DNA damage response-mediated degradation of Ho endonuclease via the ubiquitin system involves its nuclear export.
Yeast mating switch Ho endonuclease is rapidly degraded by the ubiquitin system and this depends on the DNA damage response functions, MEC1, RAD9, and CHK1. A PEST sequence marks Ho for degradation. Here we show that the novel F-box receptor, Ufo1, recruits phosphorylated Ho for degradation. Mutation of PEST residue threonine 225 stabilizes Ho, yet HoT225A still binds Ufo1 in vitro. Stable HoT225A accumulates within the nucleus, whereas HoT225E is degraded. Deletion of the nuclear exportin Msn5 traps native Ho in the nucleus and extends its half-life. These experiments suggest that Ho is degraded in the cytoplasm. In mec1 mutants stable Ho accumulates within the nucleus; Ho produced in mec1 cells does not bind Ufo1. Thus the MEC1 pathway has functions both in phosphorylation of Thr-225 for nuclear export and in additional phosphorylations for binding Ufo1. Cells with HO under its genomic promoter, but stabilized by deletion of the Msn5 exportin, proliferate, but are multibudded. These experiments elucidate some of the links between the DNA damage response and degradation of Ho by the ubiquitin system.
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D004249:DNA Damage; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*
D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific