MOLECULAR AND EXPRESSION ANALYSIS OF THE NEGATIVE REGULATORS INVOLVED IN THE TRANSCRIPTIONAL REGULATION OF ACID-PHOSPHATASE PRODUCTION IN SACCHAROMYCES-CEREVISIAE

The PHO80 and PHO85 gene products encode proteins necessary for the repression of transcription from the major acid phosphatase gene (PHO5) of Saccharomyces cerevisiae. The deduced amino acid sequences of these genes have revealed that PHO85 is likely to encode a protein kinase, whereas no potential function has been revealed for PHO80. We undertook several approaches to aid in the elucidation of the PHO80 function, including deletion analysis, chemical mutagenesis, and expression analysis. DNA deletion analysis revealed that residues from both the carboxy- and amino-terminal regions of the protein, amounting to a total of 21% of the PHO80 protein, were not required for function with respect to repressor activity. Also, 10 independent single-amino-acid changes within PHO80 which resulted in the failure to repress PHO5 transcription were isolated. Nine of the 10 missense mutations resided in two subregions of the PHO80 molecule. In addition, expression analysis of the PHO80 and PHO85 genes suggested that the PHO85 gene product was not necessary for PHO80 expression and that the PHO85 gene was expressed at much higher levels in the cell than was the PHO80 gene. Furthermore, high levels of PHO80 were shown to suppress the effect of a PHO85 deletion at a level close to full repression. Implications for the function of the negative regulators in this system are discussed.