[Phosphorylation of YLR190w by PAP1 PHO85 kinase complex]

A yeast two-hybrid screening by using PAP1 was performed to identify targets for PAP1-PHO85 cyclin-CDK complex. N-terminal fragment of protein YLR190w, a yeast gene encoding a 491 amino acids peptide, was identified, and its coding region was amplified by PCR. The interaction of PAP1 and YLR190w was confirmed by both two-hybrid assay and GST pull-down assay in vitro. The PAP1-PHO85 kinase complex obtained from the immunoprecipitates could phosphorylate GST-YLR190w expressed in E.coli, and the phosphorylation of YLR190w was affected by the phosphate concentration, and the phosphorylation sites of YLR190w were Ser/Thr-Promotif, as revealed by protein mutation assay. In another library screen, YAF9, a yeast homolog of human AF9, was isolated using the two-hybrid system with YLR190w as the bait. It was revealed that interaction of YLR190w and YAF9 was affected by phosphate concentration. When all Ser/Thr in Ser/Thr-Pro motif were mutated to Ala, the interaction of YLR190w (mutant) and YAF9 was weakened, and the effect of phosphate concentration was impaired. Ylr190w was not involved in the PHO system by the acid phosphatase activity assay. Deletion of Ylr190w was constructed by homologous recombination and the doubling time of Ylr190w mutant strain as longer than that of wild type.