The checkpoint-dependent nuclear accumulation of Rho1p exchange factor Rgf1p is important for tolerance to chronic replication stress.
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.
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.
Sofía
S
Muñoz
Elvira
E
Manjón
Patricia
P
García
Per
P
Sunnerhagen
Yolanda
Y
Sánchez
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000222:Adaptation, Physiological / Q000187:drug effects; D000595:Amino Acid Sequence; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D017353:Gene Deletion; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006918:Hydroxyurea / Q000494:pharmacology; D050296:Microbial Viability / Q000187:drug effects; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D012568:Schizosaccharomyces / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism*; D013312:Stress, Physiological / Q000187:drug effects
D020662:Guanine Nucleotide Exchange Factors; D050505:Mutant Proteins; D019913:Nuclear Localization Signals; C511265:Rgf1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D006918:Hydroxyurea