Using Yeast Two-Hybrid Screening and Structural Modeling to Identify Candidate Hrr25 Kinase Interactors at the Meiotic Kinetochore in Saccharomyces cerevisiae

In Saccharomyces cerevisiae (S. cerevisiae), sister kinetochores are mono-oriented during meiosis I to ensure accurate homolog segregation, a process dependent on Hrr25 kinase activity. However, its direct interactors remain poorly defined. To address this, we performed a yeast two-hybrid (Y2H) screen using Hrr25 as bait. HRR25 was cloned into a Y2H vector and functionally validated by complementation of a temperature-sensitive hrr25-ts mutant. Screening across three reading frames identified three putative interactors: Hed1, Cyr1, and Rep1. Additional open reading frames (ORFs), including DAD1, SYS1, and YDR015C were identified but were oppositely oriented to the GAL4 activation domain. Structural modeling and phosphorylation prediction identified high-confidence Hrr25 target residues, including S70/T73 on Hed1, S323 on Rep1, and S198/S527 on Cyr1, whereas Sys1 and YDR015C lacked favorable sites. Although Dad1 was not validated as a direct interactor from Y2H, S63 was identified as a favorable phosphorylation site, and its full-length ORF in the interacting clone and known biological role supported its inclusion. Among the meiotic candidates, Hed1 may link Hrr25 activity to homologous recombination, while Dad1 represents a plausible target for regulating kinetochore–microtubule interactions. Collectively, these findings identify new candidate interactors and substrates of Hrr25 and suggest a broader role in coordinating recombination and kinetochore function during meiosis, warranting further experimental validation.