RESUMEN
Applying real time BIA(biomolecular interaction analysis) the interaction between yeast PHO4 and PHO2 protein was analyzed. Recombinant PHO4 protein was coupled at the sensor chip via amino group. 5 &mgr;mol/L of recombinant PHO2 and PHO2 mutants which were fused with glutathione S-transferase were injected respectively. The mutant whose Ser 230 was changed into Asp (GST-2SD) showed high SPR (surface plasmon resonance) signal while wild type PHO2 and the mutant where Ser 230 was changed into Ala (GST-2SA) did not. This result indicated that there was interaction between PHO4 and GST-2SD proteins and the phosphorylation of the Ser 230 of PHO2 was essential. Five different concentration of GST-2SD (0.5 0.75 1.0 1.5 2.0&mgr;mol/L) were injected separately and the interaction kinetics was analyzed. The association rate constant is 2.4x10(4)(mol/L)(-1)s(-1) dissociation rate constant is 3.5x10(-5)s(-1) and association constant is 6.9x10(8) (mol/L)(-1).
RESUMEN
There are six ankyrin repeats in yeast transcriptional factor PHO81. The PHO81 ankyrin repeats fused with glutathione S-transferase (GST-ANK) was overexpressed in E. coli and it existed in inclusion body form. Using reduced and oxidized glutathione systems the fused protein fragment was denatured and renatured and the soluble protein was obtained. Then it was purified by affinity purification through glutathione sepharose column and its activity was analyzed. The PHO85-PHO80 and PHO85-PAP1 kinase complexes were prepared via coimmunoprecipitation respectively. With purified recombinant PHO4 protein as substrate of the kinase complexes when the purified GST-ANK was added the kinase activity of PHO85-PHO80 or PHO85-PAP1 kinase complex was inhibited. These studies indicate that the ankyrin repeats interact with PHO85-PHO80 or PHO85-PAP1 complex inhibit their kinase activities and are crucial in interaction between PHO81 and other proteins.
RESUMEN
PHO4 and PHO2 protein were overexpressed in E. coli and purified respectively. The gel retardation assays showed that PHO4 and PHO2 protein could bind to -401 -289 bp sequence of PHO81 gene promoter respectively. The assays of the mutant PHO2 proteins where the Ser-230 was changed into Asp (GST-2SD) or Ala (GST-2SA) showed the binding patterns were the same as the intact PHO2 protein. Footprinting results located the PHO4 binding site at the 354 -333 bp sequence of PHO81 gene PHO2 binding site at the -341 -334 bp. Therefore the region (-401 -289 bp) contains the upstream activation sequence (UAS) of PHO81 gene and PHO4 protein cooperates with PHO2 in acting on the upstream sequence of PHO81 gene and both of them regulate the expression of PHO81 gene.
RESUMEN
Deletion analysis on the fused PHO81-lacZ gene revealed two important regions in the upstream sequences of PHO81 gene -401 -289 bp and -1 012 -801 bp. They did not share higher similarity with the upstream regions of PHO5 and PHO84 gene, except that -401 -289 bp contains the 5'-CACGTG/T-3' motif, which was found among the upstream regions of PHO5 and PHO84 gene and was the core sequence of PHO4 binding site it also contains A/T-rich segments flanking the motif, which may be PHO2 binding sites. This suggests that the -401 -289 bp of the upstream region of PHO81 gene may contain upstream activation sequence (UAS), and -1 012 -801 bp may contain upstream enhancer sequence. The gel retardation assays of the -1 012 -801 bp was performed using yeast total protein extract, and the results showed that there was an unknown protein factor binding at the region.
RESUMEN
Yeast regulatory factor PHO85, which is a cyclin-dependent kinase (DCK), participates in the regulation of the cell cycle and the expression of the acid phosphatase gene. Using PHO85 as target, we have cloned from the yeast two-hybrid genomic library a novel gene (PAP1) with product which can asscociate with the PHO85 protein. The PAP1 gene including the 5apo; and 3apo; flanking sequence was cloned and sequenced. It codes for a protein composed of 284 amino acids, which contains a cyclin conservative region (150-263 aa), and shares 63% similarity with the conservative region of PHO80. The N-terminal of the PAP1 protein abounds in PEST sequence, which is believed to be common to rapidly degraded proteins. Using the yeast two-hybrid system, we demonstrated that both the full-length PAP1 protein and the C-terminal (99-285 aa) can interact with PHO85. The coding sequence of the PAP1 gene was cloned and sequenced, then arranged under the control of the P(L) promoter, and expressed in E. coli BL21 (DE3) pLysS. After heat induction at 42 degrees, protein band of approximately 32 kD was observed in the SDS-PAGE, which fits the molecular size of the hypothetical PAP1 protein. The 10 N-terminal amino acids of the 32 kD protein agreed with the deduced PAP1 amino acid sequence. The protein was found as inclusion body in E. coli, and accounted for 50% of the precipitant after purification.
RESUMEN
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.
