Phosphorylation of PHO4 Protein by the PHO85-PAP1 Kinase Complex

Homology comparison of the novel PAP1 protein indicated that PAP1 protein is highly homologous to PHO85-associated protein PHO80, PCL1 and PCL2. We constructed the fused HA-PAP1 gene in frame for immunoprecipitation with anti-HA monoclonal antibody. Coimmunoprecipitation of fused HA-PAP1 and PHO85 protein, which were translated in vitro, verified the association of PAP1 with PHO85 protein. Simultaneously, we purified PHO4 protein expressed in E. coli and constructed a PHO85::TRP1 strain. After arranging the fused gene under the control of yeast ADH1 promoter, we transformed the resulting plasmids into yeast YPH499 and PHO85::TRP1 respectively, then prepared the yeast lysates for immunoprecipitation with anti-HA. We found that the immunoprecipitant complex of PAP1 can phosphorylate PHO4 protein in vitro, and the kinase activity is PHO85-associated, but is not related to CDC28 protein kinase. These data suggest that PAP1 is a putative cyclin and can form cyclin-CDK complex with PHO85 protein. Northern bolt with PAP1 gene as probe indicated there was no obvious difference during the different phase of the cell-cycle in the transcription of PAP1 gene. We constructed the PAP1::TRP1 strain, and the rAPase activity analysis showed PAP1 had no function in the PHO system.