In Saccharomyces cerevisiae, the PHO pathway regulates expression of phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase (rAPase). In this pathway, Pho81p functions as an inhibitor of the cyclin-cyclin-dependent kinase (CDK) complex Pho80p-Pho85p. However, the mechanism regulating the inhibitory activity of Pho81p is poorly understood. Through use of the yeast two-hybrid system, we identified the UbL-UbA protein Ddi1p as a Pho81p-binding protein. Further, Pho81p levels were found to be low under high-phosphate condition and high during phosphate starvation, indicating that Pho81p is regulated by phosphate concentration. However, our results revealed that Ddilp and its associated protein Rad23p are not involved in the decrease in Pho81p level under high-phosphate condition. Significantly, the Delta ddi1 Delta rad23 strain exhibited a remarkable increase in rAPase activity at an intermediate-phosphate concentration of 0.4 mM, suggesting that Ddilp and Rad23p play a cooperative role as negative regulators in the PHO pathway. (C) 2007 Elsevier Inc. All rights reserved.