title: Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast.
abstract: The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s), but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1's interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA) specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and Mph1 contributes to a specific long-range chromosomal interaction required for mating-type switching, but that Fkh1-FHA must also interact with several other proteins to achieve full functionality in this process.
authors.affiliation: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Antoinette M
authors.identifier: 0000-0001-9177-0104
authors.initials: AM
authors.lastname: Dummer
authors.affiliation: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Zhangli
authors.initials: Z
authors.lastname: Su
authors.affiliation: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Rachel
authors.initials: R
authors.lastname: Cherney
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
authors.forename: Koyi
authors.initials: K
authors.lastname: Choi
authors.affiliation: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.|Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Denu
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Fox
mesh_terms: D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D049770:Genes, Mating Type, Fungal / Q000235:genetics*; D010748:Phosphopeptides / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D004718:Saccharomycetales / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; C464906:Fkh1 protein, S cerevisiae; D051858:Forkhead Transcription Factors; D010748:Phosphopeptides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D010769:Phosphothreonine; C491240:MPH1 protein, S cerevisiae; D004265:DNA Helicases; D053487:DEAD-box RNA Helicases