Phosphorylation
of YLR190w by PAP1 PHO85 Kinase Complex
SHI Xiao-Zhong, AO Shi-Zhou*
( State Key Laboratory of Molecular Biology, Institute of Biochemistry
and Cell Biology, Shanghai Institutes for Biological Sciences, the
Chinese Academy of Sciences, Shanghai 200031, China )
Abstract 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 strainw as longer than
that of wild type.
Key words PAP1；PHO85；phosphorylation；yeast two-hybrid system；YLR190w
*Corresponding author: Tel,
86-21-64374430-5256; Fax, 86-21-64338357;
e-mail, [email protected]