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. 2014 Apr 1;25(7):1137–1150. doi: 10.1091/mbc.E13-11-0689

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© 2014 Muñoz et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — Rgf1p accumulates in the nucleus during HU-induced replication stress. (A) Asynchronous cultures of exponentially growing cells expressing a functional Rgf1p-GFP from its endogenous promoter (PG40) were treated for 2 h with HU (12.5 mM) at 28ºC and then released from the drug for another 1 h. Rgf1p localization was examined before and 2 h after the addition of HU (top), and the number of cells with nuclear Rgf1p was quantitated in live cells during and after treatment (bottom). Two hundred cells were analyzed per time point. (B) The NLS is required for the nuclear accumulation of Rgf1p in HU. Red amino acids were mutated to alanines to test the functionality of the NLS sequence. The intracellular localization of the wild-type Rgf1p, Rgf1pΔN-GFP, and Rgf1p-NLS*-GFP was analyzed in log-phase cells treated with 12.5 mM HU for 2 h at 28ºC. (C) The mutation in NES1 and the crm1-809 mutation cause the nuclear accumulation of Rgf1p-GFP in the absence of HU. Red amino acids were mutated to alanines to test the functionality of NES sequences. Photos for wild-type Rgf1p-GFP, Rgf1p-NES1*-GFP, and Rgf1p-GFP in crm1-809 exponentially growing cultures are shown, and quantification of the number of cells containing nucleus-accumulated Rgf1p was obtained as means of three independent experiments. Bar, 10 μM.