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. 2009 Jan 12;184(1):21–29. doi: 10.1083/jcb.200804098

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© 2009 Scott et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 5. — 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.