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.