Muñoz Sofía, Manjón Elvira, García Patricia, Sunnerhagen Per, Sánchez Yolanda
Instituto de Biología Funcional y Genómica and Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, 37008 Salamanca, Spain Department of Chemistry and Molecular Biology, Lundberg Laboratory, University of Gothenburg, S-405 30 Gothenburg, Sweden.
Mol Biol Cell. 2014 Apr;25(7):1137-50. doi: 10.1091/mbc.E13-11-0689. Epub 2014 Jan 29.
Guanine nucleotide exchange factors control many aspects of cell morphogenesis by turning on Rho-GTPases. The fission yeast exchange factor Rgf1p (Rho gef1) specifically regulates Rho1p during polarized growth and localizes to cortical sites. Here we report that Rgf1p is relocalized to the cell nucleus during the stalled replication caused by hydroxyurea (HU). Import to the nucleus is mediated by a nuclear localization sequence at the N-terminus of Rgf1p, whereas release into the cytoplasm requires two leucine-rich nuclear export sequences at the C-terminus. Moreover, Rgf1p nuclear accumulation during replication arrest depends on the 14-3-3 chaperone Rad24p and the DNA replication checkpoint kinase Cds1p. Both proteins control the nuclear accumulation of Rgf1p by inhibition of its nuclear export. A mutant, Rgf1p-9A, that substitutes nine serine potential phosphorylation Cds1p sites for alanine fails to accumulate in the nucleus in response to replication stress, and this correlates with a severe defect in survival in the presence of HU. In conclusion, we propose that the regulation of Rgf1p could be part of the mechanism by which Cds1p and Rad24p promote survival in the presence of chronic replication stress. It will be of general interest to understand whether the same is true for homologues of Rgf1p in budding yeast and higher eukaryotes.
鸟嘌呤核苷酸交换因子通过激活Rho-GTPases来控制细胞形态发生的多个方面。裂殖酵母交换因子Rgf1p（Rho gef1）在极性生长过程中特异性调节Rho1p，并定位于皮质位点。在这里，我们报告Rgf1p在羟基脲（HU）引起的复制停滞期间重新定位于细胞核。导入细胞核是由Rgf1p N端的核定位序列介导的，而释放到细胞质中则需要C端的两个富含亮氨酸的核输出序列。此外，复制停滞期间Rgf1p的核积累依赖于14-3-3伴侣蛋白Rad24p和DNA复制检查点激酶Cds1p。这两种蛋白都通过抑制Rgf1p的核输出控制其核积累。一个将九个潜在的丝氨酸磷酸化Cds1p位点替换为丙氨酸的突变体Rgf1p-9A，在复制应激时无法在细胞核中积累，这与在HU存在下的严重生存缺陷相关。总之，我们提出Rgf1p的调节可能是Cds1p和Rad24p在慢性复制应激存在下促进生存的机制的一部分。了解芽殖酵母和高等真核生物中Rgf1p的同源物是否也是如此将具有普遍意义。
