-RFP from the experiments presented in A–D. 30 cells were analyzed for each condition in each of three independent experiments, and results are presented as a mean with SD (error bars). (H) ChIP analysis was performed on cells producing Xpo1-GFP (Y3169) growing logarithmically (asynchronous [AS]) or arrested in nocodazole (+Noc). T, total chromatin used in each IP; M, mock minus antibody IP; α-GFP, anti-GFP antibody IP. Bars, 2 μm.

Mad1p contains a functional Xpo1p-dependent NES. (A) Mad1-GFP was visualized by fluorescence microscopy in WT (Y3028, i) or nup60Δ cells (Y3040, ii and iii; Y3057, iv). Mad1-GFP intranuclear Mlp1p/Mlp2p foci in the absence of Nup60p (ii). Treatment of nup60Δ cells with nocodazole induces recruitment of Mad1-GFP to kinetochores (iii) as conferred by colocalization with Mtw1-RFP (iv). (B) An alignment of the predicted NES in Mad1p with the NESs of HIV Rev and rabbit PKI is shown. Residues required for NES function in HIV Rev and PKI are boxed together with leucine residues in the Mad1p NES (asterisks). (C) Plasmids encoding SV40-NLS/PKI-NES-GFP and SV40-NLS/MAD1^563–576-GFP₃ were introduced into a strain expressing xpo1-T539C (Y3105 + pKW711). The localization of these reporters was examined by fluorescence microscopy before or 30 min after the addition of LMB. (D) SV40-NLS/MAD1^563–576-GFP₃ was introduced into the xpo1-1 Ts strain (Y3105 + pKW457) and observed at the permissive (23°C) or restrictive temperature (30 min at 37°C). Bars: (A) 2 μm; (C and D) 5 μm.

Inactivation of the Mad1p NES dramatically reduces targeting of Mad1p to kinetochores. (A) The cellular distribution of SV40-NLS/MAD1^563-576-GFP₃ or SV40-NLS/mad1-nes^L569A-GFP₃ containing the Mad1p NES mutant in WT cells was examined by epifluorescence microscopy. (B) nup60Δ cells expressing MAD1-GFP (Y3151) or mad1^L569A-GFP (Y3154) and MTW1-RFP were arrested with nocodazole and examined by epifluorescence microscopy. The arrow points to Mad1-GFP overlap with Mtw1-RFP. The arrowhead points to kinetochores devoid of Mad1-GFP. (C) Quantification of Mad1-GFP and Mtw1-RFP colocalization from the experiments presented in B. 100 cells were analyzed at random for colocalization between Mad1-GFP and Mtw1-RFP, and the results of three experiments were combined. SD (error bars) is shown in each case. (D) ChIP was performed on cells expressing MAD1-GFP (Y3028) or mad1^L569A-GFP (Y3170) and growing logarithmically (asynchronous [AS]) or arrested in nocodazole (+Noc). (E) The indicated strains (WT [Y3162], mad1Δ [Y3165], mad1^L569A [Y3164], ndc10-1 [3166], mad1^L569Andc10-1 [Y3167], and mad1Δ ndc10-1 [Y3168]) were grown on YPD plates containing DMSO or DMSO plus benomyl for 2 or 4 d (bottom right). T, total chromatin used in each IP; M, mock minus antibody IP; α-GFP, anti-GFP antibody IP. Bars: (A) 5 μm; (B) 2 μm.

Mutations in Xpo1p, Ran, and Prp20p abrogate the turnover of Mad1p on kinetochores during SAC arrest. (A and B) MAD1-GFP and MTW1-RFP in nup60Δ strains containing an otherwise WT genotype (Y3057) or the mutant alleles kap95-14 (Y3161), msn5Δ (Y3116), or xpo1-T539C (Y3156; A) and prp20-7 (Y3091) or gsp1-G21V (Y3096; B) were arrested in nocodazole-containing media. The function of the mutant allele was inhibited by shifting to a nonpermissive growth condition (37°C or +LMB), or, in the case of gsp1-G21V, its expression was induced by the addition of galactose. WT and msn5Δ cells were examined directly after nocodazole arrest. Kinetochore-associated Mad1-GFP (prebleach; 0⁻ s) was identified by colocalization with Mtw1-RFP (not depicted). Kinetochore-associated Mad1-GFP was photobleached, and recovery was monitored by acquiring images immediately after bleaching (postbleach; 0⁺ s) and at 15-s intervals. Images from 30-s intervals are shown. The arrowheads point to bleached kinetochores (0⁻ s). Bars, 1 μm.