Phosphorylation Of Ylr190w And Yjl084c By Pcl7-Pho85 Kinase Complex

In S.cerevisiae, PHO85 was originally discovered because of its function in PHO system. When complexed with the cyclin Pho80, Pho85 phorphorylates and negatively regualtes Pho4. Pho85 has mutliple roles, incuding cell cycle, hyperaccumulation of glycogen, starvation-induced autophagy, and et al. The pleiotropic nature of Pho85 function has been ascribed to its association with multiple cyclin partners. So far, ten Pcls (Pho85 cylin) have been identified: the Pcl1/2 subfamiliy of Pcl1, Pcl2, Clg1, Pcl5, and Pcl9; and the Pho80 subfamily of Pho80, Pcl6, Pcl7, Pcl8,and Pcl10. Pcl7 (PAP1) was also isolated independent in our lab, and its function was explored primarily.The major work of this thesis is focused on identifying of the putative substrates of Pcl7-Pho85, their phosphorylation, and the significance of the phosphorylation. Ylr190w was identified as a Pcl7-binding protein in a yeast two-hybrid screening with Pcl7 as the bait. Also, Yjl084c was found to be a Pcl7-associated protein by homology analysis and database search. The interaction between Pcl7 and Ylr190w/Yjl084c was confirmed in vivo and in vitro, by two-hybrid assay or co-immunoprecipitation and GST pull-down assay respectively. Pcl7-Pho85 could phosphorylate GST-Ylr190w expressed in E.coli, Yjl084c translated invitro, and the GST fusion of Yjl084c middle fragment expressed E.coli. Their phosphorylations were affected by the phosphate concentration. Further, all of Pcl6, Pcl7, and Pho80, were found to interact with Pho81.Two mutants were constructed: Ylr190w (T12A), the Thr(12) was mutated to Ala; and Ylr190w(S/T-A), all Ser/Thr in seven Ser/Thr-Pro motif were mutated to Ala. Ylr190w(T12A) could be phosphorylated, but Ylr190w(S/T-A) not. It can be concluded that the phosphorylation site of Pcl7-Pho85 was Ser/Thr-Pro motif.Ynl107w (Yaf9), a yeast protein similar to human AF9, was isolated in the second library screen with Ylr190w as the bait. The interaction of Ylr190w and Ynl107w was affected by phosphate concentration; but that of Ylr190w (S/T-A) and Ynl107w was not.The deletion of YLR190w or YJL084c was constructed by homology recombination. It was found that: in ylr190w null mutant strain, the doubling time was prolonged, G2-M phase was reduced, and G0-G1 and S phase were increased; no significant effect was observed in yjl084c null mutant strain.